The functional interplay of low molecular weight thiols in Mycobacterium tuberculosis.

BACKGROUND: Three low molecular weight thiols are synthesized by Mycobacterium tuberculosis (M.tb), namely ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC). They are able to counteract reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In addition, the production of ERG is elevated in the MSH-deficient M.tb mutant, while the production of MSH is elevated in the ERG-deficient mutants. Furthermore, the production of GGC is elevated in the MSH-deficient mutant and the ERG-deficient mutants. The propensity of one thiol to be elevated in the absence of the other prompted further investigations into their interplay in M.tb. METHODS: To achieve that, we generated two M.tb mutants that are unable to produce ERG nor MSH but are able to produce a moderate (ΔegtD-mshA) or significantly high (ΔegtB-mshA) amount of GGC relative to the wild-type strain. In addition, we generated an M.tb mutant that is unable to produce GGC nor MSH but is able to produce a significantly low level of ERG (ΔegtA-mshA) relative to the wild-type strain. The susceptibilities of these mutants to various in vitro and ex vivo stress conditions were investigated and compared. RESULTS: The ΔegtA-mshA mutant was the most susceptible to cellular stress relative to its parent single mutant strains (ΔegtA and ∆mshA) and the other double mutants. In addition, it displayed a growth-defect in vitro, in mouse and human macrophages suggesting; that the complete inhibition of ERG, MSH and GGC biosynthesis is deleterious for the growth of M.tb. CONCLUSIONS: This study indicates that ERG, MSH and GGC are able to compensate for each other to maximize the protection and ensure the fitness of M.tb. This study therefore suggests that the most effective strategy to target thiol biosynthesis for anti-tuberculosis drug development would be the simultaneous inhibition of the biosynthesis of ERG, MSH and GGC.

Impact of human serum albumin on oritavancin in vitro activity against Staphylococcus aureus.

Human serum albumin (HSA) did not affect oritavancin MICs against non-vancomycin-intermediate Staphylococcus aureus (non-VISA) strains. In time-kill assays, oritavancin bactericidal activity in the presence of HSA was significantly more rapid than comparators against non-VISA strains. HSA increased oritavancin MICs by 4-fold for VISA strains, reflective of reduced oritavancin activity in time-kill assays with HSA.

Antagonism of phosphoramidon-induced antinociception in mice by mu- but not kappa-opioid receptor blockers.

Intracerebroventricular (i.c.v.) administration of the neutral endopeptidase 24.11-inhibitor phosphoramidon evoked a dose-dependent antinociceptive effect in the mouse acetic acid abdominal constriction test. The present study was conducted to identify the opioid receptor subtype(s) that mediate phosphoramidon antinociception in this paradigm. Mice were pretreated with different opioid antagonists prior to being challenged with phosphoramidon, i.c.v., the mu-opioid agonist sufentanil, s.c., or the kappa-opioid agonist U-50,488H, s.c. Naltrexone significantly attenuated phosphoramidon-induced antinociception at an i.c.v. dose that also blocked both sufentanil and U-50,488H. The mu-opioid antagonist beta-funaltrexamine (beta-FNA) blocked phosphoramidon and sufentanil at an i.c.v. dose that did not block U-50,488H. The kappa-opioid antagonist nor-binaltorphimine (nor-BNI) produced dose-related effects. A low dose (10 microg) of nor-BNI had no effect on either phosphoramidon or sufentanil but did reduce U-50,488H antinociception. A higher dose (30 microg) of nor-BNI blocked phosphoramidon, sufentanil, and U-50,488H, suggesting a loss of kappa-opioid receptor selectivity at this dose. These findings suggest that mu- but not kappa-opioid receptors mediate phosphoramidon-induced antinociception in the abdominal constriction test.

The mshA gene encoding the glycosyltransferase of mycothiol biosynthesis is essential in Mycobacterium tuberculosis Erdman.

