Single-Channel Kinetics,Inactivation, and Spatial Distribution of Inositol Trisphosphate (IP3) Receptors in Xenopus Oocyte Nucleus

Single-channel properties of the Xenopus inositol trisphosphate receptor (IP₃R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K⁺ as the charge carrier (cytoplasmic [IP₃] = 10 μM, free [Ca²⁺] = 200 nM), the IP₃R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and ∼300 pS at 60 mV. The channel was frequently observed with high open probability (mean P _o = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants τ < 5 ms and ∼20 ms; and at least three closed states with τ ∼1 ms, ∼10 ms, and >1 s. Higher cytoplasmic potential increased the relative frequency and τ of the longest closed state. A novel “flicker” kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F₁ and F₂. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F₁ to F₂ transitions. Channel run-down or inactivation (τ ∼ 30 s) was consistently observed in the continuous presence of IP₃ and the absence of change in [Ca²⁺]. Some (∼10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage- reversible inactivation. Mapping of functional channels indicates that the IP₃R tends to aggregate into microscopic (<1 μm) as well as macroscopic (∼10 μm) clusters. Ca²⁺-independent inactivation of IP₃R and channel clustering may contribute to complex [Ca²⁺] signals in cells.     

Structural and Mechanistic Insight into DNA Unwinding by Deinococcus radiodurans UvrD

DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement around duplexed DNA. Our structural data also improve our understanding of the molecular mechanisms that regulate DNA unwinding by Superfamily 1A (SF1A) helicases. Our biochemical data reveal that drUvrD is a DNA-stimulated ATPase, can translocate along ssDNA in the 3′-5′ direction and shows ATP-dependent 3′-5′, and surprisingly also, 5′-3′ helicase activity. Interestingly, we find that these translocase and helicase activities of drUvrD are modulated by the ssDNA binding protein. Analysis of drUvrD mutants indicate that the conserved β-hairpin structure of drUvrD that functions as a separation pin is critical for both drUvrD’s 3′-5′ and 5′-3′ helicase activities, whereas the GIG motif of drUvrD involved in binding to the DNA duplex is essential for the 5′-3′ helicase activity only. These special features of drUvrD may reflect its involvement in a wide range of DNA repair processes in vivo.     

Proinflammatory Cytokines Stimulate Mitochondrial Superoxide Flashes in Articular Chondrocytes In Vitro and In Situ

Objective

Mitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes.

Methods

Chondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1β (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by “superoxide flash” activity recorded with time lapse scanning.

Results

Cultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or “superoxide flashes” occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm²*100 s in vitro and in situ, respectively. Interleukin-1β or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation.

Conclusions

The superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases.     

Enhanced thermal tolerance in a mutant of Arabidopsis deficient in palmitic Acid unsaturation

A mutant of Arabidopsis thaliana, deficient in the activity of a chloroplast ω9 fatty acid desaturase, accumulates high amounts of palmitic acid (16:0), and exhibits an overall reduction in the level of unsaturation of chloroplast lipids. Under standard conditions the altered membrane lipid composition had only minor effects on growth rate of the mutant, net photosynthetic CO₂ fixation, photosynthetic electron transport, or chloroplast ultrastructure. Similarly, fluorescence polarization measurements indicated that the fluidity of the membranes was not significantly different in the mutant and the wild type. However, at temperatures above 28°C, the mutant grew more rapidly than the wild type suggesting that the altered fatty acid composition enhanced the thermal tolerance of the mutant. Similarly, the chloroplast membranes of the mutant were more resistant than wild type to thermal inactivation of photosynthetic electron transport. These observations lend support to previous suggestions that chloroplast membrane lipid composition may be an important component of the thermal acclimation response observed in many plant species which are photosynthetically active during periods of seasonally variable temperature extremes.     

