Advertisements
Feeds:
Posts
Comments

Posts Tagged ‘Howard Hughes Medical Institute’


Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Writer and Curator: Larry H Bernstein, MD, FCAP
and
Curator and Content Editor: Aviva Lev-Ari, PhD, RN

This article is Part V in a series of TWELVE articles, listed at the end of this article,  on the

  1. cytoskeleton,
  2. calcium calmodulin kinase signaling,
  3. muscle and nerve transduction, and
  4. calcium,
  5. Na+-K+-ATPase,
  6. neurohumoral activity and vesicles vital and essential for all functions related to
  • cell movement,
  • migration, and
  • contraction.

Calmodulin and Protein Kinase C Increase Ca–stimulated Secretion by Modulating
Membrane-attached Exocytic Machinery

YA Chen, V Duvvuri, H Schulmani, and RH.Scheller‡
From the ‡Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology,
and the Department of Neurobiology, Stanford University School of Medicine, Stanford, CA

The molecular mechanisms underlying the Ca2+ regulation of hormone and neurotransmitter release
are largely unknown.

Using a reconstituted [3H]norepinephrine release assay in permeabilized PC12 cells, we found

  • essential proteins that support the triggering stage of Ca2+-stimulated exocytosis
  • are enriched in an EGTA extract of brain membranes.
Fractionation of this extract allowed purification of two factors that stimulate secretion
  • in the absence of any other cytosolic proteins.
These are calmodulin and protein kinase Ca (PKCa). Their effects on secretion were
  • confirmed using commercial and recombinant proteins.
Calmodulin enhances secretion

  • in the absence of ATP, whereas
  • PKC requires ATP to increase secretion, suggesting that
  • phosphorylation is involved in PKC-mediated stimulation
  • but not calmodulin mediated stimulation.
  • Both proteins modulate
    • The half-maximal increase was elicited by
      3 nM PKC and 75 nM calmodulin.
These results suggest that calmodulin and PKC increase Ca2+-activated exocytosis by
  • directly modulating the membrane- or cytoskeleton-attached exocytic machinery
    downstream of Ca2+ elevation.

The abbreviations used are:

NE, norepinephrine; PKC, protein kinase C; CaM, calmodulin; SNAP-25, synaptosome-associated protein of 25 kDa; CAPS, calcium-dependent activator protein for secretion; SNARE, SNAP (soluble N-ethylmaleimide-sensitive factor attachment proteins) receptor; CaMK, Ca2+/calmodulin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; AMP-PNP, adenosine 59-(b,g- imido) triphosphate;  HA, hydroxyapatite

*This work was supported in part by Conte Center Grant MH48108. The costs of publication of
this article were defrayed in part by the payment of page charges. This article has been marked
“advertisement” in accordance with 18 U.S.C. Section 1734.

The molecular mechanisms of presynaptic vesicle release have been extensively examined
by a combination of

  • biochemical,
  • genetic, and
  • electrophysiological techniques.

A series of protein-protein interaction cascades have been proposed to lead to vesicle
docking and fusion
(1–3). The SNARE protein family, including

  • syntaxin, SNAP-25, and vesicle-associated membrane protein
    (VAMP, also called synaptobrevin),
  • plays an essential role in promoting membrane fusion, and
  • is thought to comprise the basic fusion machinery (4, 5).

In Ca2+-stimulated exocytosis, many additional proteins are important in the Ca2+ regulation
of the basic membrane trafficking apparatus.
Calcium

  • not only triggers rapid fusion of release-competent vesicles, but is also involved in
  • earlier processes which replenish the pool of readily releasable vesicles (6).

Furthermore, it appears to be critical in initiating several forms of synaptic plasticity including

  • post-tetanic potentiation (7).

The molecular mechanisms by which Ca2+ regulates these processes is not well understood.


PC12 cells have often been utilized to study Ca2+-activated exocytosis
, as

  • they offer a homogeneous cell population that possesses the same basic exocytic machinery as neurons (8).

In this study, we used an established cracked cell assay, in which

  • [3H]norepinephrine (NE)1 labeled PC12 cells are
  • permeabilized by mechanical “cracking” and
  • then reconstituted for secretion of NE in the presence of test proteins (9).

Transmitter-filled vesicles and intracellular cytoskeletal structures

  • remain intact in these cells,
  • while cytosolic proteins leak out (10).

These cracked cells readily release NE upon addition of

  • ATP,
  • brain cytosol, and
  • 1 mM free Ca2+
    • at an elevated temperature.

We term this a “composite assay,” as

  • all essential components are added into one reaction mixture.

Alternatively, cracked cells can be

  • first primed with cytosol and ATP, washed, then
  • reconstituted for NE release with cytosol and Ca2+ (11).

This sequential priming-triggering protocol is useful

  • for determining whether a protein acts early or late in the exocytic pathway, and
  • whether its effect is dependent on Ca2+ or ATP.

This semi-intact cell system serves as

  • a bridge between an in vitro system comprised of purified components, and
  • electro-physiological systems that monitor release in vivo.
  • It provides information on protein functions in a cell with an intact membrane infrastructure while being easily manipulatable.

Ca2+ regulation by membrane depolarization is no longer a concern, as
intra-cellular Ca2+ concentration can be controlled by a buffered solution.

  • Indirect readout of neurotransmitter release using a postsynaptic cell is replaced by
  • direct readout of [3H]NE released into the buffer.

Complications associated with interpreting overlapping

  • exo- and endocytotic signals are also eliminated as only one round of exocytosis is measured.

Finally, concentration estimates are likely to be accurate, since

  • added compounds do not need to diffuse long distances along axons and dendrites to their sites of action.

Using this assay, several proteins required for NE release have been purified from rat brain cytosol, including

  • phosphatidyl-inositol transfer protein (12),
  • phosphatidylinositol-4-phosphate 5-kinase (13), and
  • calcium-dependent activator protein for secretion (CAPS) (9).

The validity of the cracked cell system is confirmed by the finding that

  • phosphatidylinositol transfer protein and CAPS are mammalian homologues of
    • yeast SEC14p (12) and
    • nematode UNC31p, respectively (14),
  • both proteins involved in membrane trafficking (15, 16).

Calmodulin is the most ubiquitous calcium mediator in eukaryotic cells, yet its involvement in membrane trafficking has not been well established. Some early studies showed

  • that calmodulin inhibitors (17–19), anti-calmodulin antibodies (20,21),

or

  • calmodulin binding inhibitory peptides (22) inhibited Ca2+-activated exocytosis.

However, in other studies, calmodulin-binding peptides and an anti-calmodulin antibody led to the conclusion that

  • calmodulin is only involved in endocytosis,
  • not exocytosis (23).

More recently, it was reported that

  1. Ca2+/ calmodulin signals the completion of docking and
  2. triggers a late step of homotypic vacuole fusion in yeast,
  • thus suggesting an essential role for Ca2+/calmodulin in constitutive intracellular membrane fusion (24).

If calmodulin indeed plays an important role in exocytosis,

  • a likely target of calmodulin is
  • Ca2+/calmodulin-dependent protein kinase II (CaMKII),
    • a multifunctional kinase that is found on synaptic vesicles (25) and
    • has been shown to potentiate neurotransmitter release (26, 27).

Another Ca2+ signaling molecule, PKC, has also been implicated in regulated exocytosis.
In various cell systems, it has been shown that

  • the phorbol esters stimulate secretion (28, 29).

It is usually assumed that phorbol esters effect on exocytosis is

  • through activation of PKC,
  • but Munc13-1 was recently shown to be a presynaptic phorbol ester receptor that enhances neurotransmitter release (30, 31),

which complicates the interpretation of some earlier reports. The mode of action of PKC remains controversial. There is evidence

  • that PKC increases the intracellular Ca2+ levels by modulating plasma membrane Ca2+ channels (32, 33),
  • that it increases the size of the release competent vesicle pool (34, 35), or
  • that it increases the Ca2+ sensitivity of the membrane trafficking apparatus (36).

no consensus on these issues has been reached.

PKC substrates that have been implicated in exocytosis include

  1. SNAP-25 (37),
  2. synaptotagmin (28),
  3. CAPS (38), and
  4. nsec1 (39).

It is believed that upon phosphorylation, these PKC substrates might

  • interact differently with their binding partners, which, in turn,
  • leads to the enhancement of exocytosis.

In addition, evidence is accumulating that PKC and calmodulin interfere with each others actions, as

  • PKC phosphorylation sites are embedded in the calmodulin-binding domains of substrates such as
  • neuromodulin and
  • neurogranin (40).

It is therefore possible that PKC could modulate exocytosis via

  • a calmodulin-dependent pathway by synchronously releasing calmodulin from storage proteins.

In this study, we fractionated an EGTA extract of brain membranes in order to identify active components that could reconstitute release in the cracked cell assay system. We identified calmodulin and PKC as two active factors. Thus, we demonstrate that

  • calmodulin and PKC play a role in the Ca2+ regulation of exocytosis, and provide further insight into the mechanisms of their action.

DISCUSSION

 In this study, we first identified an EGTA extract of brain membranes as a protein source
  •  capable of reconstituting Ca2+- activated exocytosis in cracked PC12 cells.
EGTA only extracts a small pool of Ca2+-dependent membrane-associating proteins,
  • it served as an efficient initial purification step.
Further protein chromatography led to the identification of two active factors in the starting extract,
  • calmodulin and PKC,
  • which together accounted for about half of the starting activity.
Upon confirmation with commercially obtained proteins, this result unambiguously demonstrated
  • that calmodulin and PKC mediate aspects of Ca2+-dependent processes in exocytosis.
The finding that brain membrane EGTA extract alone is able
  • to replace cytosol in supporting Ca2+-triggered NE secretion
 in PC12 cells is somewhat surprising. We suggest that the likely explanation is 2-fold.
  1. some cytosolic proteins essential for exocytosis have a membrane-bound pool
    within permeabilized cells, whose activity might be sufficient for a normal level of exocytosis.
  2. although the 100,000 3 g membrane pellet was washed to remove as many cytosolic proteins as possible,
  • some cytosolic proteins that associate with membranes in a
    • Ca2+-independent manner are probably present in the membrane EGTA extract.
  • these proteins likely constitute only a small percentage of the proteins in the extract, as
    • the characteristics of the activity triggered by the membrane extract
    • are quite different to that of cytosol (Fig. 2).
 Using an unbiased biochemical purification method, we demonstrated that
  •  calmodulin and PKC directly modulate the exocytotic machinery downstream of Ca2+ entry
  • they signal through membrane-attached molecules to increase exocytosis.
 These targets include integral and peripheral membrane proteins, and cytosolic proteins that have a significant
membrane-bound pool.  The modest stimulation by calmodulin and PKC on secretion might suggest a regulatory
role. However, it is also possible that some intermediates in their signaling pathways are in limiting amounts in the
cell ghosts, so that their full effects were not observed. Half-maximal stimulation was obtained at
  • about 3 nM for PKC and
  • at about 75 nM for calmodulin.
This is consistent with an enzymatic role for PKC, and predicts a high-affinity interaction between
  • calmodulin and its substrate protein.
 Ca2+ regulates exocytosis at many different levels. Prior studies indicated that Ca2+ signaling occurs in

  • the priming steps as well
  • as in triggering steps (49, 50).
Our priming triggering protocol 
  1. does not allow Ca2+-dependent priming events to be assayed, as EGTA is present in the priming reaction.
  2. a different approach revealed the existence of both high and low Ca2+-dependent processes (Fig. 2).
  3. this analysis indicated that late triggering events require high [Ca2+], whereas
  4. early priming events require low [Ca2+]. If, as proposed, there is
a pronounced intracellular spatial and temporal [Ca2+] gradient from
  • the point of Ca2+ entry during depolarization (51),
  • perhaps triggered events occur closer to the point of Ca2+ entry,
  • while Ca2+-dependent priming events occur further away from the point of Ca2+ entry.
Fig 2A. measurements of range of [Ca2+]total - average [Ca2+]free values._page_004
Fig. 2B. measurements of range of [Ca2+] total - average [Ca2+]free values_edited-1
Distinct Ca2+ sensors at these stages might be appropriately tuned to different [Ca2+] to handle different tasks.
By analyzing the Ca2+ sensitivity of calmodulin-and PKC-stimulated release, we addressed the question of
  • whether calmodulin and PKC plays an early or a late role in vesicle release.
  •  they both require relatively high [Ca2+] (Fig. 8B),
  • implying that calmodulin and PKC both mediate late triggering events, consistent with some earlier reports
    (34, 52, 53).

In addition, it is interesting to note that PKC does not alter the calcium sensitivity of release in cracked cells, in contrast

to observations from the chick ciliary ganglion (36). Therefore, in contrast to previous electrophysiological studies (28),
we are able to limit the possible modes of PKC action in our system to an increase in the readily releasable vesicle pool or
release sites, or an enhancement of the probability of release of individual vesicles upon Ca2+ influx.
The experiments assaying the calcium sensitivity of release (Figs. 2, 5, and 8) demonstrated
  • a drop in release at very high [Ca2+].

FIG. 5 calmodulin action_page_005

FIG. 8. PKC and calmodulin stimulate... the late triggering reaction_page_006
This decline in release at high [Ca2+] has been previously reported (49, 51), and may represent
  • the true Ca2+ sensitivity of the Ca2+-sensing mechanism inside cells.

However, in our system, it could also be due to the activation of a variety of Ca2+ -activated proteases, as experiments are usually performed in the presence of crude extracts, which include unsequestered proteases.

