Biomedical subjects

G Mandel

Publications and source records attributed to G Mandel.

At least 55 records · Page 3Linked to original sources

PubMed1989

Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity.

Rat preprosomatostatin (rPPSS) is processed to two bioactive peptides, somatostatin-14 and somatostatin-28. In anglerfish islets, the two peptides are synthesized by distinct cell types and are derived from different precursors, anglerfish preprosomatostatin-1 (a(I)PPSS) and anglerfish preprosomatostatin-2 (a(II)PPSS). To determine the basis of the differential processing, we introduced a(I)PPSS or a(II)PPSS expression vectors into mammalian endocrine cell lines that can accomplish both patterns of processing. Both precursors were processed identically, indicating that cellular factors must determine the processing pattern. Although similar processing sites are present in both precursors, high levels of unprocessed anglerfish prosomatostatin-2 were secreted constitutively from the transfected cells. A hybrid protein containing the leader sequence and a portion of the pro-region of rPPSS fused to the carboxy-terminal third of a(II)PPSS was processed and secreted via a regulated pathway. We conclude that the amino-terminal 78 residues of rPPSS contain sufficient information to correct the targeting deficiency of a(II)PPSS in mammalian endocrine cell lines.

Adrenal Cortex↗

PubMed1989

Isolation and characterization of human apatite-inducing aortic proteolipid.

A proteolipid, capable of inducing hydroxyapatite in vitro, can be isolated from human aortic calcified lesions, noncalcified lesions, and nonlesion tissue obtained at autopsy. Analysis of the amino acid composition after acid hydrolysis showed all nucleators to be high in the hydrophobic amino acids glycine and alanine. Estimation of the molecular weight of the nucleator, extracted from calcified lesions, by gel filtration and lipid content showed a minimum molecular weight of 469,000 Da. All nucleators were characterized by the presence of acidic phospholipids which may have a role in the mechanism of calcification.

Adult↗

PubMed1989

Localization of a human brain sodium channel gene (SCN2A) to chromosome 2.

A DNA probe derived from a human genomic library has been used to localize on human chromosomes a gene coding for the alpha-subunit of the brain type II sodium channel (SCN2A). Hybridization of the probe to Southern blots made with DNAs from a rodent-human somatic cell hybrid panel indicates localization to the long arm of human chromosome 2. In situ hybridization to metaphase chromosomes confirms this assignment and indicates regional localization to 2q21-q33. The probe also reveals a frequent two-allele HaeIII RFLP.

Animals↗

PubMed1989

Expression of diverse Na+ channel messenger RNAs in rat myocardium. Evidence for a cardiac-specific Na+ channel.

This study examined the diversity of Na+ channel gene expression in intact cardiac tissue and purified myocardial cells. The screening of neonatal rat myocardial cell cDNA libraries with a conserved rat brain Na+ channel cDNA probe, resulted in the isolation and characterization of a putative rat cardiac Na+ channel cDNA probe (pCSC-1). The deduced amino acid sequence of pCSC-1 displayed a striking degree of homology with the eel, rat brain-1, and rat brain-2 Na+ channel, thereby identifying pCSC-1 as a related member of the family of Na+ channel genes. Northern blot analysis revealed the expression of a 7-kb CSC-1 transcript in rat cardiac tissue and purified myocardial cells, but little or no detectable expression of CSC-1 in rat brain, skeletal muscle, denervated skeletal muscle, or liver. Using RNase protection and Northern blot hybridization with specific rat brain Na+ channel gene probes, expression of the rat brain-1 Na+ channel was observed in rat myocardium, but no detectable expression of the rat brain-2 gene was found. This study provides evidence for the expression of diverse Na+ channel mRNAs in rat myocardium and presents the initial characterization of a new, related member of the family of Na+ channel genes, which appears to be expressed in a cardiac-specific manner.

Amino Acid Sequence↗

PubMed1988

Fertilization events induced by neurotransmitters after injection of mRNA in Xenopus eggs.

