Chronic liver injury drives non-traditional intrahepatic fibrin(ogen) crosslinking via tissue transglutaminase.

Essentials Fibrin clots are often implicated in the progression of liver fibrosis. Liver fibrosis was induced in transgenic mice with defects in clot formation or stabilization. Liver fibrosis and fibrin(ogen) deposition do not require fibrin polymerization or factor XIIIa. Fibrin(ogen) is an in vivo substrate of tissue transglutaminase in experimental liver fibrosis. SUMMARY: Background Intravascular fibrin clots and extravascular fibrin deposits are often implicated in the progression of liver fibrosis. However, evidence supporting a pathological role of fibrin in hepatic fibrosis is indirect and based largely on studies using anticoagulant drugs that inhibit activation of the coagulation protease thrombin, which has other downstream targets that promote fibrosis. Therefore, the goal of this study was to determine the precise role of fibrin deposits in experimental hepatic fibrosis. Methods Liver fibrosis was induced in mice expressing mutant fibrinogen insensitive to thrombin-mediated proteolysis (i.e. locked in the monomeric form), termed FibAEK mice, and factor XIII A2 subunit-deficient (FXIII-/- ) mice. Female wild-type mice, FXIII-/- mice and homozygous FibAEK mice were challenged with carbon tetrachloride (CCl4 ) twice weekly for 4 weeks or 6 weeks (1 mL kg-1 , intraperitoneal). Results Hepatic injury and fibrosis induced by CCl4 challenge were unaffected by FXIII deficiency or inhibition of thrombin-catalyzed fibrin polymer formation (in FibAEK mice). Surprisingly, hepatic deposition of crosslinked fibrin(ogen) was not reduced in CCl4 -challenged FXIII-/- mice or FibAEK mice as compared with wild-type mice. Rather, deposition of crosslinked hepatic fibrin(ogen) following CCl4 challenge was dramatically reduced in tissue transglutaminase-2 (TGM2)-deficient (TGM2-/- ) mice. However, the reduction in crosslinked fibrin(ogen) in TGM2-/- mice did not affect CCl4 -induced liver fibrosis. Conclusions These results indicate that neither traditional fibrin clots, formed by the thrombin-activated FXIII pathway nor atypical TGM2-crosslinked fibrin(ogen) contribute to experimental CCl4 -induced liver fibrosis. Collectively, the results indicate that liver fibrosis occurs independently of intrahepatic fibrin(ogen) deposition.

Randomised clinical trial: the 5-HT4 agonist revexepride in patients with gastro-oesophageal reflux disease who have persistent symptoms despite PPI therapy.

BACKGROUND: A substantial proportion of patients with gastro-oesophageal reflux disease (GERD) have only a partial response to proton pump inhibitor (PPI) therapy. Prokinetic drugs may improve reflux symptoms by enhancing oesophageal motility and gastric emptying. AIM: To evaluate the effect of revexepride, a novel prokinetic 5-hydroxytryptamine type 4 (5-HT4 ) receptor agonist, compared with placebo, in patients with GERD who have a partial response to PPIs. METHODS: A phase 2b, double-blind, parallel-group study was conducted, in which patients were randomised to one of three revexepride treatment groups (0.1, 0.5 and 2.0 mg three times daily) or placebo (1:1:1:1 ratio). Daily e-diary data captured patients' symptoms over an 8-week treatment period. The primary efficacy outcome was the weekly percentage of regurgitation-free days in the second half of the study (weeks 5-8). RESULTS: In total, 480 patients were randomised and 477 received treatment (mean age 47.9 years; 61% women). The mean percentage of regurgitation-free days increased from baseline (range, 15.0-18.8%) to week 8 (62.3-70.5%) in all four study arms; however, there were no statistically significant differences in this change between placebo and the three treatment arms. No dose-dependent relationship in treatment effect was observed for any of the study endpoints. The incidence of treatment-emergent adverse events (TEAEs) was revexepride dose-dependent. Only one serious TEAE occurred and none resulted in death. CONCLUSIONS: Revexepride was no more effective than placebo in controlling regurgitation in patients with GERD symptoms partially responsive to PPIs. Revexepride was well tolerated. ClinicalTrials.gov Identifier: NCT01472939.

