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PubMed1994

Oxygen reactions with bacterial oxidases and globins: binding, reduction and regulation.

Oxygen is favoured as terminal electron acceptor in aerobic and facultative microorganisms because of its appropriate physical state, satisfactory solubility and its desirable combinations of kinetic and thermodynamic properties. Oxygen is generally reduced by four electrons to yield oxygen, but there are important biological consequences of, and roles for, the partial reduction to superoxide and peroxide. Complex and multiple regulatory networks ensure (i) the utilization of oxygen in preference to other oxidants, (ii) the synthesis of oxygen-consuming enzymes with appropriate properties (particularly affinity for the ligand), and (iii) appropriate cellular protection in the event of oxidative stress. This contribution reviews the terminal respiratory oxidases of selected Gram-negative bacteria and microbial haemoglobin-like proteins. Recent studies of the cytochrome bd-type oxidases of Escherichia coli and Azotobacter vinelandii suggest that, despite probable similarity at the amino acid level, the reactivities of these oxidases with oxygen are strikingly different. The respiratory protection afforded to nitrogenase in the obligately aerobic diazotroph A. vinelandii by the cytochrome bd complex appears to be accompanied by, and may be the result of, a low affinity for oxygen and a high Vmax. The poorly characterized cytochrome o-containing oxidase in this bacterium is not required for respiratory protection. In E. coli, the cytochrome bd-type oxidase has a remarkably high affinity for oxygen, consistent with the view that this is an oxygen-scavenging oxidase utilized under microaerobic conditions. The demonstration of substrate (i.e. oxygen) inhibition in this complex suggests a mechanism whereby wasteful electron flux through a non-proton-pumping oxidase is avoided at higher dissolved oxygen tensions. The demonstration of two ligand-binding sites (haems d and b595) in oxidases of this type suggests plausible mechanisms for this phenomenon. In E. coli, assembly of the cytochrome bd-type oxidase (and of periplasmic cytochromes b and c) requires the presence of an ABC transporter, which may serve to export haem or some "assembly factor" to the periplasm. There is at least one additional oxygen-consuming protein in E. coli-the flavohaemoglobin encoded by the hmp gene. Globin-like proteins are also widely distributed in other bacteria, fungi and protozoa, but most have unknown functions. The function of HMP and the related chimaeric flavohaemoglobins in other bacteria and yeast is unknown; one of several possibilities for HMP is that its relatively low affinity for oxygen during turnover with NADH as substrate could enable it to function as a sensor of failing (or rising) cytoplasmic oxygen concentrations.

Bacteria↗
PubMed1993

Kidney bean lectin-induced Escherichia coli overgrowth in the small intestine is blocked by GNA, a mannose-specific lectin.

The reversible and dose-dependent hyperplastic growth of the small intestine and accelerated epithelial cell turnover caused by feeding rats with diets containing kidney bean lectin (PHA) increased the proportion of immature cells on the villi whose membrane and/or cytoplasm contained mainly simple, polymannosylated glycans. These new alpha-linked mannosyl terminals, particularly of the damaged epithelium, facilitated the preferential adherence of opportunistic Escherichia coli with mannose-sensitive Type 1 fimbriae, and other coliforms, to the glycocalyx. Accordingly, the growth of the gut was accompanied by a reversible and PHA dose-dependent overgrowth with E. coli. As expected from their common carbohydrate specificity, the inclusion in the diet of the mannose-specific agglutinin from snowdrop (Galanthus nivalis) bulbs (GNA) significantly reduced the extent of E. coli overgrowth, but abolished neither the growth nor the damage caused by PHA to the small intestine. Thus, GNA and perhaps other mannose-specific lectins, especially when used in a preventive mode, can be used to specifically block the proliferation of Type 1 E. coli in the small intestine.

Animals↗
PubMed1991

A new approach to the quantitative estimation of nitrogen metabolic pathways in the rumen.