Mycothiol is the major low-molecular-weight thiol found in actinomycetes, including Mycobacterium tuberculosis, and has important antioxidant and detoxification functions. Gene disruption studies have shown that mycothiol is essential for the growth of M. tuberculosis. Because of mycothiol's unique characteristics, inhibitors directed against mycothiol biosynthesis have potential as drugs against M. tuberculosis. Four genes have been identified in mycobacteria that are involved in the biosynthesis of mycothiol. Two genes, mshB and mshD, are not essential for growth of M. tuberculosis. Mutants in these genes produce significant amounts of mycothiol or closely related thiol compounds. A targeted gene disruption in the mshC gene is lethal for M. tuberculosis, indicating that MshC is essential for growth. The remaining gene, mshA, encodes for a glycosyltransferase. In the present study, we attempted to produce a directed knock-out of the mshA gene in M. tuberculosis Erdman but found that this was only possible when a second copy of mshA was first incorporated into the chromosome. Bacteria with only a single copy of mshA that grew after mutagenesis produced normal levels of mycothiol. We therefore conclude that the mshA gene, like the mshC gene, is essential for the growth of M. tuberculosis.

Constitutive activation of G-proteins by polycystin-1 is antagonized by polycystin-2.

Polycystin-1 (PC1), a 4,303-amino acid integral membrane protein of unknown function, interacts with polycystin-2 (PC2), a 968-amino acid alpha-type channel subunit. Mutations in their respective genes cause autosomal dominant polycystic kidney disease. Using a novel heterologous expression system and Ca(2+) and K(+) channels as functional biosensors, we found that full-length PC1 functioned as a constitutive activator of G(i/o)-type but not G(q)-type G-proteins and modulated the activity of Ca(2+) and K(+) channels via the release of Gbetagamma subunits. PC1 lacking the N-terminal 1811 residues replicated the effects of full-length PC1. These effects were independent of regulators of G-protein signaling proteins and were lost in PC1 mutants lacking a putative G-protein binding site. Co-expression with full-length PC2, but not a C-terminal truncation mutant, abrogated the effects of PC1. Our data provide the first experimental evidence that full-length PC1 acts as an untraditional G-protein-coupled receptor, activity of which is physically regulated by PC2. Thus, our study strongly suggests that mutations in PC1 or PC2 that distort the polycystin complex would initiate abnormal G-protein signaling in autosomal dominant polycystic kidney disease.

Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region.

Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21-26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca(2+) influx into cytoplasm. Mutations that prevented adenylate cyclase inhibition and Ca(2+) influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr(26); this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp(25), a part of the consensus sulfation site. The expressed CCR2B was found to be N:-glycosylated, as N:-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational modifications probably have significant biological functions.

Involvement of substance P in neutral endopeptidase modulation of carotid body sensory responses to hypoxia.

Previously, we showed that carotid bodies express neutral endopeptidase (NEP)-like enzyme activity and that phosphoramidon, a potent inhibitor of NEP, potentiates the chemosensory response of the carotid body to hypoxia in vivo. NEP has been shown to hydrolyze methionine enkephalin (Met-Enk) and substance P (SP) in neuronal tissues. The purpose of the present study is to determine whether NEP hydrolyzes Met-Enk and SP in the carotid body and if so whether these peptides contribute to phosphoramidon-induced potentiation of the sensory response to hypoxia. Experiments were performed on carotid bodies excised from anesthetized adult cats (n = 72 carotid bodies). The hydrolysis of Met-Enk and SP was analyzed by HPLC. The results showed that both SP and Met-Enk were hydrolyzed by the carotid body, but the rate of Met-Enk hydrolysis was approximately fourfold higher than that of SP. Phosphoramidon (400 microM) markedly inhibited SP hydrolysis ( approximately 90%) but had only a marginal effect on Met-Enk hydrolysis ( approximately 15% inhibition). Hypoxia (PO(2), 68 +/- 6 Torr) as well as exogenous administration of SP (10 and 20 nmol) increased the sensory discharge of the carotid body in vitro. Sensory responses to hypoxia and SP (10 nmol) were potentiated by approximately 80 and approximately 275%, respectively (P < 0.01), in the presence of phosphoramidon. SP-receptor antagonists Spantide (peptidyl) and CP-96345 (nonpeptidyl) either abolished or markedly attenuated the phosphoramidon-induced potentiation of the sensory response of the carotid body to hypoxia as well as to SP. These results demonstrate that SP is a preferred substrate for NEP in the carotid body and that SP is involved in the potentiation of the hypoxic response of the carotid body by phosphoramidon.

