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K Sen

Publications and source records attributed to K Sen.

17 recordsLinked to original sources
PubMed1992

Effects of Bacillus thuringiensis var. israelensis 20-kDa protein on production of the Bti 130-kDa crystal protein in Escherichia coli.

A 20-kDa protein of Bacillus thuringiensis var. israelensis (Bti) has been shown to be necessary for the efficient expression of the 27-kDa mosquitocidal protein gene in Escherichia coli. We have investigated the effects of this 20-kDa protein on the expression of two 130-kDa mosquitocidal protein genes (cryIVA, cryIVB) in E. coli by supplying the 20-kDa protein gene in trans. When a recombinant plasmid, pLH4BX, which was constructed to express cryIVA under the E. coli lac promoter on pUC19, coexisted with the 20-kDa protein gene, a striking increase in production of the fused CryIVA was detected. This was not accompanied by an increase in the amount of intracellular mRNA, suggesting that 20-kDa protein exerts a posttranscriptional effect. We conclude that the 20-kDa protein also stimulates the production of 130-kDa protein in E. coli.

Amino Acid Sequence↗
PubMed1991

Trimerization of an in vitro synthesized OmpF porin of Escherichia coli outer membrane.

The assembly of outer membrane proteins of Escherichia coli was examined using the OmpF porin as a model. Since this protein is made as a precursor, which is processed to a protein of Mr 37,000 before being assembled into trimers in the outer membrane, we synthesized a modified OmpF, which lacked 16 out of 22 amino acid residues from its signal sequence, in a coupled transcription-translation system. This modified protein resembled the unfolded, monomeric OmpF in its electrophoretic behavior, but much of the protein apparently existed in a more tightly folded conformation as it was recognized by a monoclonal antibody specific to a surface epitope of the native, trimeric OmpF porin. At least some conformers of this protein could be further incorporated into outer membrane or lipopolysaccharide bilayers, and assembled into trimers. The trimers formed were trypsin-resistant and heat-stable in sodium dodecyl sulfate up to 70 degrees C, thus showing the characteristics of the native trimeric protein. These results extend our earlier observation that OmpF monomer secreted by spheroplasts of E. coli can be trimerized in vitro (Sen, K., and Nikaido, H (1990) Proc. Natl. Acad. Sci. U.S. A 87, 743-747) and show that the trimerization can occur, albeit at a low efficiency, with porin monomers synthesized in vitro, presumably not contaminated by membrane fragments or other components of the cell envelope. However, comparison of trimerization efficiency of the nascent in vitro product with that of the same product already exposed to aqueous medium, as well as with that of the spheroplast-secreted product, leads us to the working hypothesis that the trimerization process in intact cells is accelerated either by accessory components or by the conformational changes accompanying the secretion through the cytoplasmic membrane and that the reactions observed in this study represent only part of the physiological process.

Amino Acid Sequence↗
PubMed1991

Cloning of a human cDNA expressing a high voltage-activating, TEA-sensitive, type-A K+ channel which maps to chromosome 1 band p21.

Over ten different mammalian genes related to the Drosophila Shaker gene (the Sh gene family) have been identified recently. These genes encode subunits of voltage-dependent K+ channels. The family consists of four subfamilies: ShI genes are homologues of Shaker; ShII, ShIII, and ShIV are homologues of three other Shaker-like genes in Drosophila, Shab, Shaw, and Shal, respectively. We report here the cloning of a human K+ channel ShIII cDNA (HKShIIIC) obtained from a brain stem cDNA library. HKShIIIC transcripts express an atypical voltage-dependent transient (A-type) K+ current in Xenopus oocytes. This current is activated by large membrane depolarizations and is extremely sensitive to the K+ channel blocker TEA unlike most A-type currents. The gene encoding HKShIIIC maps to chromosome 1p21.

Amino Acid Sequence↗
PubMed1991

Lipopolysaccharide structure required for in vitro trimerization of Escherichia coli OmpF porin.

Deep rought mutants, which produce very defective lipopolysaccharides, are unable to export normal levels of porins into the outer membrane. In this study, we showed that lipopolysaccharides from such mutants were also unable to facilitate the trimerization, in vitro, of monomeric OmpF porin secreted by spheroplasts of Escherichia coli B/r. In contrast, lipopolysaccharides containing most or all of the core oligosaccharides were able to facilitate trimerization.