Rumen nitrogen metabolism values were estimated by the use of a single injection of 15(NH4)2SO4 into the rumen of sheep and consecutive 15N enrichment measurements in the rumen ammonia pool, rumen non-NH3-N (NAN) pool, rumen purine pool and blood urea-N (BUN) pool for a period of 24 h. Synthesis and degradation of N compounds in the rumen and passage of N to and from the rumen were evaluated on a chemical rather than a microbial basis; microbial fractions were not separated. This model was examined in two experiments. In Expt 1 a ram (55 kg) was given a semi-synthetic diet (1067 g dry matter (DM), 22.8 g N) in which soya-bean meal provided over 90% of the N. In Expt 2, two rams (45 kg) were given in three consecutive periods a semi-synthetic basal diet containing: (1) roasted soya-bean meal (SBM, 725 g DM, 14.8 g N/d); or (2) fishmeal (FM, 728 g DM, 15.5 g N/d); or (3) raw soya-bean meal (RSBM, 724 g DM, 13.8 g N/d). In all these rations, the main protein source provided over 90% of the N. In Expt 1, 68.3% of N intake was degraded directly to NH3 in the rumen, 21.2% escaped rumen degradation and 10.5% was incorporated into stable N compounds in the rumen. Net NH3 transfer to the blood was 30.4%, NH3 flow from the rumen was 6.6% and rumen NAN output was 63% of N intake. In Expt 2, rumen NAN output was larger (7.67, 14.36 and 8.89 g N/d for diets containing SBM, FM and RSBM respectively; P less than 0.05) and net NH3 loss to the blood was smaller (6.1, 0.39 and 4.17 g N/d for diets SBM, FM and RSBM respectively; P less than 0.05) for diet FM as compared with the soya-bean diets. The percentage of rumen NAN that was synthesized from NH3 was larger for diet RSBM (36.4, 40.3 and 49.1 for diets SBM, FM and RSBM respectively; P less than 0.05) than for the other two rations. NH3 pool sizes (g N) were 0.463, 0.385 and 0.301 for diets SBM, FM and RSBM respectively (P less than 0.05), while their hourly turnover rates were 15.8, 26.1 and 5.12 for diets SBM, FM and RSBM respectively (P less than 0.01), indicating no correlation between pool size and its turnover rate.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonia↗
PubMed2003

Mupirocin-resistant, methicillin-resistant Staphylococcus aureus: does mupirocin remain effective?

OBJECTIVE: To determine the efficacy of mupirocin ointment in reducing nasal colonization with mupirocin-susceptible, methicillin-resistant Staphylococcus aureus (MS MRSA) as well as mupirocin-resistant MRSA (MR MRSA). DESIGN: Prospective evaluation in which patients colonized with MRSA were treated twice daily with 2% topical mupirocin ointment for 5 days. SETTING: James H. Quillen Veterans' Affairs Medical Center. PATIENTS: Forty hospitalized patients with two anterior nares cultures positive for MRSA within a 7-day period. METHODS: Treated patients had post-treatment cultures at day 3 and weeks 1, 2, and 4. Isolates underwent mupirocin-susceptibility testing and DNA typing. MRSA clearance and type turnover were assessed for isolates that were mupirocin-susceptible, low-level (LL) MR MRSA and high-level (HL) MR MRSA. RESULTS: Post-treatment nares cultures on day 3 were negative for 78.5%, 80%, and 27.7% of patients with MS MRSA, LL-MR MRSA, and HLMR MRSA, respectively. Sustained culture negativity at 1 to 4 weeks was more common in the MS MRSA group (91%) than in the LL-MR MRSA group (25%) or the HL-MR MRSA group (25%). Positive post-treatment cultures usually showed the same DNA pattern relative to baseline. Plasmid curing of 18 HL-MR MRSA resulted in 15 MS MRSA and 3 LL-MR MRSA. CONCLUSIONS: Mupirocin was effective in eradicating MS MRSA, but strains of MR MRSA often persisted after treatment. This appeared to reflect treatment failure rather than exogenous recolonization. MR MRSA is now more prevalent and it is appropriate to sample MRSA populations for mupirocin susceptibility prior to incorporating mupirocin into infection control programs.

Administration, Topical↗
PubMed1991

[Proteolytic enzymes and the destruction of articular cartilage in arthritis and chronic polyarthritis].

