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B B Kaplan

Publications and source records attributed to B B Kaplan.

At least 55 records · Page 3Linked to original sources
PubMed1989

Isolation of a rat adrenal cDNA clone encoding phenylethanolamine N-methyltransferase and cold-induced alterations in adrenal PNMT mRNA and protein.

Cold stress is known to increase the synthesis and release of catecholamines in the sympathoadrenal system. Previously, we have demonstrated that cold exposure results in a 3- to 4-fold increase in adrenomedullary tyrosine hydroxylase (TH) activity, which is mediated by concomitant alterations in TH mRNA and protein levels. To further investigate the effects of stress on the expression of the catecholamine biosynthetic enzymes, we have isolated a rat cDNA clone encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT). The cDNA clone is 905 nucleotides in length and contains a single open reading frame corresponding to 270 amino acids. The amino acid sequence predicted from this nearly full-length cDNA is 89% and 86% identical to that of bovine and human PNMT, respectively. Using the rat PNMT cDNA as a hybridization probe, we have measured the effects of cold stress on the relative abundance of adrenomedullary PNMT mRNA. Levels of PNMT protein were also estimated using an immunoblot analysis. As in the case of TH, cold exposure resulted in a rapid and prolonged increase in PNMT mRNA abundance, followed by concomitant increases in PNMT immunoreactivity. However, there appear to be quantitative and qualitative differences in the adaptive response of TH and PNMT to cold stress.

Adrenal Glands↗
PubMed1988

The complete nucleotide sequence and structure of the gene encoding bovine phenylethanolamine N-methyltransferase.

A cDNA clone for bovine adrenal phenylethanolamine N-methyltransferase (PNMT) was used to screen a Charon 28 genomic library. One phage was identified, designated lambda P1, which included the entire PNMT gene. Construction of a restriction map, with subsequent Southern blot analysis, allowed the identification of exon-containing fragments. Dideoxy sequence analysis of these fragments, and several more further upstream, indicates that the bovine PNMT gene is 1,594 base pairs in length, consisting of three exons and two introns. The transcription initiation site was identified by two independent methods and is located approximately 12 base pairs upstream from the ATG translation start site. The 3' untranslated region is 88 base pairs in length and contains the expected polyadenylation signal (AATAAA). A putative promoter sequence (TATA box) is located about 25 base pairs upstream from the transcription initiation site. Computer comparison of the nucleotide sequence data with the consensus sequences of known regulatory elements revealed potential binding sites for glucocorticoid receptors and the Sp1 regulatory protein in the 5' flanking region of the gene. Additionally, comparison of the sequence of the exons of the PNMT gene with cDNA sequences for other enzymes involved in biogenic amine synthesis revealed no significant homology, indicating that PNMT is not a member of a multigene family of catecholamine biosynthetic enzymes.

Adrenal Medulla↗
PubMed1988

Purification and partial amino acid sequence of bovine adrenal phenylethanolamine N-methyltransferase: a comparison of nucleic acid and protein sequence data.

Recently, we have reported the isolation and characterization of a putative genomic DNA clone encoding bovine adrenal phenylethanolamine N-methyltransferase (PNMT) (Batter et al., 1988). However, the lack of primary amino-acid sequence data for this enzyme precluded a definitive proof of the authenticity of this clone. To establish identity, the amino acid sequence of several peptides generated by chemical and enzymatic hydrolysis of purified PNMT was compared to that predicted from the nucleotide sequence of the exons of the putative PNMT gene. Bovine adrenomedullary PNMT was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Treatment with 70% formic acid cleaved the protein at a single Asp-Pro bond near the N-terminus. The purified protein was also cleaved at a single methionine residue near the C-terminus by treatment with cyanogen bromide. N-terminal amino acid sequence analysis identified 8 and 10 amino acid residues, respectively, following each of the scissile peptide bonds. Four tryptic peptides, generated by complete enzymatic digestion, were isolated by reverse-phase HPLC and subjected to sequence analysis. Combined, the amino acid sequences of these six peptides represent 20% of the PNMT protein. These amino acid sequences matched exactly the sequences predicted from the exons of the putative PNMT genomic clone.