Bacterial Outer Membrane Proteins↗
PubMed1990

Isolation of alpha- and beta-tubulin genes of Plasmodium falciparum using a single oligonucleotide probe.

An oligonucleotide probe (315) specific for the alpha- and beta-tubulin genes of Plasmodium falciparum was synthesized utilizing codon usage of P. falciparum determined from published gene sequences. By screening genomic and cDNA libraries with the oligonucleotide probe, alpha- and beta-tubulin clones were isolated. Positive clones were identified by partial sequencing and comparing the deduced amino acid sequence with the chicken brain alpha- and beta-tubulin amino acid sequences. The beta-tubulin gene was completely sequenced at the genomic level and partially at cDNA level. The deduced polypeptide is 445 amino acids long, shares 88% homology with chicken brain beta-tubulin, and contains two introns of 362 and 163 bp long, respectively. alpha- and beta-tubulin genes of P. falciparum are unlinked and dispersed; more than one copy of each gene may be present. Northern blot analysis of total RNA of the blood-stage parasite indicates the presence of three transcripts of alpha-tubulin (3.3 kb, 2.6 kb, 1.9 kb) and three transcripts of beta-tubulin gene (3.6 kb, 2.9 kb, 2.0 kb). The significance of these transcripts is presently unknown.

Amino Acid Sequence↗
PubMed1990

In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli.

It is not yet clear how bacterial outer membrane proteins reach their correct destination after they are secreted across the cytoplasmic membrane. We show here that porin OmpF is secreted into the medium as a water-soluble monomeric protein by spheroplasts of Escherichia coli. Furthermore, this monomeric porin is taken up by cell envelope preparations or purified lipopolysaccharides in the presence of 0.03% Triton X-100 and is converted correctly into the mature trimeric conformation. These results appear to reproduce a part of the physiological export and targeting steps of this protein.

Bacterial Outer Membrane Proteins↗
PubMed1990

Oligonucleotidase activity of phosphodiesterase from the fruit body of Flammulina velutipes.

A phosphodiesterase (EC 3.1.4.1) was purified to homogeneity from the fruit body of Flammulina velutipes. The enzyme had considerable activity toward oligonucleotides. The Km values were 0.66 mM for ApA, 2.47 mM for (Ap)2A, and 3.03 mM for (Ap)3A. The enzyme hydrolyzed oligodeoxyribonucleotides as well as oligoribonucleotides. The oligoribonucleotides bearing a phosphate residue at the 3' end were not hydrolyzed by the enzyme. The enzyme hydrolyzed the oligoribonucleotides exonucleolytically from the 3' to 5' end. Thus the PDase of F. velutipes is considered to function in vivo as an oligonucleotidase (EC 3.1.13.3), which efficiently converts oligonucleotides to 5'-mononucleotides in the cell.

Chromatography↗
PubMed1988

Porin channels in intact cells of Escherichia coli are not affected by Donnan potentials across the outer membrane.

Donnan potential (interior negative) across the outer membrane of Escherichia coli was measured by the distribution of [14C]choline in a mutant with a deletion through the genes for the active transport of choline. Calculation showed that the presence of membrane-derived oligosaccharides in the periplasm could quantitatively explain the magnitude of the Donnan potential and the periplasmic volume. By measuring the permeability of porin channels in intact cells suspended in solutions of widely different ionic strengths, it was shown that changing Donnan potential from 5 mV to approximately 100 mV had no effect on the permeability of either OmpF or OmpC porin channel toward a zwitterionic compound, cephaloridine. Thus, the "voltage-dependent gating" of porin channel, previously reported from another laboratory, is likely to be an artifact of in vitro reconstitution. The influx of negatively charged compounds, however, was affected by the Donnan potential as expected from the electrolyte diffusion theory.

Algorithms↗
PubMed1987

Biochemical studies on acetate non-utilizing mutants of Aspergillus terreus IRRL 16043.

The strain Aspergillus terreus IRRL 16043 can utilize glucose as well as acetate as a sole carbon source. Thirty-nine mutants were isolated from the wild-type by treatment with a chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNTG) which could not utilize acetate as a sole carbon source, and were designated as acetate non-utilizing (acu). By complementation and biochemical analyses they were divided into three functional groups, acu A, acu B and acu C lacking isocitrate lyase, malate synthase and acetyl-CoA synthetase activity, respectively.