The alpha-glycerophosphate cycle in Drosophila melanogaster. IV. Metabolic, ultrastructural, and adaptive consequences of alphaGpdh-l "null" mutations

"Null" mutations previously isolated at the αGpdh-1 locus of Drosophila melanogaster, because of disruption of the energy-producing α-glycerophosphate cycle, severely restrict the flight ability and relative viability of affected individuals. Two "null" alleles, αGpdh-1^BO-1-4, and αGpdh-1^BO-1-5, when made hemizygous with a deficiency of the αGpdh-1 locus, Df(2L)GdhA, were rendered homozygous by recombination with and selective elimination of the Df(2L)GdhA chromosome. After over 25 generations, a homozygous αGpdh-1^BO-1-4 stock regained the ability to fly despite the continued absence of measurable αGPDH activity. Inter se heterozygotes of three noncomplementing αGpdh-1 "null" alleles and the "adapted" αGpdh-1^BO-1-4 homozygotes were examined for metabolic enzymatic activities related to the energy-producing and pyridine nucleotide-regulating functions of the α-glycerophosphate cycle in Drosophila. The enzyme functions tested included glyceraldehyde-3-phosphate dehydrogenase, cytoplasmic and soluble malate dehydrogenase, lactate dehydrogenase, mitochondrial NADH oxidation, oxidative phosphorylation, and respiratory control with the substrates α-glycerophosphate, succinate, and pyruvate. These activities in any of the mutant genotypes in early adult life were indistinguishable from those in the wild type. There was, however, a premature deterioration and atrophy of the ultrastructural integrity of flight muscle sarcosomes observed by electron microscopy in the "null" mutants. These observations were correlated with a decrease in state 3 mitochondrial oxidation with α-glycerophosphate, succinate, and pyruvate, as well as with loss of respiratory control in adults as early as 2 wk after eclosion. Such observations, which normally are seen in aged dipterans, were accompanied by premature mortality of the mutant heterozygotes. The adapted αGpdh-1^BO-1-4 was identical with wild type in each of the aging characters with the single exception of lowered rates of mitochondrial oxidative phosphorylation.     

DdrA,DdrD, and PprA: Components of UV and Mitomycin C Resistance in Deinococcus radiodurans R1

Mutants created by deleting the ddrA, ddrB, ddrC, ddrD, and pprA loci of Deinococcus radiodurans R1alone and in all possible combinations of pairs revealed that the encoded gene products contribute to this species’ resistance to UV light and/or mitomycin C. Deleting pprA from an otherwise wild type cell sensitizes the resulting strain to UV irradiation, reducing viability by as much as eight fold relative to R1. If this deletion is introduced into a ΔddrA or ΔddrD background, the resulting strains become profoundly sensitive to the lethal effects of UV light. At a fluence of 1000 Jm^-2, the ΔddrA ΔpprA and ΔddrD ΔpprA strains are 100- and 1000-fold more sensitive to UV relative to the strain that has only lost pprA. Deletion of ddrA results in a 100 fold increase in strain sensitivity to mitomycin C, but in backgrounds that combine a deletion of ddrA with deletions of either ddrC or ddrD, mitomycin resistance is restored to wild type levels. Inactivation of ddrB also increases D. radiodurans sensitivity to mitomycin, but unlike the ddrA mutant deleting ddrC or ddrD from a ΔddrB background further increases that sensitivity. Despite the effect that loss of these gene products has on DNA damage resistance, none appear to directly affect either excision repair or homologous recombination suggesting that they participate in novel processes that facilitate tolerance to UV light and interstrand crosslinks in this species.     

Cytosolic NADPH Homeostasis in Glucose-starved Procyclic Trypanosoma brucei Relies on Malic Enzyme and the Pentose Phosphate Pathway Fed by Gluconeogenic Flux

All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H₂O₂ stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H₂O₂ stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H₂O₂ stress observed for the Δmec/Δmec/^RNAiPGI double mutant when compared with the single mutants, and (iii) the ¹³C enrichment of glycolytic and PPP intermediates from cells incubated with [U-¹³C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host.     

Active Site Mutations in Yeast Protein Disulfide Isomerase Cause Dithiothreitol Sensitivity and a Reduced Rate of Protein Folding in the Endoplasmic Reticulum

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a′, each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a′ domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.     