Fertilization initiates in the egg a dramatic increase in intracellular calcium that opens ion channels and causes exocytosis. To explore the possibility that these events might involve a receptor-mediated pathway, receptors for serotonin or acetylcholine (M1 muscarinic) were expressed in the Xenopus egg; serotonin or acetylcholine then could initiate a series of responses similar to those normally initiated by sperm. Thus, there may be an endogenous receptor in the egg membrane that is activated by sperm, and the serotonin or M1 muscarinic receptor may replace the sperm receptor in this pathway.

Animals↗

PubMed1988

Biosynthesis of thyrotropin-releasing hormone by a rat medullary thyroid carcinoma cell line.

Prepro-thyrotropin-releasing hormone (TRH) messenger RNA was detected in the rat medullary thyroid carcinoma cell line CA77. The RNA of 1.6 kilobases comigrated with that found in rat hypothalamus. Using three radioimmunoassays specific for pro-TRH-derived peptides, we demonstrated that CA77 cells synthesize high levels of immunoreactive TRH and all of the other pro-TRH-derived peptides identified in hypothalamic tissue. The relative levels of the pro-TRH-derived peptides also indicate that CA77 cells process the TRH precursor in a manner similar to hypothalamic tissue. CA77 cells provide a promising model system for further studies of prepro-TRH gene regulation and post-translational maturation.

Animals↗

PubMed1988

Serum and plasma inhibit neutrophil stimulation by hydroxyapatite crystals. Evidence that serum alpha 2-HS glycoprotein is a potent and specific crystal-bound inhibitor.

Tissue deposits of basic calcium phosphate (BCP) crystals are associated with various clinical manifestations of inflammation. We addressed the possibility that native proteins modify the ability of hydroxyapatite (HA) crystals to stimulate human inflammatory cells. Neutrophil superoxide release and chemiluminescence in response to HA crystals (0.3-4.0 mg/ml) were blunted by serum and plasma. Inhibitory activity was progressively removed from serum by sequential adsorption with HA crystals, suggesting that the inhibitors were crystal-bound proteins. Thus, we characterized HA crystal-bound plasma proteins by O'Farrell gels: Fibronectin, transferrin, albumin, alpha 2-HS glycoprotein (AHSG), alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, Gc globulin, haptoglobin, and high density lipoprotein apolipoproteins were major bound species. Of these, AHSG was the most active inhibitor of HA-induced neutrophil superoxide release, and this glycoprotein partially (60%) restored inhibitory activity to HA-adsorbed serum. AHSG also bound in vitro to the related BCP crystal, octacalcium phosphate, but only minimally to calcium pyrophosphate dihydrate crystals and monosodium urate crystals. Suppressive effects on neutrophil stimulation exhibited by AHSG were also specific for BCP crystals. AHSG was present in noninflammatory synovial fluids bound to synthetic HA crystals in vitro, and AHSG could be detected on native synovial fluid HA crystals. We conclude that the binding of AHSG may modulate the inflammatory potential of BCP crystals.

Blood Physiological Phenomena↗

PubMed1988

The effect of hydroxyapatite crystallinity on hemolysis.

Crystalline hydroxyapatite is a component of bone, teeth, and numerous pathological calcifications. The apatite crystal structure can accommodate a wide variety of atomic substitutions which gives apatite crystals an unusually high degree of variability in biochemical and physical properties. Apatite crystallites interact with numerous cellular systems in vivo, and some of these interactions may lead to altered cellular function. One measure of crystal-membrane interactions is crystal-induced membranolysis of human red blood cells. Hemolytic potentials at constant crystal surface areas were measured at 1, 2, and 4 hours for 29 different preparations of apatite. Each apatite sample was characterized by its morphology, particle size, % CO3, zeta potential, and broadening of the (211), (112), (300), (202), and (002) diffraction maxima. Only the surface area/g and the X-ray powder diffraction line broadening showed a significant inverse correlation with hemolytic potential. These parameters were related to each other, and are indications of the degree of crystallinity.