Managing acute central nervous system infections in the UK adult intensive care unit in the wake of UK encephalitis guidelines.

The acute central nervous system infections meningitis and encephalitis commonly require management on intensive care units. The clinical features often overlap and in the acute phase-altered consciousness and seizures may also need to be managed. In April 2012, the first UK national guideline for the management of suspected viral encephalitis was published by the British Infection Association and Association of British Neurologists, and other key stakeholders, and included a simple management algorithm. The new guideline results from evidence demonstrating a number of common oversights in the standard management of suspected viral encephalitis in many settings. In combination with British Infection Association meningitis guidelines, evidence-based approaches now exist to facilitate the non-expert managing patients with suspected central nervous system infections. Here we bring together these guidelines and the supporting evidence applicable for intensivists into a single resource.

Blood lactate clearance after maximal exercise depends on active recovery intensity.

AIM: High-intensity exercise is time-limited by onset of fatigue, marked by accumulation of blood lactate. This is accentuated at maximal, all-out exercise that rapidly accumulates high blood lactate. The optimal active recovery intensity for clearing lactate after such maximal, all-out exercise remains unknown. Thus, we studied the intensity-dependence of lactate clearance during active recovery after maximal exercise. METHODS: We constructed a standardized maximal, all-out treadmill exercise protocol that predictably lead to voluntary exhaustion and blood lactate concentration>10 mM. Next, subjects ran series of all-out bouts that increased blood lactate concentration to 11.5±0.2 mM, followed by recovery exercises ranging 0% (passive)-100% of the lactate threshold. RESULTS: Repeated measurements showed faster lactate clearance during active versus passive recovery (P<0.01), and that active recovery at 60-100% of lactate threshold was more efficient for lactate clearance than lower intensity recovery (P<0.05). Active recovery at 80% of lactate threshold had the highest rate of and shortest time constant for lactate clearance (P<0.05), whereas the response during the other intensities was graded (100%=60%>40%>passive recovery, P<0.05). CONCLUSION: Active recovery after maximal all-out exercise clears accumulated blood lactate faster than passive recovery in an intensity-dependent manner, with maximum clearance occurring at active recovery of 80% of lactate threshold.

Progressive multifocal leukoencephalopathy with gastrointestinal disease in a pediatric kidney transplant recipient.

PML is a demyelinating disease of the central nervous system caused by infection with JCV. Several cases of PML in bone marrow and solid organ transplant recipients have been reported in recent years. JCV has been isolated from the gastrointestinal mucosa of immunocompromised patients, but there are no published reports of PML associated with symptomatic gastrointestinal involvement in kidney transplant recipients. We report a case of a nine-yr-old girl with a kidney transplant who developed a severe gastrointestinal illness causing pseudo-obstruction in association with PML. JCV was suspected as the causative agent in this patient by the detection of high JCV titer through PCR analysis of the cerebrospinal fluid and blood and positive staining for simian virus 40 in the colon. JCV intestinal infection should be considered in kidney transplant recipients presenting with intestinal pseudo-obstruction.

Patient need at the heart of workforce planning: the use of supply and demand analysis in a large teaching hospital's acute medical unit.

Timely medical assessment is integral to the safety and quality of healthcare delivery in acute medicine. Medical staff are an expensive resource. This study aimed to develop a modelling system that facilitated efficient workforce planning according to patient need on the acute medical unit. A realistic 24-hour 'supply' of junior doctors was calculated by adjusting the theoretical numbers on the rota for leave allowances, natural breaks and other ward duties by a combination of direct observation of working practice and junior doctor interviews. 'Demand' was analysed using detailed admission data. Supply and demand were then integrated with data from a survey of the time spent on the process of clerking and assessment of medical admissions. A robust modelling system that predicted the number of unclerked patients was developed. The utility of the model was assessed by demonstrating the impact of a regulation-compliant redesign of the rota using existing staff and by predicting the most efficient use of an additional shift. This simple modelling system has the potential to enhance quality of care and efficiency by linking workforce planning to patient need.

Prevalence of oral disease among adults with primary HIV infection.

