Post-translational processing of the highly processed,secreted periplasmic carbonic anhydrase of Chlamydomonas is largely conserved in transgenic tobacco

The periplasmic carbonic anhydrase (CA) gene CAH1 of Chlamydomonas reinhardtii codes for a highly processed secreted glycoprotein. The primary translation product of the CAH1 gene is targeted to the ER, where it is proteolytically processed to yield two different subunits, glycosylated, assembled into an active heterotetramer, and secreted. After replacing the target leader sequence with that from tobacco anionic peroxidase, expression of this gene in transgenic tobacco plants was investigated. SDS-PAGE gels of the purified protein from tobacco, showed that it migrated as a series of discrete bands (two large and one small) with slightly faster mobility than the comparable bands in the purified algal protein. The expressed protein in the plant was active, and staining with thymol and sulfuric acid confirmed that it was also glycosylated. The periplasmic CA1 (peri-CA1) also was found to be enriched in the intercellular fluid of transgenic tobacco, indicating it was secreted. The specific activity of the enzyme and its sensitivity to sulfonamide inhibitors were similar to that of the native algal enzyme. These results suggest that the post translational processing of Chlamydomonas peri-CA1 is largely conserved in a higher plant.     

Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei

The Aspergillusniger and Trichodermareesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains five introns, whereas the T. reesei gene (sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70–80% identity to the SAR1 protein. Complementation of S. cerevisiaesar1 and sec12 mutants by expression vectors carrying the A. nigersarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. nigersarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger. Received: 21 January 1997 / Accepted: 21 June 1997     

Morphogenetic effects of Brefeldin A on embryogenic cell cultures of Daucus carota L.

Brefeldin A, an inhibitor of protein secretion, caused typical alterations to the endomembrane system with limited effects on viability when given to unorganized carrot cells growing in suspension. When given to the same cells during particular stages of embryogenesis, it caused similar endomembrane lesions and an almost complete arrest of the embryogenic process. Addition of conditioned medium containing extracellular secreted proteins to the embryos during treatment with Brefeldin A allowed acquisition of polarity and the continuation of a quasi-normal embryogenic process. Received: 4 December 1996 / Accepted: 4 March 1997     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

花生油对酒精所致胃粘膜损伤的保护作用

着重研究了花生油对无水酒精所引起的大白鼠胃粘膜损伤的保护作用,并对花生油作用的有效部位和机制作了初步探讨。     

Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol

Early responses of cholesterol metabolism to dietary cholesterol were compared between exogenous hypercholesterolemic (ExHC) and Sprague-Dawley rats. Both strains had a similar radioactivity of [¹⁴C]cholesterol in the serum half a day after the oral administration, but thereafter the radioactivity disappeared slowly in ExHC rats. ExHC rats promptly altered in response to the dietary cholesterol, activities of cholesterol 7α-hydroxylase and cholesterol synthesis in the liver and fecal excretion of bile acids derived from [¹⁴C]cholesterol administered orally. Lymphatic transport for 24 hr of [¹⁴C]cholesterol was similar between the strains. Triton administration resulted in a marked accumulation of cholesterol in serum d > 1.006 g/ml lipoproteins in ExHC rats; in addition, the formation of cholesteryl esters from [¹⁴C]oleic acid intravenously infused was greater in ExHC rats. These results indicate that ExHC rats increase serum cholesterol in response to exogenous cholesterol by decreasing the liver uptake and enhancing the secretion in the liver.     

CCK-resistance in Zucker obese versus lean rats

Obese Zucker rats are less sensitive to the satiety effect of CCK than lean litter mates. The present studies further characterised this CCK resistance. Subcutaneous injection of the CCK agonist caerulein dose-dependently decreased food intake in Zucker obese and lean rats whereas the CCK-B agonist gastrin-17 did not. Caerulein at 4 μg/kg, which resulted in CCK plasma bioactivity slightly above postprandial levels, decreased food intake in lean rats but not in obese rats. The decrease in food intake was also more marked at higher caerulein doses (20–100 μg/kg) in lean versus obese rats. In lean animals the satiety effects of the “near physiological” 4 μg/kg caerulein dose was abolished after blockade of vagal afferents with capsaicin, whereas the effects of higher caerulein doses were not. CCK-stimulated amylase secretion from pancreatic acini and binding capacity of ¹²⁵I- labelled CCK-8 were decreased in obese versus lean rats. The CCK-A antagonist loxiglumide at 20 mg/kg, a dose which abolished the action of all caerulein doses on food intake, failed to alter the food intake either in obese or in lean rats when given without an agonist. The results suggest that the satiety effects of “near physiological” doses of caerulein in lean rats are mediated by vagal afferents whereas pharmacological doses act via non-vagal mechanisms. The differences in CCK's satiety effect between lean and obese rats may be due to differences in CCK-receptor binding and action at peripheral vagal sites. However, the failure of the CCK-A antagonist to increase food intake questions whether any of the effects of exogenous CCK are of physiological relevance.     

