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The bacterial makeup of human milk is influenced by the mode of breastfeeding, according to a new study. Although previously considered sterile, breast milk is now known to contain a low abundance of bacteria. While the complexities of how maternal microbiota influence the infant microbiota are still unknown, this complex community of bacteria in breast milk may help to establish the infant gut microbiota. Disruptions in this process could alter the infant microbiota, causing predisposition to chronic diseases such as allergies, asthma, and obesity. While it’s unclear how the breast milk microbiome develops, there are two theories describing its origins. One theory speculates that it originates in the maternal mammary gland, while the other theory suggests that it is due to retrograde inoculation by the infant’s oral microbiome.
To address this gap in knowledge scientists carried out bacterial gene sequencing on milk samples from 393 healthy mothers three to four months after giving birth. They used this information to examine how the milk microbiota composition is affected by maternal factors, early life events, breastfeeding practices, and other milk components. Among the many factors analyzed, the mode of breastfeeding (with or without a pump) was the only consistent factor directly associated with the milk microbiota composition. Specifically, indirect breastfeeding was associated with a higher abundance of potential opportunistic pathogens, such as Stenotrophomonas and Pseudomonadaceae. By contrast, direct breastfeeding without a pump was associated with microbes typically found in the mouth, as well as higher overall bacterial richness and diversity. Taken together, the findings suggest that direct breastfeeding facilitates the acquisition of oral microbiota from infants, whereas indirect breastfeeding leads to enrichment with environmental (pump-associated) bacteria.
The researchers argued that this study supports the theory that the breast milk microbiome is due to retrograde inoculation. Their findings indicate that the act of pumping and contact with the infant oral microbiome influences the milk microbiome, though they noted more research is needed. In future studies, the researchers will further explore the composition and function of the milk microbiota. In addition to bacteria, they will profile fungi in the milk samples. They also plan to investigate how the milk microbiota influences both the gut microbiota of infants and infant development and health. Specifically, their projects will examine the association of milk microbiota with infant growth, asthma, and allergies. This work could have important implications for microbiota-based strategies for early-life prevention of chronic conditions.
Clostridium difficile-associated disease, a significant problem in healthcare facilities, causes an estimated 15,000 deaths in the United States each year. Clostridium difficile, commonly referred to as C. diff, is a bacterium that infects the colon and can cause diarrhea, fever, and abdominal pain. Clostridium difficile-associated disease (CDAD) most commonly occurs in hospitalized older adults who have recently taken antibiotics. However, cases of CDAD can occur outside of healthcare settings as well.
Although antibiotics often cure the infection, C. diff can cause potentially life-threatening colon inflammation. People with CDAD usually are treated with a course of antibiotics, such as oral vancomycin or fidaxomicin. However, CDAD returns in approximately 20 percent of people who receive such treatment, according to the Centers for Disease Control and Prevention (CDC).
Multiple research studies have indicated that fecal microbiota transplantation (FMT) is an effective method for curing patients with repeat C. diff infections. However, the long-term safety of FMT has not been established. Although more research is needed to determine precisely how FMT effectively cures recurrent CDAD, the treatment appears to rapidly restore a healthy and diverse gut microbiome in recipients. Physicians perform FMT using various routes of administration, including oral pills, upper gastrointestinal endoscopy, colonoscopy, and enema.
A research consortium recently began enrolling patients in a clinical trial examining whether FMT by enema (putting stool from a healthy donor in the colon of a recipient) is safe and can prevent recurrent CDAD, a potentially life-threatening diarrheal illness. Investigators aim to enroll 162 volunteer participants 18 years or older who have had two or more episodes of CDAD within the previous six months.
Trial sites include Emory University in Atlanta, Duke University Medical Center in Durham, North Carolina, and Vanderbilt University Medical Center in Nashville, Tennessee. Each location is a Vaccine and Treatment Evaluation Unit (VTEU), clinical research sites joined in a network funded by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health. This randomized, controlled trial aims to provide critical data on the efficacy and long-term safety of using FMT by enema to cure C. diff infections.
Volunteers will be enrolled in the trial after completing a standard course of antibiotics for a recurrent CDAD episode, presuming their diarrhea symptoms cease on treatment. They will be randomly assigned to one of two groups. The first group (108 people) will take an anti-diarrheal medication and receive a stool transplant (FMT) delivered by retention enema. The second group (54 people) will take an anti-diarrheal medication and receive a placebo solution delivered by retention enema.
Participants in either group who have diarrhea with stools that test positive for C. diff shortly after the enema will be given an active stool transplant for a maximum of two FMTs. If participants in either group have another C. diff infection after receiving two FMTs, then they will be referred to other locally available treatment options. Investigators will evaluate the stool specimens for changes in gut microbial diversity and infectious pathogens and will examine the blood samples for metabolic syndrome markers.
To learn more about the long-term outcomes of FMT, the researchers will monitor all participants for adverse side effects for three years after completing treatment for recurrent CDAD. Investigators will also collect information on any new onset of CDAD, related chronic medical conditions or any other serious health issues they may have.
The trillions of microbes in the human gut are known to aid the body in synthesizing key vitamins and other nutrients. But this new study suggests that things can sometimes be more adversarial.
Choline is a key nutrient in a range of metabolic processes, as well as the production of cell membranes. Researchers identified a strain of choline-metabolizing E. coli that, when transplanted into the guts of germ-free mice, consumed enough of the nutrient to create a choline deficiency in them, even when the animals consumed a choline-rich diet.
This new study indicate that choline-utilizing bacteria compete with the host for this nutrient, significantly impacting plasma and hepatic levels of methyl-donor metabolites and recapitulating biochemical signatures of choline deficiency. Mice harboring high levels of choline-consuming bacteria showed increased susceptibility to metabolic disease in the context of a high-fat diet.
DNA methylation is essential for normal development and has been linked to everything from aging to carcinogenesis. This study showed changes in DNA methylation across multiple tissues, not just in adult mice with a choline-consuming gut microbiota, but also in the pups of those animals while they developed in utero.