OBJECTIVE: To explore the type and prevalence of oral mucosal lesions among adults with primary HIV infection (PHI) compared with HIV-negative adults at high risk for HIV disease, and in relation to HIV viral load. METHODS: We conducted standardized oral examinations to identify specific oral mucosal lesions among adults with PHI, both pre-seroconversion and post- seroconversion-recently infected, compared with HIV-negative adults. We compared the group with oral lesions to those without oral lesions with respect to HIV-RNA load and CD4 + T-cell count. RESULTS: Among 115 adults (predominantly men), pseudomembranous candidiasis was the most common oral lesion among those with PHI, and was found in 4% of the 23 participants in pre-seroconversion and in 9% of 69 participants with post-seroconversion recent infection, compared with none found among 23 HIV negatives. Among those with PHI, the median viral load was higher and the median CD4 + T-cell count lower among the 15 participants with an oral lesion of any type than among the 77 participants without oral lesions (P = 0.02 and 0.04, respectively). CONCLUSION: This finding suggests that individuals with PHI who have oral lesions may be more likely to transmit HIV because of their higher viral load.

Conversion of a peroxiredoxin into a disulfide reductase by a triplet repeat expansion.

Pathways for the reduction of protein disulfide bonds are found in all organisms and are required for the reductive recycling of certain enzymes including the essential protein ribonucleotide reductase. An Escherichia coli strain that lacks both thioredoxin reductase and glutathione reductase grows extremely poorly. Here, we show that a mutation occurring at high frequencies in the gene ahpC, encoding a peroxiredoxin, restores normal growth to this strain. This mutation is the result of a reversible expansion of a triplet nucleotide repeat sequence, leading to the addition of one amino acid that converts the AhpC protein from a peroxidase to a disulfide reductase. The ready mutational interconversion between the two activities could provide an evolutionary advantage to E. coli.

Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures.

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.

Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis.

AhpF, a homodimer of 57 kDa subunits, is a flavoenzyme which catalyzes the NADH-dependent reduction of redox-active disulfide bonds in the peroxidase AhpC, a member of the recently identified peroxiredoxin class of antioxidant enzymes. The structure of AhpF from Salmonella typhimurium at 2.0 A resolution, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210-521) is structurally like Escherichia coli thioredoxin reductase. In addition, AhpF has an N-terminal domain (residues 1-196) formed from two contiguous thioredoxin folds, but containing just a single redox-active disulfide (Cys129-Cys132). A flexible linker (residues 197-209) connects the domains, consistent with experiments showing that the N-terminal domain acts as an appended substrate, first being reduced by the C-terminal portion of AhpF, and subsequently reducing AhpC. Modeling studies imply that an intrasubunit electron transfer accounts for the reduction of the N-terminal domain in dimeric AhpF. Furthermore, comparing the N-terminal domain with protein disulfide oxidoreductase from Pyrococcus furiosis, we describe a new class of protein disulfide oxidoreductases based on a novel mirror-image active site arrangement, with a distinct carboxylate (Glu86) being functionally equivalent to the key acid (Asp26) of E. coli thioredoxin. A final fortuitous result is that the N-terminal redox center is reduced and provides a high-resolution view of the thiol-thiolate hydrogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.

Activity of one of two engineered heterodimers of AhpF, the NADH:peroxiredoxin oxidoreductase from Salmonella typhimurium, reveals intrasubunit electron transfer between domains.

AhpF, the flavoprotein reductase component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the reduction of an intersubunit disulfide bond in the peroxidatic active site of the system's other component, AhpC, a member of the peroxiredoxin family. Previous studies have shown that AhpF can be dissected into two functional units, a thioredoxin reductase-like C-terminus (containing FAD and a redox-active disulfide, Cys345-Cys348) and an N-terminal domain containing a second redox-active disulfide center (Cys129-Cys132). The role of the N-terminal domain as the direct reductant of AhpC, mediating electron transfer from the C-terminal redox centers of AhpF, has been firmly established by several approaches. Not known, however, was whether the transfer of electrons between the C-terminal and N-terminal disulfide centers occurred as an inter- or intrasubunit process in dimeric AhpF. Two heterodimeric AhpF species were therefore created in which one of the two pathways was completely disrupted while the other was left partially intact in each construct. Only the heterodimer containing one monomer of wild type AhpF and a monomer of mutated (and truncated) AhpF exhibited peroxidase activity with AhpC indicating that electron transfer between domains of AhpF is an intrasubunit process.