Sialyl-Lewis x and Sialyl-Lewis a are associated with MUC1 in human endometrium

Endometrial epithelial cells express MUC1 with increased abundance in the secretory phase of the menstrual cycle, when embryo implantation occurs. MUC1 is associated with the apical surface of epithelial cells and is also secreted, being detectable in uterine fluid at elevated levels in the implantation phase. However, its physiological role is uncertain; it may either inhibit intercellular adhesion by steric hindrance or carry carbohydrate recognition structures capable of mediating cell-cell interaction. Here we show that endometrial epithelium expresses both Sialyl-Lewis x (SLe^x) and Sialyl-Lewis a (SLe^a), with a distribution and pattern of menstrual cycle regulation similar to that of MUC1. Using Western blotting and double determinant ELISA of uterine flushings, we demonstrate that SLe^x is associated with MUC1 core protein. The endometrial carcinoma cell lines HEC1A and HEC1B are shown to express MUC1 in a mosaic pattern, while three other cell lines express much lower amounts. HEC1A expresses both SLe^x and SLe^a while HEC1B expresses SLe^a only. Immunoprecipitation has been used to demonstrate that SLe^a is associated with MUC1 in HEC1B cells, and both SLe^x and SLe^a are associated with MUC1 in HEC1A cells.     

MUC5AC,but not MUC2, is a prominent mucin in respiratory secretions

Airway mucus was collected from healthy and chronic bronchitic subjects. The chronic bronchitic sputum was separated into gel and sol phase by centrifugation and mucins were isolated using isopycnic density-gradient centrfugation in CsCl. The presence of the MUC5AC and MUC2 mucins was investigated with antisera raised against synthetic peptides with sequences from the respective apoproteins. The gel and sol phase of chronic bronchitic sputum as well as healthy respiratory secretions were shown to contain MUC5AC whereas the MUC2 mucin could not be detected. Rate-zonal centrifugation showed that the MUC5AC mucin was large, polydisperse in size and that reduction yielded subunits. Ion-exchange HPLC revealed the presence of two subunit populations in all secretions, the MUC5AC subunits always being the more acidic. MUC5AC is thus the first large, subunit-based, gel-forming respiratory mucin identified and this glycoprotein is biochemically distinct from at least one other population of large, gel-forming mucins also composed of subunits but lacking a genetic identity.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CF cystic fibrosis - DFP diisopropylphosphofluoridate - DTT dithiothreitol - EDTA ethylenedinitrilotetraacetic acid - NEM N-ethylmaleimide - PAS periodic acid/Schiffs - PMSF phenylmethylsulphonyl fluoride - Tris Tris(hydroxymethyl)aminomethane - VNTR variable number of tandem repeats     

The role of magnesium in regulating CCK-8-evoked secretory responses in the exocrine rat pancreas

This study investigates the effect of magnesium (Mg²⁺) on the secretory responses and the mobilization of calcium (Ca²⁺) and Mg²⁺ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10^–10 M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg²⁺ [Mg²⁺]_o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg²⁺]_o Similar effects of perturbation of [Mg²⁺]_o on amylase secretion and ⁴⁵Ca²⁺ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca²⁺ concentration [Ca²⁺]_i in zero and normal [Mg²⁺]_o compared to a much reduced response in elevated [Mg²⁺]_o Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB₂ cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca²⁺]_i. In magfura-2-loaded acinar cells CCK-8 (10^–8 M) stimulated an initial transient rise in intracellular free Mg²⁺ concentration [Mg²⁺]_i followed by a more prolonged and sustained decrease. This response was abolished when sodium Na⁺ was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg²⁺ resulted in an elevation in [Mg²⁺]_i. Upon stimulation with CCK-8, [Mg²⁺]_i. decreased only slightly compared with the response obtained in normal [Mg²⁺]_o. CCK-8 caused a net efflux of Mg²⁺ in pancreatic segments; this effect was abolished when extracellular sodium [Na⁺]_o was replaced with either NMDG or choline. The results indicate that Mg²⁺ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca²⁺ mobilization. Moreover, the movement of Mg²⁺ in pancreatic acinar cells is dependent upon extracellular Na⁺.