Bacterially induced reduction of methyl-donor availability influenced global DNA methylation patterns in both adult mice and their offspring and engendered behavioral alterations. This study reveal an underappreciated effect of bacterial choline metabolism on host metabolism, epigenetics, and behavior.
The choline-deficient mice with choline-consuming gut microbes also showed much higher rates of infanticide, and exhibited signs of anxiety, with some mice over-grooming themselves and their cage-mates, sometimes to the point of baldness.
Tests have also shown as many as 65 percent of healthy individuals carry genes that encode for the enzyme that metabolizes choline in their gut microbiomes. This work suggests that interpersonal differences in microbial metabolism should be considered when determining optimal nutrient intake requirements.
Shared photosynthetic components between plant chloroplasts and cyanobacteria make these microbes ideal hosts for expressing foreign plant enzymes. Ping Xu and colleagues at the Shanghai Jiao Tong University have genetically engineered the cyanobacterium Synechococcus elongatusPC7942 with plant-derived enzymes. In total, the team created 18 bacterial strains expressing different combinations of enzymes. The different strains generate a variety of compounds with a six-carbon, phenyl group and three-carbon propene tail, called phenylpropanoids.
Phenylpropanoids perform diverse functions in plants, ranging from ultraviolet light protection to pathogen defence. One such compound, resveratrol, is made when the bacteria express the plant enzyme stilbene synthase downstream of enzymes tyrosine ammonia lyase and 4-coumarate:coenzyme A-ligase. Found in the skin of grapes and other berries, resveratrol reduces the risk of heart disease and is a valuable pharmaceutical commodity. Different versions of the engineered bacteria can also churn out the phenylpropanoid antioxidants caffeic acid, naringenin and coumaric acid.
The Shanghai Jia Tong University team genetically engineered cyanobacteria to produce compounds like flavonoids, stilbenes and curcuminoids usually only found in plants
What’s more, the team added feedback-inhibition resistant enzymes to the bacteria so that the chemical yields would surpass physiological levels. Photosynthesis within the cyanobacteria generates the chemicals from just water, carbon dioxide and a few mineral nutrients.
The bacterial growth medium houses the products, but isolating them at an industrially relevant yield is currently the biggest challenge. However, by not needing to harvest crops, generating the compounds from bacteria is potentially more sustainable. Xu stresses the potential of this point: ‘For the production of 1 tonne of natural resveratrol, our method may save about 485 hectare of farmland at its current production level.’
‘The approach deftly sidesteps major economic challenges by targeting chemicals with high intrinsic value,’ comments Paul Fowler, executive director of the Wisconsin Institute for Sustainable Technology in the US. A world-scale production plant under these circumstances is not a pre-requisite for commercialising this research.’
Many plant natural products have remarkable pharmacological activities. They are mainly produced directly by extraction from higher plants, which can hardly keep up with the surging global demand. Furthermore, the over-felling of many medicinal plants has undesirable effects on the ecological balance. In this study, we constructed a photoautotrophic platform with the unicellular cyanobacterium Synechococcus elongatus PCC7942 to directly convert the greenhouse gas CO2 into an array of valuable healthcare products, including resveratrol, naringenin, bisdemethoxycurcumin, p-coumaric acid, caffeic acid, and ferulic acid. These six compounds can be further branched to many other precious and useful natural products. Various strategies including introducing a feedback-inhibition-resistant enzyme, creating functional fusion proteins, and increasing malonyl-CoA supply have been systematically investigated to increase the production. The highest titers of these natural products reached 4.1–128.2 mg L−1 from the photoautotrophic system, which are highly comparable with those obtained by many other heterotrophic microorganisms using carbohydrates. Several advantages such as independence from carbohydrate feedstocks, functionally assembling P450s, and availability of plentiful NADPH and ATP support that this photosynthetic platform is uniquely suited for producing plant natural products. This platform also provides a green route for direct conversion of CO2 to many aromatic building blocks, a promising alternative to petrochemical-based production of bulk aromatic compounds.
Two synthetic gene circuits allow researchers to keep genetically engineered (GE) microbes alive only under specific conditions, and to kill them when their services are no longer needed. The circuits, described today (December 7) in Nature Chemical Biology,could help pave the path to safe diagnostics, therapies, or environmental remediation strategies that rely on GE bacteria.
“This is yet another step forward towards better biosafety and biocontainment based on certain aspects of existing technology,” said Guy-Bart Stan, a synthetic biologist at Imperial College London who was not involved in the study.
Study coauthor James Collins, a synthetic biologist at MIT, began to design these gene circuits, or “kill switches,” after becoming interested in using GE microbes for diagnostic and therapeutic purposes. “We were motivated to begin working on the topic as synthetic biology has moved increasingly toward real-world applications,” Collins told The Scientist. Other groups are working to engineer microbes for bioremediation and industrial processes, among other things.
But with genetic modification comes the concern that scientists will create new and uncontrollable species that outcompete or share their genes with wild-type organisms, permanently altering the environment or endangering people’s health.
Earlier this year, two research teams led by Yale bioengineer Farren Isaacs and Harvard geneticist George Church showed that they could genetically modify Escherichia coli to incorporate synthetic amino acids into essential proteins. When the bacteria are not fed the amino acids, they cannot produce these essential proteins, and so they die. This strategy yields bacteria that are very unlikely to survive without support from scientists but requires intensive engineering of the bacterial genome. (See “GMO ‘Kill Switches,’” The Scientist, January 2015.)
In contrast, Collins and his colleagues set out to create kill switches that could work in a more diverse range of microbes. “Our circuit-based safeguards can be conveniently transferred to different bacterial strains without modifying the target cell’s genome,” he wrote in an email.
First, Collins and his colleagues generated a kill switch called “Deadman,” named for a locomotive braking system in which the train will only run if the engineer is affirmatively holding down a pedal. In the microbial version of Deadman, a researcher must feed bacteria a substance called anhydrotetracycline at all times, or else the microbes will express a toxin and self-destruct.