The extracellular matrix (ECM) of articular cartilage is subject to a steady remodelling process. The collagenous components of the ECM are characterized by a very low rate of metabolism, whereas the proteoglycans exhibit an active turnover. The main proteolytic enzymes degrading the ECM components are collagenase, gelatinase and stromelysin. These enzymes undergo under pathological circumstances a remarkable enhancement of synthesis and activity. Although each of these enzymes appears to degrade one ECM component specifically, there is evidence for synergistic effects of most of them. Gelatinase acts synergistically with collagenase in degrading insoluble interstitial collagens and stromelysin activates collagenase. Thus a cascade mechanism may exist in which the cartilage-ECM is completely degraded. Yet, it is not crucial which part of the ECM (collagens or proteoglycans) is primarily degraded. The integrity of the ECM rather depends on the balance between anabolic and catabolic processes, the upset of which results in damage of the articular cartilage. Cartilage destruction in rheumatoid arthritis and osteoarthritis is considered to be a result of this imbalance in favour of the catabolic processes. This would lead to a decrease in proteoglycans which causes fibronectin deposition in the cartilage ECM. Due to chemotaxic effects of fibronectin on fibroblasts, the enrichment of this glycoprotein in the ECM gives rise to cartilage fibrosis and early degeneration.

Arthritis, Rheumatoid↗
PubMed1982

Stimulation of retinoic acid of synthesis and turnover of basement membrane in mouse embryonal carcinoma-derived endoderm cells.

The effect of retinoic acid on the synthesis and degradation of basement membrane components by endoderm cells derived from mouse embryonal carcinoma (EC) cells was studied in a serum-free, defined medium. By immunofluorescence these cells accumulate type IV collagen, laminin, and fibronectin after growth in media containing epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin, transferrin, and Pedersen fetuin. Collagen accounted for 2 to 4% of the newly synthesized proteins, of which 90% were found in the culture media. This collagen was identified as Pro-type IV be gel electrophoresis and enzymatic susceptibility. The EC cells preferentially attached to type IV collagen in vitro and such attachment was mediated by laminin. Treatment of EC cells with retinoic acid caused an increased accumulation of collagen (10 to 15% of secreted proteins) and also stimulated the elaboration of latent protease which degraded laminin and type IV collagen. The laminin-degrading activity was plasminogen dependent. The type IV collagen-degrading activity was a metal protease which could be activated by trypsin or plasmin. It is likely that at least part of the laminin degrading activity is plasmin (mediated through plasminogen activator), since highly purified plasmin is shown to degrade native laminin.

Animals↗
PubMed1992

Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome-c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit.

Cytochrome-c reductase was isolated from Saccharomyces cerevisiae GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE. Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 center (Q, ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax was 50% higher. Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.

Amino Acid Sequence↗
PubMed1985

The turnover and degradation of collagen.

The interstitial collagens are degraded predominantly extracellularly, by specific collagenases (metalloproteinases) capable of cleaving the helical region across the three chains at a similar locus, solubilizing the cleaved products from the fibril. Other neutral proteinases may also function in this role by cleaving near cross-links in the fibril. Collagen type, molecular aggregation and small changes in temperature all markedly affect rates of collagenolysis in the fibril. Regulation of collagenolysis is also modulated at the levels of (1) cellular production of latent collagenase (procollagenase), (2) activation of latent collagenase, and (3) production of collagenase inhibitors. Fibroblastic cells and certain macrophages are probably the predominant sources of collagenases in inflammation; an enzyme in polymorphonuclear leucocytes (neutrophils) is distinct from the tissue enzyme. Molecules such as mononuclear cell factor (MCF), homologous with interleukin 1, which augment cellular collagenase production in inflammation, are derived from monocytes. The mechanisms of augmented collagenase production involve new protein synthesis and, if this augmentation is analogous to that produced by urate crystals, it is probably associated with increased levels of procollagenase mRNA. MCF production is itself controlled by products of lymphocytes as well as by interactions of monocytes with the Fc portion of immunoglobulins and components of the extracellular matrix. Activation of latent (pro)collagenase probably occurs in vivo through the action of neutral proteinases such as plasmin (through plasminogen activator). These effects may be indirect and exerted through proteolytic activation of a procollagenase activator. Tissue inhibitors act to regulate the active collagenase.

Animals↗
PubMed2002

The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures.

