Chemical coding of endocrine cells of the airways: presence of helodermin-like peptides

Summary The epithelium of the airways is rich in endocrine cells containing serotonin and/or a wide variety of regulatory peptides. These cells usually occur in clusters in the lungs but are also found scattered in the larynx and trachea. In the present study, endocrine cells in the airways of mouse, rat, hamster, guinea pig, pig, sheep and squirrel monkey were examined for the presence of serotonin, helodermin-like peptides and other regulatory peptides using immunocytochemistry and radioimmunoassay. In addition, we looked for the protein gene product 9.5 (PGP), which occurs in many peptide hormone-producing endocrine cells in the body. Both clustered and scattered endocrine cells in the airways were found to display coexistence of serotonin and peptides, such as a helodermin-like peptide, calcitonin and calcitonin gene-related peptide (CGRP). The PGP-immunoreactive cells were numerous and included elements containing serotonin and/or regulatory peptides. An additional PGP-immunoreactive endocrine cell population lacked serotonin and regulatory peptides. Helodermin-immunoreactive material was demonstrated in endocrine cells of the airways in the mouse and hamster but not in any of the other species studied. Serotonin was an endocrine cell constituent in all the species studied. Calcitonin and CGRP could be demonstrated by immunocytochemistry in the mouse, rat, and hamster, but not in the guinea pig, sheep, pig and monkey. In the hamster airways double immunostaining indicated that the helodermin-like peptide occurred in a subpopulation of the CGRP- and serotonin-containing cells. Most of the CGRP-containing cells stored serotonin; some of them also contained calcitonin. The chemical coding of these cells resembled that of the thyroid C cells.     

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

Comparison of salt- and heat-induced alterations of protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803

Protein synthesis of the cyanobacterium Synechocystis spec. PCC 6803 decreases after a 684 mM NaCl salt shock. Qualitative changes were observed during the shock and the subsequent adaptation process using one-dimensional polyacrylamide electrophoresis. Proteins of apparent molecular masses of 13.0, 14.2, 16.6, 20.0, 21.0, 23.0, 33.0, 47.0, 52.0, 65.0 and 72.0 kDa are synthesized at enhanced rates after salt stress. The proteins of 14.2, 21.1 and 52.0 kDa are transiently induced during the first hours of the adaptation phase, while the other proteins are also synthesized at enhanced rates in salt-adapted cells. The proteins of 14.2, 23.0, 33.0 and 65.0 kDa are also induced by heat shock (43°C). Heat shock proteins of about 88.0, 75.0, 58.0, 17.5 and 13.8 kDa, in contrast, are induced by heat shock but not by salt. Two-dimensional polyacrylamide electrophoresis showed that the induced salt and heat shock proteins in some cases consisted of isoforms of different isoelectric points.Abbreviations IP isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride     

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart,and inferences on evolution of the isoenzymes

Summary We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme fromEscherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the geneduplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10⁹ years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme fromE. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3×10⁹ years ago.     

Effect of base substitutions in the colicin E1 gene on colicin E1 export and bacteriocin activity

Summary Base substitutions have been introduced into the segment of the colicin E1 gene corresponding to the polypeptide region between the 404th and the 502nd residues which was considered to participate in colicin E1 export and bacteriocin activity. The methods used were in vitro localized mutagenesis with sodium bisulphite and in vivo mutagenesis using either nitrosoguanidine or ethyl methane sulphonate. Cells carrying mutagenized plasmids were screened by their inability to form a clear zone on a lawn of colicin E1 sensitive cells. Mutation sites were determined from the nucleotide sequence analysis and the altered amino acid residues were reduced. The mutant proteins were analysed for their ability to be exported to the periplasmic space and for their bacteriocin activity. Out of eight mutants obtained, three had a single amino acid replacement. Mutant proteins that had Ser and Glu in place of Pro-462 and Gly-502, respectively, showed a decrease in both the export and the bacteriocin activity. A mutant protein having Arg in place of Gly-439 showed a decrease only in the bacteriocin activity. These results suggest that the target region of colicin E1 contributes to the export as well as the bacteriocin activity but the two functions are supported in part by different amino acid residues of the protein.     

Effects of seasonal changes of soil salinity and soil nitrogen on the N-metabolism of the halophyteArthrocnemum fruticosum (L.) Moq.

Summary The N-metabolism ofArthrocnemum fruticosum (L.) Moq., growing in a saline area north-east of the Dead Sea in Jordan, was studied over its vegetative growth period from March to September 1981. Plant and soil samples were taken at monthly intervals. Water content, Na⁺, K⁺, Cl⁻, NH ₄ ⁺ , NO ₂ ⁻ and NO ₃ ⁻ concentrations were determined in the soil extracts, and the same determinations plus ash weight, soluble carbohydrates, proline, proteins andin vivo nitrate reductase in the plant roots and shoots. Soil humidity decreased and salinity increased from March to August, with re-wetting occurring in late July. K⁺ and Cl⁻ were much lower in the soils than Na⁺. Plant relative dry weight increased during summer due to the absorption of Na⁺ in addition to increased organic dry weight. The uptake of Na⁺ was not balanced by a similar uptake of Cl⁻. Ammonium and nitrate decreased in soil and plants in parallel with increasing salinity. Nitrite was only found in the roots and always in very low quantities. Proline was found only in March. The total soluble carbohydrates in the roots showed a short increase in June when the sodium in the plants also increased. It was concluded that carbohydrates may be used to balance osmotic shocks, but that another compatible compounds is necessary to maintatin long-term osmotic equilibrium. The nitrate reductase activity, measuredin vivo, and the soluble protein changed roughly in parallel with the internal nitrate from May to August, suggesting that nitrogen uptake and reduction in the plant is inhibited during summer when the soil is dry and very saline. This could be a direct effect of drought and/or salinity on the plants, or an indirect onevia an inhibition of nitrifying bacteria.     

Release of Endogenous Amino Acids from Striatal Neurons in Primary Culture

Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.     

雄激素对大鼠储精囊分泌蛋白mRNA的调节作用

本文报道了应用DNA重组和分子克隆技术研究雄激素与大鼠储精囊分泌蛋白基因的相互作用。大鼠储精囊总mRNA在逆转录酶作用下合成dsc-DNA,并克隆于pBR322/X1776中。经原位杂交法筛选含有互补于雄激素调节的mRNA的三个克隆株,其中二株经信使选择杂交翻译法证实它们是编码54K和16.6K道尔顿的分泌蛋白质的基因。同时应用后者的cDNA作为探针进一步研究雄激素对处于不同生理态状下的大鼠储精囊mRNA水平的影响。     

A linkage study of protein-coding loci in Macaca mulatta and Macaca fascicularis

Genealogical and gene marker data from the closely related species Macaca mulatta and Macaca fascicularis have been used to search for linkage between genes coding for the blood proteins albumin, carbonic anhydrase 1 and 2, diaphorase 1 and 2, group-specific component, glucose phosphate isomerase, hemoglobin alpha chains, isocitrate dehydrogenase, prealbumin, and transferrin. The results are consistent with conservation of the linkage between the loci coding for albumin and group-specific component and loci coding for the two carbonic anhydrase isozymes, as observed in other species. Among the 38 possible pairwise comparisons, no new linkage groups were identified. Tight linkage can be excluded for most pairs of loci.