The researchers generated a genetic circuit containing genes for the proteins LacI and TetR, a toxin that is only expressed in the absence of LacI, and a protease that degrades LacI. Under normal circumstances, TetR is preferentially expressed over LacI. TetR expression also triggers expression of the protease, which degrades any LacI that has been expressed. Without LacI, cells express the toxin and die. But when the cells are fed anhydrotetracycline, TetR is inhibited and LacI is expressed. LacI represses the toxin and keeps the cells alive.
Other versions of the Deadman circuit can be designed to degrade essential proteins in the absence of anhydrotetracycline, said Collins.
A second kill switch, “Passcode,” similarly requires that researchers maintain a specific environment for cells lest they express a toxin. Passcode requires a combination of input molecules for cells to survive. The system relies on hybrid transcription factors, each with one component that recognizes a specific DNA sequence, and one component that is sensitive to specific small molecules, such as galactose or cellobiose. One hybrid transcription factor, factor C, turns off expression of a toxin. Two other hybrid transcription factors, factors A and B, suppress expression of factor C. But specific small molecules can keep them from interacting with C. Another small molecule could prevent C from repressing the toxin. Therefore, to keep the cells alive, researchers must provide them with two small molecules that keep factors A and B in check, and make sure not to give them a third small molecule that will interfere with C.
Scientists designing Passcode kill-switches could make hybrid transcription factors respond to whatever combination of small molecules they desired, said Collins. “The strength of our kill switches lies in their flexibility and their ability to detect complex environmental signals for biocontainment.” He noted that companies hoping to keep others from using their cells could keep the recipe for their feed a secret.
“The great advantage is that you can effectively scale this and create different combinations of environments that contain different cocktails of these small molecules, thereby allowing you to effectively create a suite of cells that are going to be viable in different environments,” said Isaacs.
But Church warned that Collins’s circuit-based approach might not as effectively contain bacteria as an amino acid-based method, like one his group developed, since the cells are not fundamentally dependent on foreign biology to survive.
“If you need to have the ability to really scale your containment across a number of different species, then I could see the Passcode kill switches would be incredibly valuable,” said Isaacs. “If you are very concerned about escape frequencies and your degree of biocontainment, maybe you’d opt for something where the organism has been recoded and it relies on a synthetic amino acid.”
Still, Stan said the new paper is a demonstration that creating easy-to-insert kill switches based on genetic circuits is feasible. “I think what they wanted to show in the paper is basically that using some existing genetic circuitry . . . you can obtain biosafety for the here and now.”
C.T.Y. Chan et al., “‘Deadman’ and ‘Passcode’ microbial kill switches for bacterial containment,” Nature Chemical Biology, doi:10.1038/nchembio.1979, 2015.
One of the biggest concerns about genetically modified organisms (GMOs) is that they can infiltrate wild populations and spread their altered genes among naturally occurring species. In Nature today (January 21), two groups present a new method of containing GMOs: by making some of their essential proteins reliant upon synthetic amino acids not found outside of the laboratory.
“What really makes this a valuable step change is that kill switches beforehand were very susceptible to mutation or other conditions, such as metabolic cross feeding, from basically inactivating them,” said Tom Ellis, a synthetic biologist at Imperial College London who was not involved in the studies. The new approach circumvents some of those problems by making it extremely unlikely for the genetically modified bacteria to be able to survive outside of the conditions dictated by their custom-designed genomes.
Both research teams—one led by George Church at Harvard Medical School and the other by Farren Isaacsat Yale University—based their work on so-called genetically recoded organisms (GROs), bacterial genomes that have had all instances of a particular codon replaced by another. Church and Isaacs, along with their colleagues, had previously developed this concept in collaboration. Since then, their respective groups designed the replacement codons to incorporate a synthetic amino acid, and engineered proteins essential to the organism to rely upon the artificial amino acid for proper function.
“Here, for the first time, we’re showing that we’re able to engineer a dependency on synthetic biochemical building blocks for these proteins,” Isaacs told reporters during a conference call.
Both teams found that the cells perished in environments lacking the synthetic amino acid. Although the technology is not ready for industrial-scale deployment, the scientists suggested that such an approach could be applied as a safeguard against the escape of GMOs.
…..
‘Deadman’ and ‘Passcode’ microbial kill switches for bacterial containment
To demonstrate active control over host cell viability, cells grown under survival conditions (with ATc) were exposed to 1 mM IPTG to directly induce EcoRI and mf-Lon expression. Cell viability was measured by CFU count and is displayed…
Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the ‘Deadman’ and ‘Passcode’ kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently killEscherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.
Nontoxic antimicrobials that evade drug resistance
Drugs that act more promiscuously provide fewer routes for the emergence of resistant mutants. This benefit, however, often comes at the cost of serious off-target and dose-limiting toxicities. The classic example is the antifungal amphotericin B (AmB), which has evaded resistance for more than half a century. We report markedly less toxic amphotericins that nevertheless evade resistance. They are scalably accessed in just three steps from the natural product, and they bind their target (the fungal sterol ergosterol) with far greater selectivity than AmB. Hence, they are less toxic and far more effective in a mouse model of systemic candidiasis. To our surprise, exhaustive efforts to select for mutants resistant to these more selective compounds revealed that they are just as impervious to resistance as AmB. Thus, highly selective cytocidal action and the evasion of resistance are not mutually exclusive, suggesting practical routes to the discovery of less toxic, resistance-evasive therapies.
the antigenic characteristics expected for the synthetic virus.
The cDNA was transcribed
within the nucleus, and
the RNA translocated to the cytoplasm.
Interestingly, the recovered virus had
essentially the same sequence as the original one, and
no splicing was observed.
During the engineering of the infectious cDNA,
the spike gene of the virus was replaced by
the spike gene of an enteric isolate.
The synthetic virus
replicated abundantly in the enteric tract and was fully virulent, demonstrating that
the tropism and virulence of the recovered coronavirus can be modified.
the application of two strategies,
cloning of the cDNAs into a bacterial artificial chromosome and
nuclear expression of RNAs that are typically produced within the cytoplasm,
is useful for the engineering of large RNA molecules.
