Biomedical subjects

J C Kandala

Publications and source records attributed to J C Kandala.

13 recordsLinked to original sources

PubMed1992

Involvement of tyrosine kinase and protein kinase C in platelet-activating-factor-induced c-fos gene expression in A-431 cells.

In A-431 cells, platelet-activating factor (PAF) induces the expression of c-fos and TIS-1 genes in both the absence and the presence of cycloheximide in a structurally specific and receptor-coupled manner. We have now investigated the molecular mechanisms of this response, particularly in relation to the role of protein kinases. Pretreatment of cells with genistein or methyl-2,5-dihydroxycinnamate (tyrosine kinase inhibitors) or staurosporine (a protein kinase C inhibitor) for 20 min abolished the c-fos expression induced by PAF. Interestingly, when genistein was added 90 s after addition of PAF, no inhibition was observed. Similarly, staurosporine did not inhibit c-fos expression when added 8 min after PAF addition to the cells. These inhibitions were dose-dependent (IC50 for staurosporine was 180 nM, and for genistein 50 microM). Simultaneous addition of PAF and phorbol 12-myristate 13-acetate (PMA) did not give a synergistic effect on c-fos expression. Pretreatment of cells with PMA had no effect on [3H]PAF binding, but abolished the PAF-induced gene expression. PAF-stimulated gene expression was desensitized if cells were pretreated with PAF. Interestingly, epidermal growth factor was able to stimulate c-fos expression in PAF-desensitized cells, and thus indicated involvement of distinct mechanisms for the two stimuli. Forskolin, an activator of adenylate cyclase, did not induce c-fos expression and had no effect on the PAF response. Exposure of cells to PAF for as little as 1 min, followed by its removal, was sufficient to activate the gene expression and demonstrated the rapidity and the exquisite nature of the signalling involved in this process. It is concluded that activation of PAF receptor (a proposed G-protein-coupled receptor) causes rapid production of signals which induce the expression of c-fos gene and that this is mediated via tyrosine kinase and protein kinase C.

Alkaloids↗

PubMed1992

Cloning and characterization of a highly conserved HMG-like protein (PF16) gene from Plasmodium falciparum.

A novel gene encoding a protein of 147 amino acids (Pf16) has been cloned from Plasmodium falciparum and expressed in E. coli. The protein contains 19 methionines, all of which are localized in the NH2-terminal 35 amino acid residues, and it is also rich in lysine. Pf16 is highly basic, contains a polyacidic domain consisting of aspartic acid and is related to the non-histone high mobility group proteins of higher eukaryotes. The gene is conserved among eight different species of Plasmodium so far examined, suggesting an important function for this gene product in the parasite's life cycle.

Amino Acid Sequence↗

PubMed1992

Improved method for screening cDNA expression libraries for DNA-binding proteins.

The ability to successfully screen a lambda gt11 cDNA expression library for specific gene products that can bind to selected sequences of DNA depends on radioactive double-stranded DNA probes with high specific activity. We demonstrate here that probes labeled by the PCR are superior to probes made by the Klenow reaction. The use of these PCR-generated probes have facilitated our efforts to isolate recombinant phage containing putative DNA-binding gene products that recognized a 246-base pair transcriptional enhancer region of Rous sarcoma virus long terminal repeat.

Animals↗

PubMed1991

Platelet activating factor induces expression of early response genes c-fos and TIS-1 in human epidermoid carcinoma A-431 cells.

The effect of platelet activating factor (PAF) on the induction of early response genes was investigated in A-431 cells (human epidermal carcinoma cells). PAF induced a transient expression of c-fos and TIS-1 mRNA in a time- and dose-dependent manner. As low as 10(-10) M PAF caused detectable expression of these genes with a maximum observed at 10(-7) M. In the presence of cycloheximide, increases in the gene expression were noticeable at 20 min and peaked between 30-60 min. A lack of induction with lyso-PAF, an inactive PAF metabolite, confirmed the specificity of PAF towards this expression. The cells pretreated with CV-6209, a PAF receptor antagonist, did not show any induction of these genes by PAF. It is concluded that PAF causes induction of the early response genes c-fos and TIS-1 in a structurally specific and receptor dependent manner. This finding offers a new role for PAF at the nuclear level and may have important implications in the long term effects of PAF in pathophysiological conditions.