What might the molecular targets of PKC and calmodulin be? An obvious calmodulin target molecule is CaMKII.
  • but calmodulin’s effect on exocytosis is ATP-independent, rendering the involvement of a kinase unlikely.
 Calmodulin has also been shown to associate with
  • synaptic vesicles in a Ca2+-dependent fashion through synaptotagmin (54),
  • probably by binding to its C-terminal tail (55), and to promote Rab3A dissociation from synaptic vesicles (56).
  • However, there was little calcium-dependent binding of calmodulin to synaptotagmin
    • either on synaptic vesicles, in a bead binding assay with recombinant proteins,
    • or in a calmodulin overlay (data not shown).

In addition, using immobilized calmodulin, we did not see

  • significant Ca2+-dependent pull-down of synaptotagmin or Rab3A from rat brain extract (data not shown).
Recent work has suggested three other candidate targets for calmodulin, Munc13, Pollux, and CRAG (57).
  • Pollux has similarity to a portion of a yeast Rab GTPase-activating protein, while
  • CRAG is related to Rab3 GTPase exchange proteins.
Further work is required to investigate the role of their interactions with calmodulin in vivo.
The recent report that calmodulin mediates yeast vacuole fusion (24) is intriguing, as it raises the possibility that
  • calmodulin, a highly conserved ubiquitous molecule,
    • may mediate many membrane trafficking events.

It is not yet known if

  • the effector molecule of calmodulin is conserved or variable across species and different trafficking steps.

It is enticing to propose a model for Ca2+ sensing whereby

  • calmodulin is a high affinity Ca2+ sensor for both constitutive and regulated membrane fusion.
  1. In the case of constitutive fusion, calmodulin may be the predominant Ca2+ sensor.
  2. In the case of slow, non-local exocytosis of large dense core granules, an additional requirement for
  3. the concerted actions of other molecule(s) that are better tuned to intermediate rises in [Ca2+] might exist.
At the highly localized sites of fast exocytosis of small clear vesicles where high [Ca2+] is reached,
  • specialized low affinity sensor(s) are likely required
  • in addition to calmodulin to achieve membrane fusion.

Therefore, although calmodulin participates in multiple types of vesicle fusion,

  • the impact of Ca2+ sensing by calmodulin on vesicle release likely varies.
Due to the fact that calmodulin binding to some proteins can be modulated by PKC phosphorylation, one might suspect
  • PKC action on exocytosis proceeds through a calmodulin-dependent pathway.
  • but the effects of calmodulin and PKC are additive within our system,
    • suggesting that PKC does not act by releasing calmodulin from a substrate
      • that functions as a calmodulin storage protein.
How Ca2+ regulates presynaptic vesicle release has been an open question for many years. By

  • identifying calmodulin and PKC as modulators of Ca21-regulated exocytosis and clarifying their functions,
  • we have extended our knowledge of the release process.

While the basic machinery of membrane fusion is becoming better understood,

  • the multiple effects of Ca2+ on exocytosis remain to be elucidated at the molecular level.

In addition, the ways that Ca2+ regulation may be important to

  • the mechanisms of synaptic plasticity in the central nervous system

EXPERIMENTAL PROCEDURES

Materials
Rat Brain Cytosol Preparation
Membrane EGTA Extract Preparation

Cracked Cell Assay

PC12 cells were maintained and [3H]NE labeled as described previously (11). Labeled cells were harvested by pipetting with ice-cold potassium glutamate buffer (50 mM Hepes, pH 7.2, 105 mM potassium glutamate, 20 mM potassium acetate, 2 mM EGTA) containing 0.1% bovine serum albumin. Subsequent manipulations were carried out at 0–4 °C. Labeled cells (1–1.5 ml/dish) were mechanically permeabilized passage through a stainless steel homogenizer. The cracked cells were adjusted to 11 mM EGTA and

  • incubated on ice for 0.5–3 h, followed by three washes in which
  • the cells were centrifuged at 800 3 g for 5 min and
  • resuspended in potassium glutamate buffer containing 0.1% bovine serum albumin.

Composite Assay 

Each release reaction contains 0.5–1 million cracked cells, 1.5 mM free Ca2+, 2 mM MgATP,
and the protein solution to be tested in potassium glutamate buffer. Release reactions were initiated
by incubation at 30 °C and terminated by returning to ice. The supernatant of each reaction was
isolated by centrifugation at 2,500 3 g for 30 min at 4 °C, and the

  • released [3H]NE was quantified by scintillation counting (Beckman LS6000IC).

Cell pellets were dissolved in 1% Triton X-100, 0.02% azide and similarly counted. NE release

  • was calculated as a percentage of total [3H] in the supernatant.

Priming Assay

A priming reaction contains about

  • 1–2 million cracked cells,
  • 2 mM MgATP, and
  • the protein solution to be tested.
  • Ca2+ is omitted.

The primed cells were spun down, washed once with fresh potassium glutamate buffer, and

  • distributed into two triggering reactions, each containing
  • rat brain cytosol and free Ca2+
  • The triggering reaction was performed at 30 °C for 3 min, and
  • the NE release was measured
    • as in a composite assay.

Triggering Assay

Cracked cells were primed …, centrifuged, washed …, and

distributed into triggering reactions containing

  • 1.5 mM free Ca2+ and the protein solution 

To inhibit any ATP dependent activity in the triggering reaction,  an

  • ATP depletion system of
    1. hexokinase
    2. MgCl2,
    3. glucose or
  • a non-hydrolyzable ATP analogue AMPPNP

was added into the triggering reaction. NE release was measured as above.

Free Ca2+ Concentration Determination

The range of Ca2+free in the release reaction (Fig. 2B) was achieved

  • by adding Ca2+ into potassium glutamate buffer to reach final [Ca2+] total values of
    • 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, and 2.0 mM.
  • The pH of the reaction was 7.24 when no Ca2+ was added and
  • 7.04 when 2.0 mM Ca2+ was added
    • in the absence of protein extracts or cracked cells.
Fig. 2B. measurements of range of [Ca2+] total - average [Ca2+]free values_edited-1
Fig. 2B.   The range of [Ca21]free in the release reaction (Fig. 2B)
Free Ca2+ concentrations were determined using video microscopic
measurements of fura-2 fluorescence
 (41). [Ca2+]free was calculated from the equation
  • [Ca2+]free 5 Kd*3 (R 2 Rmin)/(Rmax 2 R) (42).
The values of Rmin, Rmax, and Kd* were determined in the following solutions: 
potassium glutamate buffer (PGB) containing
  • 8 x 3 10^6 cracked cells/ml, 2 mM MgATP (PGB+CC)
1) Rmin:  PGB+CC and 10 mM additional EGTA;
2) Rmax: PBG+CC, and 10 mM total Ca2+;
3) Kd*: PGB+CC, 28 mM additional EGTA, and 18 mM total Ca2+, pH 7.2
([Ca2+]free 5 = 169 nM, determined in the absence of cells and MgATP
  • based on fura-2 calibration in cell-free solutions).
These solutions were
  1. incubated at 37 °C ,
  2. mixed with fura-2 pentapotassium salt
    (100 mM; Molecular Probes, Eugene, OR), and
  3. imaged.
This procedure allowed us to take into account
  • changes in fura-2 properties
  • caused by the presence of
    • permeabilized cells.
Duplicate measurements of the above range of [Ca2+] total gave
  • the following average [Ca2+] free values:
  • 106, 146, 277, 462, 971, 1468, 1847, and 2484 nM.

Purification of Active Proteins

All procedures were carried out at 4 °C or on ice. Membrane
EGTA extract of one or two bovine brain(s) was

  1. filtered through cheesecloth and
  2. loaded overnight onto a column packed with DEAE-Sepharose
    CL-6B beads (Amersham Pharmacia Biotech).

The column was then

  1. washed with
    (20 mM Hepes, pH 7.5, 0.25 mM sucrose, 2 mM EGTA, 1 mM dithiothreitol) and 
  2. step eluted with 10 column volumes of elution buffer
    (20 mM Hepes, pH 7.5, 2 mM EGTA, 400 mM KCl, 1 mM dithiothreitol).
    100 ml of every other fraction was
  3. dialyzed overnight into PGB, and
  4. tested in a composite release assay for activity.
  • The active fractions were pooled and dialyzed into zero salt buffer
    (20 mM Hepes, pH 7.5, 2 mM EGTA) and
  • batch bound to 10 ml of Affi-Gel Blue beads (Bio-Rad) or DyeMatrex-Green A beads (Amicon)

Blue beads were used in earlier experiments, and Green beads were used later to

  • specifically deplete CAPS, which was known to bind to Green beads (9).

The unbound material was

  1. collected,
  2. concentrated to about 2 ml using a Centriprep-10 (Amicon), and
  3. loaded onto a 120-ml HiPrep Sephacryl S-200 gel filtration column
    (Amersham Pharmacia Biotech).
Samples were run on the S-200 column in PGB at a flow rate of 7 ml/h.
  • 10–50 ml of every other fraction was tested for
    • activity in the cracked cell composite assay, and
  • two peaks of activity were observed (Fig. 3).

FIG. 3. Gel filtration chromatography reveals two stimulatory_page_004

The first peak of activity had a predicted molecular mass of 85 kDa.
The corresponding material was

  • adjusted to 10 mM potassium phosphate concentration (pH 7.2) and
  • loaded onto a 1-ml column packed with hydroxyapatite Bio-Gel HT
    (Bio-Rad).

The bound material was

  • eluted with a linear K-PO4 gradient from 10 to 500 mM (pH 7.2)
  •  at a flow rate of about 0.1 ml/min, and
  • 0.4–0.5-ml fractions were collected.
  •  each fraction was dialyzed into PGB and
  • tested for activity.

The fractions were also analyzed by

  • SDS-PAGE and silver staining (Sigma silver stain kit).

The active material was concentrated and resolved

  • on an 8% poly-acrylamide gel.

Two Coomassie-stained protein bands that matched the activity profile (Fig. 6)

  • were excised from the gel,
  • sequenced by the Stanford PAN facility.

FIG. 6. Purification of the high molecular weight active factor_page_001

The two polypeptide sequences obtained from the upper band were:

  1. LLNQEEGEYYNVPIXEGD
  2. IRSTLNPRWDESFT.

The only bovine protein that contains both polypeptides is PKCa.
The four polypeptide sequences obtained from the lower band were:

  1. YELTGKFERLIVGLMRPPAY,
  2. LIEILASRTNEQIHQLVAA,
  3. MLVVLLQGTREEDDVVSEDL, and
  4. EMSGDVRDVFVAIVQSVK.

Based on these sequences, the protein band was

  • unambiguously identified to be bovine annexin VI.

The second S-200 peak has a predicted molecular mass of 25 kDa.
The corresponding material was

  • dialyzed into zero salt buffer
    (20 mM Tris, pH 7.5, 1 mM EGTA) and
  • injected onto a Mono-Q HR 5/5 FPLC column
    (Pharmacia).

The FPLC run was performed at 18 °C at 1 ml/min and

  • 1-ml fractions were collected
  • with a linear salt gradient from 0 to 1 M KCl over 71 ml.

The fractions containing proteins (determined by A280) were

  • dialyzed into PGB and
  • tested in the cracked cell assay.

Western Blot

Anti-calmodulin antibody and anti-PKC antibody were used, and

  • ECL (Amersham) was used for detection.

RESULTS

A Membrane EGTA Extract Supports NE Release 

Brain cytosol, prepared as the supernatant of the brain homogenate,

  • effectively stimulates NE release
  • in the cracked cell assay (Fig. 1)
    as previously shown (9). 

Fig. 1 EGTA extract can support NE release_page_003_edited-2

We wondered whether crude extracts other than cytosol
  • could support NE release, and we focused on
  • extractable peripheral membrane proteins.
We found that a salt or EGTA extract of brain membranes,
membranes defined as the
  • 100,000 3 g pellet of the crude homogenate,
  • reconstituted secretion in the absence of cytosol.
  • the salt extract only slightly enhanced NE release
    above background (data not shown), the 
EGTA extract not only stimulated NE release to a high level,
  • similar to that supported by cytosol, but also
  • had a higher specific activity than cytosol (Fig. 1). 
Fig. 1 EGTA extract can support NE release_page_003_edited-3
FIG. 1. The EGTA extract of brain membranes can support NE release in the absence of cytosol. Rat brain membrane EGTA extract (closed triangles) and rat brain cytosol (closed squares) were prepared as described under “Experimental Procedures.” NE release was measured in a composite reaction mixture of cracked cells, MgATP, Ca2+, and the indicated amount of crude extracts.
The ability of the membrane EGTA extract to support secretion is consistent with the fact that
  • following cracking, the cells are immediately extracted with EGTA, and are presumably
  • devoid of most membrane EGTA-extractable factors.

This also suggests that these factors, some of which are probably

  • Ca2+-dependent membrane-associating proteins,
  • participate in Ca2+- triggered exocytosis.

The Membrane EGTA Extract Is Enriched in Triggering Fators

NE release in cracked cells can be resolved into two sequential stages,
  • an ATP-dependent priming stage and
  • an ATP-independent Ca21-dependent triggering stage (11), and
  • proteins can be tested for activity in either stage.
An effect in priming indicates
  1. an early role for the protein, and
  2. an effect in triggering a late ATP-independent role.
Since the protein composition of the
  • membrane EGTA extract and cytosol are different,
we tested whether they had different activities
  • in the priming stage versus the triggering stage.
We found that the membrane EGTA extract is enriched in factors that
  • act during triggering stage of NE releaseas
  • the same amount of protein from the membrane EGTA extract as cytosol
  • gave a higher stimulation in the triggering assay, but
  • not in the priming assay (Fig. 2A). 