A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus
conserved all of the genetic markers introduced throughout the sequence and
showed a standard mRNA pattern and
the antigenic characteristics expected for the synthetic virus.
The cDNA was transcribed within the nucleus, and
the RNA translocated to the cytoplasm.
Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus
Over the past few years, the viruses of prokaryotes have been transformed in the view of microbiologists from simply being convenient experimental model systems into being a major component of the biosphere. They are
the global champions of diversity,
they constitute a majority of organisms on the planet,
they have large roles in the planet’s ecosystems,
they exert a significant—some would say dominant—force on
the evolution of their bacterial and archaeal hosts, and
they have been doing this for billions of years,
possibly for as long as there have been cells.
This transformation in status or, rather, our expanded appreciation of the importance of these viruses in the biosphere is due to a few significant developments in both understanding and technology.
(i) It has become clear that the population sizes of these viruses are astoundingly large. This realization grew out of electron microscopic enumerations of tailed phage virions in costal seawater, and numerous measurements in other environments have been made since then. A current estimate based on these measurements is that
there are 1031 individual tailed phage virions in the global biosphere—
enough to reach for 200 million light years if laid end to end—and measurements of population turnover suggest that
it takes roughly 1024 productive infections per second to maintain the global population.
(ii) Advances in DNA sequencing technology have led to dramatic qualitative improvements in how we understand the
The majority of newly determined gene and protein sequences of these viruses has no relatives detectable in the public sequence databases, and
analysis of metagenomic data provides strong evidence that
there is more genetic diversity in the genes of the viruses of prokaryotes
than in any other compartment of the biosphere.
(iii) Facilitated by these conceptual and technical advances, studies of bacterial and archaeal viruses as important components of global biology have flourished. These viruses are revealed as important players in
carbon and energy cycling in the oceans and other natural environments and
as major agents in the ecology and evolution of their cellular hosts.
(iv) The isolation and characterization of new viruses have accelerated. This has been especially important for the archaeal viruses, where the discovery of new viruses and of new virus types had lagged behind bacteriophage discovery. For the bacteriophages, the isolation of newly discovered viruses has helped improve the still extremely sparse coverage of sequence diversity and the narrow phylogenetic range of hosts represented by current data.
In 1980, geneticists used the relatively new technique of gene splicing, which we will describe in this chapter, to introduce
the human gene that encodes interferon into a bacterial cell’s genome.
Interferon is a rare blood protein that increases human resistance to viral infection, and medical scientists have been interested in its possible usefulness in cancer therapy. Purification of the large amounts of interferon required for clinical testing would have been prohibitively expensive at the time. Introducing the gene responsible for its production into a bacterial cell made that possible. The cell that had
acquired the human interferon gene proceeded to produce interferon at a rapid rate, and to grow and divide.
The millions of interferon-producing bacteria growing in the culture were all descendants of the cell that had originally received the human interferon gene.
The human insulin gene has also been cloned in bacteria, and now insulin can be manufactured at little expense. Furthermore, cloning and related molecular techniques are needed to provide basic information about how genes are put together and regulated.
The essence of genetic engineering is
the ability to cut DNA into recognizable pieces and rearrange those pieces in different ways.
In the interferon experiment,
a piece of DNA carrying the interferon gene was
inserted into a plasmid,which
carried the gene into a bacterial cell.
Most other genetic engineering approaches bring the gene of interest into the target cell by first incorporating it into a plasmid or an infective virus.
This cutting is performed
by enzymes that recognize and
cleave specific sequences of nucleotides in DNA.
Discovery of Restriction Endonucleases
Scientific discoveries often have their origins in seemingly unimportant observations that receive little attention by researchers before their general significance is appreciated. In the case of genetic engineering, the original observation was that bacteria use enzymes to defend themselves against viruses.
Most organisms eventually evolve means of defending themselves from predators and parasites, and bacteria are no exception. Among the natural enemies of bacteria are bacteriophages, viruses that infect bacteria and multiply within them. At some point, they cause the bacterial cells to burst, releasing thousands more viruses.
Some types of bacteria have acquired powerful weapons against these viruses: they contain enzymes called restriction endonucleases
that fragment the viral DNA as soon as it enters the bacterial cell.
Why don’t restriction endonucleases cleave the bacterial cells’ own DNA as well as that of the viruses?
bacteria modify their own DNA, using other enzymes known as methylases to add methyl (CH3) groups
to some of the nucleotides in the bacterial DNA.
When nucleotides within a restriction endonuclease’s recognition sequence have been methylated,
the endonuclease cannot bind to that sequence.
the bacterial DNA is protected from being degraded at that site.
but viral DNA has not been methylated, and therefore
is not protected from enzymatic cleavage.
How Restriction Endonucleases Cut DNA
The sequences recognized by restriction endonucleases are
typically four to six nucleotides long, and
they are often palindromes.
the nucleotides at one end of the recognition sequence are complementary to those at the other end, so that
the two strands of the DNA duplex have the same nucleotide sequence running in opposite directions for the length of the recognition sequence.
Two important consequences arise from this arrangement of nucleotides to be discussed.
Biochemistry. 5th edition.
Berg JM, Tymoczko JL, Stryer L.
Section 9.3 Restriction Enzymes: Performing Highly Specific DNA-Cleavage Reactions
Bacteria and archaea have evolved mechanisms to protect themselves from viral infections so that viruses inject their DNA genomes into cells and the viral DNA hijacks the cell’s machinery A major protective strategy for the host is to use restriction endonucleases (restriction enzymes) to degrade the viral DNA. These particular base sequences the enzymes recognize are called recognition sequences or recognition sites.
theycleave that DNA at defined positions.
The most well studied class are the so-called type II restriction enzymes.
Restriction endonucleases must show tremendous specificity at two levels.
First, they must cleave only DNA molecules that contain recognition sites (hereafter referred to as cognate DNA) without cleaving DNA molecules that lack these sites.
endonucleases must cleave cognate DNA molecules much more than 5000 times as efficiently as they cleave nonspecific sites.
Second, restriction enzymes must not degrade the host DNA.
How do these enzymes manage to degrade viral DNA while sparing their own?
