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Y Singh

Publications and source records attributed to Y Singh.

At least 73 records · Page 4Linked to original sources
PubMed1994

The chymotrypsin-sensitive site, FFD315, in anthrax toxin protective antigen is required for translocation of lethal factor.

The protective antigen (PA) component of anthrax toxin contains two sites that are uniquely sensitive to proteolytic cleavage. Cleavage at the sequence RKKR167 by the cellular protease furin is absolutely required for toxicity, whereas cleavage by chymotrypsin or thermolysin at the sequence FFD315 inactivates the protein, apparently by blocking the ability of PA to translocate the catalytic moieties of the toxins, lethal factor (LF) and edema factor (EF), to the cytosol of eukaryotic cells. To specify the role of the chymotrypsin-sensitive site of PA in the translocation of LF, we altered residues 313-315. None of the mutations in this region interfered with the ability of PA to bind to its cellular receptor, be cleaved by cell surface furin, and bind LF. Substitution of Ala for Asp315 or for both Phe313 and Phe314 reduced the ability of PA to intoxicate cells in the presence of LF by 3- and 7-fold, respectively. Substitution of Phe313 by Cys greatly reduced the rate of LF translocation and delayed toxicity. The rate at which the Cys-substituted PA killed cells was increased significantly by blocking the sulfhydryl group with iodoacetamide, suggesting that this added Cys interacts with cellular proteins and slows translocation of LF. Deletion of the 2 Phe rendered PA completely non-toxic. This deleted PA protein lacked the ability shown by native PA to form oligomers on cells and in solution and to induce release of 86Rb from Chinese hamster ovary cells. These results suggest that the chymotrypsin-sensitive site in PA is required for membrane channel formation and translocation of LF into the cytosol. PA double mutants were constructed that cannot be cleaved at either the furin or chymotrypsin sites. These PA proteins were more stable in Bacillus anthracis culture supernatants and may therefore be useful as a replacement for PA in anthrax vaccines.

Amino Acid Sequence↗
PubMed1994

Expression and mutagenesis of recombinant cholera toxin A subunit.

ADP-ribosylating protein exotoxins from Vibrio cholerae (CT) and Escherichia coli (LT-I) share two short regions of sequence similarity with Bordetella pertussis toxin (PT). Previous studies have indicated that substitution of arginine for lysine 7 within the first region of CT drastically decreases ADP ribosyltransferase activity. We have more closely defined the role of other amino acids in this region by generating modified proteins in which arginine 7 was replaced with lysine (R7K), aspartate 9 was replaced with arginine (D9R), glycine was substituted for proline 12 (P12G), amino acids 6 to 13 were deleted (delta 613) or the C-terminal KDEL sequence was changed to NEDL. The modified proteins R7K, D9R and delta 613 exhibited undetectable ADP ribosyltransferase activity. Comparison of the tryptic digest of R7K with native CT suggested that changes in protein conformation may be responsible for the loss of ADP-ribosylation activity.

Adenosine Diphosphate Ribose↗
PubMed1994

Adaptation of a strain of Spirulina platensis to grow in cobalt- and iodine-enriched media.

Cobalt- and iodide-enriched (adapted, tolerant) strains of the protein-rich cyanobacterium, Spirulina platensis, were produced by repeated sub-culturing in increasing concentrations of the two trace elements. The strains enriched with cobalt and iodide showed higher uptake of these elements than the controls. The LD50 values for the parent and cobalt-adapted strains were 95 and 231 mumol l-1 CO2+, respectively. Likewise, the LD50 values for parent and iodide-adapted strains were 12 and 42 mmol l-1 I-. The carotenoid:chlorophyll a ratio of the parent strains increased after cobalt addition. The cobalt-adapted strain showed a much higher ratio than the cobalt-grown parent (sensitive) cells which remained unchanged after cobalt addition. Intracellular CO2+ uptake by the cells was concentration-dependent and followed Michaelis-Menten kinetics with saturation in uptake occurring in the parent and adapted strains at 126 and 189 mumol l-1 Co2+, respectively. At saturating concentrations, the maximum CO2+ uptake was 39.73 and 158.43 nmol CO2+ mg-1 protein, respectively for the parent and adapted strains. The adapted strain also showed greater cobalt adsorption. The Km of intracellular CO2+ uptake was lower in the case of adapted cells as compared with the parent, whereas Vmax showed an opposite trend. Thus, the adapted cells appear to be more efficient than the parent strain in intracellular uptake of cobalt. Differences between kinetic constants of both the strains suggest that the strains may be physiologically different. Likewise, iodide uptake was significantly higher in iodide-adapted cells than in controls.

Adaptation, Physiological↗
PubMed1993

Characterization of macrophage sensitivity and resistance to anthrax lethal toxin.