Adrenal Medulla↗
PubMed1988

Isolation and nucleotide sequence of a cDNA clone encoding bovine adrenal tyrosine hydroxylase: comparative analysis of tyrosine hydroxylase gene products.

Investigations into the structure and mechanisms regulating the expression of the genes involved in catecholamine biosynthesis have led to the isolation of a cDNA coding for bovine adrenal tyrosine hydroxylase (TH). The 1,722 bp cDNA contains the complete coding sequence and 3' untranslated region of the TH mRNA. The nucleotide sequence of the cDNA and the deduced amino acid sequence were compared to those reported for rat and human TH. Bovine TH shares 85% and 84% amino acid sequence identity with that of rat and human TH, respectively. Alignment of the amino acid sequences of rat, bovine, and human TH reveals that 79% of the residues are identical in all three species, indicating a strong evolutionary conservation of enzyme structure. Moreover, three of the four putative phosphorylation sites located in the N-terminal region of TH are conserved in these animal species. There are, however, some interspecies differences in TH gene products. The 3' untranslated region of bovine TH mRNA is 56 and 97 nucleotides shorter than rat and human TH mRNA, respectively. Additionally, the bovine protein is 7 and 6 amino acids smaller than its rat and human homologues. All of the absent amino acid residues of bovine TH are missing from an alanine-rich region in the N-terminal portion of the rat and human proteins (amino acids 51-68). Comparison of the size of bovine and rat TH mRNA and protein by northern blot and immunoblot analyses yielded differences consistent with those predicted from the nucleotide sequence data.

Amino Acid Sequence↗
PubMed1988

A cholinergic antagonist blocks cold stress-induced alterations in rat adrenal tyrosine hydroxylase mRNA.

The role of nicotinic cholinergic transmission in cold stress-induced alterations in rat adrenomedullary tyrosine hydroxylase (TH) mRNA was investigated by RNA dot-blot hybridization, using a cloned TH cDNA probe. Chlorisondamine, a ganglionic blocking agent, greatly attenuated the induction of TH mRNA levels caused by cold exposure, whereas carbachol and nicotine, cholinergic agonists, increased TH mRNA in control animals. These results suggest that cholinergic nicotinic receptors play a key role in the transsynaptic induction of adrenal TH gene expression.

Adrenal Medulla↗
PubMed1987

Occurrence and sequence complexity of polyadenylated RNA in squid axoplasm.

Axoplasmic RNA from the giant axon of the squid (Loligo pealii) comprises polyadenylated [poly (A)+] RNA, as judged, in part, by hybridization to [3H]polyuridine and by in situ hybridization analyses using the same probe. The polyadenylate content of axoplasm (0.24 ng/microgram of total RNA) suggests that the poly(A)+ RNA population makes up approximately 0.4% of total axoplasmic RNA. Axoplasmic poly(A)+ RNA can serve as a template for the synthesis of cDNA using a reverse transcriptase and oligo(deoxythymidine) as primer. The size of the cDNA synthesized is heterogeneous, with most fragments greater than 450 nucleotides. The hybridization of axoplasmic cDNA to its template RNA reveals two major kinetic classes: a rapidly hybridizing component (abundant sequences) and a slower-reacting component (moderately abundant and rare sequences). The latter component accounts for approximately 56% of the total cDNA mass. The rapidly and slowly hybridizing kinetic components have a sequence complexity of approximately 2.7 kilobases and 3.1 X 10(2) kilobases, respectively. The diversity of the abundant and rare RNA classes is sufficient to code for one to two and 205, respectively, different poly(A)+ RNAs averaging 1,500 nucleotides in length. Overall, the sequence complexity of axoplasmic poly(A)+ RNA represents approximately 0.4% that of poly(A)+ mRNA of the optic lobe, a complex neural tissue used as a standard. Taken together, these findings indicate that the squid giant axon contains a heterogeneous population of poly(A)+ RNAs.

Animals↗
PubMed1986

Molecular adaptations in catecholamine biosynthesis induced by cold stress and sympathectomy.