Cycloheximide↗

PubMed1990

Isolation of a genomic clone encoding the rat histone variant, H1d.

Mammals contain a family of five closely related H1 histone variants (H1a-e) as well as two less closely related forms, H10 and H1t. We have sequenced a rat genomic clone that encodes one of the standard H1 variants. An RNA transcript of the gene was made with bacteriophage SP6 RNA polymerase and translated in a cell-free system. The protein synthesized in vitro was identified as variant H1d by its electrophoretic mobility.

Amino Acid Sequence↗

PubMed1990

2-Chloroacetaldehyde and 2-chloroacetal are potent inhibitors of DNA synthesis in animal cells.

The effect of 2-chloroacetaldehyde, CAA, a metabolite of vinyl chloride and 2-chloroacetal, CAC, an ethyl diester of chloroacetaldehyde, on DNA synthesis in animal cells has been investigated. Both compounds drastically inhibited DNA synthesis at 10 to 20 microM. The inhibitory effect of the chemicals appears to be directly on DNA synthesis rather than on the uptake of thymidine or the formation of nucleotides. Residual DNA made in the presence of CAA had an average chain length of 300 nucleotides compared to a length of several thousand nucleotides in the absence of CAA. Synchronization experiments revealed that the inhibitory effect is reversible if 2-chloroacetaldehyde is removed within two hours but not after longer exposures.

Acetaldehyde↗

PubMed1990

A transformation-competent recombinant between v-src and Rous-associated virus RAV-1.

The LTR, v-src, LTR provirus, which arose by the reverse transcription and integration of src mRNA in the H-19 hamster tumor, has been successfully rescued by fusion with chicken fibroblasts infected with Rous-associated virus RAV-1. One rescued virus, E6, acquired 1 kilobase of the 5' end of the gag gene structure. Recombination took place in the region of 15-nucleotide homology exactly between v-src exon (position 7054) and gag (position 1417). This recombination resulted in the alteration of src splice acceptor site sequences, but this site is maintained as a functional splice acceptor site. The nucleotide structure of the long terminal repeat of recombinant E6 virus suggests that it arose by the intermolecular jump of reverse transcription from RAV-1 to src mRNA and then the switch of templates between already depicted regions of homology. The second jump of reverse transcription was apparently an intramolecular event. The acquisition of 1 kilobase of the 5' gag by E6 resulted in maintaining the balance of unspliced and spliced E6 RNAs and assured the replication advantage of rescued E6 virus over rescued F6 virus, the genome of which corresponds to that present in ancestral H-19 cells.

Animals↗

PubMed1987

Liver cell turnover in rats fed a choline-devoid diet.

Liver DNA was labeled in a group of weanling Fischer-344 male rats by placing mini-osmotic pumps, loaded with [methyl-3H]thymidine, under the dorsal skin for 14 days. The animals were then placed on either a choline-supplemented or a choline-devoid diet, and subgroups on each diet were killed after 1, 2, 4, 8 and 16 weeks. Liver DNA total and specific radioactivities were determined in all rats. The results were used to estimate the half-life (t1/2) of liver cells, and the fractional rates of liver cell death (Kd) and proliferation (Kp). In rats fed the control choline-supplemented diet, liver cells were estimated to die at an overall Kd of 0.16% per day, and to have a t1/2 of 439.5 days; Kp was higher in younger than in older rats. In rats fed the choline-devoid diet, liver cells diet at a Kd ranging from 4.82 down to 0.93% per day, as the length of the feeding period increased; corresponding t1/2S were 14.3 and 74.6 days. In these rats, Kp was more than sufficient to compensate for cell loss. The results show that liver-cell death represents a major consequence of feeding a choline-devoid diet to rats, and that the necrogenic action of the diet is a major factor responsible for the highly increased turnover of liver cells present in these animals. However, evidence was obtained that the diet has also a primary mitogenic action, beyond those related to replacement of dead cells, and to cell accretion due to normal growth of the animals and the liver.

