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D Shields

Publications and source records attributed to D Shields.

At least 73 records · Page 4Linked to original sources
PubMed1985

Somatostatin-like immunoreactivity within neuritic plaques.

Alzheimer's disease or senile dementia of the Alzheimer type (SDAT) is a progressive neurodegenerative disease that is characterized pathologically by two types of microscopic lesions in the neocortex: the neurofibrillary tangle and neuritic plaque. The concentration of neuritic plaques is correlated with significant reductions in the level of specific neurotransmitter and neuropeptide systems in autopsied brains of patients with SDAT, including decreased amounts of the tetradecapeptide, somatostatin. The clinical effects of reduced cortical somatostatin activity in patients with SDAT is unclear, nor is it known whether somatostatinergic neurons participate in either lesion. In the present study we employed light microscopic immunocytochemistry to determine whether somatostatin-containing neurons participate in the formation of neuritic plaques. Examination of selected cortical regions from autopsied brains revealed 20-50% of all neuritic plaques contained somatostatin-positive profiles indicating that processes of somatostatinergic neurons are associated with neuritic plaque formation.

Aged↗
PubMed1984

Cell-free biosynthesis of multiple preprosomatostatins: characterization by hybrid selection and amino-terminal sequencing.

In vitro translation of mRNA isolated from islets of Langerhans results in the synthesis of three major preprosomatostatins of Mr 19 000, 18 000, and 16 000, each of which can be resolved into several isoelectric forms [Warren, T. G., & Shields, D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3729-3733]. Here we present further characterization of the somatostatin precursors by (i) hybrid selection translation of specific preprosomatostatin mRNAs, (ii) in vitro proteolytic processing of the nascent preprosomatostatins synthesized from hybrid-selected mRNAs, (iii) comparison of their tryptic peptides, and (iv) partial amino-terminal sequence analysis of the signal peptide regions. Hybrid selection experiments using specific cDNA clones demonstrated which preprosomatostatin species corresponded to previously characterized precursor cDNAs [Hobart, P., Crawford, R., Shen, L. P., Picket, R., & Rutter, W. J. (1980) Nature (London) 288, 137-141]; thus, the polypeptide encoded by plasmid pLaS1 corresponds to one form of the Mr 18 000 preprosomatostatins while one form of the Mr 16 000 preprosomatostatins is encoded by pLaS2. Analysis of the tryptic peptides demonstrated that the Mr 16 000 molecule possessed the mature hormone sequence at the carboxyl terminus, as had been shown for the Mr 19 000 and 18 000 precursors. Partial NH2-terminal sequence analysis (a) confirmed the data from hybrid selection and (b) demonstrated that the Mr 18 000 precursor contained a signal peptide manifesting amino acid heterogeneity at certain positions in the signal peptides of each preprosomatostatin. It is suggested that this heterogeneity might account, in part, for variants of the preprosomatostatin molecules.

Amino Acid Sequence↗
PubMed1984

Expression of preprosomatostatin in heterologous cells: biosynthesis, posttranslational processing, and secretion of mature somatostatin.

Somatostatin is a 14 amino acid peptide hormone that is synthesized as part of a larger precursor, preprosomatostatin, which comprises about 120 amino acids. The overall organization of the precursor is conserved in many species in that it consists of a signal peptide followed by a proregion of 90-100 amino acids and the mature hormone is located at the carboxyl terminus of the molecule. To understand the role of the propeptide in generating the mature hormone, we have used gene-transfer experiments to introduce angler fish preprosomatostatin into mammalian cells. Here we report the results of transfection of COS-7 cells with an SV40 expression vector containing preprosomatostatin cDNA cloned into the VP-1 late gene. Analysis of the parameters of somatostatin gene expression showed that COS cells synthesized prosomatostatin, which was detected intracellularly; the prosomatostatin, was proteolytically processed to mature somatostatin; and the mature hormone was secreted by the COS cells into the tissue culture medium. Our results suggest that COS cells, which do not normally secrete polypeptide hormones, contain the necessary proteolytic processing enzymes to convert preprosomatostatin to the mature hormone and the cellular apparatus necessary for its secretion.

Animals↗
PubMed1984

Mouse myeloma cells that make short immunoglobulin heavy chains: pleiotropic effects on glycosylation and chain assembly.

Two variants in immunoglobulin heavy chain production, derived from the MPC 11 mouse myeloma cell line, make short heavy (H) chains with identical precise deletions of the CH3 domain. The CH3 domain is expressed in the H chain mRNA from both variants. Although in vitro translation of this mRNA produces one H chain species, deleted heavy chains are secreted as heavy-light (HL) and H2L2 moieties in contrast to MPC 11, which secretes only H2L2 . The heavy chains of HL apparently contain more carbohydrate (CHO+) than do the H chains of H2L2 , and inhibition of N-linked glycosylation results in the secretion of relatively more H2L2 . Here we present evidence suggesting that (a) the absence of the CH3 domain has led to conformational changes in these molecules, (b) these changes permit posttranslational glycosylation, and (c) unrestrained glycosylation can frequently yield unusual CHO+ structures that make complete assembly unlikely.