Chronic cold stress and chemical sympathectomy are known to increase the synthesis and release of catecholamines in the adrenal medulla. Chromaffin cells adapt to altered functional requirements by increasing the synthesis of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. In this study, we investigated the molecular genetic mechanisms underlying these changes in enzyme activity. Estimates of TH mRNA levels were obtained by RNA dot-blot analysis with a cloned TH cDNA hybridization probe. Exposure to cold produced a 4.3-fold increase in the relative abundance of adrenomedullary TH mRNA. Increases in TH mRNA levels (90%) also were observed in the brainstem of cold-stressed animals. The relative amount of TH synthesized in vitro in a rabbit reticulocyte cell-free system, programmed with adrenal poly(A)+RNA, increased 4.3 times in cold-stressed rats. Alteration in TH mRNA abundance appears to be specific, as we observed no significant difference in the levels of total RNA or poly(A)+RNA in this tissue. In addition, the relative abundance of adrenomedullary TH mRNA increased by 60% 4 days after systemic administration of the neurotoxin 6-hydroxydopamine. This increase was transient and disappeared 2 weeks after the lesion. Changes in TH mRNA levels after cold stress or sympathectomy were eliminated by denervation of the adrenal gland. These results indicate that alterations in the relative abundance of TH mRNA mediate changes in TH activity induced by chronic stress or sympathectomy, and that these changes require an intact sympathetic input.

Adrenal Medulla↗
PubMed1986

Diversity of gene expression in goldfish brain.

The sequence complexity of nuclear and polysomal RNA from goldfish brain and kidney was measured by RNA-driven hybridization reactions with single-copy [3H]DNA. At saturation, brain nuclear and polysomal RNA were complementary to 23.2 and 6.7% of the DNA probe, respectively. In contrast to these findings, nuclear and polysomal RNA from kidney hybridized to 16.1 and 3.1% of the single-copy DNA, values that were significantly lower than that obtained in the CNS. Taken together, the results focus attention on the striking diversity of gene expression in goldfish brain and extend to lower vertebrates the observation that nervous tissue expresses significantly more genetic information than other somatic tissues or organs.

Animals↗
PubMed1986

Complexity of nuclear and polysomal RNA from squid optic lobe and gill.

The sequence complexity of nuclear and polysomal RNA from squid optic lobe and gill was measured by RNA-driven hybridization reactions with single-copy [3H]DNA. At saturation, brain nuclear and polysomal RNAs were complementary to 22.8 and 7.9% of the DNA probe, respectively. Assuming asymmetric transcription, the complexity of nuclear and polysomal RNA was equivalent to 2.5 X 10(8) and 8.8 X 10(7) nucleotides, respectively. Approximately 80-85% of the sequence complexity of brain total polysomal RNA was found in the polyadenylated RNA fraction. In contrast to these findings, nuclear and polysomal RNAs from gill hybridized to 9.1 and 2.9%, respectively, of the single-copy DNA, values that were 2.5-fold lower than those obtained in the CNS. Taken together, the results focus attention on the striking diversity of gene expression in the squid CNS and extend to the cephalopod mollusks the observation that nervous tissue expresses significantly more genetic information than other somatic tissues or organs.

Animals↗
PubMed1985

Effect of chronic cold exposure on tyrosine hydroxylase mRNA in rat adrenal gland.

The effect of chronic stress on the levels of tyrosine hydroxylase (TH) RNA in rat adrenal gland was investigated by RNA-DNA hybridization using a cloned TH cDNA probe. Results of dot-blot hybridization experiments and northern analysis demonstrate that exposure of animals to cold for 1 week results in a 4-5-fold increase in the relative abundance of TH mRNA. This increase in TH mRNA level may underlie the increase in adrenal TH activity that is known to occur when rats are exposed to such cold stress.

Adrenal Medulla↗
PubMed1983

Changes in gene expression during postnatal development of the rat cerebellum.