Anthrax lethal toxin, which consists of two proteins, protective antigen and lethal factor, is cytolytic for macrophages. Macrophages from different mouse strains were found to vary in their sensitivities to toxin. C3H mouse macrophages lysed by lethal factor concentrations of 0.001 micrograms/ml were 100,000 times more sensitive than those from resistant A/J mice. We analyzed various stages of the intoxication process to determine the basis for this resistance. Direct binding studies with radioiodinated protective antigen revealed that the affinity (Kd, approximately 0.5 nM) and number of receptors per cell (25,000 to 33,000) were the same in sensitive and resistant cells. Proteolytic activation of protective antigen by a cell surface protease and subsequent binding of lethal factor were also the same in both sensitive and resistant macrophages. Resistant A/J macrophages were not cross-resistant to other toxins and a virus which, like lethal toxin, require vesicular acidification for activity, implying that resistance is not due to a defect in vesicular acidification. When introduced into the cytosol by osmotic lysis of pinosomes, lethal factor in the absence of protective antigen was cytolytic for the sensitive macrophages while resistant cells were unaffected. Thus, lethal factor by itself possesses the toxic activity of lethal toxin. These results suggest that macrophage resistance is due to a defect at a stage occurring after toxin internalization. A/J macrophages may lack the putative lethal factor target in the cytosol or be defective in the further processing or activation of lethal factor in the cytosol or in endocytic vesicles.

Animals↗
PubMed1993

Is a long delay necessary before appendectomy after appendiceal mass formation? A preliminary report.

The standard treatment for an appendiceal mass is conservative therapy followed by appendectomy after 6 to 10 weeks. With the advent of antibiotics designed to prevent the growth of anaerobes, early appendectomy can now be carried out without complication. The authors studied 56 patients with appendiceal mass formation, 26 (group A) treated conventionally and 30 (group B) treated by early appendectomy. In group B, the infection rate was 17%, the mean operating time was 38.7 minutes, the mean hospital stay was 15 days and there was an early return to work. The corresponding parameters for group A were an infection rate of 8%, a mean operating time of 35.2 minutes, a hospital stay of 19.1 days and a late return to work. Furthermore, 15% of the patients in group A had a recurrent acute episode during the waiting period. Overall, early appendectomy appears to be a safe and cost-effective treatment for appendiceal mass formation.

Abscess↗
PubMed1992

Fusions of anthrax toxin lethal factor to the ADP-ribosylation domain of Pseudomonas exotoxin A are potent cytotoxins which are translocated to the cytosol of mammalian cells.

The lethal factor (LF) and edema factor (EF) components of anthrax toxin are toxic to animal cells only if internalized by interaction with the protective antigen (PA) component. PA binds to a cell surface receptor and is proteolytically cleaved to expose a binding site for LF and EF. To study how LF and EF are internalized and trafficked within cells, LF was fused to the translocation and ADP-ribosylation domains (domains II and III, respectively) of Pseudomonas exotoxin A. LF fusion proteins containing Pseudomonas exotoxin A domains II and III were less toxic than those containing only domain III. Fusion proteins with a functional endoplasmic reticulum retention sequence, REDLK, at the carboxyl terminus of domain III were less toxic than those with a nonfunctional sequence, LDER. The most potent fusion protein, FP33, had an EC50 = 2 pM on Chinese hamster ovary cells, exceeding that of native Pseudomonas exotoxin A (EC50 = 420 pM). Toxicity of all the fusion proteins required the presence of PA and was blocked by monensin. These data suggest that LF and LF fusion proteins are efficiently translocated from acidified endosomes directly to the cytosol without trafficking through other organelles, as is required for Pseudomonas exotoxin A. This system provides a potential vehicle for importing diverse proteins into the cytosol of mammalian cells.

ADP Ribose Transferases↗
PubMed1992

Species differences in membrane susceptibility to lipid peroxidation.

The susceptibility of liver microsomes to lipid peroxidation was evaluated in seven species: rat, rabbit, trout, mouse, pig, cow, and horse. Lipid peroxidation was measured as thiobarbituric acid reactive substances formed in the presence of either FeCl3-ADP/ascorbate or FeCl2/H2O2 initiating systems. For rat, rabbit, and trout microsomes, the order of susceptibility to peroxidation was rat greater than rabbit much greater than trout. The lack of peroxidation in trout microsomes could be explained by high microsomal vitamin E levels. Membrane fatty acid levels differed between species. Docosahexaenoic acid predominated in the trout, arachidonic acid in the rat, and linoleic acid in the rabbit. The contribution of individual fatty acids to lipid peroxidation reflected the degree of unsaturation with docosahexaenoic greater than arachidonic much much greater than linoleic. For all species except trout, the predicted susceptibility to peroxidation, based on the response of individual fatty acids, agreed well with directly measured microsomal peroxidation. With the exception of the trout, vitamin E content ranged from 0.083-0.311 nmol/mg microsomal protein between species, and low levels did not influence susceptibility to peroxidation. Trout microsomes peroxidized only after vitamin E depletion by prolonged incubation. The data indicate that below a vitamin E threshold, species differences in membrane susceptibility to peroxidation can be reasonably predicted based only on content of individual peroxidizable fatty acids.