Acetate-CoA Ligase↗
PubMed1986

Cytochrome b is necessary for the effective processing of core protein I and the iron-sulfur protein of complex III in the mitochondria.

The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in a mutant of yeast (W-267, Box 6-2) that lacks a spectrally detectable cytochrome b and synthesizes a shortened form of apocytochrome b. We recently reported that several cytochrome b-deficient mutants contained significantly diminished amounts of core proteins I and II as well as the iron-sulfur protein, but contained equal amounts of cytochrome c1 compared to the wild type (K. Sen and D. S. Beattie, Arch. Biochem. Biophys. 242, 393-401, 1985). In the present study, the time course of processing of precursors of both core protein I and the iron-sulfur protein which had accumulated in cells treated with the uncoupler carbonyl m-chlorophenyl hydrazone (CCCP) was noted to be significantly lower in the mutant compared to the wild type. The amounts of the mature forms of these proteins in mitochondria pulse labeled under different conditions was also considerably decreased at all times studied. The synthesis of both proteins appeared to be unaffected in the mutant, as the precursor forms of both proteins accumulated to the same extent when processing in vivo was blocked by CCCP. Furthermore, translation of RNA in a reticulocyte lysate in vitro indicated that the messenger RNAs for both proteins were present in the mutant and translated with equal efficiency. The import into isolated mitochondria of the precursor forms of the iron-sulfur protein synthesized in the cell-free system was also decreased in the mutant mitochondria. In addition, the precursor form was bound to the exterior of the mitochondrial membrane where it was sensitive to digestion with proteases. By contrast, the synthesis and processing of cytochrome c1 appeared to be unaffected in these mutants. These results suggest that cytochrome b is necessary for the proper processing and assembly of both core protein I and the iron-sulfur protein, but not for cytochrome c1, into complex III of the inner mitochondrial membrane.

Biological Transport↗
PubMed1985

Decreased amounts of core proteins I and II and the iron-sulfur protein in mitochondria from yeast lacking cytochrome b but containing cytochrome c1.

The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in four mutants of yeast that lack a spectrally detectable cytochrome b and do not synthesize apocytochrome b. Quantitative analysis of intact mitochondria by immunoprecipitation or immunoblotting techniques with specific antisera revealed that the core proteins and the iron-sulfur protein were decreased 50% or more in the mitochondria from the mutants as compared to the wild type. Sonication of wild-type mitochondria did not result in any decrease in any of these proteins from the membrane; however, sonication of mitochondria from the four mutants resulted in a further decrease in the amount of these proteins suggesting that they are not as tightly bound to the mitochondrial membrane in the absence of cytochrome b. By contrast, the amounts of cytochrome c1 in the mitochondria, as determined both spectroscopically and immunologically, were not significantly affected by the absence of cytochrome b. In addition, no loss of cytochrome c1 was observed after sonication of the mitochondria suggesting that this protein is tightly bound to the membrane. These results suggest that the processing and/or assembly of these subunits of complex III into the mitochondrial membrane is affected by the absence of cytochrome b.

Cytochrome b Group↗
PubMed1978

Effect of respiratory deficiency and temperature on the mitochondrial lipid metabolism of Aspergillus niger.

A comparative study of the mitochondrial lipid composition of a wild strain (V 35) and a respiratory-deficient mutant (rd3) of Aspergillus niger grown at different temperatures (25, 30, 35, and 40 degrees C) has been performed. The lipid spectrum, though qualitatively similar, differs quantitatively in both cases. At the optimum growth temperature (30 degrees C) depletion in ergosterol (40%) and cardiolipin (52%) was observed. This probably indicates the formation of defective mitochondria in the mutant with a resultant impaired respiratory system. Complete depletion of cardiolipin species containing fatty acid (20:5omega3) in the respiratory-deficient mutant suggests a possible role of this lipid in mitochondriogenesis at least in A. niger. The effect of temperature is predominantly on the degree of unsaturation and sterol ester formation. The linoleic acid (18:2omega6) content decreases with a concomitant increase in oleic acid (18:1omega9) content as the growth temperature increases for both cell types. Some morphological changes and effects on the vegetative life cycle have been observed with variation in growth temperature in the wild type and also in the mutant form.

Aspergillus niger↗