Amino Acid Sequence↗
PubMed1984

Somatostatin discriminates between the intracellular pathways of secretory and membrane proteins.

Somatostatin is a14-amino acid peptide hormone that inhibits the secretion of a variety of other polypeptide hormones, including growth hormone. Here we describe an experimental system used to determine whether somatostatin can discriminate in its inhibition between secretory and plasma membrane proteins. Growth hormone-secreting cells (GH3) were infected with vesicular stomatitis virus and pulse-chased with [35S]methionine to follow the simultaneous intracellular transit of growth hormone and the viral membrane glycoprotein, G protein. Secretion of growth hormone was monitored by immunoprecipitation of chase media, while appearance of G protein on the plasma membrane was detected by cell surface labeling and virus purification. In the presence of somatostatin (10 micrograms/ml), the secretion of growth hormone was inhibited by 80%. In contrast, G protein appeared on the plasma membrane with slightly enhanced kinetics. When cells were treated with the ionophore monensin (0.2 microM), there was a dramatic inhibition of both the secretion of growth hormone and the incorporation of G protein into plasma membranes. Our results on the differential effect of somatostatin provide evidence for sorting of secretory and membrane proteins into distinct compartments in the secretory pathway. The data further suggest that this sorting event occurs late in the Golgi complex or after proteins exit from that organelle.

Animals↗
PubMed1984

Sodium butyrate stimulates somatostatin production by cultured cells.

Expression of the neuropeptide SRIF can be enhanced by sodium butyrate, a known modulator of gene transcription. Two different cell lines, RIN cells (derived from a rat pancreatic islet tumor) as well as HeLa cells, showed basal secretion of SRIF immunoreactive material that was induced between 5- and 15-fold by sodium butyrate. The induction was dose and time dependent, fully reversible, and specific for sodium butyrate. Analysis of the induced mRNAs by Northern blot hybridization using a SRIF specific cDNA probe revealed a 12-fold increase in the level of SRIF mRNA caused by butyrate treatment. These results suggest that sodium butyrate may be a useful tool for studying the regulation of SRIF gene expression.

Adenoma, Islet Cell↗
PubMed1983

Cell-free processing and segregation of insulin precursors.

The biosynthesis, segregation, and processing of preproinsulin (116 amino acids) was investigated to determine the mechanism(s) by which it is translocated across the endoplasmic reticulum membrane. Islet mRNA was translated in the wheat germ cell-free system, and at various times during preproinsulin synthesis, puromycin was added, followed by addition of microsomal membranes. Neither processing of preproinsulin nor translocation of proinsulin into microsomal membranes occurred in the presence of puromycin. Synchronization of preproinsulin translation by addition of 7-methylguanosine 5'-phosphate enabled the timing of preproinsulin synthesis and proinsulin (91 amino acids) segregation into microsomal membranes to be determined. Membrane binding occurs when about 60 amino acids have been polymerized, i.e. prior to the completion of the polypeptide chain. The binding of signal recognition particle to the nascent signal is demonstrated to be an absolute requirement for translocation and processing of preproinsulin. The results indicate that segregation and processing of preproinsulin are co-translational events; no evidence for a post-translational mechanism was found. Furthermore, this work, together with similar studies, suggests that presecretory polypeptides must be synthesized as part of a precursor with a minimum size of 60-80 amino acids in order to effect membrane binding and translocation of the polypeptide chain within the intracisternal space of the endoplasmic reticulum.

Animals↗
PubMed1982

Cell-free biosynthesis of somatostatin precursors: Evidence for multiple forms of preprosomatostatin.

It has been demonstrated [Shields, D. (1980) J. Biol. Chem. 255, 11625-11628] that mRNA isolated from the islets of Langerhans codes for two preposomatostatin molecules of apparent molecular weights 18,000 and 19,000, respectively. Here evidence is presented that in vitro translation of pancreatic islet mRNA in two different cell-free protein-synthesizing systems directs the synthesis of up to nine distinct forms of somatostatin-immunoreactive polypeptides. The multiplicity of the preprosomatostatin molecules was the result of initiation of translation from separate species of mRNA as demonstrated by amino-terminal labeling with N-formyl-[35S]Met-tRNAMetf. Translation of islet mRNA isolated from different individual animals showed that all of the preposomatostatin polypeptides were present amongst the cell-free products, which implies that the multiple forms were not due to genetic variation in the wild population. Based on their apparent molecular weights and distinctly different isoelectric points, the different preprosomatostatin molecules could be classified into two major families. These results suggest that the anglerfish preprosomatostatins are encoded by separate mRNA species and are consistent with the existence of a multigene family for somatostatin.

Animals↗
PubMed1981

Cell-free synthesis and processing of multiple precursors to glucagon.