Animals↗
PubMed1992

Methotrexate: clinical and immunological effects in refractory rheumatoid arthritis.

Thirty five patients with refractory rheumatoid arthritis were given 7.5 mg of methotrexate (Mtx) every week. Eleven patients had to discontinue treatment either because of adverse effects or unresponsiveness. Twenty four patients showed clinical response and significant improvement in ESR and they continued Mtx for a mean of 25.24 months. Seven patients achieved clinical remission as defined by ARA criteria. Immunological parameeters including IgG, IgM, IgA, lymphocyte subsets (CD3+, CD4+, CD8+ and B), C3 and C4 however, did not show any change during this treatment in any of the groups upto 6 months. There was a significant fall in the erythrocyte sedimentation rate (ESR), c-reactive protein (CRP) and rheumatoid factor (RF) levels in responders only.

Administration, Oral↗
PubMed1991

Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis.

Linker insertion mutagenesis was employed to create structural disruptions of the lethal factor (LF) protein of anthrax toxin to map functional domains. A dodecameric linker was inserted at 17 blunt end restriction enzyme sites throughout the gene. Paired MluI restriction sites within the linker allowed the inserts to be reduced from four to two amino acids. Shuttle vectors containing the mutated genes were transformed into the avirulent Bacillus anthracis UM23C1-1 for expression and secretion of the gene products. Mutations at five sites in the central one-third of the sequence made the protein unstable, and purified protein could not be obtained. Mutated LF proteins with insertions at the other sites were purified and assessed for toxic activity in a macrophage lysis assay and for their ability to bind to the protective antigen (PA) component of anthrax toxin, the receptor binding moiety. Most insertions located in the NH2-terminal one-third of the LF protein eliminated both toxicity and binding to PA, while all four insertions in the COOH-terminal one-third of the protein eliminated toxicity without affecting binding to PA. These data support the hypothesis that the NH2-terminal domain contains the structures required for binding to PA and the COOH-terminal domain contains the putative catalytic domain of LF.

Antigens, Bacterial↗
PubMed1991

The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity.

Anthrax toxin consists of three separate proteins produced by Bacillus anthracis: protective antigen (PA), lethal factor (LF), and edema factor (EF). Previous work showed that the process by which these proteins damage eukaryotic cells begins with binding of PA (83 kDa) to cell surface receptors. PA is then cleaved by a cell surface protease so as to expose a high-affinity binding site for LF or EF on the COOH-terminal, receptor-bound, 63-kilodalton fragment. In this report we more closely define a region of PA involved in receptor binding. The gene encoding PA was mutagenized so as to delete 3, 5, 7, 12, or 14 amino acids from the carboxyl terminus of the protein, and the truncated PA variants were purified from Bacillus subtilis or Escherichia coli. Deletion of 3, 5, or 7 amino acids reduced the binding of PA to cells and the subsequent toxicity of the PA.LF complex to J774A.1 cells and also the ability to cause EF binding to cells. Deletion of 12 or 14 amino acids completely eliminated all these activities. These results show that the carboxy terminus comprises or is part of the receptor-binding domain of PA.

Amino Acid Sequence↗
PubMed1991

Renal metallothionein and platinum levels in diabetic and nondiabetic rats injected with cisplatin.

This study was designed to investigate the relationship between the attenuation of cisplatin-induced nephrotoxicity in experimental diabetes and the increased level of renal metallothionein (MT) reported to occur in this condition. Two groups of male Sprague-Dawley rats were used: 42-day streptozotocin diabetics and age-matched nondiabetics. Half of each group was injected with a nephrotoxic dose of cisplatin (5 mg/kg, ip) and half with vehicle. Four hours after injection, renal MT and platinum (Pt) content were quantified. Mean renal MT concentration in vehicle-injected diabetics was about triple that found in nondiabetics. Comparison of renal MT concentrations in cisplatin-injected diabetics and nondiabetics with their vehicle-injected counterparts suggested an inducing effect of the drug. In contrast to the marked elevations of MT in diabetic kidney, mean renal Pt concentration in the cisplatin-injected diabetic group was only about one-fourth that of the nondiabetic group. No difference was evident in the intracellular distribution Pt between cytosolic and particulate fractions from diabetic and nondiabetic kidneys. It was concluded that: (i) Sequestration of Pt by MT cannot account for the resistance of diabetic kidney to cisplatin toxicity. (ii) Rather, the resistance is due to a significant decrease in renal uptake/retention of cisplatin or derivatives during the critical first few hours after injection.

Animals↗