Biomedical subjects
Publications and source records attributed to B K De.
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A cloned cDNA copy of the haemagglutinin (HA) gene of A/Chicken/Scotland/59 (H5N1) influenza virus has been expressed in vaccinia virus. This pox virus is poorly infectious or non-infectious for chickens. However, immunization of chickens with lysates of cell cultures infected with the recombinant vaccinia virus, that had been emulsified with adjuvant and which contained an estimated 0.5 microgram influenza HA, elicited a substantial neutralizing antibody response to influenza virus. Challenges of immunized and non-immunized adult chickens with virulent A/Chicken/Scotland/59 influenza virus showed that the immunized animals were highly protected while the non-immunized controls died. Immunized birds were also protected against infection with the recent virulent H5 avian influenza virus, A/Chicken/Pennsylvania/83 (H5N2).
We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, can serve as a substrate for the epidermal growth factor (EGF) receptor/tyrosine kinase (Fava, R.A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). We now report the detection of an antigenically related 35-kDa protein in a number, but not all, of rat, pig, and human tissues. These antigenically related proteins also can serve as substrates for the EGF receptor/kinase in the presence of Ca2+. All of these proteins share the property of reversible, Ca2+-dependent binding to the particulate fraction (presumably membranes) of cell homogenates. We have isolated the 35-kDa substrate from porcine lung and have demonstrated that it is a Ca2+-binding protein. The amino-terminal sequence and the site of tyrosine phosphorylation therein have been determined. The positions of the acidic amino acid residues amino-terminal to the tyrosine phosphorylation site bear a distinct resemblance to the sequence in the homologous region of a number of other substrates for tyrosine kinases. Based on available data, the 35-kDa protein clearly differs from the protein I complex derived from intestinal mucosa and thought to be related to the proteins isolated herein (Gerke, V., and Weber, K. (1985) J. Biol. Chem. 260, 1688-1695). Finally, we report a striking sequence homology between the porcine 35-kDa described herein and human lipocortin, a phospholipase A2 inhibitor.
A cDNA clone encoding the nonstructural, 30-kDa protein of the common (U1) strain of tobacco mosaic virus (TMV) was isolated and characterized. cDNA clones representing the intact gene as well as deletions from the 5' end of the gene were subcloned into SP6 vectors. Capped RNAs produced by in vitro transcription reactions were translated in a wheat germ cell-free system. The resultant proteins were compared to proteins obtained from the in vitro translation of intermediate length (I2) rods of TMV. Transcripts of the cDNA clones encoded polypeptides of 30, 28, or 18 kDa that were immunoprecipitated by antibody prepared against a synthetic peptide representing the carboxy terminus of the 30-kDa protein. cDNA clones containing the intact 30-kDa sequence coded for 30-kDa polypeptides while clones lacking the 30-kDa initiation codon produced 28-kDa polypeptides. Surprisingly, translation of a transcript from a cDNA clone containing the 30-kDa gene plus 390 nucleotides 5' of the initiator AUG yielded a polypeptide with an approximate molecular mass of 18 kDa. The results indicate that an intact and functional 30-kDa protein gene has been cloned. The significance of these results, with respect to determining the function of the 30-kDa protein, is discussed.
Electrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [3H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanide m-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Non-electrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (delta psi). The data could be explained by a delta psi threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K+, influx of Na+ and reduction of delta psi preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of delta psi induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP acts via specific sites on the surface of transformed cells.
External ATP causes a rapid increase in passive permeability to nucleotides and phosphate esters in transformed cell lines, such as 3T6 mouse fibroblasts. However, untransformed lines, such as 3T3, do not show a similar sensitivity to external ATP. Ca2+ inhibits permeabilization, but only at concentrations approaching those of external ATP. In contrast, La3+ and Tb3+ inhibit ATP-dependent permeabilization at one-fifth the concentration of external ATP. Considering reports that lanthanides can substitute for calcium ion in many enzymatic reactions, often with a higher affinity, it would appear that Ca2+ plays a specific role in the maintenance of a passive membrane permeability barrier and in opposing the effects of external ATP. Other data suggest a regulatory role for the Ca2+-calmodulin complex in the permeabilization process. Trifluoperazine, chlorpromazine and W-7, compounds which inhibit cellular functions dependent on the Ca2+-calmodulin complex, are able to enhance the effect of external ATP. Thus, a dramatic stimulation of nucleotide permeability occurs with concentrations of external ATP and inhibitor that are ineffective when added alone. Calmodulin antagonists and low concentrations of external ATP increased membrane permeability to Na+ and K+ as was previously shown for permeabilization with ATP alone. Earlier studies have shown that energy inhibitors which reduce intracellular ATP levels greatly increase the sensitivity of transformed cells to external ATP. However, the Ca2+-calmodulin antagonists used in the present study exert their effects at concentrations which do not alter intracellular ATP levels.
Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [gamma-32P]ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [gamma-32P]ATP resulted in substantial formation of 32P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the alpha-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.
Transformed mouse fibroblasts, such as 3T6, exhibit an increase in plasma membrane permeability to nucleotides and other normally impermeant molecules when incubated with external ATP in an alkaline medium low in divalent cations. Increased nucleotide permeability, induced by external ATP, occurs after a 3- to 5-min lag period. Prior to this event, there is a dramatic Na+ influx and K+ efflux, a significant reduction in the levels of intracellular ATP and organic phosphates, and a reduction in the plasma membrane potential. Accordingly, we postulate that these cellular responses to external ATP play a role in the efflux of nucleotides. Ouabain, a specific inhibitor of the plasma membrane (Na+,K+)-ATPase, acts together with low concentrations of external ATP to increase nucleotide permeability in 3T6 cells. This effect occurs at concentrations of ouabain and ATP which alone do not increase nucleotide permeability. In addition, ouabain and low concentrations of ATP alone have little effect on the level of intracellular ATP. This is in contrast to energy inhibitors and uncouplers which appear to enhance nucleotide permeability by lowering the intracellular ATP concentration. Ouabain alone causes a threefold increase in intracellular Na+ levels and a similar reduction in intracellular K+ levels under our experimental conditions, supporting the idea that ion fluxes are involved in the mechanism of permeabilization.
We have determined the nucleotide sequence at the 3' end of the vesicular stomatitis virus (VSV) polymerase or L gene and compared it to that obtained from a defective interfering particle (DI) RNA generated by this virus. The latter (DI 0.50) contains a large internal deletion within this gene. The deletion begins exactly 253 residues from the transcription start of the gene and extends to approximately 300 bases from the 5' end of the standard RNA genome. The flanking sequences bear no homology to the eukaryotic consensus splice sequence. The sequence immediately preceding the deletion is complementary to the ribosome binding site of the L gene transcript and also resembles genome transcription termination sites. In addition, we present the results of nuclease protection experiments which show that this DI RNA retains an exact copy of the 3' end half of the standard genome (leader, N, NS, M and G genes), although its own 3' end is non-genomic. The implications of these findings regarding mechanisms of DI generation are discussed.
WSN (H0N1) influenza virus upon undiluted passages in different species of cells, namely, bovine kidney (MDBK), chicken embryo (CEF), and HeLa cells, produced a varying amount of defective interfering (DI) virus which correlated well with the ability of the species of cell to produce infectious virus. However, the nature of the influenza DI viral RNA produced from a single clonal stock was essentially identical in all three cells types, suggesting that these cells do not exert a great selective pressure in the amplification of specific DI viral RNAs either at early or late passages. DI viruses produced from one subtype (H0N1) could interfere with the replication of infectious viruses belonging to other subtypes (H1N1, H3N2). DI viral RNAs could also replicate with the helper function of other subtype viruses. The persistent infection of MDBK and HeLa cells could be initiated by coinfecting cells with both temperature-sensitive mutants (ts-) and DI influenza viruses. Persistently infected cultures cultures at early passages (up to passage 7) showed a cyclical pattern of cell lysis and virus production (crisis), whereas, at later passages (after passage 20), they produced little or no virus and were resistant to infection by homologous virus but not by heterologous virus. The majority of persistently infected cells, however, contained the complete viral genome since they expressed viral antigens and produced infectious centers. Selection of a slow-growing temperature-sensitive variant rather than the presence of DI virus or interferon appears to be critical in maintaining persistent influenza infection in these cells.