Essential thioredoxin-dependent peroxiredoxin system from Helicobacter pylori: genetic and kinetic characterization.

Helicobacter pylori, an oxygen-sensitive microaerophile, contains an alkyl hydroperoxide reductase homologue (AhpC, HP1563) that is more closely related to 2-Cys peroxiredoxins of higher organisms than to most other eubacterial AhpC proteins. Allelic replacement mutagenesis revealed ahpC to be essential, suggesting a critical role for AhpC in defending H. pylori against oxygen toxicity. Characterization of the ahpC promoter region divulged two putative regulatory elements and identified the transcription initiation site, which was mapped to 96 and 94 bp upstream of the initiation codon. No homologue of ahpF, which encodes the dedicated AhpC reductase in most eubacteria, was found in the H. pylori genome. Instead, homologues of Escherichia coli thioredoxin (Trx) reductase (TrxR, HP0825) and Trx (Trx1, HP0824) formed a reductase system for H. pylori AhpC. A second Trx homologue (Trx2, HP1458) was identified but was incapable of AhpC reduction, although Trx2 exhibited disulfide reductase activity with other substrates [insulin and 5,5'-dithiobis(2-nitrobenzoic acid)]. AhpC interactions with each substrate, Trx1 and hydroperoxide, were bimolecular and nonsaturable (infinite V(max) and K(m) values) but rapid enough (at 1 x 10(5) to 2 x 10(5) M(-1) s(-1)) to suggest an important role for AhpC in cellular peroxide metabolism. AhpC also exhibited a wide specificity for hydroperoxide substrates, which, taken together with the above results, suggests a minimal binding site for hydroperoxides composed of little more than the cysteinyl (Cys49) active site. H. pylori AhpC was not reduced by Salmonella typhimurium AhpF and was slightly more active with E. coli TrxR and Trx1 than was S. typhimurium AhpC, demonstrating the specialized catalytic properties of this peroxiredoxin.

PEPP: collaborating to improve quality.

By collecting and analyzing information about Medicare payment errors, PEPP initiatives are making a contribution to healthcare quality efforts nationwide. Here's how one organization takes a collaborative approach to improving quality.

Low doses vs. high doses of the angiotensin converting-enzyme inhibitor lisinopril in chronic heart failure: a cost-effectiveness analysis based on the Assessment of Treatment with Lisinopril and Survival (ATLAS) study. The ATLAS Study Group.

OBJECTIVE: A cost-effectiveness analysis of high and low doses of the angiotensin-converting enzyme (ACE) inhibitor lisinopril in the treatment of chronic heart failure. METHODS: A cost-effectiveness analysis using data from a randomized controlled trial, ATLAS, where 3164 patients with chronic heart failure were allocated to a high-dose (daily target dose 32.5-35 mg) or low-dose strategy (daily target dose 2.5-5.0 mg) of lisinopril. Differential costs were based on resource use data collected in the trial costed using UK unit costs. Cost-effectiveness analysis related differential costs to differential life-years during a 4-year trial follow-up. RESULTS: The mean total number of hospital in-patient days per patient was 18. 5 in the high dose group and 22.5 in the low dose group. Over the whole duration of the trial, the mean (S.D.) daily dose of lisinopril in the high-dose group was 22.5 mg (15.7 mg) compared to 3.2 mg (2.5 mg) in the low-dose group. The mean difference in cost per patient was pound sterling 397 lower in the high-dose group [95% CI (high-dose-low-dose) - pound sterling 1263 to pound sterling 436]. Mean life-years per patient were 0.085 years higher in the high-dose group [95% CI (high-dose-low-dose) -0.0074 to 0.1706). Based on mean costs and life-years, high-dose therapy dominates low-dose (less costly and more effective). Allowing for uncertainty in mean costs and life-years, the probability of high-dose therapy being less costly than low dose was 82%. If a decision maker is willing to pay at least pound sterling 3600 per life-year gained, the probability of high-dose being more cost-effective was 92%. CONCLUSIONS: The ATLAS Study showed that the treatment of heart failure with high-doses of lisinopril has a high probability of being more cost-effective than low-dose therapy.

AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase.

A group of bacterial flavoproteins related to thioredoxin reductase contain an additional approximately 200-amino-acid domain including a redox-active disulfide center at their N-termini. These flavoproteins, designated NADH:peroxiredoxin oxidoreductases, catalyze the pyridine-nucleotide-dependent reduction of cysteine-based peroxidases (e.g. Salmonella typhimurium AhpC, a member of the peroxiredoxin family) which in turn reduce H2O2 or organic hydroperoxides. These enzymes catalyze rapid electron transfer (kcat > 165 s-1) through one tightly bound FAD and two redox-active disulfide centers, with the N-terminal-most disulfide center acting as a redox mediator between the thioredoxin-reductase-like part of these proteins and the peroxiredoxin substrates. A chimeric protein with the first 207 amino acids of S. typhimurium AhpF attached to the N-terminus of Escherichia coli thioredoxin reductase exhibits very high NADPH:peroxiredoxin oxidoreductase and thioredoxin reductase activities. Catalytic turnover by NADH:peroxiredoxin oxidoreductases may involve major domain rotations, analogous to those proposed for bacterial thioredoxin reductase, and cycling of these enzymes between two electron-reduced (EH2) and four electron-reduced (EH4) redox states.

Hind limb malformations in free-living northern leopard frogs (Rana pipiens) from Maine, Minnesota, and Vermont suggest multiple etiologies.

BACKGROUND: Reports of malformed frogs have increased throughout the North American continent in recent years. Most of the observed malformations have involved the hind limbs. The goal of this study was to accurately characterize the hind limb malformations in wild frogs as an important step toward understanding the possible etiologies. METHODS: During 1997 and 1998, 182 recently metamorphosed northern leopard frogs (Rana pipiens) were collected from Minnesota, Vermont, and Maine. Malformed hind limbs were present in 157 (86%) of these frogs, which underwent necropsy and radiographic evaluation at the National Wildlife Health Center. These malformations are described in detail and classified into four major categories: (1) no limb (amelia); (2) multiple limbs or limb elements (polymelia, polydactyly, polyphalangy); (3) reduced limb segments or elements (phocomelia, ectromelia, ectrodactyly, and brachydactyly; and (4) distally complete but malformed limb (bone rotations, bridging, skin webbing, and micromelia). RESULTS: Amelia and reduced segments and/or elements were the most common finding. Frogs with bilateral hind limb malformations were not common, and in only eight of these 22 frogs were the malformations symmetrical. Malformations of a given type tended to occur in frogs collected from the same site, but the types of malformations varied widely among all three states, and between study sites within Minnesota. CONCLUSIONS: Clustering of malformation type suggests that developmental events may produce a variety of phenotypes depending on the timing, sequence, and severity of the environmental insult. Hind limb malformations in free-living frogs transcend current mechanistic explanations of tetrapod limb development.

Identification of a new gene responsible for the oxygen tolerance in aerobic life of Streptococcus mutans.

Alkyl hydroperoxide reductase in Streptococcus mutans consists of two components, Nox-1 and AhpC. Deletion of nox-1 and ahpC in a double mutant as well as the wild-type of Streptococcus mutans can form colonies in the presence of air to the same extent. The evidence suggested the presence of some other antioxidant system(s) independent of the Nox-1/AhpC system in the bacterium. Here we identified a new antioxidant gene (dpr) and the gene product (Dpr) which complements the defect of peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. The dpr-disruption mutant of S. mutans could form colonies anaerobically but not aerobically.

Attachment of the N-terminal domain of Salmonella typhimurium AhpF to Escherichia coli thioredoxin reductase confers AhpC reductase activity but does not affect thioredoxin reductase activity.