The restriction endonuclease EcoRV (from E. coli) cleaves double-stranded viral DNA molecules that contain the sequence 5′-GATATC-3′ but leaves intact host DNA containing hundreds of such sequences. The host DNA is protected by other enzymes called methylases, which methylate adenine bases within host recognition sequences (Figure 9.32). For each restriction endonuclease, the host cell produces a corresponding methylase that marks the host DNA and prevents its degradation.
These pairs of enzymes are referred to as restriction-modification systems.
All restriction enzymes catalyze the hydrolysis of DNA phosphodiester bonds, leaving a phosphoryl group attached to the 5′ end. The bond that is cleaved is shown in red.
Mechanism Type 1 (covalent intermediate)
Mechanism Type 2 (direct hydrolysis)
Each postulates a different nucleophile to carry out the attack on the phosphorus. In either case, each reaction takes place by an in-line displacement path:
The incoming nucleophile attacks the phosphorus atom, and
a pentacoordinate transition state is formed.
This species has a trigonal bipyramidal geometry centered at the phosphorus atom, with
the incoming nucleophile at one apex of the two pyramids and the group that is displaced (the leaving group, L) at the other apex.
The two mechanisms differ in the number of times the displacement occurs in the course of the reaction.
The incoming nucleophile attacks the phosphorus atom, and
a pentacoordinate transition state is formed.
The analysis revealed that the stereochemical configuration at the phosphorus atom was inverted only once with cleavage. This result is consistent with a direct attack of water at phosphorus and
rules out the formation of any covalently bound intermediate (Figure 9.35).
Cleavage of DNA by EcoRV endonuclease results in overall inversion of the stereochemical configuration at the phosphorus atom.
9.3.2 Restriction Enzymes Require Magnesium for Catalytic Activity
Restriction endonucleases as well as many other enzymes that act on phosphate-containing substrates require Mg2+ or some other similar divalent cation for activity. What is the function of this metal?
It has been possible to examine the interactions of the magnesium ion when it is bound to the enzyme. Crystals have been produced of EcoRV endonuclease
bound to oligonucleotides that contain the appropriate recognition sequences.
These crystals are grown in the absence of magnesium to prevent cleavage; then,
when produced, the crystals are soaked in solutions containing the metal.
No cleavage takes place, allowing the location of the magnesium ion binding sites to be determined (Figure 9.36).
The magnesium ion was found to be bound to six ligands:
three are water molecules,
two are carboxylates of the enzyme’s aspartate residues, and
one is an oxygen atom of the phosphoryl group at the site of cleavage.
The magnesium ion holds a water molecule in a position from which the water molecule can attack the phosphoryl group and,
in conjunction with the aspartate residues,
helps polarize the water molecule toward deprotonation.
Cleavage does not take place within these crystals. But a second magnesium ion must be present in an adjacent site for EcoRV endonuclease to cleave its substrate.
Magnesium Ion Binding Site in ECORV Endonuclease. The magnesium ion helps to activate a water molecule and positions it so that it can attack the phosphate.
9.3.3 The Complete Catalytic Apparatus Is Assembled Only Within Complexes of Cognate DNA Molecules, Ensuring Specificity
Specificityis the defining feature of restriction enzymes. The recognition sequences for most restriction endonucleases are inverted repeats.
This arrangement gives the three-dimensional structure of the recognition site
a twofold rotational symmetry (Figure 9.37).
The restriction enzymes display a corresponding symmetry to facilitate recognition:
they are dimers whose two subunits are related by twofold rotational symmetry.
The matching symmetry of the recognition sequence and the enzyme has been confirmed
by the determination of the structure of the complex between EcoRV endonuclease and DNA fragments containing its recognition sequence (Figure 9.38).
Structure of the ECORV – Cognate DNA Complex.
This view of the structure of EcoRV endonuclease bound to a cognate DNA fragment is down the helical axis of the DNA. The two protein subunits are in yellow and blue, and the DNA backbone is in red.
A unique set of interactions occurs between the enzyme and a cognate DNA sequence. Within the 5′-GATATC-3′ sequence,
the G and A bases at the 5′ end of each strand and their Watson-Crick partners directly contact the enzyme
by hydrogen bonding with residues that are located in two loops,
one projecting from the surface of each enzyme subunit (Figure 9.39).
The most striking feature of this complex is the distortion of the DNA, which is substantially kinked in the center (Figure 9.40). The central two TA base pairs in the recognition sequence play a key role in producing the kink. They do not make contact with the enzyme but appear to be required because of their ease of distortion. 5′-TA-3′ sequences are known to be among the most easily deformed base pairs.
The distortion of the DNA at this site has severe effects on the specificity of enzyme action.
Hydrogen Bonding Interactions between ECORV Endonuclease and Its DNA Substrate.
One of the DNA-binding loops (in green) of EcoRV endonuclease is shown interacting with the base pairs of its cognate DNA binding site. Key amino acid residues are shown.
The DNA is represented as a ball-and-stick model. The path of the DNA helical axis, shown in red, is substantially distorted on binding to the enzyme. For the B form of DNA, the axis is straight (not shown).
Specificity is often determined by an enzyme’s binding affinity for substrates. In regard to EcoRV endonuclease, however, binding studies performed in the absence of magnesium have demonstrated that
the enzyme binds to all sequences, both cognate and noncognate, with approximately equal affinity.
the structures of complexes formed with noncognate DNA fragments are strikingly different from those formed with cognate DNA:
the noncognate DNA conformation is not substantially distorted (Figure 9.41).
This lack of distortion has important consequences with regard to catalysis. No phosphate is positioned sufficiently close to the active-site aspartate residues to complete a magnesium ion binding site (see Figure 9.36). Hence, the nonspecific complexes do not bind the magnesium ion and
the complete catalytic apparatus is never assembled.
The distortion of the substrate and the subsequent binding of the magnesium ion account for
the catalytic specificity of more than 1,000,000-fold that is observed for EcoRV endonuclease
Nonspecific and Cognate DNA within ECORV Endonuclease.