It is generally known that cofactors play a major role in the production of different fermentation products. This paper is part of a systematic study that investigates the potential of cofactor manipulations as a new tool for metabolic engineering. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ and producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. We have previously investigated a genetic means of increasing the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase and have demonstrated that this manipulation provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically (Berríos-Rivera et al., 2002, Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229). The current work explores further the effect of substituting the native cofactor-independent formate dehydrogenase (FDH) by an NAD(+)-dependent FDH from Candida boidinii on the NAD(H/+) levels, NADH/NAD+ ratio, metabolic fluxes and carbon-mole yields in Escherichia coli under anaerobic chemostat conditions. Overexpression of the NAD(+)-dependent FDH provoked a significant redistribution of both metabolic fluxes and carbon-mole yields. Under anaerobic chemostat conditions, NADH availability increased from 2 to 3 mol NADH/mol glucose consumed and the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol to acetate ratio and a decrease in the flux to lactate. It was also found that the NADH/NAD+ ratio should not be used as a sole indicator of the oxidation state of the cell. Instead, the metabolic distribution, like the Et/Ac ratio, should also be considered because the turnover of NADH can be fast in an effort to achieve a redox balance.

Anaerobiosis↗
PubMed1990

The effect of phenytoin on collagenase and gelatinase activities in UMR 106-01 rat osteoblastic osteosarcoma cells.

Phenytoin (PHT), a widely used anticonvulsant, has been shown to inhibit bone resorption in rodent organ cultures. The drug also has complex effects on bone metabolism including chronic clinical symptoms of osteomalacia. However, the precise mechanism of PHT action in bone is still unclear. Neutral collagenases that specifically cleave native collagen have been implicated in the turnover of connective tissue. The effect of PHT was assessed on collagenase and gelatinase activities from UMR 106-01 rat osteoblastic osteosarcoma cells. Semiconfluent cells were treated with PHT (50 and 10 micrograms/ml) in the presence of bovine parathyroid hormone, b-PTH-(1-34), at 10(-7) M for 24, 48, 72 and 96 h. The media were assayed following concentration, APMA activation, and incubation with native or denatured [3H]-methyl collagen substrate (approximately 100,000 dpm) at 27 degrees C for 18 h and 35 degrees C for 2 h, respectively. Enzyme activities were presented as primary counts per minute for each time point and calculated as % activity of PTH at 10(-7) M. Parathyroid hormone (10(-7) M) stimulated collagenase activity (approximately 65-fold) and gelatinase activity (approximately 400-fold). PHT (50 micrograms/ml) reduced the PTH-stimulated collagenase activity by 18-53% and the gelatinase activity by 58-72%. SDS PAGE and fluorography following PHT treatment indicated a PHT-induced partial inhibition of PTH-stimulated degradation to alpha A chains of Type I collagen. Phenytoin may inhibit bone resorption through its action on the transcription, synthesis, and/or secretion of the collagenolytic enzymes, collagenase and gelatinase.

Animals↗
PubMed1991

Expression of genes related to the extracellular matrix in human endothelial cells. Differential modulation by elevated glucose concentrations, phorbol esters, and cAMP.

To identify agents and mechanisms responsible for the thickened basement membranes characteristic of diabetic angiopathy we examined the effects of high glucose (30 mM) on the expression of genes related to extracellular matrix composition and turnover and investigated whether the changes induced by high glucose were mimicked and sustained by activation of protein kinase C or A. In human umbilical vein endothelial cells high glucose increased fibronectin, collagen IV, tissue plasminogen activator (tPA), and plasminogen activator-inhibitor 1 (PAI-1) mRNA levels 2-fold but did not affect type IV and interstitial collagenase expression. Acute treatment with phorbol esters resulted in increased collagen IV, tPA, PAI-1, and interstitial collagenase mRNAs; the type IV collagenase mRNA levels were instead suppressed to 50% of control. Upon longer exposure to phorbol esters (48 h) suppression of fibronectin and PAI-1 mRNAs also occurred. Intracellular elevation of cAMP led to over-expression of fibronectin and type IV collagenase and potentiated the effects of phorbol esters on collagen IV, tPA, and interstitial collagenase expression. The mRNA changes induced by high glucose occurred in the absence of protein kinase C activation or cAMP elevation. These studies indicate that events other than activation of protein kinase C or A bridge high ambient glucose to changes in endothelial cell gene expression that may contribute to diabetic angiopathy.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Gastrin and CCK-8 induce inositol 1,4,5-trisphosphate formation in rabbit gastric parietal cells.

