Protein synthesis in the presynaptic endings of the squid photoreceptor neuron: in vitro and in vivo modulation.
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Animals↗
Biomedical subjects
B B Kaplan
Publications and source records attributed to B B Kaplan.
Association and linkage studies of the TAQI A1 allele at the dopamine D2 receptor gene in samples of female and male alcoholics.
To address the controversy surrounding DRD2 and alcoholism, we performed linkage and association studies utilizing alcoholic men from high-density families largely uncontaminated by other psychopathology and female alcoholics for whom secondary drug dependence (averaging 10 years later onset) was a prominent feature. The males and females were combined for a total of 52 alcoholics, and compared to 30 controls screened for the absence of alcoholism and other psychopathology, revealing a significant association between the frequency of the TaqI A1 allele and alcoholism. However, linkage and family-based association studies conducted on 20 families of male alcoholics found no evidence for association or linkage between Taq A and alcoholism. The results of our population-based association study, placed in the context of the literature, suggest that minimizing psychopathology in control groups is probably a more important explanation for divergent results than either sampling error or population stratification. When combined with the complete lack of within-family evidence, we concluded that the association, while not appearing to be artifactual, is not specific to the alcoholism phenotype, per se.
Alcoholism↗
What can the DRD2/alcoholism story teach us about association studies in psychiatric genetics?
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Alcoholism↗
Characterization of squid enolase mRNA: sequence analysis, tissue distribution, and axonal localization.
Enolase is a glycolytic enzyme whose amino acid sequence is highly conserved across a wide range of animal species. In mammals, enolase is known to be a dimeric protein composed of distinct but closely related subunits: alpha (non-neuronal), beta (muscle-specific), and gamma (neuron-specific). However, little information is available on the primary sequence of enolase in invertebrates. Here we report the isolation of two overlapping cDNA clones and the putative primary structure of the enzyme from the squid (Loligo pealii) nervous system. The composite sequence of those cDNA clones is 1575 bp and contains the entire coding region (1302 bp), as well as 66 and 207 bp of 5' and 3' untranslated sequence, respectively. Cross-species comparison of enolase primary structure reveals that squid enolase shares over 70% sequence identity to vertebrate forms of the enzyme. The greatest degree of sequence similarity was manifest to the alpha isoform of the human homologue. Results of Northern analysis revealed a single 1.6 kb mRNA species, the relative abundance of which differs approximately 10-fold between various tissues. Interestingly, evidence derived from in situ hybridization and polymerase chain reaction experiments indicate that the mRNA encoding enolase is present in the squid giant axon.
Amino Acid Sequence↗
Kinesin mRNA is present in the squid giant axon.
Recently, we reported the construction of a cDNA library encoding a heterogeneous population of polyadenylated mRNAs present in the squid giant axon. The nucleic acid sequencing of several randomly selected clones led to the identification of cDNAs encoding beta-actin and beta-tubulin, two relatively abundant axonal mRNA species. To continue characterization of this unique mRNA population, the axonal cDNA library was screened with a cDNA probe encoding the carboxy terminus of the squid kinesin heavy chain. The sequencing of several positive clones unambiguously identified axonal kinesin cDNA clones. The axonal localization of kinesin mRNA was subsequently verified by in situ hybridization histochemistry. In addition, the presence of kinesin RNA sequences in the axoplasmic polyribosome fraction was demonstrated using PCR methodology. In contrast to these findings, mRNA encoding the squid sodium channel was not detected in axoplasmic RNA, although these sequences were relatively abundant in the giant fiber lobe. Taken together, these findings demonstrate that kinesin mRNA is a component of a select group of mRNAs present in the squid giant axon, and suggest that kinesin may be synthesized locally in this model invertebrate motor neuron.
Actins↗
Protein synthesis in nerve endings from squid brain: modulation by calcium ions.
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Animals↗
Lack of variation in the nucleotide sequence corresponding to the transmembrane domain of the beta-amyloid precursor protein in Alzheimer's disease.
The nucleotide sequence corresponding to the APP transmembrane domain and flanking regions of charged amino acids was determined for 91 patients with histologically confirmed Alzheimer's disease, 9 patients with dementias of other etiologies, and 14 controls who had no identifiable brain disease. Twenty-eight of the AD patients had a first-degree relative with dementia. No mutations were detected among the 100 demented patients. However, one of the 14 controls exhibited a change in the 3' base of codon 716 which would not be expected to result in an amino acid substitution at this position.
