Molybdate reduction by Escherichia coli K-12 and its chl mutants.

During anaerobic growth, Escherichia coli can reduce phosphomolybdate. The reduction can also be carried out by washed cells suspended in buffer at pH 5.7. Phosphate, molybdate, glucose, cells, and anaerobic conditions are required. Reduction is inhibited by 200 microM chromate, 290 microM nitrite, 10 mM tungstate, or 20 mM cysteine. Wild-type (chl+) cells are inhibited by addition of 200 microM nitrate, but chlA, chlB, and chlE mutants are not. The inhibition of chl+ cells results from reduction of nitrate to nitrite. This nitrate reduction is not catalyzed by nitrate reductase. Wild-type cells are more sensitive than chl mutants to inhibition by nitrite and cysteine but more resistant to chromate. Pregrowth of chlD cells in 1 mM Na2MoO4 increases their sensitivity to nitrite and cysteine, and pregrowth of chl+ cells in 1 mM Na2MoO4 increases their resistance to these agents. Assays of biotin sulfoxide reductase show that the tightness of the chlD block depends on growth conditions; chlD cells grown aerobically in tryptone broth make about 50% as much active enzyme as chl+ cells, whereas chlD cells grown anaerobically with tryptone plus glucose make less than 10%. The effect of anaerobic pregrowth on the inhibition of molybdate reduction by added nitrate indicates that in vivo nitrate reduction responds to growth conditions in the same manner as biotin sulfoxide reductase does.     

Helicobacter pylori erradication and its relation to antibiotic resistance and CYP2C19 status]

OBJECTIVE: to assess the efficacy of rabeprazole (RPZ), amoxicillin (Am), and clarithromycin (Cla) (7 vs. 14 days) in the eradication of H. pylori, and to determine the effect of strain-specific antibiotic resistance and host CYP2C19 status. MATERIAL AND METHODS: first, we determined the CYP2C19 status of 100 healthy subjects to establish a sample size for the clinical trial. Then, 59 H. pylori-infected patients were randomized to receive RPZ (20 mg daily) plus Cla (500 mg b.d.) and Am (1,000 mg b.d.) for 7 vs. 14 days. The MIC for Am and Cla were determined using the agar dilution method. The CYP2C19 genotype was determined by the PCR-RFLP method. RESULTS: In the per-protocol analysis (PP) eradication rates were 89.7 and 72% for the 7- and 14-day groups (p = 0.159). In the intention to-treat analysis (ITT) eradication rates were 86.7 and 62.1% in the 7- and 14-day groups, respectively (p = 0.06).None of the strains was resistant to Am, and 4 strains were resistant to Cla: 3 (11.1%) in the 14-day group and 1 (4%) in the 7-day group. Neither strain-specific antibiotic resistance nor host CYP2C19 status influenced eradication rates. CONCLUSIONS: both 7- and 14-day therapies were effective for H. pylori eradication. Strain resistance and CYP2C19 status do not seem to influence eradication rates in the studied population.     

In vitro stimulation of placental progesterone production by 19-nortestosterone and C19 steroids in early human pregnancy.

To explore the regulatory mechanism at the critical period of the luteal-placental shift, the effects of various steroids and peptides on the production of progesterone by placental explants at 7-10 weeks were studied. Androstenedione increased progesterone production 3-fold at a concentration of 1 mumol and more than 20-fold at 18 mumol. 19-Nortestosterone (1-18 mumol) stimulated progesterone production 10- to 100-fold. 5 alpha-Androstane-3 beta,17 beta diol (1-18 mumol) stimulated progesterone production about 2- to 5-fold while its 3 alpha isomer (1-6 mumol) increased it 2-fold. Estrone, estradiol, and estriol up to a concentration of 30 mumol had no effect. Dehydroepiandrosterone sulfate (to 36 mumol), androst-5-ene-3 beta,17 beta diol (1-6 mumol), 5 beta-androstane-3 alpha,17 beta diol (1-6 mumol), and dihydrotestosterone (1-12 mumol) had no effect. Cortisol and dexamethasone (up to 12 mumol), hCG (20,000 IU/L), GnRH (4 mumol), and ACTH 1-24 (20 mumol) also had no effect. Thus, of all the compounds tested, only 19-nortestosterone and, to a lesser extent, androstenedione, 5 alpha-androstane-3 beta,17 beta diol, and 5 alpha-androstane-3 alpha,17 beta diol stimulated progesterone production in early pregnancy; at term, only 5 alpha-androstane-3 beta,17 beta diol was stimulatory. 19-Nortestosterone was found to be less efficiently aromatized compared to other androgens; since it is also known to be present in blood from pregnant women and thought to be made in the placenta, the stimulation observed may be a paracrine effect. These observations suggest that C18 and C19 steroids may be important in the regulation of progesterone synthesis by the human placenta in early pregnancy.     