Insights into the Role of the Unusual Disulfide Bond in Copper-Zinc Superoxide Dismutase

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at ∼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30–50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.     

Glucose 6-Phosphate Accumulation in Mycobacteria: IMPLICATIONS FOR A NOVEL F420-DEPENDENT ANTI-OXIDANT DEFENSE SYSTEM*

Glucose 6-phosphate (G6P) is a metabolic intermediate with many possible cellular fates. In mycobacteria, G6P is a substrate for an enzyme, F₄₂₀-dependent glucose-6-phosphate dehydrogenase (Fgd), found in few bacterial genera. Intracellular G6P levels in six Mycobacterium sp. were remarkably higher (∼17–130-fold) than Escherichia coli and Bacillus megaterium. The high G6P level in Mycobacterium smegmatis may result from 10–25-fold higher activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase when grown on glucose, glycerol, or acetate compared with B. megaterium and E. coli. In M. smegmatis this coincided with up-regulation of the first gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, when acetate was the carbon source, suggesting a cellular program for maintaining high G6P levels. G6P was depleted in cells under oxidative stress induced by redox cycling agents plumbagin and menadione, whereas an fgd mutant of M. smegmatis used G6P less well under such conditions. The fgd mutant was more sensitive to these agents and, in contrast to wild type, was defective in its ability to reduce extracellular plumbagin and menadione. These data suggest that intracellular G6P in mycobacteria serves as a source of reducing power and, with the mycobacteria-specific Fgd-F₄₂₀ system, plays a protective role against oxidative stress.     

Asp-52 in Combination with Asp-398 Plays a Critical Role in ATP Hydrolysis of Chaperonin GroEL

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼150 h (∼6 days), providing a good model to characterize the football-shaped complex.     

Inactivation of Factor VIIa by Antithrombin In Vitro,Ex Vivo and In Vivo: Role of Tissue Factor and Endothelial Cell Protein C Receptor

Recent studies have suggested that antithrombin (AT) could act as a significant physiologic regulator of FVIIa. However, in vitro studies showed that AT could inhibit FVIIa effectively only when it was bound to tissue factor (TF). Circulating blood is known to contain only traces of TF, at best. FVIIa also binds endothelial cell protein C receptor (EPCR), but the role of EPCR on FVIIa inactivation by AT is unknown. The present study was designed to investigate the role of TF and EPCR in inactivation of FVIIa by AT in vivo. Low human TF mice (low TF, ∼1% expression of the mouse TF level) and high human TF mice (HTF, ∼100% of the mouse TF level) were injected with human rFVIIa (120 µg kg⁻¹ body weight) via the tail vein. At varying time intervals following rFVIIa administration, blood was collected to measure FVIIa-AT complex and rFVIIa antigen levels in the plasma. Despite the large difference in TF expression in the mice, HTF mice generated only 40–50% more of FVIIa-AT complex as compared to low TF mice. Increasing the concentration of TF in vivo in HTF mice by LPS injection increased the levels of FVIIa-AT complexes by about 25%. No significant differences were found in FVIIa-AT levels among wild-type, EPCR-deficient, and EPCR-overexpressing mice. The levels of FVIIa-AT complex formed in vitro and ex vivo were much lower than that was found in vivo. In summary, our results suggest that traces of TF that may be present in circulating blood or extravascular TF that is transiently exposed during normal vessel damage contributes to inactivation of FVIIa by AT in circulation. However, TF’s role in AT inactivation of FVIIa appears to be minor and other factor(s) present in plasma, on blood cells or vascular endothelium may play a predominant role in this process.     

Static and Dynamic Factors Limit Chromosomal Replication Complexity in Escherichia coli,Avoiding Dangers of Runaway Overreplication

We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC∼6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the “natural” CRC limit of ∼8 (cells divide more slowly); the “functional” CRC limit of ∼22 (cells divide extremely slowly); and the “tolerance” CRC limit of ∼64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter.     

Modulation of Conotoxin Structure and Function Is Achieved through a Multienzyme Complex in the Venom Glands of Cone Snails

The oxidative folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a “non-native” peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the oxidative folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.     