Alzheimer Disease↗
Cold-induced increases in phenylethanolamine N-methyltransferase (PNMT) mRNA are mediated by non-cholinergic mechanisms in the rat adrenal gland.
Previously, we reported that cold stress induces a rapid increase in adrenomedullary PNMT mRNA levels, followed by concomitant increases in PNMT immunoreactivity (10). In the present study, the extracellular signals mediating this adaptive response to stress were investigated using northern analysis and RNA slot-blot hybridization. Although adrenal denervation significantly diminished cold-induced increments in adrenomedullary PNMT mRNA levels, it did not completely abolish the cold stress response. In contrast to these results, splanchnectomy completely inhibited cold-induced increments in TH mRNAs in the same tissue samples. These findings indicate that the effects of cold exposure on PNMT mRNA levels are mediated by both neural and non-neural mechanisms, and that adrenal PNMT and TH are differentially regulated in response to cold stress. Surprisingly, the neural component of the PNMT stress response could not be attenuated by peripheral administration of chlorisondamine, a powerful nicotinic ganglionic blocking agent. In contrast, chlorisondamine was effective in inhibiting sympathetic neural activity, as judged by the drug's ability to completely block increases in blood pressure, heart rate, and plasma catecholamines resulting from spinal cord stimulation in pithed rats. The administration of atropine, a muscarinic receptor antagonist, also failed to inhibit cold-induced alterations in adrenal PNMT mRNA. These results suggest that the trans-synaptic induction of adrenal PNMT mRNA involves a non-cholinergic component, and that cold-induced increases in PNMT mRNA are not coupled to acetylcholine-mediated adrenal catecholamine release.
Adrenal Glands↗
Neurofilament proteins are synthesized in nerve endings from squid brain.
It is generally believed that the proteins of the nerve endings are synthesized on perikaryal polysomes and are eventually delivered to the presynaptic domain by axoplasmic flow. At variance with this view, we have reported previously that a synaptosomal fraction from squid brain actively synthesizes proteins whose electrophoretic profile differs substantially from that of the proteins made in nerve cell bodies, axons, or glial cells, i.e., by the possible contaminants of the synaptosomal fraction. Using western analyses and immunoabsorption methods, we report now that (a) the translation products of the squid synaptosomal fraction include neurofilament (NF) proteins and (b) the electrophoretic pattern of the synaptosomal newly synthesized NF proteins is drastically different from that of the NF proteins synthesized by nerve cell bodies. The latter results exclude the possibility that NF proteins synthesized by the synaptosomal fraction originate in fragments of nerve cell bodies possibly contaminating the synaptosomal fraction. They rather indicate that in squid brain, nerve terminals synthesize NF proteins.
Animals↗
Discordant estimates of heterologous promoter activity as determined by reporter gene mRNA levels and enzyme activity.
In this study, the human cytomegalovirus (CMV) promoter fused to the lacZ (beta-gal) reporter gene was transfected into neuroblastoma SK-N-BE(2)-C cells, and phorbol ester-stimulated promoter activity assessed by both PCR quantitation of reporter gene mRNA levels and enzyme activity. Surprisingly, significant differences were observed in the induction profile of CMV promoter activity as judged by these two independent methods of analysis. For example, at 24 hrs post-transfection beta-gal activity was elevated 7.3-fold in phorbol ester-treated cells, whereas 2.4-fold increases were observed in the cognate mRNA levels. These findings demonstrate the efficacy of quantitative PCR methodology to evaluate promoter activity in DNA-mediated cell transfection analyses, and raise a cautionary note on the reliance of reporter gene enzyme activity to estimate the transcriptional activity of heterologous promoters.
Base Sequence↗
Cold-induced alterations in the binding of adrenomedullary nuclear proteins to the promoter region of the tyrosine hydroxylase gene.