The role of phosphoinositide turnover in the mediation of acid secretion was examined in an enriched preparation of isolated rabbit parietal cells (75%). Both gastrin and CCK-8 (octapeptide of cholecystokinin) stimulated [14C]aminopyrine (AP) uptake by cells (EC50 0.07 +/- 0.03 nM (gastrin) and 0.093 +/- 0.065 nM (CCK-8] and increased [3H]inositol phosphates cellular contents (EC50 0.142 +/- 0.016 nM (gastrin) and 0.116 +/- 0.027 nM (CCK-8] in a parallel fashion. In addition, the EC50 values for both phenomenon were quite similar to the Kd values obtained from binding experiments. HPLC analysis of the different [3H]inositol phosphates produced under gastrin or CCK-8 stimulation showed a 2-fold increase in [3H]Ins(1,4,5)P3 levels within 5 s with a concomitant increase in [3H]Ins(1,4)P2 content within 15 s. A low but significant rise in [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 cellular contents was also observed. No difference between gastrin- and CCK-8-induced inositol phosphates production could be shown. We can conclude that gastrin and CCK-8 display an identical profile of action, suggesting that they stimulate the acid secretory function of parietal cells through the same receptor site coupled to the Ins(1,4,5)P3 production.

Aminopyrine↗
PubMed1992

Structural characterization of the mesangial cell type IV collagenase and enhanced expression in a model of immune complex-mediated glomerulonephritis.

Secretion of glomerular cell-derived matrix metalloproteinases (MMPs) and their specific inhibitors, TIMP-1,2, may play an important role in the turnover of the glomerular extracellular matrix under basal and pathologic conditions. A 66-68 kd MMP secreted by cultured mesangial cells (MC) with activity against Type IV collagen and gelatin was purified and shown by amino-acid sequence analysis to be identical with a Type IV collagenase/gelatinase secreted by certain transformed tumor cell lines. The expression of the mesangial MMP in vivo was limited within the kidney to a small subset of the intrinsic glomerular mesangial cell population. After induction of acute anti-Thy 1.1 glomerulonephritis, there was a large increment in the number of Type IV collagenase-secreting MC, temporally coincident with the development of mesangial hypercellularity. The expression of the MMP inhibitor protein, TIMP-1, was not changed over this period. Ultrastructural studies localized the mesangial MMP to areas of evolving mesangiolysis and at sites of glomerular basement membrane disruption. Enhanced expression of the mesangial cell-derived Type IV collagenase may contribute to the evolution of glomerular injury in this model of immune complex-mediated glomerulonephritis or may be involved in the extensive matrix remodeling process that accompanies this form of glomerular injury.

Amino Acid Sequence↗
PubMed1991

Expression of matrix metalloproteinases and inhibitor by human trabecular meshwork.

Extracellular matrix (ECM) turnover and remodeling are initiated, at least in part, by the regulated secretion of members of a family of matrix metalloproteinases. Human and bovine trabecular mesh-work in culture secrete interstitial collagenase, both the 72- and the 92-kD forms of type IV collagenase (gelatinases) and stromelysin, and the tissue inhibitor of metalloproteinases (TIMP). These proteinases and TIMP were identified by immunoblotting western transfers from sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using several specific antiprotein and antipeptide polyclonal antibodies. Gelatinase and stromelysin enzymatic activities were also analyzed by substrate SDS-PAGE, in which proteinase substrates were polymerized into the gels before electrophoresis to allow subsequent activity assays. These matrix metalloproteinases and TIMP are secreted at low basal levels into trabecular culture medium; their secretion levels are increased several-fold by treatment of the cultures with the phorbol mitogen. 12-O-tetradecanoylphorbol-13-acetate (TPA). Characteristics of the trabecular matrix metalloproteinases and TIMP are similar to those secreted by numerous other tissues, including the retinal pigment epithelium. These proteinases may serve an important role in the maintenance and regulation of the trabecular extracellular matrix and, subsequently, of the aqueous humor outflow pathway in normal and glaucomatous eyes.

Animals↗
PubMed1983

Turnover and functions of glutathione studied with isolated hepatic and renal cells.

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.

Animals↗
PubMed1997

Current knowledge of HIV-1 reverse transcriptase mutations selected during nucleoside analogue therapy: the potential to use resistance data to guide clinical decisions.