Cell-free synthesis of fish preproinsulin, and processing by heterologous mammalian microsomal membranes.

Poly(A)-containing mRNA isolated from the islets of Langerhans obtained from two species of fish, angler fish (Lophius americanus) and sea raven (Hemitripterus americanus), stimulated protein synthesis 16-fold in a wheat germ cell-free system. Characterization of the translation products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed a major polypeptide weighing 11,500 daltons that was specifically precipitated by an antibody against angler fish insulin. Partial sequence analysis of the amino terminal revealed that this polypeptide is preproinsulin, in which the amino terminus of proinsulin is preceded by either 23 (angler fish) or 25 (sea raven) amino acid residues. Translation of fish islet mRNA in a wheat germ cell-free system in the presence of dog pancreas microsomal membranes led to the correct cleavage of the nascent preproinsulin, resulting in the synthesis of authentic fish proinsulin, as verified by partial sequence analysis. Moreover, the synthesized fish proinsulin was segregated, presumably into the luminal space of the dog pancreas microsomal vesicles, because it was found to be resistant to proteolysis by added trypsin and chymotrypsin. Our data thus suggest that the mechanisms and information for the transfer of secretory proteins across the microsomal membrane are highly conserved during evolution.     

Degradation of penicillin G to phenylacetylglycine by D-alanine carboxypeptidase from Bacillus stearothermophilus.

D-Alanine carboxypeptidase from Bacillus stearothermophilus is a membrane-bound enzyme which is inhibited by covalent interaction with penicillin G. The penicilloyl enzyme spontaneously reactivates and simultaneously releases a penicillin G degradation product; 0.2 mumol of the latter was isolated after incubation of 4.2 mumol of [8-14C]penicillin G with 10 g of membrane protein. It was identified as phenylacetylglycine by chromatographic techniques, infrared spectroscopy, and mass spectrometry. A mechanism for the degradation is proposed in which the remaining part of penicillin G would be released as 5,5-dimethyl-delta2-thiazoline-4-carboxylic acid. The implications of this finding are discussed.     

Frequency of infection by hepatitis B virus and its surface mutants in a northern Indian population.

INTRODUCTION: The reported prevalence of hepatitis B virus (HBV) infection in the Indian general population varies from 2% to 11%. Epidemiological studies conducted so far have selection biases, since these included populations of defined age group, gender, social class, high-risk group, etc. The present study was designed to look for the molecular epidemiology of HBV infection in the rural and urban general populations in India. METHODS: Sera obtained from healthy volunteers during college and social service camps from parts of northern India were tested for HBsAg and anti-HBc using enzyme immunoassays and for HBV DNA using polymerase chain reaction and Southern blot hybridization. The amplification products were cloned and sequenced, and nucleotide and deduced amino acid sequences of the surface and polymerase genes were analyzed for mutations. RESULTS: Of the 730 subjects (rural 543, urban 187), 15 (2.1%) tested positive for HBsAg and 143 (19.5%) for anti-HBc; 10 were positive for both. The overall HBV exposure rate in the population was 20.3% (148/730). The HBsAg carrier rate was similar in the urban and rural populations (1.5% and 2.3%; p=ns), and anti-HBc positivity was lower in the urban population (8.5% vs. 23.3%; p<0.01). History of parenteral interventions or blood transfusion was associated with markers of exposure to HBV (10.2% vs. 4.6%; p=0.01). Among the 220 representative samples tested for HBV DNA, 14 (6.4%) were positive; of these, only four were positive for HBsAg or anti-HBc. Sequencing of a 388-nt segment of the S-gene from three individuals (two adw and one ayw subtype) revealed four mutations. Two and three of these led to amino acid changes in the HBV surface and polymerase genes, respectively; alterations in known cytotoxic T cell epitopes of HBV surface and polymerase proteins were observed in one individual each. None had the G587A mutation, which is known to be associated with loss of the 'a' determinant of HBsAg. CONCLUSION: Our study shows a high frequency of exposure to HBV infection in the Indian general population; a proportion of HBV infected persons were detectable only by molecular methods. The positivity rate was higher in the rural population.     

Palmitoylation of bovine opsin and its cysteine mutants in COS cells.