Crystallization↗

PubMed1988

The CGTCA sequence motif is essential for biological activity of the vasoactive intestinal peptide gene cAMP-regulated enhancer.

cAMP-regulated transcription of the human vasoactive intestinal peptide gene is dependent upon a 17-base-pair DNA element located 70 base pairs upstream from the transcriptional initiation site. This element is similar to sequences in other genes known to be regulated by cAMP and to sequences in several viral enhancers. We have demonstrated that the vasoactive intestinal peptide regulatory element is an enhancer that depends upon the integrity of two CGTCA sequence motifs for biological activity. Mutations in either of the CGTCA motifs diminish the ability of the element to respond to cAMP. Enhancers containing the CGTCA motif from the somatostatin and adenovirus genes compete for binding of nuclear proteins from C6 glioma and PC12 cells to the vasoactive intestinal peptide enhancer, suggesting that CGTCA-containing enhancers interact with similar transacting factors.

Base Sequence↗

PubMed1988

Selective induction of brain type II Na+ channels by nerve growth factor.

Cells derived from a rat pheochromocytoma (PC12 cells) can generate an action potential only upon treatment with nerve growth factor. Using electrophysiological methods, we found that the appearance of action potentials in nerve growth factor-treated PC12 cells can be explained by an increase in the density of Na+ channels. The functional properties of Na+ channels in PC12 cells are similar to those described for peripheral nerves but appear to be different from Na+ channels synthesized in Xenopus oocytes injected with brain type II Na+ -channel mRNA. To determine if PC12 cells express the brain type II Na+ -channel gene, we performed RNase-protection analyses using probes that can distinguish between the brain type I and type II Na+ -channel mRNAs. The results from these studies indicate that undifferentiated PC12 cells express the type II but not the type I Na+ -channel gene. Treatment with nerve growth factor increases expression of the type II Na+ -channel gene but has no effect on type I gene expression. Our findings suggest that Na+ -channel excitability in PC12 cells is due to the specific induction of the brain type II gene by nerve growth factor.

Action Potentials↗

PubMed1988

A Moloney MLV-rat somatotropin fusion gene produces biologically active somatotropin in a transgenic pig.

Expression of a Moloney murine leukemia virus (MLV) rat somatotropin fusion gene was examined in a transgenic pig. The fusion gene was integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated MLV-rat somatotropin fusion gene produced high levels of circulating rat somatotropin and resulted in an elevation in the circulating levels of insulin-like growth factor I. Although there was no increase in the rate of growth of the transgenic animal during the rapid growth phase, several phenotypic changes were evident. Skeletal growth was markedly increased and fat deposition was reduced throughout the animal. Blood glucose levels were elevated without ketosis. Northern blot analyses of rat somatotropin RNA revealed that expression of the fusion gene was highest in the spleen, lung, intestine, lymph nodes, and bone marrow. These results show that the MLV promoter can be used to express high levels of biologically active rat somatotropin in transgenic swine.

Animals↗

PubMed1988

Calcium pyrophosphate crystal deposition in model systems.