It is well documented that cold stress induces a rapid trans-synaptically mediated increase in the relative abundance of rat adrenomedullary tyrosine hydroxylase (TH) mRNA. To investigate the transcriptional mechanisms regulating the cold stress response, we have employed a gel mobility shift assay, using DNA fragments prepared from the proximal 5' flanking region of the bovine TH gene as a heterologous molecular probe. In pilot studies, this region of the bovine TH promoter (nucleotides -246 to +21) was fused to the bacterial reporter gene, chloramphenicol acetyltransferase, and the chimeric construct transfected into human neuroblastoma SK-N-BE(2)-C, hepatoma HepG2, and rat pheochromocytoma PC-12 cells. Results of this analysis indicate that the proximal 5' flanking region of the bovine TH gene contains sufficient information to drive transient reporter gene expression in both human and rat catecholaminergic clonal cell lines. The findings derived from the gel mobility shift studies demonstrate that cold exposure causes rapid and selective alterations in the binding of adrenomedullary nuclear proteins to the proximal 5' flanking region of the TH gene. The most striking cold stress-induced alteration in DNA/nucleoprotein binding occurs in a region of the TH promoter (nucleotides -246 to -189) which contains an element bearing marked sequence similarity to an AP1 binding site and is highly conserved among animal species. This alteration occurs within 1 hr of cold exposure and persists for up to 48 hr after the onset of stress. The results of adrenal denervation experiments indicate that the cold-induced change in DNA/nucleoprotein binding is neurally mediated, requiring intact sympathetic innervation of the gland.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenal Medulla↗
Clinically-silent mutation in the putative iron-responsive element in exon 17 of the beta-amyloid precursor protein gene.
Three missense mutations in exon 17 of the beta-amyloid precursor protein (APP) gene have been reported to cosegregate in families with early onset Alzheimer's disease (AD). All three mutations result in amino acid substitutions at codon 717 and may produce AD by altering the structure of the transmembrane domain of APP. Alternatively, the mutations may destabilize the stem of a putative iron-responsive element (IRE) in which they lie and confer pathogenicity by inactivating this negative regulatory element. We have detected a clinically-silent mutation in codon 716 that would also be expected to disrupt the putative IRE but results in no amino acid substitution. This result strongly suggests that the missense mutations at codon 717 produce AD by altering the amino acid sequence of APP rather than the IRE. Furthermore, the identification of a clinically-silent mutation among four point mutations that span only three nucleotides of exon 17 suggests that this region may be a mutational "hot" spot.
Alzheimer Disease↗
Selective activation of norepinephrine- and epinephrine-secreting chromaffin cells in rat adrenal medulla.
The differential effects of insulin-induced hypoglycemia and cold exposure on adrenal medullary epinephrine (Epi) and norepinephrine (NE) cells were investigated in male Sprague-Dawley rats. In rats fasted overnight, insulin produced a marked hypoglycemia that resulted in a 70% decrease in adrenal medullary Epi content 3 h after the insulin was administered. No change in NE content was observed. Plasma Epi concentration was increased markedly after insulin, with a smaller increment in NE. In contrast, exposure to a 4 degrees C environment selectively reduced adrenal NE content, with the effect reaching statistical significance at 18 h. Cold exposure also led to a significant rise in plasma NE but not Epi. Both insulin-induced hypoglycemia and cold exposure significantly elevated adrenal dopamine, indicating that catecholamine synthesis was stimulated. Further evidence of enhanced catecholamine formation was the observation that inhibition of synthesis with alpha-methyl-p-tyrosine (AMT) greatly augmented the ability of insulin-induced hypoglycemia to selectively reduce adrenal medullary Epi content. Similarly, in cold-exposed animals, AMT pretreatment accelerated the NE depletion so that a significant decline was observed at 3 h. These results support the conclusion that the two major populations of adrenal catecholamine-secreting cells may be preferentially stimulated by different stressors. Moreover, augmented synthetic activity functions to maintain catecholamine stores in both Epi- and NE-secreting cells.
Adrenal Medulla↗
Active polysomes in the axoplasm of the squid giant axon.
Axons and axon terminals are widely believed to lack the capacity to synthesize proteins, relying instead on the delivery of proteins made in the perikaryon. In agreement with this view, axoplasmic proteins synthesized by the isolated giant axon of the squid are believed to derive entirely from periaxonal glial cells. However, squid axoplasm is known to contain the requisite components of an extra-mitochondrial protein synthetic system, including protein factors, tRNAs, rRNAs, and a heterogeneous family of mRNAs. Hence, the giant axon could, in principle, maintain an endogenous protein synthetic capacity. Here, we report that the squid giant axon also contains active polysomes and mRNA, which hybridizes to a riboprobe encoding murine neurofilament protein. Taken together, these findings provide direct evidence that proteins (including the putative neuron-specific neurofilament protein) are also synthesized de novo in the axonal compartment.
Animals↗
Evidence against close linkage of unipolar affective illness to human chromosome 11p markers HRAS1 and INS and chromosome Xq marker DXS52.