The base sequence complexity of total and polysomal poly(A +)RNA from rat cerebellum was measured during postnatal development by RNA-DNA hybridization. At saturation, total and polysomal poly(A +)RNA from neonate cerebellum hybridized to 12.7% and 5.0% of the single-copy genomic DNA, respectively. Assuming asymmetric transcription, the sequence complexity of these RNA populations is sufficient to code for greater than 100,000 different gene transcripts. The percentage of single-copy DNA expressed as total and polysomal poly(A +)RNA declined during postnatal development, reaching adult values of 10.0% and 4.1%, respectively. These results indicate that cerebellar maturation is accompanied by significant reductions in the diversity of genetic information expressed in the tissue.

Animals↗
PubMed1982

Different forms of adrenal phenylethanolamine N-methyltransferase: species-specific posttranslational modification.

Phenylethanolamine N-methyltransferase was purified from rat and cow adrenal glands. The enzymes from the two species have the same molecular weight of 31,000, but differ in electrophoretic mobility. During polyacrylamide gel electrophoresis, the rat form migrates faster than the bovine form. Antibodies to bovine enzyme precipitated equally well the rat and cow form of the enzyme, but antibodies against rat enzyme precipitated poorly the bovine form. In contrast, both antibodies recognized a similar protein in the in vitro translation products of poly(A+)mRNA isolated from cow adrenal glands. The results suggest that the primary protein structure of rat and bovine enzyme is similar and that differences in electrophoretic mobility are due to posttranslational modification of the enzyme molecule.

Adrenal Glands↗
PubMed1981

Translation of tyrosine hydroxylase from poly(A)-mRNA in pheochromocytoma cells is enhanced by dexamethasone.

Polysomal poly(A)-mRNA was purified from a clonal cell line of rat pheochromocytoma (PC 12) and translated in a reticulocyte cell-free protein-synthesizing system. Tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahyropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] was isolated from other protein by immunoprecipitation and NaDodSO4/polyacrylamide gel electrophoresis. The molecular weight and relative proportion of tyrosine hydroxylase to other proteins synthesized in vitro were identical to those of the enzyme synthesized in vivo in cultured cells. Incubation of PC 12 cells with 10 microM dexamethasone increased the activity and amount of tyrosine hydroxylase 2.5-fold. The ratio of tyrosine hydroxylase to total protein translated from poly(A)-mRNA isolated from cells treated with dexamethasone was 2.5 times higher than the ratio of enzyme to total protein translated from an equal amount of poly(A)-mRNA from untreated cells. The dexamethasone-elicited induction of tyrosine hydroxylase in PC 12 cells therefore is a result of an increased "relative" amount or activity of tyrosine hydroxylase mRNA.

Adrenal Gland Neoplasms↗
PubMed1979

An improved method for the rapid isolation of brain ribonucleic acid.

An improved one-step method for the extraction of RNA from rat brain is described. Fresh or frozen tissue is disrupted in the powerful protein denaturant guanidine thiocyanate, and RNA isolated by ultracentrifugation through CsCl. The procedure is advantageous in that it is relatively simple, is rapid and does not expose the sample to enzyme treatments or repeated organic extractions.

Animals↗
PubMed1975

Macromolecules and behavior II. Training induced alteration in leucine transfer RNA of goldfish brain.

Goldfish were trained to swim upright after a polystyrene float had been attached ventrally. The effects of training on the amino acid acceptor activity and methylated albumin-kieselguhr column chromatography profile of several brain isoaccepting tRNA species were studied. Comparison of the kinetics of aminoacylation and chromatography profile of tRNAs from trained and control fish showed no differences for Arginyl-, glutamyl-, histidyl-, lysyl-, phenylalanyl-, seryl, threonyl- or valyl-tRNA. In contrast, training induced alterations in tRNA-Leu activity and activity profile. Training had no effect on brain amino acid pools or liver tRNA-Leu activity. Together, behavioral control experiments and plasma cortisol titers indicated that the training-induced increase in tRNA-Leu activity was not caused by stress, exertion, random disoriented swimming or the minor surgical procedures required. Rather, this activity change seems to correlate with some, as yet unidentified, aspect of behavioral training. The implications of these findings with respect to learning are discussed.

Adaptation, Biological↗