Inhalation of phosphoramidon, a neutral endopeptidase inhibitor, induces cough in awake guinea-pigs.

In the present study, we explored whether or not a neutral endopeptidase inhibitor provokes cough in awake guinea-pigs. Inhalation of phosphoramidon at a concentration of 10(-6) M did not cause cough, but increasing the concentration to 10(-5) M caused cough with a latency of about 10 to 12 min. Inhalation of enalapril, an angiotensin-converting enzyme inhibitor, did not cause cough, even at high concentrations of 10(-5) M and 10(-4) M. Pretreatment with phosphoramidon (10(-5) M) significantly increased the number of coughs caused by substance P and capsaicin. Capsaicin-induced coughs were more easily produced in bronchitic guinea-pigs than in normal guinea-pigs. However, there was no significant difference in the number of phosphoramidon-induced coughs between normal and bronchitic guinea-pigs. Phosphoramidon-induced coughs were significantly depressed by codeine (20 mg/kg, p.o.) and CP96345 (2 mg/kg, i.v.). The present results provide new evidence for the proposed idea that neutral endopeptidase may regulate the occurrence of cough.

A multicentre study: Staphylococcus and Enterococcus susceptibility to antibiotics.

A multicentre study to evaluate the susceptibility of Gram-positive cocci isolated from clinical samples, was performed by six centres working in different areas of Italy. We examined 4,544 strains of Staphylococcus aureus, 4,381 strains of coagulase-negative staphylococci and 2,478 strains of enterococci. The following antibiotics were tested: penicillin G, ampicillin, amoxicillin, piperacillin, imipenem, oxacillin, ofloxacin, pefloxacin, ciprofloxacin, gentamicin, tobramycin, amikacin, netilmicin, rifampicin, clindamycin, tetracycline, cotrimoxazole, erythromycin, chloramphenicol, vancomycin and teicoplanin. Oxacillin-susceptible staphylococci confirmed their susceptibility to many other antimicrobial agents while oxacillin-resistant strains confirmed their multiple and frequent resistance to antibiotics. Resistance to oxacillin, cotrimoxazole and chloramphenicol was more frequent in coagulase-negative staphylococci than in Staphylococcus aureus. Aminoglycosides, rifampicin and quinolones were more active against coagulase-negative staphylococci than against Staphylococcus aureus. Enterococci were susceptible to penicillins and imipenem, and moderately susceptible to ciprofloxacin. Susceptibility of 70-79% was observed with high levels of aminoglycosides. Excellent results against staphylococci and enterococci were observed with vancomycin and teicoplanin.

In vitro suppression of fibroblast growth inhibitory lymphokine production by asbestos.

Human peripheral blood mononuclear leucocytes (PBML) stimulated with concanavalin A (Con A) or phytohaemagglutinin (PHA) produced a soluble factor which inhibits lung fibroblast DNA synthesis and growth. Lymphocyte enriched preparations produced significant growth inhibitory activity in the presence of PHA whereas media from adherent mononuclear cells incubated in the presence of the mitogen did not contain similar activity. This fibroblast growth inhibitory factor (FGIF) was non-dialysable, heat stable and resistant to pH 5. FGIF was also resistant to treatment with chymotrypsin and phosphodiesterase but partially sensitive to treatment with trypsin. Interestingly, there was significant suppression of FGIF production by PBML cultured with PHA in the presence of low concentrations of chrysotile asbestos (5-25 micrograms/ml). In this regard, asbestos (25 micrograms/ml) was not cytotoxic for lymphocytes but had a damaging effect on monocytes as evidenced by the release of lactate dehydrogenase (LDH) a cytoplasmic enzyme, in their culture media. These findings indicate that stimulated lymphocytes have the ability to inhibit fibroblast proliferation by releasing FGIF and that asbestos interfere with this process. Thus, while FGIF may regulate the extent of connective tissue proliferation during normal repair process, suppression of its production by asbestos may contribute to excessive fibroblast accumulation and fibrosis.