A comparison of the positions of the nonspecific (orange) and the cognate DNA (red) within EcoRV reveals that,
in the nonspecific complex, the DNA backbone is too far from the enzyme
We can now see the role of binding energy in this strategy for attaining catalytic specificity.
In binding to the enzyme, the DNA is distorted in such a way that
additional contacts are made between the enzyme and the substrate, increasing the binding energy.
However, this increase is canceled by the energetic cost of distorting the DNA from its relaxed conformation (Figure 9.42). Thus, for EcoRV endonuclease,
there is little difference in binding affinity for cognate and nonspecific DNA fragments.
However, the distortion in the cognate complex dramatically affects catalysis by completing the magnesium ion binding site.
This example illustrates how enzymes can utilize available binding energy to deform substrates and poise them for chemical transformation.
Interactions that take place within the distorted substrate complex
stabilize the transition state leading to DNA hydrolysis.
Greater Binding Energy of EcoRV Endonuclease Bound to Cognate Versus Noncognate Dna.
The additional interactions between EcoRV endonuclease and cognate DNA increase the binding energy, which can be used to drive DNA distortions.
The distortion in the DNA explains how methylation blocks catalysis and protects host-cell DNA. When a methyl group is added to the amino group of the adenine nucleotide at the 5′ end of the recognition sequence,
the methyl group’s presence precludes the formation of a hydrogen bond between the amino group and the side-chain carbonyl group of asparagine 185 (Figure 9.43).
This asparagine residue is closely linked to the other amino acids that form specific contacts with the DNA.
The absence of the hydrogen bond disrupts other interactions between the enzyme and the DNA substrate, and
the distortion necessary for cleavage will not take place.
The methylation of adenine blocks the formation of hydrogen bonds
between EcoRV endonuclease and cognate DNA molecules and
prevents their hydrolysis.
9.3.4 Type II Restriction Enzymes Have a Catalytic Core in Common and Are Probably Related by Horizontal Gene Transfer
Type II restriction enzymes are prevalent in Archaea and Eubacteria. What can we tell of the evolutionary history of these enzymes?
Comparison of the amino acid sequences of a variety of type II restriction endonucleases did not reveal significant sequence similarity between most pairs of enzymes. However, a careful examination of three-dimensional structures, taking into account the location of the active sites, revealed
the presence of a core structure conserved in the different enzymes.
This structure includes β strands that contain the aspartate (or, in some cases, glutamate) residues forming the magnesium ion binding sites (Figure 9.44).
A Conserved Structural Core in Type II Restriction Enzymes.
Four conserved structural elements, including the active-site region (in blue), are highlighted in color in these models of a single monomer from each dimeric enzyme.
These observations indicate that many type II restriction enzymes are indeed evolutionary related. Analyses of the sequences in greater detail suggest that bacteria may have obtained genes encoding these enzymes
from other species by horizontal gene transfer, the passing between species of pieces of DNA (such as plasmids) that provide
a selective advantage in a particular environment.
For example, EcoRI (from E. coli) and RsrI (from Rhodobacter sphaeroides) are 50% identical in sequence over 266 amino acids, clearly
indicative of a close evolutionary relationship.
these species of bacteria are not closely related,
as is known from sequence comparisons of other genes and other evidence.
Thus, it appears that these species obtained the gene for this restriction endonuclease from a common source
more recently than the time of their evolutionary divergence.
the gene encoding EcoRI endonuclease uses particular codons to specify given amino acids that are
strikingly different from the codons used by most E. coli genes, which
suggests that the gene did not originate in E. coli.
Horizontal gene transfer may be a relatively common event.
genes that inactivate antibiotics are often transferred, leading to the transmission of antibiotic resistance from one species to another.
For restriction-modification systems,
protection against viral infections may have favored horizontal gene transfer.
Biochemistry. 5th edition.
Berg JM, Tymoczko JL, Stryer L.
New York: W H Freeman; 2002.
Cleavage Is by In-Line Displacement of 3′ Oxygen from Phosphorus by Magnesium-Activated Water
Restriction Enzymes Require Magnesium for Catalytic Activity
The Complete Catalytic Apparatus Is Assembled Only Within Complexes of Cognate DNA Molecules, Ensuring Specificity
Type II Restriction Enzymes Have a Catalytic Core in Common and Are Probably Related by Horizontal Gene Transfer
By Richard Wheeler (Zephyris) 2007. Image of EcoRV homodimer in complex with a DNA substrate. From . (Photo credit: Wikipedia)
HindIII restriction endonuclease in complex with cognate DNA (Photo credit: Wikipedia)
English: 3d surface model of HindIII dimer complexed with a DNA fragment from PDB 2E52. Ref.: Watanabe, N., Sato, C., Takasaki, Y., Tanaka, I. Crystal structural analysis of HindIII restriction endonuclease in complex with cognate DNA at 2.0 angstrom resolution to be published (Photo credit: Wikipedia)
English: BglII active site containing residues that coordinate to a metal ion and water molecules including the nucleophilic water that breaks the scissile phosphodiester bond at the recognition site. (Photo credit: Wikipedia)
This post is in continuation to Part 1 by the same title.
In part one I covered the basics of role of redox chemistry in immune reactions, the phagosome cauldron, and how bacteria bacteria, virus and parasites trigger the complex pathway of NO production and its downstream effects. While we move further in this post, the previous post can be accessed here.
Regulation of the redox immunomodulators—NO/RNS and ROS
In addition to eradicating pathogens, NO/RNS and ROS and their chemical interactions act as effective immunomodulators that regulate many cellular metabolic pathways and tissue repair and proinflammatory pathways. Figure 3 shows these pathways.