Animals↗

PubMed1987

Genes for chromosomal proteins expressed before and after meiosis.

Cloned gene sequences have been isolated for two testis-specific chromosomal proteins, one of which, histone (H1t), appears during meiosis, whereas the other, transition protein 1 (TP1), appears only during the later steps of spermatid development. Aspects of the regulation of each gene have been examined. In the case of H1t, analysis of its promoter region shows that it contains excellent matches to each of the four sequence homologies identified for the usual somatic H1 variants, so that the factor(s) that restrict H1t expression to spermatocytes remain a mystery. In the case of TP1, a cDNA clone allowed identification of its message by Northern blots as well as by in situ hybridization. The message appears postmeiotically in late round spermatids but is translationally repressed until the spermatid nucleus begins to condense.

Amino Acid Sequence↗

PubMed1986

Isolation of the gene for the testis-specific H1 histone variant H1t.

H1t is a testis-specific H1 variant found in pachytene spermatocytes and round spermatids of mammals. The H1t gene was isolated from the Sargent-Bonner library of recombinant lambda bacteriophage containing EcoRI fragments of rat liver DNA using a hybridization probe derived from a chicken H1 variant. The rat H1t gene encodes a 207-amino acid protein (ignoring the initiating methionine) that matches perfectly what is known of the sequence and composition of H1t isolated from rat testes. The gene lacks introns and has good matches to all the consensus sequences known to lie upstream from a variety of H1 genes from diverse organisms. It also has the standard downstream palindromic sequence that specifies the 3'-end of most histone messages. Accordingly, the features of the gene or its environs that restrict its expression to a particular phase of spermatogenesis are not yet evident.

Amino Acid Sequence↗

PubMed1985

Methylation of the rat seminal vesicle secretory protein IV gene. Extensive demethylation occurs in several male sex accessory glands.

Seminal vesicle secretory protein IV (SVS IV) is perhaps the most abundant protein made by the epithelial cells of the rat seminal vesicle. In this report, we have asked whether the methylation of its gene correlates with its tissue-specific pattern of expression. Using methylation-sensitive restriction endonucleases HpaII and AvaI, we could examine seven potential methylation sites in or near the gene. All seven sites were largely unmethylated in the seminal vesicle, while in most nonexpressing organs, all sites were heavily modified. The correlation of demethylation with expression was broken by finding that both ventral prostate and coagulating gland (anterior prostate) DNA showed the same demethylation pattern seen for the seminal vesicle. SVS IV protein has not been reported in either of these two organs, and we could not detect mature SVS IV message in prostate RNA or SVS IV transcription by in vitro incubation of prostate nuclei. We conclude that demethylation of the SVS IV gene accompanies the differentiation of several androgen-dependent sex accessory glands but confirm that demethylation per se is not a sufficient signal to bring about gene transcription.

Animals↗

PubMed1985

Androgen regulated genes from prostate and seminal vesicle share upstream sequence homologies.

The genes for seminal vesicle secretory protein IV and prostate steroid binding protein component 3 of the rat share upstream homologies of which the most striking is a 30 nucleotide sequence located between position -190 and -330 relative to the major transcriptional initiation sites. This sequence does not appear to be a common repetitive element and deserves consideration as a potential site involved in the androgen regulated expression of these genes.

Androgens↗

PubMed1981

Initiation of Bacillus subtilis sporulation by the stringent response to partial amino acid deprivation.

We have controlled the rates at which three different amino acids were available to auxotrophs of Bacillus subtilis by avoiding active transport of the respective substrate. The active transport of oxomethylvalerate, a precursor of isoleucine, was prevented by a kauA mutation, the uptake of L-aspartate was competed by 20 mM L-glutamate, and D-methionine was used instead of L-methionine. When in this way conditions of partial amino acid deprivation were achieved, a partial "stringent response" occurred which included the increase of ppGpp and pppGpp, and the decrease of GTP; such conditions initiated sporulation. In the corresponding relaxed (relA) mutants, the changes of guanine nucleotides were greatly reduced and no sporulation was observed at any substrate concentration; but addition of decoyinine produced a further decrease of GTP and caused sporulation.

Amino Acids↗