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A temperature-sensitive group II mutant of influenza virus, ts-52, with a presumed defect in viral RNA synthesis, readily produced von Magnus-type defective interfering virus (DI virus) when passed serially (four times) at high multiplicity in MDBK cells. The defective virus (ts-52 DI virus) had a high hemagglutinin and a low infectivity titer, and strongly interfered with the replication of standard infectious viruses (both ts-52 and wild-type ts+) in co-infected cells. Progeny virus particles produced by co-infection of DI virus and infectious virus were also defective and also had low infectivity, high hemagglutinating activity, and a strong interfering property. Infectious viruses ts+ and ts-52 were indistinguishable from ts-52 DI viruses by sucrose velocity or density gradient analysis. Additionally, these viruses all possessed similar morphology. However, when the RNA of DI viruses was analyzed by use of polyacrylamide gels containing 6 M urea, there was a reduction in the amount of large RNA species (V1 to V4), and a number of new smaller RNA species (D1 to D6) with molecular weights ranging from 2.9 X 10(5) to 1.05 X 10(5) appeared. Since these smaller RNA species (D1 to D6) were absent in some clones of infectious viruses, but were consistently associated with DI viruses and increased during undiluted passages and during co-infection of ts-52 with DI virus, they appeared to be a characteristic of DI viruses. Additionally, the UV target size of interfering activity and infectivity of DI virus indicated that interfering activity was 40 times more resistant to UV irradiation than was infectivity, further implicating small RNA molecules in interference. Our data suggest that the loss of infectivity observed among DI viruses may be due to nonspecific loss of a viral RNA segment(s), and the interfering property of DI viruses may be due to interfering RNA segments (DIRNA, D1 to D6). ts-52 DI virus interfered with the replication of standard virus (ts+) at both permissive (34 degrees C) and nonpermissive temperatures. The infectivity of the progeny virus was reduced to 0.2% for ts+ and 0.05% for ts-52 virus without a reduction in hemagglutinin titer. Interference was dependent on the concentration of DI virus. A particle ratio of 1 between DI virus (0.001 PFU/cell) and infectious virus (1.0 PFU/cell) produced a maximal amount of interference. Infectious virus yield was reduced 99.9% without any reduction of the yield of DI viruses Interference was also dependent on the time of addition of DI virus. Interference was most effective within the first 3 h of infection by infectious virus, indicating interference with an early function during viral replication.
The purpose of this study was to investigate the interaction of phosphoglycerate kinase with the human erythrocyte ghost membrane. Ghosts prepared in 0.1 mM EDTA and 17 mM Tris buffer (pH 7.5) have about 230 molecules of phosphoglycerate kinase/ghost. No additional binding is observed after incubating soluble enzyme with these leaky ghosts. This binding is tight but reversible with Kd = 7.1 X 10(-10) M. The enzyme can be eluted significantly from the membrane by incubation with 0.15 M NaCl and it rebinds to the membrane when the depleted ghosts are incubated with rabbit muscle phosphoglycerate kinase. Ligand binding studies show that NADH and NAD have opposite effects on the binding of the enzyme to the membrane; NAD (1.0 MM) favors binding while NADH (0.25 MM) does not. Similarly, ADP (0.2 mM) favors binding while ATP does not. ATP elutes the membrane-bound enzyme with Kd = 0.058 mM. MgSO4 also stimulates dissociation of the membrane-bound phosphoglycerate kinase (Kd = 0.36 mM), an effect which appears to be due to the magnesium ion. ADP (0.2 mM) can counteract the negative effect of MgSO4 (1.0 mM) on binding of phosphoglycerate kinase to the membrane. We have been unable so far to find tight coupling of the (Na+-K+)-ATPase with the membrane-bound phosphoglycerate kinase.
Soil supports the growth of a jute pathogen Colletotrichum gloeosporioides but only to a limited extent that of its antagonist Aspergillus versicolor. The growth of the sensitive pathogen is considerably checked by the antagonist in mixed soil culture although versicolin production could not be demonstrated within the limits of assay. Both the sensitive and the antagonistic organisms grow well in soil-compost medium and versicolin production by the latter is also enhanced. The antagonistic effect of Aspergillus versicolor on Colletotrichum gloeosporioides is expectedly more marked in soil-compost medium than in soil medium.
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1. The growth of rats treated with 3,5-dimethyoxy salicylic acid was retarded in comparison to that of normal rats. 2. The free cholesterol level of plasma of rats treated with 3,5-dimethoxy salicylic acid diminished while the pyruvate level of erythrocyte under the same condition increased in comparison with their normal levels; but on administration of thiamine both the cholesterol and pyruvate levels became normal. 3. The growth of a thiamine dependent strain of S. aureus was retarded when the organism was incubated in the medium containing 3,5-dimethoxy salicylic acid and this growth can be restored with the supplementation of thiamine in the medium.
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