AhpF of Salmonella typhimurium, the flavoprotein reductase required for catalytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroperoxide reductase system, is a 57 kDa protein with homology to thioredoxin reductase (TrR) from Escherichia coli. Like TrR, AhpF employs tightly bound FAD and redox-active disulfide center(s) in catalyzing electron transfer from reduced pyridine nucleotides to the disulfide bond of its protein substrate. Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-terminal part of AhpF; a stretch of about 200 amino acids at the N-terminus of AhpF contains an additional redox-active disulfide center and is required for catalysis of AhpC reduction. We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k(cat)/K(m)) as AhpF in AhpC reductase assays; both k(cat) and the K(m) for AhpC are decreased about 3-4-fold for Nt-TrR compared with AhpF. In addition, Nt-TrR retains essentially full TrR activity. Based on results from two mutants of Nt-TrR (C129, 132S and C342,345S), AhpC reductase activity requires both centers while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR. The high catalytic efficiency with which Nt-TrR can reduce thioredoxin implies that the attached N-terminal domain does not block access of thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is proposed to undergo during catalysis. These studies indicate that the C-terminal part of AhpF and bacterial TrR have very similar mechanistic properties. These findings also confirm that the N-terminal domain of AhpF plays a direct role in AhpC reduction.

AhpF can be dissected into two functional units: tandem repeats of two thioredoxin-like folds in the N-terminus mediate electron transfer from the thioredoxin reductase-like C-terminus to AhpC.

AhpF, the flavin-containing component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the NADH-dependent reduction of an active-site disulfide bond in the other component, AhpC, which in turn reduces hydroperoxide substrates. The amino acid sequence of the C-terminus of AhpF is 35% identical to that of thioredoxin reductase (TrR) from Escherichia coli. AhpF contains an additional 200-residue N-terminal domain possessing a second redox-active disulfide center also required for AhpC reduction. Our studies indicate that this N-terminus contains a tandem repeat of two thioredoxin (Tr)-like folds, the second of which contains the disulfide redox center. Structural and catalytic properties of independently expressed fragments of AhpF corresponding to the TrR-like C-terminus (F[208-521]) and the 2Tr-like N-terminal domain (F[1-202]) have been addressed. Enzymatic assays, reductive titrations, and circular dichroism studies of the fragments indicate that each folds properly and retains many functional properties. Electron transfer between F[208-521] and F[1-202] is, however, relatively slow (4 x 10(4) M(-)(1) s(-)(1) at 25 degrees C) and nonsaturable up to 100 microM F[1-202]. TrR is nearly as efficient at F[1-202] reduction as is F[208-521], although neither the latter fragment, nor intact AhpF, can reduce Tr. An engineered mutant AhpC substrate with a fluorophore attached via a disulfide bond has been used to demonstrate that only F[1-202], and not F[208-521], is capable of electron transfer to AhpC, thereby establishing the direct role this N-terminal domain plays in mediating electron transfer between the TrR-like part of AhpF and AhpC.

Role of the dpr product in oxygen tolerance in Streptococcus mutans.

We have previously identified and characterized the alkyl hydroperoxide reductase of Streptococcus mutans, which consists of two components, Nox-1 and AhpC. Deletion of both nox-1 and ahpC had no effect on the sensitivity of S. mutans to cumene hydroperoxide or H(2)O(2), implying that the existence of another antioxidant system(s) independent of the Nox-1-AhpC system compensates for the deficiency. Here, a new antioxidant gene (dpr for Dps-like peroxide resistance gene) was isolated from the S. mutans chromosome by its ability to complement an ahpCF deletion mutant of Escherichia coli with a tert-butyl hydroperoxide-hypersensitive phenotype. The dpr gene complemented the defect in peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. Under aerobic conditions, the dpr disruption mutant carrying a spectinomycin resistance gene (dpr::Spc(r) mutant) grew as well as wild-type S. mutans in liquid medium. However, the dpr::Spc(r) mutant could not form colonies on an agar plate under air. In addition, neither the dpr::Spc(r) ahpC::Em(r)::nox-1 triple mutant nor the dpr::Spc(r) sod::Em(r) double mutant was able to grow aerobically in liquid medium. The 20-kDa dpr gene product Dpr is an iron-binding protein. Synthesis of Dpr was induced by exposure of S. mutans cells to air. We propose a mechanism by which Dpr confers aerotolerance on S. mutans.