Previously, bovine rhodopsin has been shown to be palmitoylated at cysteine residues 322 and 323. Here we report on palmitoylation of bovine opsin in COS-1 cells following expression of the synthetic wild-type opsin gene and several of its cysteine mutants in the presence of [3H]palmitic acid. Two moles of palmitic acid are introduced per wild-type opsin molecule in thioester linkages. Palmitoylation is abolished when both Cys-322 and Cys-323 are replaced by serine residues. Replacement of Cys-322 by serine prevents palmitoylation at Cys-323, whereas replacement of the latter with serine allows palmitoylation at Cys-322. Opsin mutants that evidently do not contain a Cys-110/Cys-187 disulfide bond and presumably remain in the endoplasmic reticulum are not palmitoylated. Replacement of Cys-140 or Cys-185 reduces the extent of palmitoylation of the opsin. Lack of palmitoylation at Cys-322 and/or Cys-323 does not affect 11-cis-retinal binding, absorption maximum or extinction coefficient of the chromophore, the bleaching behavior of the chromophore, or the light-dependent binding and activation of transducin. Mutants containing serine substitutions at Cys-140 or Cys-323 showed reduced light-dependent phosphorylation by rhodopsin kinase.     

Infective endocarditis caused by Streptococcus bovis resistant to the lethal effect of penicillin G.

Penicillin G alone is generally recommended for the treatment of infective endocarditis caused by Streptococcus bovis because clinical isolates of S bovis are represented as being uniformly and markedly susceptible to penicillin G. However, two strains of S bovis recovered from two patients with bacterial endocarditis were resistant to the lethal effect of penicillin G. Combination therapy, cefazolin sodium and gentamicin sulfate in patient 1 and penicillin G and gentamicin in patient 2, was necessary; synergy, as manifested by lethal activity against the infecting strains, was demonstrated in the laboratory. We stress the need to determine the minimal lethal concentration of penicillin G for clinical isolates of S bovis. Until such information is available, particularly in life-threatening infections, combination drug therapy, consisting of an aminocyclitol added to a beta-lactam antimicrobic, should be used.     

Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates.

Several lines of evidence suggest that hepatitis C virus (HCV) core protein may modulate cellular transduction signals and alter lipid metabolism. We have investigated the binding of HCV core protein to cellular proteins by combining 2 yeast hybrid, confocal, and surface plasmon resonance assays. Our results show the direct binding of the viral protein to apolipoprotein AII (apoAII) and map the interaction domain to the C-terminal of HCV core protein. To investigate the biological relevance of the interaction between HCV core and lipid metabolism, we took advantage of the well-established increase in apoAII expression caused by fibrates in HepG2 cells. After fenofibric acid treatment, we show a parallel increase in apoAII and core protein secretion, this effect being abolished by brefeldin A. Our study identifies apoAII as one of the cellular targets for HCV core protein. We also show that the intervention of fenofibric acid in cellular lipid metabolism directly affects the expression pattern of HCV core protein.     

Phosphorylation by protein kinase C of a 20-kDa cytoskeletal polypeptide enhances its susceptibility to digestion by calpain.

Incubation of the cytoskeletal fraction from human neutrophils with the proteolytically activated form of protein kinase C results in the phosphorylation of several components, including a 20-kDa polypeptide, probably consisting of myosin light chains. The 20-kDa polypeptide is also specifically phosphorylated by activated protein kinase C in a solubilized 20-kDa/80-kDa complex that was obtained after sonication of the insoluble cytoskeletal fraction. Phosphorylation of this polypeptide, in either the insoluble cytoskeletal fraction or the soluble 20-kDa/80-kDa complex, greatly enhances its susceptibility to digestion by the Ca2+-requiring proteinase (calpain, EC 3.4.22.17) of human neutrophils. Thus, signals that activate calpain by mobilizing intracellular calcium would lead to proteolytic activation of protein kinase C, phosphorylation of cytoskeletal proteins, and remodeling of the cytoskeleton by proteolysis of at least one cytoskeletal component.     

Structural and dynamic aspects related to oligomerization of apo SOD1 and its mutants

The structural and dynamical properties of the metal-free form of WT human superoxide dismutase 1 (SOD1) and its familial amyotrophic lateral sclerosis (fALS)-related mutants, T54R and I113T, were characterized both in solution, through NMR, and in the crystal, through X-ray diffraction. We found that all 3 X-ray structures show significant structural disorder in 2 loop regions that are, at variance, well defined in the fully-metalated structures. Interestingly, the apo state crystallizes only at low temperatures, whereas all 3 proteins in the metalated form crystallize at any temperature, suggesting that crystallization selects one of the most stable conformations among the manifold adopted by the apo form in solution. Indeed, NMR experiments show that the protein in solution is highly disordered, sampling a large range of conformations. The large conformational variability of the apo state allows the free reduced cysteine Cys-6 to become highly solvent accessible in solution, whereas it is essentially buried in the metalated state and the crystal structures. Such solvent accessibility, together with that of Cys-111, accounts for the tendency to oligomerization of the metal-free state. The present results suggest that the investigation of the solution state coupled with that of the crystal state can provide major insights into SOD1 pathway toward oligomerization in relation to fALS.