Fig 2A. measurements of range of [Ca2+]total - average [Ca2+]free values._page_004

Regular cytosol is prepared in a buffer containing 2 mM EGTA, and thus

  • presumably contains some of the proteins present in the membrane EGTA extract.
Cytosol prepared in the absence of EGTA showed an even lower specific activity
  • in the triggering assay compared with regular cytosol (Fig. 2A).

Identification of Calmodulin as an Active Triggering Factor in the EGTA Extract

Biochemical fractionation of the bovine brain membrane EGTA extract was carried out

  • to identify the active components capable of reconstituting NE release.

Activity was assayed in a composite reaction mixture containing

  • cracked cells,
  • ATP,
  • Ca2+, and
  • the test protein(s).

Except for the presence of bovine serum albumin in the basal buffer,

  • no other proteins were added to the cell ghosts except for the test protein(s).

Initial tests indicated that at least

  1. part of the activity in the membrane EGTA extract binds to and
  2. can be efficiently eluted from an anion exchanger and hydroxyapatite resin,
  3. but does not bind to Amicon color resins.

The starting material was, therefore, sequentially purified using

  • DEAE, Affi-Gel Blue (or Matrex Green-A), and gel filtration chromotography.

Gel filtration fractionation indicated the presence of two peaks of activity with

  • predicted molecular masses of 25 and 85 kDa, respectively (Fig. 3).

FIG. 3. Gel filtration chromatography reveals two stimulatory_page_004

FIG. 3. Gel filtration chromatography reveals two stimulatory factors in the membrane EGTA extract.

In order to purify the active component(s) in the membrane EGTA extract, the crude extract from one bovine brain was fractionated chromatographically (see Experimental Procedures” for details). Fractions from a Sephacryl S-200 gel filtration column were tested for their activity in stimulating NE release in the composite assay. The two activity peaks have predicted molecular masses of 85 and 25 kDa, respectively. The arrows indicate the retention volume of standard proteins run on the same column.

The low molecular weight active factor was purified to homogeneity, as judged by a

  • Coomassie-stained SDS-PAGE gel, after a subsequent Mono-Q fractionation (Fig. 4).

FIG. 4. The low molecular weight active factor is calmodulin_page_004

FIG. 4. The low molecular wen.ight active factor is calmodulin

A, the  membrane EGTA extract from one bovine brain (Start) was subjected to sequential fractionation on DEAE, Blue A, and
Sephacryl S-200 columns. The pooled material containing the activity after each chromotographic step was analyzed by SDS-
PAGE and Coomassie staining. The arrowheads indicate the presence of calmodulin in all the lanes. Calmodulin shows a
mobility shift depending on whether or not Ca2+ is present during electrophoresis (see panel C).
B, the active material  pooled from Sephacryl S-200 was fractionated on a Mono-Q FPLC column and the fractions
(5 ml/fraction) were tested for activity in a composite assay. The activity peak is shown.
C, the active Mono-Q fractions (5 ml/fraction) were subjected to SDS-PAGE in the presence of 1 mM EGTA or 0.1 mM Ca2+,
and the gels stained with Coomassie Blue.
D, fraction 47 (1 ml) was probed by Western blotting with a monoclonal anti-calmodulin antibody. No Ca2+ or EGTA was
added during SDS-PAGE.

We reasoned that the protein might be calmodulin (43) based on the following:

1) It is a relatively small protein (14–18 kDa) that is abundant in the
starting extract (Fig. 4A).
2) It elutes at a very high salt concentration (0.41 M KCl) on the
Mono-Q column.
3) It stains negatively in silver stain (data not shown).
4) Its electrophoretic mobility shifts depending on the presence or
absence of Ca21 (Fig. 4C).

A Western blot with an anti-calmodulin monoclonal antibody gave a
positive signal (Fig. 4D), confirming our prediction.

Properties of Calmodulin-stimulated Exocytosis

We used commercial calmodulin or bacterially expressed recombinant calmodulin to confirm our purification result; both sources of authentic calmodulin stimulated NE release as expected. Moreover, we found that calmodulin stimulates secretion in a triggering assay as well as in a composite assay (Fig. 5A).

FIG. 5A calmodulin action_page 5

The half-maximal increase was at 75 nM (250 ng/200 ml) final calmodulin concentration. This is within the broad
range of affinities between calmodulin and its various targets and suggests that the interaction between
calmodulin and its target molecule in exocytosis is in the physiological range. When the triggering reaction was
performed at different Ca2+ concentrations, calmodulin increased NE release only at high [Ca2+] (0.4 – 2 mM)
similar to the crude EGTA extract (Fig. 5B),

FIG. 5B calmodulin action_page_5

suggesting that calmodulin contributes to the triggering activity of the membrane EGTA extract.  Calmodulin’s affinity for Ca2+ has
been  reported to be around 1 mM (25),

  • consistent with the Ca2+ requirement for
  • calmodulin-stimulated secretion that we observed.

FIG. 5 calmodulin action_page_005

FIG. 5. Calmodulin stimulates NE release in the triggering stage.
A, calmodulin (obtained from Sigma) increased NE release in the
triggering assay in a dose-dependent fashion, in the absence of ATP
or any other cytosolic proteins. In this particular experiment, the
maximal release achieved by addition of rat brain cytosol was 46.5%.

B, the triggering assay was performed with different concentrations
of free Ca2+. Calmodulin (3 mg bacterially expressed recombinant
protein; closed squares) increased NE release with a similar Ca2+
sensitivity to rat brain membrane EGTA extract (10 mg; closed
triangles), as compared with conditions in which no protein was
added (open squares).

Western analysis with commercial protein as standards indicated that calmodulin 

  •  constitutes about 5% of total proteins in the rat brain membrane EGTA extract
  • and about 2% of total proteins in the rat brain cytosol (data not shown).

In addition, a significant amount of calmodulin appears to be left

  • in the washed cell ghosts (data not shown).

Based on the activity of saturating levels of

  • pure calmodulin (releasing 6–10% of total [3H]NE)
  • and crude EGTA extract (releasing ;45% of total [3H]NE),

we estimated that

  • calmodulin accounts for 13–22% of total activity of the extract.

Consistent with this,

  • a high affinity calmodulin-binding peptide
    (CaMKIIa(291–312) (44), used at 5 mM) and
  • an anti-calmodulin antibody (2 mg/200 ml)
  • inhibited about 20% of the membrane EGTA extract-stimulated release
    (6.7 mg of extract added; data not shown).

We showed that calmodulin increased NE release

  • in the triggering stage.

Since regular triggering reactions were performed

  • in the absence of any added ATP,

this suggests that

  • calmodulin enhanced secretion in an ATP-independent fashion.

Furthermore, residual ATP in the cell ghosts did not play a role, since

  •  addition of a hexokinase ATP depletion system that
  • can deplete millimolar concentrations of ATP
    • within a few minutes (11) had little effect, as did
    • addition of 5 mM AMPPNP,
  • which blocks ATP-dependent enzymatic activity (Fig.8A).

Therefore, we ruled out the possibility that a kinase mediates calmodulin’s effect.

FIG. 8. PKC and calmodulin stimulate... the late triggering reaction_page_006

FIG. 8. PKC and calmodulin stimulate the late triggering reaction in
an ATP-dependent and ATP-independent manner respectively.
A, triggering assays were performed to test the activity of calmodulin
(recombinant; black bars) and PKC (purified rat brain PKC from
Calbiochem; shaded bars) in the absence of ATP. A regular triggering
assay is done in the absence of ATP (2ATP). To deplete residual ATP
in the cells, hexokinase-based ATP depletion was employed (1Hexo).
Alternatively, 5 mM AMP-PNP (1AMP-PNP) was added in the triggering
reaction. Under all three conditions, calmodulin increased release
as compared with the background (buffer only; white bars), whereas
PKC did not.
B, NE release in a composite assay was measured with varying
concentrations of free Ca2+ in the presence of 10 mg of calmodulin
(recombinant; closed triangles), 70 ng of PKC  (purified rat brain PKC
from Calbiochem; closed squares), or buffer only (open squares).

A series of calmodulin mutants from Paramecium and chicken were tested

  • for their ability to enhance Ca2+-stimulated secretion, and
  • none of the mutations abolished the calmodulin effect (data not shown).

These mutations include

  • S101F, M145V, E54K, G40E/D50N, V35I/D50N within Paramecium
  • calmodulin (45), and M124Q, M51A/V55A, and M51A/V55A/L32A
    within chicken calmodulin (46, 47).

The Paramecium calmodulin mutants are the result of

  • naturally occurring mutations that result in aberrations in their behavior.

These mutants can be grouped into two categories according to their
behavior, reflecting their loss of either

  1. a Ca2+-dependent Na1 current
     (calmodulin N-terminal lobe mutants: E54K, G40E/D50N, and
     V35I/D50N) or
  2. a Ca21-dependent K1 current
    (calmodulin C-terminal lobe mutants: S101F and M145V) (45).

The chicken calmodulin mutants have been shown to

  • differentially activate myosin light chain kinase
    (M124Q, M51A/V55A, and M51A/V55A/L32A),
    CaMKII (M124Q),  
    and CaMKIV (M124Q),

and the mutated residues are thought to be important in

  • defining calmodulin’s binding specificity (46, 47).

Our finding that these mutant calmodulins can stimulate exocytosis suggests that

  • calmodulin-binding domains similar to those of Paramecium Ca2+/calmodulin-dependent
    ion channels, myosin light chain kinase, CaMKII, and CaMKIV,
  • are unlikely to mediate release utilizing the conserved SNARE fusion machinery, as they
  • could be completely abolished by addition of exogenous syntaxin H3 domains (data not shown).
  • the same molecular pathway was not activated, since their effects were additive (data not shown).

 

Acknowledgments
We thank Diana Bautista and Dr. Richard S.Lewis for generous help
with [Ca21]free determination; Dr. Ching Kung for providing the Paramecium calmodulin
mutants, and Dr. Anthony R. Means for providing the chicken calmodulin mutants. We also
thank Dr. Jesse C. Hay for the initial setup of the cracked cell assay, and Dr. Suzie J.
Scales for helpful comments on the manuscript.

REFERENCES

1. Calakos, N., and Scheller, R. H. (1996) Physiol. Rev. 76, 1–29
2. Su¨ dhof, T. C. (1995) Nature 375, 645–653
3. Zucker, R. S. (1996) Neuron 17, 1049–1055
4. Hanson, P. I., Heuser, J. E., and Jahn, R. (1997) Curr. Opin. Neurobiol. 7, 310–315
5. Chen, Y. A., Scales, S. J., Patel, S. M., Doung, Y.-C., and Scheller, R. H. (1999) Cell 97, 165–174
6. Neher, E., and Zucker, R. S. (1993) Neuron 10, 21–30
7. Kamiya, H., and Zucker, R. S. (1994) Nature 371, 603–606
SOURCE

Other related articles published in this Open Access Online Scientific Journal include the following:

The role of ion channels in Na(+)-K(+)-ATPase: regulation of ion transport across the plasma membrane has been studies by our Team in 2012 and 2013. Chiefly, our sources of inspiration were the following:

1. 2013 Nobel work on vesicles and calcium flux at the neuromuscular junction Machinery Regulating Vesicle Traffic, A Major Transport System in our Cells The 2013 Nobel Prize in Physiology or Medicine is awarded to Dr. James E. Rothman, Dr. Randy W. Schekman and Dr. Thomas C. Südhof

  • for their discoveries of machinery regulating vesicle traffic,
  • a major transport system in our cells.

This represents a paradigm shift in our understanding of how the eukaryotic cell, with its complex internal compartmentalization, organizes

  • the routing of molecules packaged in vesicles
  • to various intracellular destinations,
  • as well as to the outside of the cell

Specificity in the delivery of molecular cargo is essential for cell function and survival.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/2013/advanced-medicineprize2013.pdf

Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with
cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-
how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

2. Perspectives on Nitric Oxide in Disease Mechanisms

available on Kindle Store @ Amazon.com

http://www.amazon.com/dp/B00DINFFYC

https://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/
perspectives-on-nitric-oxide-in-disease-mechanisms-v2/

3. Professor David Lichtstein, Hebrew University of Jerusalem, Dean, School of Medicine

Lichtstein’s main research focus is the regulation of ion transport across the plasma membrane of eukaryotic cells.

His work led to the discovery that specific steroids that have crucial roles, as

  • the regulation of cell viability,
  • heart contractility,
  • blood pressure and
  • brain function.

His research has implications for the fundamental understanding of body functions,

  • as well as for several pathological states such as
    • heart failure, hypertension
    • and neurological and psychiatric diseases.