Functional Characterization of the Role of the Chromosome I Partitioning System in Genome Segregation in Deinococcus radiodurans

Deinococcus radiodurans, a radiation-resistant bacterium, harbors a multipartite genome. Chromosome I contains three putative centromeres (segS1, segS2, and segS3), and ParA (ParA1) and ParB (ParB1) homologues. The ParB1 interaction with segS was sequence specific, and ParA1 was shown to be a DNA binding ATPase. The ATPase activity of ParA1 was stimulated when segS elements were coincubated with ParB1, but the greatest increase was observed with segS3. ParA1 incubated with the segS-ParB1 complex showed increased light scattering in the absence of ATP. In the presence of ATP, this increase was continued with segS1-ParA1B1 and segS2-ParA1B1 complexes, while it decreased rapidly after an initial increase for 30 min in the case of segS3. D. radiodurans cells expressing green fluorescent protein (GFP)-ParB1 produced foci on nucleoids, and the ΔparB1 mutant showed growth retardation and ∼13%-higher anucleation than the wild type. Unstable mini-F plasmids carrying segS1 and segS2 showed inheritance in Escherichia coli without ParA1B1, while segS3-mediated plasmid stability required the in trans expression of ParA1B1. Unlike untransformed E. coli cells, cells harboring pDAGS3, a plasmid carrying segS3 and also expressing ParB1-GFP, produced discrete GFP foci on nucleoids. These findings suggested that both segS elements and the ParA1B1 proteins of D. radiodurans are functionally active and have a role in genome segregation.     

Perturbation of Human Coronary Artery Endothelial Cell Redox State and NADPH Generation by Methylglyoxal

Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH). We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC) were incubated with high glucose (25 mM, 24 h, 37°C), or methylglyoxal (MGO), glyoxal, or glycolaldehyde (100–500 µM, 1 h, 37°C), before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05) decreased total thiols (∼35%), further experiments with MGO showed significant losses of GSH (∼40%) and NADPH (∼10%); these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10%) NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE) formed; lower levels of N ^ε-(carboxyethyl)lysine (CEL) and N ^ε-(carboxymethyl)lysine (CML) were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis.     

The Vibrio cholerae Extracellular Chitinase ChiA2 Is Important for Survival and Pathogenesis in the Host Intestine

In aquatic environments, Vibrio cholerae colonizes mainly on the chitinous surface of copepods and utilizes chitin as the sole carbon and nitrogen source. Of the two extracellular chitinases essential for chitin utilization, the expression of chiA2 is maximally up-regulated in host intestine. Recent studies indicate that several bacterial chitinases may be involved in host pathogenesis. However, the role of V. cholerae chitinases in host infection is not yet known. In this study, we provide evidence to show that ChiA2 is important for V. cholerae survival in intestine as well as in pathogenesis. We demonstrate that ChiA2 de-glycosylates mucin and releases reducing sugars like GlcNAc and its oligomers. Deglycosylation of mucin corroborated with reduced uptake of alcian blue stain by ChiA2 treated mucin. Next, we show that V. cholerae could utilize mucin as a nutrient source. In comparison to the wild type strain, ΔchiA2 mutant was 60-fold less efficient in growth in mucin supplemented minimal media and was also ∼6-fold less competent to survive when grown in the presence of mucin-secreting human intestinal HT29 epithelial cells. Similar results were also obtained when the strains were infected in mice intestine. Infection with the ΔchiA2 mutant caused ∼50-fold less fluid accumulation in infant mice as well as in rabbit ileal loop compared to the wild type strain. To see if the difference in survival of the ΔchiA2 mutant and wild type V. cholerae was due to reduced adhesion of the mutant, we monitored binding of the strains on HT29 cells. The initial binding of the wild type and mutant strain was similar. Collectively these data suggest that ChiA2 secreted by V. cholerae in the intestine hydrolyzed intestinal mucin to release GlcNAc, and the released sugar is successfully utilized by V. cholerae for growth and survival in the host intestine.