Figure 3. Schematic overview of interactive connections between NO and ROS-mediated metabolic pathways. Credit: (Wink et al., 2011)
Regulation of iNOS enzyme activity is critical to NO production. Factors such as the availability of arginine, BH4, NADPH, and superoxide affect iNOS activity and thus NO production. In the absence of arginine and BH4 iNOS becomes a O2_/H2O2 generator (Vásquez-Vivar et al., 1999). Hence metabolic pathways that control arginine and BH4 play a role in determining the NO/superoxide balance. Arginine levels in cells depend on various factors such as type of uptake mechanisms that determine its spatial presence in various compartments and enzymatic systems. As shown in Fig3 Arginine is the sole substrate for iNOS and arginase. Arginase is another key enzyme in immunemodulation. AG is also regulated by NOS and NOX activities. NOHA, a product of NOS, inhibits AG, and O2–increases AG activity. Importantly, high AG activity is associated with elevated ROS and low NO fluxes. NO antagonises NOX2 assembly that in turn leads to reduction in O2_ production. NO also inhibits COX2 activity thus reducing ROS production. Thus, as NO levels decline, oxidative mechanisms increase. Oxidative and nitrosative stress can also decrease intracellular GSH (reduced form) levels, resulting in a reduced antioxidant capability of the cell.
Immune-associated redox pathways regulate other important metabolic cell functions that have the potential for widespread impact on cells, organs, and organisms. These pathways, such as mediated via methionine and polyamines, are critical for DNA stabilization, cell proliferation, and membrane channel activity, all of which are also involved in immune-mediated repair processes.
NO levels dictate the immune signaling pathway
NO/RNS and ROS actively control innate and adaptive immune signaling by participating in induction, maintenance, and/or termination of proinflammatory and anti-inflammatory signaling. As in pathogen eradication, the temporal and spatial concentration profiles of NO are key factors in determining immune-mediated processes.
Brune and coworkers (Messmer et al., 1994) first demonstrated that p53 expression was associated with the concentrations of NO that led to apoptosis in macrophages. Subsequent studies linked NO concentration profiles with expression of other key signaling proteins such as HIF-1α and Akt-P (Ridnour et al., 2008; Thomas et al., 2008). Various levels of NO concentrations trigger different pathways and expectedly this concentration-dependent profile varies with distance from the NO source.NO is highly diffucible and this characteristic can result in 1000 fold reduction in concentration within one cell length distance travelled from the source of production. Time course studies have also shown alteration in effects of same levels of NO over time e.g. NO-mediated ERK-P levels initially increased rapidly on exposure to NO donors and then decreased with continued NO exposure (Thomas et al., 2004), however HIF-1α levels remained high as long as NO levels were elevated. Thus some of the important factors that play critical role in NO effects are: distance from source, NO concentrations, duration of exposure, bioavailability of NO, and production/absence of other redox molecules.
Figure and legend credits: (Wink et al., 2011)
Fig 4: The effect of steady-state flux of NO on signal transduction mechanisms.
This diagram represents the level of sustained NO that is required to activate specific pathways in tumor cells. Similar effects have been seen on endothelial cells. These data were generated by treating tumor or endothelial cells with the NO donor DETANO (NOC-18) for 24 h and then measuring the appropriate outcome measures (for example, p53 activation). Various concentrations of DETANO that correspond to cellular levels of NO are: 40–60 μM DETANO = 50 nM NO; 80–120 μM DETANO = 100 nM NO; 500 μM DETANO = 400 nM NO; and 1 mM DETANO = 1 μM NO. The diagram represents the effect of diffusion of NO with distance from the point source (an activated murine macrophage producing iNOS) in vitro (Petri dish) generating 1 μM NO or more. Thus, reactants or cells located at a specific distance from the point source (i.e., iNOS, represented by star) would be exposed to a level of NO that governs a specific subset of physiological or pathophysiological reactions. The x-axis represents the different zone of NO-mediated events that is experienced at a specific distance from a source iNOS producing >1 μM. Note: Akt activation is regulated by NO at two different sites and by two different concentration levels of NO.
Species-specific NO production
The relationship of NO and immunoregulation has been established on the basis of studies on tumor cell lines or rodent macrophages, which are readily available sources of NO. However in humans the levels of protein expression for NOS enzymes and the immune induction required for such levels of expression are quite different than in rodents (Weinberg, 1998). This difference is most likely due to the human iNOS promotor rather than the activity of iNOS itself. There is a significant mismatch between the promoters of humans and rodents and that is likely to account for the notable differences in the regulation of gene induction between them. The combined data on rodent versus human NO and O2– production strongly suggest that in general, ROS production is a predominant feature of activated human macrophages, neutrophils, and monocytes, and the equivalent murine immune cells generate a combination of O2– and NO and in some cases, favor NO production. These differences may be crucial to understanding how immune responses are regulated in a species-specific manner. This is particularly useful, as pathogen challenges change constantly.
The next post in this series will cover the following topics:
The impact of NO signaling on an innate immune response—classical activation
NO and proinflammatory genes
NO and regulation of anti-inflammatory pathways
NO impact on adaptive immunity—immunosuppression and tissue-restoration response
Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012
Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012
Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN
Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk
July 2, 2012
An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery
Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography
Nitric oxide (NO), reactive nitrogen species (RNS) and reactive oxygen species (ROS) perform dual roles as immunotoxins and immunomodulators. An incoming immune signal initiates NO and ROS production both for tackling the pathogens and modulating the downstream immune response via complex signaling pathways. The complexity of these interactions is a reflection of involvement of redox chemistry in biological setting (fig. 1)
Fig 1. Image credit: (Wink et al., 2011)
Previous studies have highlighted the role of NO in immunity. It was shown that macrophages released a substance that had antitumor and antipathogen activity and required arginine for its production (Hibbs et al., 1987, 1988). Hibbs and coworkers further strengthened the connection between immunity and NO by demonstrating that IL2 mediated immune activation increased NO levels in patients and promoted tumor eradication in mice (Hibbs et al., 1992; Yim et al., 1995).
In 1980s a number of authors showed the direct evidence that macrophages made nitrite, nitrates and nitrosamines. It was also shown that NO generated by macrophages could kill leukemia cells (Stuehr and Nathan, 1989). Collectively these studies along with others demonstrated the important role NO plays in immunity and lay the path for further research in understanding the role of redox molecules in immunity.