Physiologist, Professor Lichtstein, Chair in Heart Studies at The Hebrew University elected
Dean of the Faculty of Medicine at The Hebrew University of Jerusalem

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/18/physiologist-professor-lichtstein-chair-in-heart-studies-
at-the-hebrew-university-elected-dean-of-the-faculty-of-medicine-at-the-hebrew-university-of-jerusalem/

4. Professor Roger J. Hajjar, MD at Mount Sinai School of Medicine

Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension
and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-for-
pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

5.            Seminal Curations by Dr. Aviva Lev-Ari on Genetics and Genomics of Cardiovascular Diseases with a focus on Conduction and Cardiac Contractility

Aviva Lev-Ari, PhD, RN

Aviva Lev-Ari, PhD, RN

Aviva Lev-Ari, PhD, RN and Larry H. Bernstein, MD, FCAP

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

6. Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

This study presents  the possible correlation between Myocardial Ischemia (Coronary Artery Disease (CAD)) aka Ischemic Heart Disease (IHD) and single-nucleotide polymorphisms (SNPs) genes encoding several regulators involved in Coronary Blood Flow Regulation (CBFR), including

  • ion channels acting in vascular smooth muscle and/or
  • endothelial cells of coronary arteries.

They completely analyzed exon 3 of both KCNJ8 and KCNJ11 genes (Kir6.1 and Kir6.2 subunit, respectively) as well as

  • the whole coding region of KCN5A gene (Kv1.5 channel).

The work suggests certain genetic polymorphisms may represent a non-modifiable protective factor that could be

  • used to identify individuals at relatively low-risk for cardiovascular disease
    • an independent protective role of the
    • rs5215_GG against developing CAD and
    • a trend for rs5219_AA to be associated with protection against coronary microvascular dysfunction

Other related articles published on this Open Access Online Scientific Journal include the following:

ION CHANNEL and Cardiovascular Diseases

https://pharmaceuticalintelligence.com/?s=Ion+Channel

Calcium Role in Cardiovascular Diseases

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-
that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-
skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease
Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-
exchange-mechanism-in-health-and-disease/

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-
involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-
post-ischemic-arrhythmia-similarities-and-differen/

Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
and
Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary

Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-
for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure –
Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-
and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells:
The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-
muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction
(Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-
and-calcium-release-related-contractile-dysfunction-ryanopathy/

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of
vesicles with cell membranes during Neurotransmission
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-
regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Part XI: Sensors and Signaling in Oxidative Stress
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

 

Mitochondria and its Role in Cardiovascular Diseases

Mitochondria and Oxidative Stress Role in Cardiovascular Diseases Reversal of Cardiac Mitochondrial Dysfunction
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

Calcium Signaling, Cardiac Mitochondria and Metabolic Syndrome
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/11/09/calcium-signaling-cardiac-mitochondria-and-metabolic-syndrome/

Mitochondrial Dysfunction and Cardiac Disorders
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

Mitochondrial Metabolism and Cardiac Function
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix Identified
Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/02/03/mit-scientists-on-proteomics-all-the-proteins-
in-the-mitochondrial-matrix-identified/

Mitochondrial Dynamics and Cardiovascular Diseases
Ritu Saxena, Ph.D.
https://pharmaceuticalintelligence.com/2012/11/14/mitochondrial-dynamics-and-cardiovascular-diseases/

Mitochondrial Damage and Repair under Oxidative Stress
Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Nitric Oxide has a Ubiquitous Role in the Regulation of Glycolysis -with a Concomitant Influence on Mitochondrial Function
Larry H. Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-
glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Mitochondrial Mechanisms of Disease in Diabetes Mellitus
Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/08/01/mitochondrial-mechanisms-of-disease-in-diabetes-mellitus/

Mitochondria Dysfunction and Cardiovascular Disease – Mitochondria: More than just the “Powerhouse of the Cell”
Ritu Saxena, PhD
https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Advertisements

Read Full Post »


Nitric Oxide Synthase Inhibitors (NOS-I)

Author: Larry H Bernstein, MD, FCAP

Curator: Stephen J. Williams, PhD

and

Co-Curator: Aviva Lev-Ari, PhD, RN

 

This recent article sheds a new light on nitric oxide and the activity of NOS in reactive oxygen species generation and the effect of NOS inhibitors in bacteria.

Structural and Biological Studies on Bacterial Nitric Oxide Synthase Inhibitors

Jeffrey K. Holdena, Huiying Lia, Qing Jingb, Soosung Kangb, Jerry Richoa, Richard B. Silvermanb,1, and Thomas L. Poulosb,1
Agman@chem.northwestern.edu
Author contributions: J.K.H. designed research; J.K.H. and J.R. performed research; Q.J. and S.K. contributed new reagents/analytic tools; J.K.H., H.L., R.B.S., and T.L.P. analyzed data; and J.K.H., R.B.S., and T.L.P. wrote the paper.

PNAS Oct 21, 2013;       http://dx.doi.org/10.1073/pnas.1314080110
This article is a PNAS Direct Submission
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank
Edited by Douglas C. Rees, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, and approved September 23, 2013 (received for review July 29, 2013)
Keywords:  crystallography, antibiotics, nitric oxide, NOS inhibitors, Bacillus subtilis, gram positive bacteria

Significance

Nitric oxide (NO) produced by bacterial nitric oxide synthase has recently been shown to

Using Bacillus subtilis as a model system, we identified

  • two NOS inhibitors that work in conjunction with an antibiotic to kill B. subtilis.

Moreover, comparison of inhibitor-bound crystal structures between the bacterial NOS and mammalian NOS revealed an unprecedented

  • mode of binding to the bacterial NOS that can be further exploited for future structure-based drug design.

Overall, this work is an important advance in developing inhibitors against gram-positive pathogens.

Abstract

Nitric oxide (NO) produced by bacterial NOS functions as

  • a cytoprotective agent against oxidative stress in Staphylococcus aureusBacillus anthracis, and Bacillus subtilis.

The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds

  • impede the growth of B. subtilis under oxidative stress, and
  • crystal structures show that each compound exhibits a unique binding mode.

Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria.

Read Full Post »


Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Reporters: Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

This article is the Part X in a series of articles on Activation and Dysfunction of the Calcium Release Mechanisms in Cardiomyocytes and Vascular Smooth Muscle Cells.

The Series consists of the following articles:

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
 and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-exchange-mechanism-in-health-and-disease/

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/

Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
and
Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmiasand Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Part XI: Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

Introduction

Author: Larry H Bernstein, MD, FCAP 

This introduction is based on two sources:

#1:

Michael J. Berridge, Smooth muscle cell calcium activation mechanisms

The Babraham Institute, Babraham, Cambridge CB22 4AT, UK

J Physiol 586.21 (2008) pp 5047–5061

http://jp.physoc.org/content/586/21/5047.full.pdf

and

#2

Thomas C Südhof, A molecular machine for neurotransmitter release: synaptotagmin and beyond

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Part IX of this series of articles discussed the mechanism of the signaling of smooth muscle cells by the interacting parasympathetic neural innervation that occurs by calcium triggering neurotransmitter release by initiating synaptic vesicle fusion.   It involves the interaction of soluble N-acetylmaleimide-sensitive factor (SNARE) and SM proteins, and in addition, the discovery of a calcium-dependsent Syt1 (C) domain of protein- kinase C isoenzyme, which binds to phospholipids.  It is reasonable to consider that it differs from motor neuron activation of skeletal muscles, mainly because the innervation is in the involuntary domain.   The cranial nerve rooted innervation has evolved comes from the spinal ganglia at the corresponding level of the spinal cord.  It is in this specific neural function that we find a mechanistic interaction with adrenergic hormonal function, a concept intimated by the late Richard Bing.  Only recently has there been a plausible concept that brings this into serious consideration.  Moreover, the review of therapeutic drugs that are used in blocking adrenergic receptors are closely related to the calcium-channels.  Interesting too is the participation of a phospholipid bound protein-kinase isoenzyme C calcium-dependent domain Syt1.  The neurohormonal connection lies in the observation by Katz in the 1950’s that the vesicles of the neurons hold and eject fixed amounts of neurotransmitters.

In Sudhof’s Lasker Award presentation he refers to the biochemical properties of synaptotagmin were found to precisely correspond to the extraordinary calcium-triggering properties of release, and to account for a regulatory pathway that also applies to other types of calcium-triggered fusion, for example fusion observed in hormone secretion and fertilization. At the synapse, finally, these interdependent machines — the fusion apparatus and its synaptotagmin-dependent control mechanism — are embedded in a proteinaceous active zone that links them to calcium channels, and regulates the docking and priming of synaptic vesicles for subsequent calcium-triggered fusion. Thus, work on neurotransmitter release revealed a hierarchy of molecular machines that mediate the fusion of synaptic vesicles, the calcium-control of this fusion, and the embedding of calcium-controlled fusion in the context of the presynaptic terminal at the synapse.  The neural transmission is described as a biological relay system. Neurotransmission kicks off with an electrical pulse that runs down a nerve cell, or neuron. When that signal reaches the tip, calcium enters the cell. In response, the neuron liberates chemical messengers—neurotransmitters—which travel to the next neuron and thus pass the baton.

He further stipulates that synaptic vesicle exocytosis operates by a general mechanism of membrane fusion that revealed itself to be a model for all membrane fusion, but that is uniquely regulated by a calcium-sensor protein called synaptotagmin.  Neurotransmission is thus a combination of electrical signal and chemical transport.

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Several SMC types illustrate how signaling mechanisms have been adapted to control different contractile functions with particular emphasis on how Ca2+ signals are activated.

[1] Neural activation of vas deferens smooth muscle cells

Noradrenaline (NA) acts by stimulating α1-adrenoreceptors to produce InsP3, which then releases Ca2+ that may induce an intracellular Ca2+ wave similar to that triggered by the ATP-dependent entry of external Ca2+. In addition, the α1-adrenoreceptors also activate the smooth muscle Rho/Rho kinase signalling pathway that serves to increase the Ca2+ sensitivity of the contractile machinery.

[2] Detrusor smooth muscle cells

The bladder, which functions to store and expel urine, is surrounded by layers of detrusor SMCs. The latter have two operational modes: during bladder filling they remain relaxed but contract vigorously to expel urine during micturition. The switch from relaxation to contraction, which is triggered by neurotransmitters released from parasympathetic nerves, depends on the acceleration of an endogenous membrane oscillator that produces the repetitive trains of action potentials that drive contraction.

This mechanism of activation is also shared by [1], and uterine contraction.  SMCs are activated by membrane depolarization (ΔV) that opens L-type voltage-operated channels (VOCs) allowing external Ca2+ to flood into the cell to trigger contraction. This depolarization is induced either by ionotropic receptors (vas deferens) or a membrane oscillator (bladder and uterus). The membrane oscillator, which resides in the plasma membrane, generates the periodic pacemaker depolarizations responsible for the action potentials that drive contraction.

The main components of the membrane oscillator are the Ca2+ and K+ channels that sequentially depolarize and hyperpolarize the membrane, respectively. This oscillator generates the periodic pacemaker depolarizations that trigger each action potential. The resulting Ca2+ signal lags behind the action potential because it spreads into the cell as a slower Ca2+ wave mediated by the type 2 RYRs.

Neurotransmitters such as ATP and acetylcholine (ACh), which are released from parasympathetic axonal varicosities that innervate the bladder, activate or accelerate the oscillator by inducing membrane depolarization (ΔV).

[3]  The depolarizing signal that activates gastrointestinal, urethral and ureter SMCs is as follows:

A number of SMCs are activated by pacemaker cells such as the interstitial cells of Cajal (ICCs) (gastrointestinal and urethral SMCs) or atypical SMCs (ureter). These pacemaker cells have a cytosolic oscillator that generates the repetitive Ca2+ transients that activate inward currents that spread through the gap junctions to provide the depolarizing signal (ΔV) that triggers contraction.

[4]  Our greatest interest has been in this mechanism.  The rhythmical contractions of vascular, lymphatic, airway and corpus cavernosum SMCs depend on an endogenous pacemaker driven by a cytosolic Ca2+ oscillator that is responsible for the periodic release of Ca2+ from the endoplasmic reticulum. The periodic pulses of Ca2+ often cause membrane depolarization, but this is not part of the primary activation mechanism but has a secondary role to synchronize and amplify the oscillatory mechanism. Neurotransmitters and hormones act by modulating the frequency of the cytosolic oscillator.

Vascular or airway SMCs are driven by a cytosolic oscillator that generates a periodic release of Ca2+ from the endoplasmic reticulum that usually appears as a propagating Ca2+ wave.

Step 1. The initiation and/or modulation of this oscillator depends upon the action of transmitters and hormones such as ACh, 5-HT, NA and endothelin-1 (ET-1) that increase the formation of InsP3 and diacylglycerol (DAG), both of which promote oscillatory activity.

Step 2. The oscillator is very dependent on Ca2+ entry to provide the Ca2+ necessary to charge up the stores for each oscillatory cycle. The nature of these entry mechanisms vary between cell types.

Step 3. The entry of external Ca2+ charges up the ER to sensitize the RYRs and InsP3 receptors prior to the next phase of release. An important determinant of this sensitivity is the luminal concentration of Ca2+ and as this builds up the release channels become sensitive to Ca2+ and can participate in the process of Ca2+-induced Ca2+ release (CICR), which is responsible for orchestrating the regenerative release of Ca2+ from the ER. The proposed role of cyclic ADP-ribose (cADPR) in airway SMCs is consistent with this aspect of the model on the basis of its proposed action of stimulating the SERCA pump to enhance store loading and such a mechanism has been described in colonic SMCs.

Step 4. The mechanism responsible for initiating Ca2+ release may depend either on the RYRs or the InsP3 receptors (I). RYR channels are sensitive to store loading and the InsP3 receptors will be sensitized by the agonist-dependent formation of InsP3.