The chemistry and molecular bonding characteristics of the CaPPi family of compounds are very complex. The unique molecular flexibility of the PPi anion and the potential variability of Ca coordination geometries have allowed for a broad spectrum of CaPPi type structures. The structure of t-CPPD has the smallest P-OB-P angle of the known CaPPi structures, both Ca atoms are 7 coordinate which is the maximum allowable contacts for Ca atoms, and the two water molecules of crystallization not only serve to fill molecular space, but they are also involved in direct contact to the PPi anions and the Ca atoms. The structure of t-CPPD appears to be very stable and the structural characteristics support the observation that the crystals are sparingly soluble in an aqueous environment. Unfortunately, the structure of m-CPPD is not known and comparisons cannot be made. The solution model studies have resulted in the observation that t-CPPD and m-CPPD crystals can be grown in an aqueous environment at conditions far less harsh than those required for the standard synthetic procedure. However, the synthetic procedure, in contrast to the solution models, yields the prismatic crystal growth morphology of t-CPPD and the rod morphology of m-CPPD observed in vivo. The solution models showed that increasing Mg or Pi retarded crystal formation. At physiologic levels of Mg and Pi, a-CaPPi formed, but neither t-CPPD nor m-CPPD would form. In all solution studies, the final Ca and PPi were not determined and therefore a correlation could not be made between the ionic concentrations and crystal type formed. The gel models using silica, polyacrylamide, and biologic grade gelatin all highlighted that the time of incubation of Ca and PPi ions was a critical parameter in determining the type of crystal formed. The biologic grade gelatin model studies that we conducted indicated that the formation of the two in vivo crystals was mediated by the formation of intermediate crystalline materials and the subsequent dissolution of those species. This formation/dissolution/reformation mechanism allows for a very localized ionic concentrating process to occur. In our model system, we measured the final Ca and PPi levels at all points of crystallization and could map the ionic concentration gradients and compare them to the crystal type formed with respect to the time of incubation. However, the crystal growth morphologies for t-CPPD and m-CPPD still did not match the morphologies observed in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Anions↗

PubMed1987

Peptide YY. Structure of the precursor and expression in exocrine pancreas.

Peptide YY is a 36-residue gastrointestinal hormone which inhibits both pancreatic and gastric secretion. We have isolated a cDNA encoding the peptide YY precursor by screening a rat intestinal lambda gt11 cDNA library with an antiserum directed against the porcine hormone. The nucleotide sequence of the cDNA encodes a 98-residue protein (molecular weight, 11, 121) which has an amino acid sequence identical to that of porcine peptide YY. Rat peptide YY is preceded immediately by a signal sequence and followed by a cleavage-amidation sequence Gly-Lys-Arg plus 31 additional amino acids. Thus the peptide YY precursor is similar in structure to that of two related peptides, pancreatic polypeptide and neuropeptide Y. RNA blot hybridizations reveal that the peptide YY gene is much more actively expressed in pancreas than previously realized. In situ hybridizations localized peptide YY cells exclusively to the exocrine pancreas. The abundance of peptide YY in one of its target organs, the pancreas, suggests a paracrine mechanism for peptide YY in regulating pancreatic enzyme secretion.

Amino Acid Sequence↗

PubMed1987

Genomic organization and deduced amino acid sequence of a putative sodium channel gene in Drosophila.

The deduced amino acid sequence of a Drosophila gene isolated with a vertebrate sodium channel complementary DNA probe revealed an organization virtually identical to the vertebrate sodium channel protein; four homologous domains containing all putative membrane-spanning regions are repeated in tandem with connecting linkers of various sizes. All areas of the protein presumed to be critical for channel function show high evolutionary conservation. These include those proposed to function in voltage-sensitive gating, inactivation, and ion selectivity. All 24 putative gating charges of the vertebrate protein are in identical positions in the Drosophila gene. Ten introns interrupt the coding regions of the four homology units; introns with positions conserved among homology units bracket a region hypothesized to be the selectivity filter for the channel. The Drosophila gene maps to the right arm of the second chromosome in region 60D-E. This position does not coincide with any known mutations that confer behavioral phenotypes, but is close to the seizure locus (60A-B), which has been hypothesized to code for a voltage-sensitive sodium channel.

Amino Acid Sequence↗

PubMed1987

Secretion of apolipoprotein A-I in lipoprotein particles following transfection of the human apolipoprotein A-I gene into 3T3 cells.