NO is produced by different forms of nitric oxide synthase (NOS) enzymes such as eNOS (endothelial), iNOS (inducible) and nNOS (neuronal). The constitutive forms of eNOS generally produce NO in short bursts and in calcium dependent manner. The inducible form produces NO for longer durations and is calcium independent. In immunity, iNOS plays a vital role. NO production by iNOS can occur over a wide range of concentrations from as little as nM to as much as µM. This wide range of NO concentrations provide iNOS with a unique flexibility to be functionally effective in various conditions and micro-environements and thus provide different temporal and concentration profiles of NO, that can be highly efficient in dealing with immune challenges.
Redox reactions in immune responses
NO/RNS and ROS are two categories of molecules that bring about immune regulation and ‘killing’ of pathogens. These molecules can perform independently or in combination with each other. NO reacts directly with transition metals in heme or cobalamine, with non-heme iron, or with reactive radicals (Wink and Mitchell, 1998). The last reactivity also imparts it a powerful antioxidant capability. NO can thus act directly as a powerful antioxidant and prevent injury initiated by ROS (Wink et al., 1999). On the other hand, NO does not react directly with thiols or other nucleophiles but requires activation with superoxide to generate RNS. The RNS species then cause nitrosative and oxidative stress (Wink and Mitchell, 1998).
The variety of functions achieved by NO can be understood if one looks at certain chemical concepts. NO and NO2 are lipophilic and thus can migrate through cells, thus widening potential target profiles. ONOO-, a RNS, reacts rapidly with CO2 that shortens its half life to <10 ms. The anionic form and short half life limits its mobility across membranes. When NO levels are higher than superoxide levels, the CO2-OONO–intermediate is converted to NO2 and N2O3 and changes the redox profile from an oxidative to a nitrosative microenvironment. The interaction of NO and ROS determines the bioavailability of NO and proximity of RNS generation to superoxide source, thus defining a reaction profile. The ROS also consumes NO to generate NO2 and N2O3 as well as nitrite in certain locations. The combination of these reactions in different micro-environments provides a vast repertoire of reaction profiles for NO/RNS and ROS entities.
The Phagosome ‘cauldron’
The phagosome provides an ‘isolated’ environment for the cell to carry out foreign body ‘destruction’. ROS, NO and RNS interact to bring about redox reactions. The concentration of NO in a phagosome can depend on the kind of NOS in the vicinity and its activity and other localised cellular factors. NO and is metabolites such as nitrites and nitrates along with ROS combine forces to kill pathogens in the acidic environment of the phagosome as depicted in the figure 2 below.
Fig 2. The NO chemistry of the phagosome. (image credit: (Wink et al., 2011)
This diagram depicts the different nitrogen oxide and ROS chemistry that can occur within the phagosome to fight pathogens. The presence of NOX2 in the phagosomes serves two purposes: one is to focus the nitrite accumulation through scavenging mechanisms, and the second provides peroxide as a source of ROS or FA generation. The nitrite (NO2−) formed in the acidic environment provides nitrosative stress with NO/NO2/N2O3. The combined acidic nature and the ability to form multiple RNS and ROS within the acidic environment of the phagosome provide the immune response with multiple chemical options with which it can combat bacteria.
Bacteria
There are various ways in which NO combines forces with other molecules to bring about bacterial killing. Here are few examples
E.coli: It appears to be resistant to individual action of NO/RNS and H2O2 /ROS. However, when combined together, H2O2 plus NO mediate a dramatic, three-log increase in cytotoxicity, as opposed to 50% killing by NO alone or H2O2 alone. This indicates that these bacteria are highly susceptible to their synergistic action.
Staphylococcus: The combined presence of NO and peroxide in staphylococcal infections imparts protective effect. However, when these bacteria are first exposed to peroxide and then to NO there is increased toxicity. Hence a sequential exposure to superoxide/ROS and then NO is a potent tool in eradicating staphylococcal bacteria.
Mycobacterium tuberculosis: These bacterium are sensitive to NO and RNS, but in this case, NO2 is the toxic species. A phagosome microenvironment consisting of ROS combined with acidic nitrite generates NO2/N2O3/NO, which is essential for pathogen eradication by the alveolar macrophage. Overall, NO has a dual function; it participates directly in killing an organism, and/or it disarms a pathway used by that organism to elude other immune responses.
Parasites
Many human parasites have demonstrated the initiation of the immune response via the induction of iNOS, that then leads to expulsion of the parasite. The parasites include Plasmodia(malaria), Leishmania(leishmaniasis), and Toxoplasma(toxoplasmosis). Severe cases of malaria have been related with increased production of NO. High levels of NO production are however protective in these cases as NO was shown to kill the parasites (Rockett et al., 1991; Gyan et al., 1994). Leishmania is an intracellualr parasite that resides in the mamalian macrophages. NO upregulation via iNOS induction is the primary pathway involved in containing its infestation. A critical aspect of NO metabolism is that NOHA inhibits AG activity, thereby limiting the growth of parasites and bacteria including Leishmania, Trypanosoma, Schistosoma, Helicobacter, Mycobacterium, and Salmonella, and is distinct from the effects of RNS. Toxoplasma gondii is also an intracellular parasite that elicits NO mediated response. INOS knockout mice have shown more severe inflammatory lesions in the CNS that their wild type counterparts, in response to toxoplasma exposure. This indicates the CNS preventative role of iNOS in toxoplasmosis (Silva et al., 2009).
Virus
Viral replication can be checked by increased production of NO by induction of iNOS (HIV-1, coxsackievirus, influenza A and B, rhino virus, CMV, vaccinia virus, ectromelia virus, human herpesvirus-1, and human parainfluenza virus type 3) (Xu et al., 2006). NO can reduce viral load, reduce latency and reduce viral replication. One of the main mechanisms as to how NO participates in viral eradication involves the nitrosation of critical cysteines within key proteins required for viral infection, transcription, and maturation stages. For example, viral proteases or even the host caspases that contain cysteines in their active site are involved in the maturation of the virus. The nitrosative stress environment produced by iNOS may serve to protect against some viruses by inhibiting viral infectivity, replication, and maturation.
Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012
Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012
Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN
Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk
July 2, 2012
An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery
Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography
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