Step 5. This initial release of Ca2+ is then amplified by regenerative Ca2+ release by either the RYRs or InsP3 receptors, depending on the cell type.

Step 6. The global Ca2+ signal then activates contraction.

Step 7. The recovery phase depends on the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), that pumps some of the Ca2+ back into the ER, and the plasma membrane Ca2+-ATPase (PMCA), that pumps Ca2+ out of the cell.

Step 8. One of the effects of the released Ca2+ is to stimulate Ca2+-sensitive K+ channels such as the BK and SK channels that will lead to membrane hyperpolarization. The BK channels are activated by Ca2+ sparks resulting from the opening of RYRs.

Step 9.  Another action of Ca2+ is to stimulate Ca2+-sensitive chloride channels (CLCA) (Liu & Farley, 1996; Haddock & Hill, 2002), which result in membrane depolarization to activate the CaV1.2 channels that introduce Ca2+ into the cell resulting in further membrane depolarization (ΔV).

Step 10. This depolarization can spread to neighbouring cells by current flow through the gap junctions to provide a synchronization mechanism in those cases where the oscillators are coupled together to provide vasomotion.

SOURCE

Smooth muscle cell calcium activation mechanisms. Berridge MJ.
J Physiol. 2008; 586(Pt 21):5047-61.   http://dx.doi.org/10.1113/jphysiol.2008.160440

Synaptotagmin functions as a Calcium Sensor

Thomas C. Südhof is at the Department of Molecular and Cellular Physiology and the Howard Hughes Medical Institute, Stanford University School of Medicine, Palo Alto, California, USA

Prof.  Thomas C. Südhof explains:

Fifty years ago, Bernard Katz’s seminal work revealed that calcium triggers neurotransmitter release by stimulating ultrafast synaptic vesicle fusion. But how a presynaptic terminal achieves the speed and precision of calcium-triggered fusion remained unknown. My colleagues and I set out to study this fundamental problem more than two decades ago.

How do the synaptic vesicle and the plasma membrane fuse during transmitter release? How does calcium trigger synaptic vesicle fusion? How is calcium influx localized to release sites in order to enable the fast coupling of an action potential to transmitter release? Together with contributions made by other scientists, most prominently James Rothman, Reinhard Jahn and Richard Scheller, and assisted by luck and good fortune, we have addressed these questions over the last decades.

As he described below, we now know of a general mechanism of membrane fusion that operates by the interaction of SNAREs (for soluble N-ethylmaleimide–sensitive factor (NSF)-attachment protein receptors) and SM proteins (for Sec1/Munc18-like proteins). We also have now a general mechanism of calcium-triggered fusion that operates by calcium binding to synaptotagmins, plus a general mechanism of vesicle positioning adjacent to calcium channels, which involves the interaction of the so-called RIM proteins with these channels and synaptic vesicles. Thus, a molecular framework that accounts for the astounding speed and precision of neurotransmitter release has emerged. In describing this framework, I have been asked to describe primarily my own work. I apologize for the many omissions of citations to work of others; please consult a recent review for additional references1.

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Outlook

Our work, together with that of other researchers, uncovered a plausible mechanism explaining how membranes undergo rapid fusion during transmitter release, how such fusion is regulated by calcium and how the calcium-controlled fusion of synaptic vesicles is spatially organized in the presynaptic terminal. Nevertheless, many new questions now arise that are not just details but of great importance. For example, what are the precise physicochemical mechanisms underlying fusion, and what is the role of the fusion mechanism we outlined in brain diseases? Much remains to be done in this field.

How calcium controls membrane fusion

The above discussion describes the major progress that was made in determining the mechanism of membrane fusion. At the same time, my laboratory was focusing on a question crucial for neuronal function: how is this process triggered in microseconds when calcium enters the presynaptic terminal?

While examining the fusion machinery, we wondered how it could possibly be controlled so tightly by calcium. Starting with the description of synaptotagmin-1 (Syt1)5, we worked over two decades to show that calcium-dependent exocytosis is mediated by synaptotagmins as calcium sensors.

Synaptotagmins are evolutionarily conserved transmembrane proteins with two cytoplasmic C2 domains (Fig. 3a)5,6. When we cloned Syt1, nothing was known about C2 domains except that they represented the ‘second constant sequence’ in protein-kinase C isozymes. Because protein kinase C had been shown to interact with phospholipids by an unknown mechanism, we speculated that Syt1 C2 domains may bind phospholipids, which we indeed found to be the case5. We also found that this interaction is calcium dependent6,7 and that a single C2 domain mediates calcium-dependent phospholipid binding (Fig. 3b)8. In addition, the Syt1 C2 domains also bind syntaxin-1 and the SNARE complex6,9. All of these observations were first made for Syt1 C2 domains, but they have since been generalized to other C2 domains.

As calcium-binding modules, C2 domains were unlike any other calcium-binding protein known at the time. Beginning in 1995, we obtained atomic structures of calcium-free and calcium-bound Syt1 C2 domains10 in collaboration with structural biologists, primarily Jose Rizo (Fig. 3c). These structures provided the first insights into how C2 domains bind calcium and allowed us to test the role of Syt1 calcium binding in transmitter release11.

The biochemical properties of Syt1 suggested that it constituted Katz’s long-sought calcium sensor for neurotransmitter release. Initial experiments in C. elegans and Drosophila, however, disappointingly indicated otherwise. The ‘synaptotagmin calcium-sensor hypothesis’ seemed unlikely until our electrophysiological analyses of Syt1 knockout mice revealed that Syt1 is required for all fast synchronous synaptic fusion in forebrain neurons but is dispensable for other types of fusion (Fig. 4)12. These experiments established that Syt1 is essential for fast calcium-triggered release, but not for fusion as such.

Although the Syt1 knockout analysis supported the synaptotagmin calcium-sensor hypothesis, it did not exclude the possibility that Syt1 positions vesicles next to voltage-gated calcium channels (a function now known to be mediated by RIMs and RIM-BPs; see below),

with calcium binding to Syt1 performing a role unrelated to calcium sensing and transmitter release. To directly test whether calcium binding to Syt1 triggers release, we introduced a point mutation into the endogenous mouse Syt1 gene locus. This mutation decreased the Syt1 calcium-binding affinity by about twofold11. Electrophysiological recordings revealed that this mutation also decreased the calcium affinity of neurotransmitter release approximately twofold, formally proving that Syt1 is the calcium sensor for release (Fig. 5). In addition to mediating calcium triggering of release, Syt1 controls (‘clamps’) the rate of spontaneous release occurring in the absence of action potentials, thus serving as an essential mediator of the speed and precision of release by association with SNARE complexes and phospholipids (Fig. 6a,b).

It was initially surprising that the Syt1 knockout produced a marked phenotype because the brain expresses multiple synaptotagmins6. However, we found that only three synaptotagmins—Syt1, Syt2 and Syt9—mediate fast synaptic vesicle exocytosis13. Syt2 triggers release faster, and Syt9 slower, than Syt1. Most forebrain neurons express only Syt1, but not Syt2 or Syt9, accounting for the profound Syt1 knockout phenotype. Syt2 is the predominant calcium sensor of very fast synapses in the brainstem14, whereas Syt9 is primarily present in the limbic system13. Thus, the kinetic properties of Syt1, Syt2 and Syt9 correspond to the functional needs of the synapses that contain them.

Parallel experiments in neuroendocrine cells revealed that, in addition to Syt1, Syt7 functions as a calcium sensor for hormone exocytosis. Moreover, experiments in olfactory neurons uncovered a role for Syt10 as a calcium sensor for insulin-like growth factor-1 exocytosis15, showing that, even in a single neuron, different synaptotagmins act as calcium sensors for distinct fusion reactions. Viewed together with results by other groups, these observations indicated that calcium-triggered exocytosis generally depends on synaptotagmin calcium sensors and that different synaptotagmins confer specificity onto exocytosis pathways.

We had originally identified complexin as a small protein bound to SNARE complexes (Fig. 6b)16. Analysis of complexin-deficient neurons showed that complexin represents a cofactor for synaptotagmin that functions both as a clamp and as an activator of calcium-triggered fusion17. Complexin-deficient neurons exhibit a phenotype milder than that of Syt1-deficient neurons, with a selective suppression of fast synchronous exocytosis and an increase in spontaneous exocytosis, which suggests that complexin and synaptotagmins are functionally interdependent.

How does a small molecule like complexin, composed of only ~130 amino acid residues, act to activate and clamp synaptic vesicles for synaptotagmin action? Atomic structures revealed that, when bound to assembled SNARE complexes, complexin contains two short a-helices flanked by flexible sequences (Fig. 6c). One of the a-helices is bound to the SNARE complex and is essential for all complexin function18. The second a-helix is required only for the clamping, and not for the activating function of complexin17. The flexible N-terminal sequence of complexin, conversely, mediates only the activating, but not the clamping, function of the protein. Our current model is that complexin binding to SNAREs activates the SNARE–SM protein complex and that at least part of complexin competes with synaptotagmin for SNARE complex binding. Calcium-activated synaptotagmin displaces this part of complexin, thereby triggering fusion-pore opening (Fig. 6a)1,18.

REFERENCES

1. Südhof, T.C. & Rothman, J.E. Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477 (2009).

2. Hata, Y., Slaughter, C.A. & Südhof, T.C. Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature 366, 347–351 (1993).

3. Burré, J. et al. a-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science 329, 1663–1667 (2010).

4. Khvotchev, M. et al. Dual modes of Munc18–1/SNARE interactions are coupled by functionally critical binding to syntaxin-1 N-terminus. J. Neurosci. 27, 12147–12155 (2007).

5. Perin, M.S., Fried, V.A., Mignery, G.A., Jahn, R. & Südhof, T.C. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature 345, 260–263 (1990).

6. Li, C. et al. Ca2+-dependent and Ca2+-independent activities of neural and nonneural synaptotagmins. Nature 375, 594–599 (1995).

7. Brose, N., Petrenko, A.G., Südhof, T.C. & Jahn, R. Synaptotagmin: a Ca2+ sensor on the synaptic vesicle surface. Science 256, 1021–1025 (1992).

8. Davletov, B.A. & Südhof, T.C. A single C2-domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid-binding. J. Biol. Chem. 268, 26386–26390 (1993).

9. Pang, Z.P., Shin, O.-H., Meyer, A.C., Rosenmund, C. & Südhof, T.C. A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent SNARE-complex binding in synaptic exocytosis. J. Neurosci. 26, 12556–12565 (2006).

10. Sutton, R.B., Davletov, B.A., Berghuis, A.M., Südhof, T.C. & Sprang, S.R. Structure of the first C2-domain of synaptotagmin I: a novel Ca2+/phospholipid binding fold. Cell 80, 929–938 (1995).

11. Fernández-Chacón, R. et al. Synaptotagmin I functions as a Ca2+-regulator of release probability. Nature 410, 41–49 (2001).

12. Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994).

13. Xu, J., Mashimo, T. & Südhof, T.C. Synaptotagmin-1, -2, and -9: Ca2+-sensors for fast release that specify distinct presynaptic properties in subsets of neurons. Neuron 54, 567–581 (2007).

14. Sun, J. et al. A dual Ca2+-sensor model for neuro-transmitter release in a central synapse. Nature 450, 676–682 (2007).

15. Cao, P., Maximov, A. & Südhof, T.C. Activity-dependent IGF-1 exocytosis is controlled by the Ca2+-sensor synaptotagmin-10. Cell 145, 300–311 (2011).

16. McMahon, H.T., Missler, M., Li, C. & Südhof, T.C. Complexins: cytosolic proteins that regulate SNAP-receptor function. Cell 83, 111–119 (1995).

17. Maximov, A., Tang, J., Yang, X., Pang, Z. & Südhof, T.C. Complexin controls the force transfer from SNARE complexes to membranes in fusion. Science 323, 516–521 (2009).

18. Tang, J. et al. Complexin/synaptotagmin-1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175–1187 (2006).

19. Wang, Y., Okamoto, M., Schmitz, F., Hofman, K. & Südhof, T.C. RIM: a putative Rab3-effector in regulating synaptic vesicle fusion. Nature 388, 593–598 (1997).

20. Kaeser, P.S. et al. RIM proteins tether Ca2+-channels to presynaptic active zones via a direct PDZ-domain interaction. Cell 144, 282–295 (2011).

21. Schoch, S. et al. RIM1a forms a protein scaffold for regulating neurotransmitter release at the active zone. Nature 415, 321–326 (2002).