Massive viral turnover and the high error rate of reverse transcriptase create the potential for drug-resistant viral variants to appear rapidly under the selective pressure of antiretroviral therapy. Loss of antiviral effect in treatment compliant persons is most commonly coincident with the appearance of viral mutants with reduced drug sensitivity. Thus, detection of viral resistance may represent an early marker of therapy failure. Similarly, substantial reduction in viral replication in the plasma compartment, to below quantification of a viral load assay, is associated with a sustained therapeutic response and delayed development of viral resistance. Information on patterns of resistance to and cross-resistance between antiretroviral agents are increasingly well characterized and represents an important consideration when deciding how to combine and/or sequence antiretrovirals to achieve optimal antiviral effects. When switching therapy, the change of several agents in the treatment regimen is currently recommended. The use of novel means of evaluating resistance, such as a genotypic probe, may guide clinicians in choosing an agent to which the patient's dominant viral quasispecies remains sensitive, potentially increasing the chances of achieving a therapeutic response. However, no studies using resistance to guide clinical decision making have yet been reported and available sequencing studies have focused largely on switching or adding therapies to patients who have received zidovudine monotherapy. Thus, no resistance driven treatment algorithm is currently available.

Anti-HIV Agents↗
PubMed1995

Purification, characterization, and investigation of the mechanism of aminoglycoside 3'-phosphotransferase type Ia.

Aminoglycoside 3'-phosphotransferases [APH(3')s] are the most common cause of bacterial high-level resistance to aminoglycoside antibiotics in clinical isolates. A one-step affinity chromatography was used to purify APH(3') type Ia. The kinetic parameters for turnover of seven aminoglycosides and the corresponding minimum inhibitory concentrations for a strain of Escherichia coli harboring APH-(3')-Ia were determined. The enzyme phosphorylates its substrates with kcat/Km values of 10(6)-10(8) M-1 s-1, including substrates such as amikacin and butirosin A which traditionally have been considered poor substrates for this enzyme. The optimal pH for the phosphotransferase activity was observed to be 7.0-7.5. The purified enzyme was found to be prone to dimerization in the absence of a reducing agent. Treatment of the enzyme with trypsin excised a 4 kDa fragment from the N-terminus which contained the amino acid residue Cys-10. The 27 kDa proteolyzed APH(3')-Ia did not dimerize, suggesting that Cys-10 was involved in dimerization via a disulfide bond. The phosphorylated kanamycin A was isolated, and the phosphorylation was confirmed to occur at the 3'-hydroxyl. Furthermore, both APH(3')-Ia and APH(3')-IIa were shown to phosphorylate water ("ATP hydrolase" activity) at a rate of ca. 10(4)-10(6)-fold slower (effect on kcat/Km) than that for the phosphoryl transfer to a typical aminoglycoside. The results of product-inhibition and alternative substrate diagnostics indicate an equilibrium-random mechanism for phosphorylation of aminoglycosides by APH(3')-Ia.

Adenosine Diphosphate↗
PubMed1990

Immunohistochemical distribution of type IV collagenase in normal, benign, and malignant breast tissue.

Production of type IV collagenase by tumor cells has been linked to their metastatic potential in several experimental models. A possible role for this enzyme in basement membrane type IV collagen turnover has also been suggested. Two recently developed affinity-purified, monospecific antibodies directed against the amino terminus (H1), or an internal active site domain (metal binding region [MBR]) of human type IV collagenase, were employed in the avidin-biotin-immunoperoxidase technique in formalin-fixed, paraffin-embedded breast tissue samples from 55 patients. Intense cytoplasmic immunostaining of myoepithelial cells was found in normal and hyperplastic tissue, and discontinuous staining was noted in intraductal carcinomas. Luminal epithelial cells were negative or weakly positive in large- or medium-sized ducts but reacted frequently in normal terminal ducts and hyperplastic lesions. Epithelial cells in intraductal carcinomas exhibited immunoreactivity in 20 of 23 cases. Invasive carcinomas were positive in 36 of 40 cases, and metastatic cells in lymph nodes stained in 10 of 12 cases. These results support a role for type IV collagenase in the basement membrane remodeling of normal breast. Our findings suggest that myoepithelial cells play a pivotal role in this enzymatic activity. The high percentage of positive cells in invasive carcinomas and the strong immunoreactivity of lymph node metastases support the role of the enzyme in tumor invasion and metastasis and suggest that tumor cells are the essential source of the enzyme in these processes.

Antibodies↗