Apolipoprotein A-I (apoA-I) is the major protein constituent of plasma high density lipoproteins (HDL). To examine apoA-I processing and secretion, the human apoA-I gene (2.2-kilobase PstI-PstI fragment) linked to the mouse metallothionein promoter was transfected by electroporation into NIH 3T3 fibroblasts along with the plasmid pSV2 neo, which confers neomycin resistance. Transfected cells were selected for neomycin resistance and screened for the ability to produce apoA-I by enzyme-linked immunosorbent assay. In the absence of lipids in the medium, selected 3T3 cells secreted apoA-I, mainly in the proprotein form, at density greater than 1.25 g/ml. Following incubation of cells with lipids, and subsequent washing with lipid-free medium, apoA-I was recovered in the HDL region (1.063-1.21 g/ml) as well as in the 1.21 g/ml infranatant. Examination of the HDL fraction by electron microscopy revealed round particles, 10-21 nm in diameter. These data indicate that human apoA-I secreted by transfected 3T3 fibroblasts can assemble into lipoprotein particles under the appropriate conditions.

Animals↗

PubMed1987

Identification of a region in the human vasoactive intestinal polypeptide gene responsible for regulation by cyclic AMP.

Transcription of the vasoactive intestinal polypeptide (VIP) gene is regulated by cAMP. To identify the nucleotide sequences in the human VIP gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5'-flanking region of the human VIP gene fused to the structural sequence encoding the bacterial reporter enzyme chloramphenicol acetyltransferase (CAT). The transcriptional activities of the fusion genes introduced into the rat pheochromocytoma cell line PC12 were assayed by measuring CAT activity in the cell lysates. Forskolin, an adenylate cyclase-activating agent, stimulated the expression of VIP-CAT fusion genes. Deletional analysis demonstrated that a region between -86 and -70 nucleotides upstream from the transcriptional origin of the human VIP gene was responsible for stimulation by forskolin. This region was able to confer cAMP-responsiveness to a gene that is not normally regulated by cAMP. Two copies of a 5 base pair motif, 5'-CGTCA-3', are required for activity of the VIP cAMP regulatory region. This motif is also present in the cAMP regulatory region of several other eukaryotic genes.

Acetyltransferases↗

PubMed1987

Cell-specific processing of preprosomatostatin in cultured neuroendocrine cells.

We have previously found that preprosomatostatin is processed accurately to both somatostatin-14 and somatostatin-28 in pituitary gonadotrophs of transgenic mice. The foreign somatostatin peptides have been shown to enter the regulated secretory pathway of these cells. To determine whether accurate preprosomatostatin processing can occur in any neuroendocrine cell, we introduced preprosomatostatin cDNA expression vectors into several different neuroendocrine cell lines. We found that prosomatostatin was cleaved efficiently to somatostatin-14 and somatostatin-28 in RIN 5F and AtT20 cells, but not in GH4 or PC12 cells. The ability of a particular cell type to process prosomatostatin did not correlate with cellular storage capacity and was independent of the level of biosynthesis of the precursor. These data suggest that prosomatostatin processing requires specific pathways which are present in some neuroendocrine cells, but not in others.

Adrenal Gland Neoplasms↗

PubMed1987

Expression of influenza hemagglutinin-polyoma T-antigen fusion proteins in a rat embryo fibroblast cell line.

Plasmids encoding the amino terminal portion of an influenza virus hemagglutinin (HA) fused to polyoma virus middle T (mT) or large T (lT) sequences have been constructed. Stable expression of the chimeric proteins was obtained in established rat embryo fibroblasts following plasmid co-transfection and selection for G418 resistance. The synthesis and localization of the proteins was followed by metabolic labeling with [35S]methionine and [3H]mannose, cell fractionation, and immunoprecipitation with anti-polyoma T antibody. The HA leader and amino terminal peptide direct the synthesis of the lT and mT proteins into the endoplasmic reticulum where they undergo glycosylation, but this occurs with a very low efficiency. Most of the HA-mT and HA-lT fusion protein molecules do not enter completely into the endoplasmic reticulum, but rather achieve their normal locations in the cell as slightly higher molecular weight proteins, presumably due to the extra sequences derived from HA at their amino termini. HA-mT fusion protein is found to have associated tyrosine-specific protein kinase activity precipitable with anti-src as well as anti-T antibody, and cells expressing this fusion protein have a transformed phenotype.

Animals↗