22. Verhage, M. et al. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287, 864–869 (2000).

 

SOURCE

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

NATURE MEDICINE | SPOONFUL OF MEDICINE

Lasker Awards go to rapid neurotransmitter release and modern cochlear implant

09 Sep 2013 | 13:38 EDT | Posted by Roxanne Khamsi | Category: 

Lasker_logo 2Posted on behalf of Arielle Duhaime-RossA very brainy area of research has scooped up one of this year’s $250,000 Lasker prizes, announced today: The Albert Lasker Basic Medical Research Award has gone to two researchers who shed light on the molecular mechanisms behind the rapid release of neurotransmitters—findings that have implications for understanding the biology of mental illnesses such as schizophrenia, as well the cellular functions underlying learning and memory formation.By systematically analyzing proteins capable of quickly releasing chemicals in the brain, Genentech’s Richard Scheller and Stanford University’s Thomas Südhofadvanced our understanding of how calcium ions regulate the fusion of vesicles with cell membranes during neurotransmission. Among Scheller’s achievements is the identification of three proteins—SNAP-25, syntaxin and VAMP/synaptobrevin—that have a vital role in neurotransmission and molecular machinery recycling. Moreover, Südhof’s observations elucidated how a protein called synaptotagmin functions as a calcium sensor, allowing these ions to enter the cell. Thanks to these discoveries, scientists were later able to understand how abnormalities in the function of these proteins contribute to some of the world’s most destructive neurological illnesses. (For an essay by Südhof on synaptotagmin, click here.)The Lasker-DeBakey Clinical Medical Research Award went to three researchers whose work led to the development of the modern cochlear implant, which allows the profoundly deaf to perceive sound. During the 1960s and 1970s Greame Clark of the University of Melbourne and Ingeborg Hochmair, CEO of cochlear implant manufacturer MED-EL, independently designed implant components that, when combined, transformed acoustical information into electrical signals capable of exciting the auditory nerve. Duke University’s Blake Wilson later contributed his “continuous interleaved sampling” system, which gave the majority of cochlear implant wearers the ability to understand speech clearly without visual cues. (For a viewpoint by Graeme addressing the evolving science of cochlear implants, click here.)Bill and Melinda Gates were also honored this year with the Lasker-Bloomberg Public Service Award. Through their foundation, the couple has made large investments in helping people living in developing countries gain access to vaccines and drugs. The Seattle-based Bill & Melinda Gates Foundation also runs programs to educate women about proper nutrition for their families and themselves. The organization has a broad mandate in public health; one of its most well known projects is the development of a low-cost toilet that will have the ability to operate without water.The full collection of Lasker essays, as well as a Q&A between Lasker president Claire Pomeroy and the Gateses, can be found here.

Summary

Author: Larry H Bernstein, MD, FCAP

Chapter IX focused on VSM of the artery and related the action of calcium-channel blockers (CCMs) to the presynaptic interruption of synaptic-vesicle fusion necessary for CA+ release that leads to neurotransmitter secretion.  Under the circumstance neurotransmitter activation, the is VSM contraction (associated with tone).  The effect of CCB action on neurotransmitter action, there is a resultant vascular dilation facilitating flow.    In this section, we extend the mechanism to other smooth muscle related action in various organs.

[1] Neural activation of vas deferens smooth muscle cells

Noradrenaline (NA) acts by stimulating α1-adrenoreceptors to produce InsP3, which then releases Ca2+ that may induce an intracellular Ca2+ wave similar to that triggered by the ATP-dependent entry of external Ca2+. In addition, the α1-adrenoreceptors also activate the smooth muscle Rho/Rho kinase signaling pathway that serves to increase the Ca2+ sensitivity of the contractile machinery.

[2]  Urinary bladder and micturition

The bladder, which functions to store and expel urine, is surrounded by layers of detrusor SMCs. The latter have two operational modes: during bladder filling they remain relaxed but contract vigorously to expel urine during micturition. The switch from relaxation to contraction, which is triggered by neurotransmitters released from parasympathetic nerves, depends on the acceleration of an endogenous membrane oscillator that produces the repetitive trains of action potentials that drive contraction.

SMCs are activated by membrane depolarization (ΔV) that opens L-type voltage-operated channels

This mechanism of activation is also shared by [1], and uterine contraction. SMCs are activated by membrane depolarization (ΔV) that opens L-type voltage-operated channels (VOCs) allowing external Ca2+ to flood into the cell to trigger contraction. This depolarization is induced either by ionotropic receptors (vas deferens) or a membrane oscillator (bladder and uterus). The membrane oscillator, which resides in the plasma membrane,  generates the periodic pacemaker depolarizations responsible for the action potentials that drive contraction.

The main components of the membrane oscillator are the Ca2+ and K+ channels that sequentially depolarize and hyperpolarize the membrane, respectively. This oscillator generates the periodic pacemaker   depolarizations that trigger each action potential. The resulting Ca2+ signal lags behind the action potential because it spreads into the cell as a slower Ca2+ wave mediated by the type 2 RYRs.   Neurotransmitters such as ATP and acetylcholine (ACh), which are released from parasympathetic axonal varicosities that innervate the bladder, activate or accelerate the oscillator by inducing membrane depolarization (ΔV).

[3] The depolarizing signal that activates gastrointestinal, urethral and ureter SMCs is as follows:

A number of SMCs are activated by pacemaker cells such as the interstitial cells of Cajal (ICCs) (gastrointestinal and urethral SMCs) or atypical SMCs (ureter). These pacemaker cells have a cytosolic oscillator that generates the repetitive Ca2+ transients that activate inward currents that spread through the gap junctions to provide the depolarizing signal (ΔV) that triggers contraction. Our greatest interest has been in this mechanism. The rhythmical contractions of vascular, lymphatic, airway and corpus cavernosum SMCs depend on an endogenous pacemaker driven by a cytosolic Ca2+ oscillator that is responsible for the periodic release of Ca2+ from the endoplasmic reticulum. The periodic pulses of Ca2+ often cause membrane depolarization, but this is not part of the primary activation mechanism but has a secondary role to synchronize and amplify the oscillatory mechanism. Neurotransmitters and hormones act by modulating the frequency of the cytosolic oscillator.

Vascular or airway SMCs are driven by a cytosolic oscillator that generates a periodic release of Ca2+ from the endoplasmic reticulum that usually appears as a propagating Ca2+ wave.

The following points are repeated:

Step 1. The initiation and/or modulation of this oscillator depends upon the action of transmitters and hormones such as ACh, 5-HT, NA and endothelin-1 (ET-1) that increase the formation of InsP3 and diacylglycerol (DAG), both of which promote oscillatory activity.

Step 2. The oscillator is very dependent on Ca2+ entry to provide the Ca2+ necessary to charge up the stores for each oscillatory cycle. The nature of these entry mechanisms vary between cell types.

Step 3. The entry of external Ca2+ charges up the ER to sensitize the RYRs and InsP3 receptors prior to the next phase of release.

The proposed role of cyclic ADP-ribose (cADPR) in airway SMCs is consistent with this aspect of the model on the basis of its proposed action of stimulating the SERCA pump to enhance store loading and such a mechanism has been described in colonic SMCs.

Step 4. The mechanism responsible for initiating Ca2+ release may depend either on the RYRs or the InsP3 receptors (I). RYR channels are sensitive to store loading and the InsP3 receptors will be sensitized by the agonist-dependent formation of InsP3.

The global Ca2+ signal then activates contraction

Smooth muscle cell calcium activation mechanisms. Berridge MJ.
J Physiol. 2008; 586(Pt 21):5047-61. http://dx.doi.org/10.1113/jphysiol.2008.160440

Read Full Post »


Zinc-Finger Nucleases (ZFNs) and Transcription Activator–Like Effector Nucleases (TALENs)

Reporter: Larry H Bernstein, MD, FCAP

 

TALENs and ZFNs are associated with different mutation signatures

Y Kim,  J Kweon  & Jin-Soo Kim

Zinc-finger nucleases (ZFNs) and transcription activator–like effector nucleases (TALENs) are of great interest for genome engineering in higher eukaryotic cells and organisms. These enzymes

  1. contain the same FokI nuclease domain and
  2. induce site-specific DNA cleavage.

http://www.nature.com/nmeth/journal/v10/n3/extref/nmeth.2364-S1.pdf

http://www.nature.com/nmeth/journal/v10/n3/full/nmeth.2364.html?WT.ec_id=NMETH-201303

English: Bacterial multi-drug resistance syste...

English: Bacterial multi-drug resistance system: complex of dimeric transcription-activator protein BmrR with bound TPP, untwisting the DNA to position the two promoter sites (top) for transcription. Coordinates from PDB file 1R8E, Brennan lab; displayed in KiNG. (Photo credit: Wikipedia)

                                   

English: Diagram of a typical rAAV vector

English: Diagram of a typical rAAV vector (Photo credit: Wikipedia)

Splicing activation

Splicing activation (Photo credit: Allen Gathman)

Read Full Post »


Reporter: Aviva Lev-Ari, PhD, RN

UPDATED ON 6/17/2013

UCSC Designing Social Network-type Model for Analyzing Cancer Data

June 17, 2013

NEW YORK (GenomeWeb News) – Seeking to make the masses of cancer sequence data that is being generated more useful for researchers, investigators at University of California, Santa Cruz, plan to use a $3.5 million grant from the National Cancer Institute to create a new platform for organizing and accessing these data.

The UCSC group plans to create a method for making the raw sequence information in repositories like the university’s Cancer Genomics Hub more useful for investigators seeking to make clinical predictions about how cancer mutations respond to drugs, for example.

The aim of the project will be to develop a new database called the Biomedical Evidence Graph, or BMEG, which will use a graph database structure, like Facebook does, to enable swift access to complex and interconnected datasets.

Principal investigator Joshua Stuart, a UCSC associate professor of engineering, likened the difficulty for many investigators of using raw sequence data to average computer users trying to work directly with binary code.

“Your web browser doesn’t understand zeros and ones. There are layers and layers of software programs between that and what you see on a web page. We need to do the same thing for DNA sequences to reach the higher levels of interpretation needed for scientific discovery,” Stuart said in a statement.

Stuart said that a platform similar to what social networks like Facebook use offer a “natural way” to represent data from tumor samples based upon the connections between their molecular profiles.

CGHub, which launched last year to house data from The Cancer Genome Atlas consortium and similar projects, holds thousands of genome sequences from individual patients and access is highly controlled and limited to approved projects.

BMEG, however, will not require such security because it will host higher-level data from analyses of the raw genome sequencing. This will enable a broader group of investigators to use and analyze these datasets without having to download massive files to their computers.

“TCGA researchers have built a lot of great tools for data analysis, and we need to get those installed in the BMEG so the rest of the world can engage in that higher level analysis,” Stuard said. “The idea is to build a shared knowledge base and create a playground where lots of researchers can interact, test their algorithms, and compare results.”

The BMEG will be located with the CGHub servers at the San Diego Supercomputer Center, and investigators will be able to run their analyses as apps on the BMEG, UCSC said.

SOURCE

http://www.genomeweb.com//node/1242591?hq_e=el&hq_m=1590835&hq_l=3&hq_v=e1df6f3681

 

Five3, maker of cancer genomics software, takes off from UCSC labs

October 29, 2012 | By 

A group from the University of California, Santa Cruz (UCSC), has embarked on a new project to commercialize cancer genomics software through a new startup company called Five3 Genomics. The company has attracted a few of the biggest names in genomics and biotech to serve as advisers.

Recent software applications have enabled scientists to analyze cancer genomic data to track molecular changes in cells, spotting some of the triggers that cause tumors to grow. Led by CEO and co-founder Steve Benz, Five3 Genomics plans to sell its cancer genomics software to healthcare companies and pharmaceutical firms. Drugmakers could use the company’s software to discover new targets for cancer therapies, while hospitals could use the technology to put patients on existing drugs that home in on the molecular triggers of their cancer.

Benz and his fellow co-founders have a crack group of bioinformatics and biotech experts to help guide their startup. They’ve called on their UCSC mentors, David Haussler and Joshua Stuart. Haussler’s lab has participated in some of the most pioneering efforts in genomics over the past couple of decades, including the Human Genome Project that raced to decode an entire human genome. Also, Dr. Patrick Soon-Shiong, who has made billions of dollars in biotech, is serving as a scientific adviser.

“We’re working with academic collaborators to build out the platform and starting conversations with pharmaceutical companies and insurance companies,” Benz, who recently wrapped up his doctorate at UCSC, told the Santa Cruz Sentinel newspaper. “It’s a great opportunity to be able to take this technology and commercialize it so that it can be used to help patients.”

SOURCE:

http://www.fiercebiotechit.com/story/five3-maker-cancer-genomics-software-takes-ucsc-labs/2012-10-29

UCSC grad students launch cancer genomics company in Santa Cruz

By Sentinel Staff Report

Santa Cruz Sentinel

Posted:   10/24/2012 04:11:37 PM PDT

SANTA CRUZ — The co-founders of Five3 Genomics, a new biotech company based in Santa Cruz, are former graduate students in the Baskin School of Engineering at UC Santa Cruz, where they helped develop innovative cancer genomics software.

Their company, which has signed a license agreement with UCSC, offers software and services for cancer researchers, pharmaceutical companies, and health-care organizations. Its goal is to provide the data processing and analysis required for personalized cancer therapy, in which treatments are matched to the specific genetic aberrations found in an individual patient’s cancer cells.

“We’re working with academic collaborators to build out the platform and starting conversations with pharmaceutical companies and insurance companies,” said CEO Steve Benz, who completed his doctorate in bioinformatics this year. “It’s a great opportunity to be able to take this technology and commercialize it so that it can be used to help patients.”

In addition to Benz, the co-founders of Five3 Genomics include Chief Technical Officer Zachary Sanborn and Chief Scientific Officer Charles Vaske. All three of them worked as graduate students with UC Santa Cruz bioinformatics experts David Haussler and Joshua Stuart, who are doing pioneering work in the field of cancer genomics. Haussler, a professor of biomolecular engineering and Howard Hughes Medical Institute investigator, said that Benz, Sanborn, and Vaske were “brilliant gradstudents.”

“Working at UCSC they were exposed to the cutting edge in computational genomics,” Haussler said. “They played a key role in developing our cancer genomics program.”