Glucagon, a polypeptide hormone of 29 amino acids, is synthesized in the islets of Langerhans and immunoreactive forms of the molecule have been found in several tissues. Like many other polypeptide hormones, glucagon is synthesized via a larger precursor molecular, proglucagon; however, estimates of its size vary considerably and the biosynthetic relationship between some of the putative precursors and authentic secreted glucagon is unclear. Consequently it was of interest to investigate the primary translation product of glucagon mRNA to relate its size to that of previously described glucagon precursors. Here we provide evidence for three distinct immunoreactive preproglucagon molecules, two of which have an apparent molecular weight (MW) of approximately 16,000 (16K). Furthermore, when microsomal membranes were present during translation, the nascent 14K preproglucagon polypeptides were processed to proglucagon with a higher apparent MW of 15,000. In contrast, the nascent 16K preproglucagon was co-translationally processed to a slightly smaller polypeptide. The data indicate that the 14K and 16K preproglucagons undergo different types of post-translational modification.

Animals↗
PubMed1980

In vitro biosynthesis of somatostatin. Evidence for two distinct preprosomatostatin molecules.

mRNA isolated from angler fish islets of Langerhans was translated in the wheat germ cell-free protein-synthesizing system and the products identified by immunoprecipitation with specific antibodies to somatostatin followed by sodium dodecyl sulfate gel electrophoresis. As previously shown (Shields, d. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4074), a major polypeptide of 18,000 dalton, designated preprosomatostatin, was immunoprecipitable. Here, evidence is presented for an additional somatostatin-immunoreactive polypeptide of apparent Mr = 19,000. The 19 kilodalton polypeptide was similar, but not identical with the 18 kilodalton preprosomatostatin, as determined by tryptic peptide analysis. Comparison of the tryptic peptides of the 19,000 dalton polypeptide with those of unlabeled somatostatin demonstrated that it contained the authentic somatostatin sequence. Like the 18,000 dalton precursor, the 19,000 dalton polypeptide had the mature somatostatin sequence located at its COOH terminus; it is proposed that this molecule is a minor species of preprosomatostatin.

Animals↗
PubMed1980

In vitro biosynthesis of fish islet preprosomatostatin: evidence of processing and segregation of a high molecular weight precursor.

Evidence is presented for a precursor to somatostatin that is 10-12 times larger than the authentic secreted hormone. mRNA from angler fish (Lophius americanus) islets of Langerhans was translated in the wheat germ cell-free system and the products were identified by immunoprecipitation with specific antibodies to somatostatin followed by sodium dodecyl sulfate gel electrophoresis. One 18,000-dalton polypeptide was specifically immunoprecipitable. Competition experiments showed that authentic somatostatin competed with the 18,000-dalton molecule for antibody binding. When dog pancreas microsomal membranes were present during translation, an additional polypeptide of 16,000 daltons was also immunoprecipitable. Comparison of their tryptic peptides demonstrated that the 16,000-dalton polypeptide was derived from the 18,000-dalton one. Tryptic peptide analysis of somatostatin and the 18,000-dalton precursor demonstrated that the 18,000-dalton polypeptide contains the authentic somatostatin amino acid sequence and suggests that it is located at the carboxyl terminus of the precursor molecule and is preceded by a basic amino acid.

Animals↗
PubMed1979

Association of messenger ribonucleic acid with mammalian microsomal membranes: characterization by analysis of cell-free translation products.

Total rough microsomes, isolated from the dog pancreas, were stripped of membranes-bound polysomes by treatment with either EDTA or puromycin and 0.5 M KCl. The stripped microsomal membranes were isolated relatively free from contamination, by using buoyant density centrifugation, and mRNA was isolated from both the membrane fraction and the released material. Depending on the method used to strip the rough microsomes, we found a variable but small percentage (3--15%) of the cellular poly(A)-containing mRNA attached to the microsomal membranes. Reextraction of isolated microsomal membranes with puromycin and 0.5 M KCl reduced the content of membrane-associated mRNA by approximately 50%, resulting in less than 2% of the total membrane-bound polysomal mRNA remaining associated with the microsomal membranes. The membrane-associated mRNA was characterized by translation in the wheat germ cell-free protein synthesizing system, and the products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The translation products of the membrane-associated mRNA were identical with those from the total pancreas mRNA and also with those obtained by using mRNA isolated from material released directly from the rough microsomes.

Animals↗
PubMed1979

Discontinuous translation of silk fibroin in a reticulocyte cell-free system and in intact silk gland cells.

Silk fibroin mRNA was translated in a rabbit reticulocyte cell-free system. Addition of tRNA from silk glands was essential for complete translation of the fibroin polypeptide. (Mr approximately 400,000). Synthesis of full-sized product took at least 85 min. In addition to full-size product, a large number of smaller polypeptides were observed upon analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Evidence is presented that these smaller polypeptides are growing fibroin chains that transiently accumulate as discrete size classes due to discontinuities in the translation process. These discontinuities, or pauses, occur at specific sites in the fibroin mRNA template. The relative duration of the pauses can be experimentally modulated by changing the source of the supplementary tRNA added to the in vitro system. Silk glands were incubated in organ culture under conditions where essentially exclusive labeling of newly synthesized fibroins was attained. Analysis in sodium dodecyl sulfate gels showed that the labeling pattern of nascent silk fibroins is similar to the pattern observed in the reticulocyte cell-free system. This result suggests that discontinuities or pauses in polypeptide chain elongation also occur in vivo under conditions of organ culture.

Animals↗