The genetic basis of various subtypes of the affective disorders has been investigated by family, twin, and adoption studies, as well as by segregation and linkage analysis. Linkage analyses of bipolar disorder with the chromosome 11p15 DNA markers HRAS1 and INS, and the chromosome Xq28 markers for color blindness and G6PD have been reported. We have used restriction fragment length polymorphisms as markers to examine linkage in three extended families with unipolar affective illness, ascertained through probands with either recurrent unipolar or bipolar II illness. Using an inclusive definition of the affected phenotype, linkage could be excluded up to 28cM around the HRAS1-INS linkage group on chromosome 11p15, and up to 5 cM around the DNA marker DXS52 on Xq28. Negative linkage results were also obtained for two more restrictive definitions of affective illness. Thus, we find no evidence for the involvement of the chromosomal regions 11p15 and Xq28 with unipolar affective disorder in these three families.
Bipolar Disorder↗
Effects of cold exposure on rat adrenal tyrosine hydroxylase: an analysis of RNA, protein, enzyme activity, and cofactor levels.
Long-term cold exposure (5-7 days) is known to induce concomitant increases in the levels of adrenomedullary tyrosine hydroxylase (TH) RNA, protein, and enzyme activity. In this report, we compare the time courses of these changes and investigate the effects of cold exposure on the levels of biopterin, the cofactor required for tyrosine hydroxylation. After only 1 h of cold exposure, TH mRNA abundance increased 71% compared with nonstressed controls. Increases in total cellular TH RNA levels were maximal (threefold over control values) within 3-6 h of cold exposure and remained elevated throughout the duration of the experiment (72 h). TH protein levels increased rapidly after 24 h of cold exposure and reached a maximal value threefold above that of controls at 48-72 h. Despite the relatively rapid and large elevations in TH RNA and protein content, only modest increases in TH activity were detected during the initial 48 h of cold exposure. Adrenomedullary biopterin increased rapidly after the onset of cold exposure, rising to a level approximately twofold that of the nonstressed controls at 24 h, and remained at this level throughout the duration of the stress period. Taken together, the results of this time course study indicate that cold-induced alterations in adrenal TH activity are mediated by multiple cellular control mechanisms, which may include pre- and posttranslational regulation. Our findings also suggest that cold stress-induced increases in the levels of the TH cofactor may represent another key event in the sympathoadrenal system's response to cold stress.
Adrenal Glands↗
Effect of cold stress on cholinergic receptors in the rat adrenal gland.
The effects of cold exposure on cholinergic binding sites in the rat adrenal gland were assessed by examining the binding of [125I]alpha-bungarotoxin (BTX), a nicotonic receptor antagonist and [3H]quinuclidinyl benzilate (QNB), a muscarinic receptor antagonist, to adrenal tissue homogenates. Cold exposure resulted in significant alterations in both nicotinic and muscarinic binding. Exposure to cold for 4 and 7 days resulted in a significant decrease in QNB binding. Scatchard analysis indicates that this alteration is due to a decrease in binding sites (Bmax) rather than a change in ligand affinity (Kd). In contrast, chronic cold exposure produced a significant increase in BTX binding sites. These results indicate that adrenal cholinergic receptors are altered in reciprocal fashion by chronic cold exposure, and that this change may represent a key event in the sympathoadrenal system's adaptive response to chronic cold stress.
Adrenal Glands↗
Isolation and structural characterization of the bovine tyrosine hydroxylase gene.
A bovine tyrosine hydroxylase (TH) cDNA probe was used to screen a charon 30 genomic library. Screening of approximately 1 million recombinant phage resulted in the identification of one clone, lambda B1, containing the entire bovine TH gene. Results derived from restriction endonuclease mapping and sequence analysis reveal that the bovine gene contains 13 exons spanning approximately 7 kb of genomic DNA. Determination of the transcription initiation site indicates that the TH gene has a 5' untranslated region of 27 bp. A TATA-box sequence is located between positions-29 and -24 from the transcription initiation site and a cyclic AMP regulatory element (CRE) between-45 and -38. Although the TH gene appears to be glucocorticoid responsive in vitro, no regions bearing identity to the consensus sequence for the glucocorticoid regulatory element (GRE) were detected within approximately 1.5 kb of 5' flanking sequence. A cross-species comparison of the 5' flanking sequences of the bovine, rat, and human TH genes reveals strong sequence and positional conservation of seven sequence elements. An analysis of the nucleotide sequence within these elements reveals similarity to the consensus sequences reported for known cis-acting regulatory elements and transcription factor binding sites, suggesting that they may play a role in the regulation of TH gene expression.
Adrenal Medulla↗