Vaske, who earned his doctorate in 2009, and Benz were lead developers of a software program from Stuart’s lab called Paradigm. Stuart, a professor of biomolecular engineering, has been a close collaborator with Haussler on cancer genomics projects, including The Cancer Genome Atlas funded by the National Institutes of Health and two cancer research “Dream Teams” funded by Stand Up To Cancer and other organizations.

Paradigm, one of the core technologies for Five3 Genomics, is used to understand which molecular pathways are affected by the genetic changes in a patient’s cancer cells. This information can be used in a clinical setting to guide therapeutic decisions and by pharmaceutical companies to identify new targets for drug development.

“On the pharmaceutical side, we can provide indications for new uses for drugs that are already out there, as well as identify targets for new drugs,” Benz said.

Sanborn, who will finish his doctorate this year, worked in Haussler’s lab on a DNA sequence analysis program called BamBam, which is used to identify the genetic changes in cancer cells. Sanborn and Benz also contributed to the development of the UCSC Cancer Genome Browser in Haussler’s lab.

The scientific advisers for Five3 Genomics include Haussler and Stuart, as well as Dr. Patrick Soon-Shiong, a surgeon, medical researcher, and biotechnology entrepreneur, and Dr. Margaret Tempero, deputy director and director of research programs at the UCSC Helen Diller Family Comprehensive Cancer Center.

“It’s particularly gratifying to see this UCSC research transition to a commercial product, so these cutting-edge techniques can begin to benefit the public as quickly as possible,” said Bruce Margon, vice chancellor for research at UCSC.

SOURCE:

http://www.santacruzsentinel.com/localnews/ci_21846767

Biotech billionaire’s supercomputer cuts cancer analysis to 47 seconds

October 4, 2012 | By 

Dr. Patrick Soon-Shiong, a surgeon and biotech mogul, has spotlighted a supercomputer-based system and network to rapidly transfer and analyze cancer genetic data in mere seconds as opposed to the weeks or months of previous approaches. The supercomputer crunches genetic data from a tumor with results on abnormalities in 47 seconds, and the high-speed fiber-optic network Soon-Shiong has championed transfers samples in shy of 18 seconds, according to an announcement Wednesday.

Soon-Shiong’s L.A.-based company NantHealth has joined forces with Verizon, Intel, Hewlett-Packard, Blue Shield of California and other players to advance a national system to enable rapid sharing of genomic information among cancer doctors, aiding physicians in making the right call on treatments for patients based on the characteristics of their tumors. It’s a big deal because lack of such information contributes to misdiagnoses.

Via NantHealth and other vehicles, Soon-Shiong has worked on integrating a variety of digital technologies to revolutionize scientific research and medicine. As Reuters reports, he’s poured more than $400 million from his estimated fortune of more than $7 billion into building the fiber-optic network. His nonprofit is working on connecting sequencing centers, medical research hubs and hospitals to the network to create an infrastructure for these groups to share data from big science endeavors such as The Cancer Genome Atlas.

Soon-Shiong built most of his fortune with the sales of Abraxis BioScience to Celgene ($CELG) in 2010 for $2.9 billion and APP Pharmaceuticals to Germany’s Fresenius two years earlier for billions. (Abraxis developed Celgene’s anti-cancer drug Abraxane.) He’s now reportedly the richest man in Los Angeles, where he owns a piece of the NBA’s Los Angeles Lakers and has been connected with efforts to bring an NFL franchise back to the city.

SOURCE:

http://www.fiercebiotechit.com/story/biotech-billionaires-supercomputer-cuts-cancer-analysis-47-seconds/2012-10-04

Bringing genomic medicine into clinical practice by placing supercomputers in the hands of physicians at point of care

WASHINGTON—-Dr. Patrick Soon-Shiong, Chairman of NantHealth and the Chan Soon-Shiong Institute for Advanced Health announced a revolutionary advance in cancer treatment that will reduce the necessary time for analysis from 8 weeks to an unprecedented 47 seconds per patient. For the first time, oncologists can compare virtually every known treatment option on the basis of genetics, risk, and cost – before treatment begins, not after.

Alongside Senator Bill Frist, MD, of the Bipartisan Policy Center and J. Michael McGinnis, MD of the Institute of Medicine and Doctors Helping Doctors, Dr. Soon-Shiong reported on the successful real-time analysis of the largest collection of tumor genomes in the United States, of 6,017 cancer genomes from 3,022 patients with 19 different cancer types, in the record time of 69 hours. Genomic analysis has taken an average of 8 to 10 weeks to complete. That delay leads not just to less efficient, more costly care, but sometimes to the wrong course of treatment altogether – and, thus, higher mortality. “Incorrect care that leads to loss of life is unacceptable,” said Dr. Soon-Shiong, “and from today onward, it will no longer be necessary.”

Oncologists currently prescribe a course of cancer treatment based on the anatomical location of the cancer. Yet a patient with breast cancer could benefit from the positive results discovered from a patient with lung cancer, if the underlying molecular pathways involving both cancers were the same. The inability to utilize genomic sequencing to guide treatment has been due to the inability to convert a patient’s DNA into actionable information in actionable time.

But by collaborating with Blue Shield of California, the Chan Soon-Shiong Institute for Advanced Health, the National LambdaRail, Doctors Helping Doctors, Verizon, Bank of America, AT&T, Intel, and Hewlett-Packard, NantHealth has built a supercomputer-based high-speed fiber network that will not only provide thousands of oncology practices with life-saving information, but do so in exponentially faster time. “Doctors will finally be able to provide higher-quality treatment in a dramatically more efficient, effective, and affordable manner,” says Dr. Soon-Shiong.

“It currently takes approximately two months and tens of thousands of dollars to perform the sequencing and analysis of a single cancer patient’s genome. We can’t reduce the cost of care and improve outcomes in cancer if we don’t have the capability to know the right treatment for the right patient before treatment begins. We needed a national supercomputing infrastructure that brings genomic medicine into clinical practice. By placing supercomputers in the hands of physicians, that need is now a reality,” said Dr. Soon-Shiong.

Accuracy will also be radically improved. Among NantHealth’s partner oncologists utilizing its fact-based software platform (eviti – http://www.eviti.com) the number of cases where doctors have made incorrect recommendations has dropped from 32% to virtually zero“With this patient-centered, fact-based approach to collecting and analyzing data, millions more patients will have a better chance of beating cancer,” Dr. Soon-Shiong emphasized. Over the past 12 months over 2,000 oncology practices representing 8,000 oncologists and nurses have successfully installed and utilized this fact-based (eviti) software platform, positively impacting thousands of cancer patients lives.

THE RESEARCH PROCESS

In July 2012, NantWorks’ scientific team (Five3 Genomics – http://www.Five3Genomics.com) collected 6,017 tumor and germline exomes, representing 3,022 cancer patients with 19 unique cancer types. The sample collection included: 999 breast cancer; 1.156 kidney and bladder cancer; 985 gastrointestinal cancer; 744 brain cancer; 745 lung cancer; 670 ovarian, uterine and cervical caner; 436 head and neck cancer; 177 prostate cancer; 70 melanoma cancer; and 35 blood tumor samples.

This massive amount of data totaled 96,512 gigabytes and was successfully transferred and processed via our supercomputing, high-speed fiber netowrk in 69 hours. This overall transfer speed represents a stream of one sample every 17.4 seconds, and the supercomputer analysis for genetic and protein alterations between the tumor and normal sample completed every 47 seconds per patient.

Given the nation’s estimated cancer rate of 1.8 million new cases in 2012, this infrastructure now brings the capability of analyzing 5,000 patients per day.

He noted that medicine has continued to make dramatic advances, but the delivery of medicine has lagged far behind, stuck in a world where information is trapped, patterns get missed, and patients suffer. Powered by advanced supercomputing technology and wireless mobile health, the network has become one of country’s fastest genomic platforms with connectivity to over 8000 practicing oncologists and nurses. “This revolution in healthcare is long overdue – converging 21st century medical science with 21st century technology,” Dr. Soon-Shiong concluded.

Through NantHealth’s genomic analysis network, doctors can finally make cancer treatment more efficient, more effective, and more affordable for more patients. And with public and private partners equally as committed to reshaping the way doctors deliver healthcare and treat cancer, there are no limits to what this health information breakthrough might lead to for all cancer patients.

A network of major cancer centers including those at City of Hope, John Wayne Cancer Institute, and Methodist Hospital in Houston, have contributed to this collection of over 6,000 genomes, which also included the entire collection of exome samples from The Cancer Genome Atlas.

About NantWorks

The core mission of NantWorks, LLC, is to converge a wide range of technologies to accelerate scientific discoveries, enhance research and improve healthcare treatment and outcomes. Founded and led by Dr. Patrick Soon-Shiong, NantWorks is building an integrated fact-based, genomically-informed, personalized approach to the delivery of care and the development of next generation diagnostics and therapeutics. For more information, see http://www.nantworks.com.

Contacts

NantWorks, LLC
Jen Hodson
310.405.7539
jhodson@nantworks.com

SOURCE:

http://www.fiercebiotechit.com/press-releases/launch-nations-fastest-genomic-supercomputing-platform-reduces-cancer-genom

Research cache in works

by Emily Gersema – Jan. 28, 2012 01:29 PM

The Republic | azcentral.com

Supercomputing supports genetic, cancer research in Arizona: compare patient cases to tailor care

A massive building near Phoenix Sky Harbor International Airport is now home to a supercomputer that one day is expected to store clinical-research reports, medical records and the decoded genetic makeup of millions of patients and their cancers.

Having this vault of medical information is a dream for doctors, specialists and researchers who are trying to tailor medical care to the individual needs of their cancer patients. Despite huge advances in research and medicine, doctors have no one-stop shop for up-to-date clinical-trial results, other medical cases and genetic maps of their patients.

With access to this massive library, cancer doctors potentially could specify with precision the dosages of medicines, chemotherapy and radiation therapy for their patients by comparing those cases to those of other patients with similar genetic makeups and similar cancers.

In effect, this supercomputer could be a gateway to personalized medical care, as its creator, billionaire scientist Patrick Soon-Shiong, envisions it. His staff at CSS Institute for Advanced Health in California, which owns the project, and supporters of personalized medicine said the vault also could help reduce doctor error in the diagnosis and treatment of patients.

Better treatments and more accurate diagnoses could help lower the cost of medical care and enable patients to get treatment at home instead of at the hospital, they said.

The presence of the supercomputer could put Phoenix on the cutting edge of medical research and treatment. The path to these potential medical breakthroughs, however, is fraught with privacy concerns. Patient advocates fear the project could open a pathway to exploitation if patient information isn’t confidential. They want assurances that the institute would require patient consent to obtain records, the records would be kept private and the project would be under close regulatory oversight.

The engine: A supercomputer

While the word “supercomputer” evokes an image of a giant computer, the machine located in the Phoenix storage site resembles a large herd of smaller computers that have been linked to one another.

“It used to be a one big monolithic thing,” said Anoj Willy, of the CSS Institute. “But now what we’re able to do is take lots of general-purpose computers and band them to create a big, superprocessing engine.”

The CSS Institute project, which involves equipment and products from Hewlett-Packard and Intel Corp., is in its earliest stages, Willy said. The institute plans to focus data collection on genetic research and cancer.

The endeavor would create at least 50 jobs with annual salaries of about $75,000. Soon-Shiong also would invest at least $200 million in development, construction, machinery and equipment to build the electronic-data-storage facility.

The institute is in the process of signing agreements with various institutions that have been sequencing genomes — the maps of DNA strands that make up living things.

Bob Peirce, senior vice president of Soon-Shiong’s Nant Holdings in Los Angeles, said that while scientists have made strides in human genomic sequencing, the maps of these sequences are scattered at different sites around the world, depending on which institution decoded them.

Researchers have not yet decoded the whole human genome, Peirce said. They have each decoded snippets.

The lack of a complete map and a one-stop shop for the genomic information for doctors and researchers impedes their progress in personalized medical treatment, he said.

This means genomic sequences currently aren’t “relevant to the average patient or the average doctor,” Peirce said.

Creating a complete map of the human genome would require a massive, computerized data center, like the one being built by Soon-Shiong in Phoenix — to decode what scientists estimate are 3 billion pairs of DNA strands.

In addition, Soon-Shiong wants the supercomputer and its data centers, including one planned for Scottsdale, to aid in mapping the genetic makeup of individual patients’ cancerous cells.

“We need to be in a position where we can analyze the genome of the cancer and determine the genome of the host patient (to treat them),” Peirce said.

Peirce offered assurances that the data would be highly secured to guard against hackers. The data could be accessed by people who are deemed “authorized users,” he said, which could include the patients themselves who are trying to monitor their conditions and care. The institute has been working with a “chief technical officer,” who worked at the Pentagon, on securing the data centers and information they contain, Peirce said. He declined to name the officer.

The concern: Privacy

Edward Abrahams, president of the Personalized Medicine Coalition, a non-profit group in Washington, D.C., said researchers are on the cusp of creating medical care tailored to each person’s needs, and they can reach that with a supercomputer.

But they are faced with several challenges. Chief among them is patient privacy, he said.

The federal Health Insurance Portability and Accountability Act guards patient privacy, but its reach is limited. Patient information is kept private within the realm of health care — at the doctor’s office, the hospital and with the patient’s insurance company, said Bob Gellman, a privacy expert in Washington, D.C.

“An institution like this (CSS Institute) is not covered by health-privacy laws,” Gellman said. “It’s not a health-care provider. It’s not an insurer.”

Gellman said a worst-case scenario would involve a patient sharing genetic information with a company or organization, only to have it misused or exploited by another party.

“The information when it sat in the health-care system — when it sat in your doctor’s office — had all kinds of protections,” Gellman said. “But if you give the information with your consent to somebody else, then someone could just go to that third party and say, ‘Give me all your information.’ ”

In that scenario, the records and data are out of the patient’s control and are unprotected.

Individuals trying to solve the health problems of their autistic children, for example, may want to participate.

“That may be a perfectly rational decision.” Gellman said. “But for people who don’t know or aren’t aware of that (institution’s) motivation … you might agree to give this information, and 20 years later, you’re in litigation with somebody or you’re applying for a job and it comes up.”

Read more: http://www.azcentral.com/arizonarepublic/business/articles/2012/01/26/20120126medical-research-cache-in-works.html?nclick_check=1#ixzz2AjfTgdsf

http://www.azcentral.com/arizonarepublic/business/articles/2012/01/26/20120126medical-research-cache-in-works.html

Cancer Research targets human genome breathrough with supercomputer

Platform Computing LSF integrated with genetic sequencing technology

By Antony Savvas | Published: 16:04 GMT, 09 December 11 | Computerworld UK


A new supercomputing workload management system is aiding scientific work by Cancer Research and the Cambridge Research Institute’s human genome project.

Cancer Research UK is using Platform Computing’s LSF software to improve cluster efficiency and reduce IT costs on the CRI genome research.

By integrating Platform LSF with a new advanced genetic sequencing platform, the institute has already gained greater insight into genetic cancer mutations that will lead to scientific breakthroughs in the areas of cancer diagnosis, treatment and prevention, said Cancer Research.

“Platform LSF gives us the means to produce and manage a wealth of gene sequencing data that we could only have dreamed about previously,” said Peter Maccallum, head of IT and scientific computing at Cancer Research UK in Cambridge. “This has already lead to tangible published work looking into breast cancer, and is proving its worth in helping our researchers further the understanding of how cancers progress.”

Prior to implementing Platform LSF, CRI’s 21 research groups employed separate computing resources in separate locations, which drove up server costs, reduced utilisation rates and increased server maintenance.

By orchestrating workloads and managing CRI’s research applications in a single data centre, Platform LSF has enabled CRI to save approximately £50,000 by removing hardware and maintenance duplication across each location, while increasing the amount of data processed. Cancer Research says the institute can now direct more computing resources directly to its research teams “to use in a more timely and cost efficient manner”.

CRI has already saved the equivalent in man hours of one full-time employee by integrating Platform LSF, says Cancer Research. As a result, the institute plans to scale Platform LSF internally by adding more servers as compute requirements increase.

CRI is also collaborating with Platform Computing to architecturally support cross-organisation systems for HPC (high performance computing) clusters, that will enable CRI to collaborate with other research organisations in order to meet the growing demand for genomics research.

In other recent medical technology news, scientists at Cambridge University are developing a computer system that can read vast amounts of scientific literature, make rapid connections between facts and develop hypotheses. Cambridge University said most biomedical scientists cannot keep on top of reading all of the publications in their field, let alone an adjacent field. As a first step to solving the problem, Cambridge has developed its CRAB text-mining tool.

SOURCES:

http://www.cio.co.uk/news/3324141/cancer-research-targets-human-genome-breathrough-with-supercomputer/


Read Full Post »


 

Reporter: Aviva Lev-Ari, PhD, RN

Public release date: 18-Oct-2012
Contact: Lauren Woods
law2014@med.cornell.edu
212-821-0560
New York- Presbyterian Hospital/Weill Cornell Medical Center/Weill Cornell Medical College

 

New study shows reprogrammed amniotic fluid cells could treat vascular diseases

Weill Cornell Researchers discover a new effective approach for converting amniotic fluid-derived cells into endothelial cells to repair damaged blood vessels in heart disease, stroke, diabetes and trauma

NEW YORK (Oct. 18, 2012) — A research team at Weill Cornell Medical College has discovered a way to utilize diagnostic prenatal amniocentesis cells, reprogramming them into abundant and stable endothelial cells capable of regenerating damaged blood vessels and repairing injured organs.

Their study, published online today in Cell, paints a picture of a future therapy where amniotic fluid collected from thousands of amniocentesis procedures yearly, during mid-pregnancy to examine fetal chromosomes, would be collected with the permission of women undergoing the test. These cells, which are not embryonic, would then be treated with a trio of genes that reprogram them quickly into billions of endothelial cells — the cells that line the entire circulatory system. The new endothelial cells could be frozen and banked the same way blood is, and patients in need of blood vessel repair would be able to receive the cells through a simple injection.

If proven in future studies, this novel therapy could dramatically improve treatment for disorders linked to a damaged vascular system, including heart disease, stroke, lung diseases such as emphysema, diabetes, and trauma, says the study’s senior investigator, Dr. Shahin Rafii, the Arthur B. Belfer Professor in Genetic Medicine at Weill Cornell Medical College and co-director of its Ansary Stem Cell Institute.

“Currently, there is no curative treatment available for patients with vascular diseases, and the common denominator to all these disorders is dysfunction of blood vessels, specifically endothelial cells that are the building blocks of the vessels,” says Dr. Rafii, who is also a Howard Hughes Medical Institute investigator.

But these cells do much more than just provide the plumbing to move blood. Dr. Rafii has recently led a series of transformative studies that show endothelial cells in blood vessels produce growth factors that actively participate in organ maintenance, repair and regeneration. So while damaged vessels cannot repair the organs they nurture with blood, he says an infusion of new endothelial cells could.

“Replacement of the dysfunctional endothelial cells with transplantation of normal, properly engineered cultured endothelial cells could potentially provide for a novel therapy for many patients,” says study co-author Dr. Sina Rabbany, adjunct associate professor of bioengineering in genetic medicine at Weill Cornell. “In order to engineer tissues with clinically relevant dimensions, endothelial cells can be assembled into porous three-dimensional scaffolds that, once introduced into a patient’s injured organ, could form true blood vessels.”

Dr. Rafii says that this study will potentially create a new field of translational vascular medicine. He estimates that as few as four years are needed for the preclinical work to seek FDA approval to start human clinical trials to advance the potential of reprogrammed endothelial cells for treatment of vascular disorders.

As part of their study, the research team proved, in mice, that endothelial cells reprogrammed from human amniotic cells could engraft into an injured liver to form stable, normal and functional blood vessels. “We have shown that these engrafted endothelial cells have the capacity to produce unique growth factors to promote regeneration of the liver cells,” says the study’s lead investigator, Dr. Michael Ginsberg, a senior postdoctoral associate in Dr. Rafii’s laboratory.

“The novelty of this technique is that, from 100,000 amniotic cells — a small amount — we grew more than six billion new authentic endothelial cells within a matter of weeks,” Dr. Ginsberg says. “And when we injected these cells into mice, a substantial amount of them engrafted into regenerating vessels. It was remarkable to see that these cells went right to work building new blood vessels in the liver as well as producing the right growth factors that could potentially regenerate and repair injured organs.”

The Goldilocks of Cellular Reprogramming

To date, there have been many failed attempts to clinically produce endothelial cells that can be used to treat patients. Isolation of endothelial cells from adult organs so they can be grown in the laboratory is not efficient, according to Dr. Daylon James, study co-author and an assistant professor of stem cell biology in reproductive medicine at Weill Cornell Medical College. Attempts to produce the cells from the body’s master pluripotent stem cells have also not worked out. Experiments have shown that prototypical pluripotent stem cells, such as embryonic stem cells, which have the potential to become any cell in the body, produce endothelial cells but often grow poorly, and if not fully differentiated could potentially cause cancer. “Coaxing adult cells to revert to a stem-like state so they can then be pushed to form endothelial cells is, at this point, not clinically feasible, and ongoing studies in my lab are focused on achieving this goal,” says Dr.

James, who is also assistant professor of stem cell biology in obstetrics and gynecology and genetic medicine at Weill Cornell. Therefore, Dr. Rafii’s team searched for a new source of cells that they could turn into a vast supply of stable endothelial cells. They probed human amniotic fluid-derived cells, which some studies had suggested have the potential to become differentiated cell types, if stimulated in the right way — which no one had yet identified.

In their first experiments with these cells three years ago, Dr. Ginsberg used cells taken from an amniocentesis given at 16 weeks of gestation. Researchers found that amniotic cells are the “Goldilocks” of cellular programming. “They are not as plastic and unstable as endothelial cells derived from embryonic cells or as stubborn as those produced from reprogramming differentiated adult cells,” Dr. Ginsberg says. Instead, he says amniotic cells provide conditions that are just right — the so-called “Goldilocks Principle” — for producing endothelial cells.

But in order to make that discovery, the researchers had to know how to reprogram the amniotic cells. To this end, they looked for the genes that embryonic stem cells use to differentiate into endothelial cells. Dr. Rafii’s group identified three genes that are expressed during vascular development, all of which are members of the E-twenty six (ETS) family of transcription factors known to regulate cellular differentiation, especially blood vessel formation.

Next, they used gene transfer technology to insert the three genes into mature amniotic cells, and then shut one of them off after a brief and critical period of activity by using a special molecular inhibitor. Remarkably, 20 percent of the amniotic cells could efficiently be reprogrammed into endothelial cells. “This is quite an achievement since current strategies to reprogram adult cells result less than one percent of the time in successful reprogramming into endothelial cells,” says Dr. Rafii.

“These transcription factors do not cause cancer, and the endothelial cells reprogrammed from human amniotic cells are not tumorigenic and could in the future be infused into patients with a large margin of safety,” Dr. Ginsberg says.

The findings suggest that other transcription factors could be used to reprogram the amniotic cells into many other tissue-specific cells, such as those that make up muscles, the brain, pancreatic islet cells and other parts of the body.

“While our work focused primarily on the reprogramming of amniotic cells into endothelial cells, we surmise that through the use of other transcription factors and growth conditions, our group and others will be able to reprogram mouse and human amniotic cells virtually into every organ cell type, such as hepatocytes in the liver, cardiomyocytes in heart muscle, neurons in the brain and even chondrocytes in cartilage, just to name a few,” Dr. Ginsberg says.

“Obviously, the implications of these findings would be enormous in the field of translational regenerative medicine,” emphasizes study co-author Dr. Zev Rosenwaks, the Revlon Distinguished Professor of Reproductive Medicine in Obstetrics and Gynecology at Weill Cornell Medical College and director and physician-in-chief of the Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine at NewYork-Presbyterian Hospital/Weill Cornell Medical Center. “The greatest obstacle to overcome in the pursuit to regenerate specific tissues and organs is the requirement for substantial levels of cells — in the billions — that are stable, safe and durable. Our approach will bring us closer to this milestone.”

“Most importantly, these endothelial cells could be reprogrammed from amniotic cells from genetically diverse individuals,” says co-author Dr. Venkat R. Pulijaal, director of the Cytogenetic Laboratory, associate professor of clinical pathology and laboratory medicine at Weill Cornell. What endothelial cells a patient receives would depend on their human leukocyte antigen (HLA) type, which is a set of self-recognition molecules that enable doctors to match a patient with potential donors of blood or tissue.

“Selecting the proper immunologically matched endothelial cells for each patient would be akin to blood typing. There are only so many varieties, which are well represented across the amniotic fluid cells that could be obtained, frozen and banked from wide variety of ethnic groups around the world,” Dr. Rafii says.

A patent has been filed on the discovery.

 

Other study co-authors from Weill Cornell Medical College include: Dr. Bi-Sen Ding, Dr. Daniel Nolan, Dr. Fuqiang Geng, Dr. Jason M. Butler, Dr. William Schachterle, Dr. Susan Mathew, Dr. Stephen T. Chasen, Dr. Jenny Xiang, Dr. Koji Shido and Dr. Olivier Elemento.

Dr. Rafii’s research is funded by the Howard Hughes Medical Institute, the National Heart, Lung, and Blood Institute, the Ansary Stem Cell Institute at Weill Cornell Medical College, the Empire State Stem Cell Board and New York State Department of Health grants, and the Qatar National Priorities Research Foundation.

Weill Cornell Medical College

Weill Cornell Medical College, Cornell University’s medical school located in New York City, is committed to excellence in research, teaching, patient care and the advancement of the art and science of medicine, locally, nationally and globally. Physicians and scientists of Weill Cornell Medical College are engaged in cutting-edge research from bench to bedside, aimed at unlocking mysteries of the human body in health and sickness and toward developing new treatments and prevention strategies. In its commitment to global health and education, Weill Cornell has a strong presence in places such as Qatar, Tanzania, Haiti, Brazil, Austria and Turkey. Through the historic Weill Cornell Medical College in Qatar, the Medical College is the first in the U.S. to offer its M.D. degree overseas. Weill Cornell is the birthplace of many medical advances — including the development of the Pap test for cervical cancer, the synthesis of penicillin, the first successful embryo-biopsy pregnancy and birth in the U.S., the first clinical trial of gene therapy for Parkinson’s disease, and most recently, the world’s first successful use of deep brain stimulation to treat a minimally conscious brain-injured patient. Weill Cornell Medical College is affiliated with NewYork-Presbyterian Hospital, where its faculty provides comprehensive patient care at NewYork-Presbyterian Hospital/Weill Cornell Medical Center. The Medical College is also affiliated with the Methodist Hospital in Houston. For more information, visit weill.cornell.edu.

Source:

 

Read Full Post »