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31.
1. Kinetic, saturation and inhibition radioligand binding experiments with [3H]-N-methylscopolamine and [3H]quinuclidinyl benzilate were used to characterize the muscarinic cholinergic receptor in opossum (Didelphis virginiana, Kerr) submandibular salivary gland membranes. 2. The receptor density in opossum submandibular gland was found to be more than 3-fold higher than in rat, and 22-fold higher than in human, submandibular glands. 3. Inhibitor equilibrium dissociation constants for the antagonists pirenzepine, dicyclomine, atropine, N-methylscopolamine and AF-DX 116 revealed that the muscarinic receptor present in opossum submandibular gland appears to be the M1 subtype rather than the M3 subtype found in human and rat. 相似文献
32.
Incubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes sensitive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: 1) in vitro endosomes were osmotically insensitive; and 2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled by incubating Chinese hamster ovary (CHO) cells for 1-10 min at 37 degrees C with horseradish peroxidase (HRP) and/or fluorescein isothiocyanate-conjugated dextran (FITC-dextran). Cell fractions prepared in 0.25 M sucrose were hypotonically shocked by dilution with 5 mM Na phosphate buffer, pH 6.7, to a final sucrose concentration of 0.05 M. After hypotonic shock, endocytized HRP and FITC-dextran pelleted with membrane while lysosomal hydrolases did not. The HRP activity in the pellet was latent, suggesting that endosomes were resistant to osmotic shock. Uptake in the presence of hypertonic sucrose had little effect on the subsequent osmotic sensitivity of the endosomes. Uptake in the presence of hypertonic sucrose and PEG 1,000 rendered endosomes fragile to cell homogenization. Unexpectedly, the inclusion of hypertonic sucrose in the uptake and chase media inhibited the appearance of HRP in lysosomes. HRP internalized during a 10-min uptake appeared as if it were present in two physically distinct compartments, one accessible to transport inhibition by exogenous sucrose ("very early" endosomes) and the other not ("early" endosomes). After a brief uptake (1-3 min), postincubation of CHO cells in 0.25 M sucrose-containing media completely blocked transport of internalized HRP to lysosomes. This blockage could be partially relieved by cointernalization of invertase with HRP. These results suggest that transport between multiple early endosome populations is sensitive to intraorganellar osmotic conditions. 相似文献
33.
Patrick Sullivan 《CMAJ》1988,138(2):160-161
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Summary Previous studies of primary production in Antarctic seas have concluded that microalgae associated with sea ice make only a minor contribution to the carbon budget; however, production estimates for sea ice algae have been based almost exclusively on microalgae from nearshore fast ice. We measured biomass and rates of photosynthesis (at saturating irradiances) in microalgae collected from offshore pack ice during four cruises to the Weddell-Scotia Sea and the region west of the Antarctic Peninsula. Chlorophyll a concentrations in pack ice (0.089 to 260 g 1-1) were as high as reported from fast ice. Photosynthetic rates typically ranged (median 75%) from 0.3 to 3.6 C g chl a
-1 h-1 (n=127; arithmetic mean = 1.7, S D =1.9). These photosynthetic capacities are approximately an order of magnitude greater than previously reported for fast ice microalgae, but are similar to rates reported for Antarctic phytoplankton. Because pack ice constitutes more than 90% of the ice cover in Antarctic seas and indigenous microalgae have a higher photosynthetic capacity than previously realized, we raise the question: has the importance of sea ice algae to primary product: on in the southern ocean been underestimated? 相似文献
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Immature rats were injected with pregnant mares' serum gonadotrophin followed by human chorionic gonadotrophin (hCG). Ovaries were removed 0, 2, 5 or 8 days after hCG and either prepared for morphometric analysis or perifused with 0, 5 or 30 ng luteinizing hormone (LH)/min. In a second study, ovaries were removed on Day 2 or 8 and perifused with 0.1 mg 8-br-cyclic adenosine 5'-phosphate/ml (8-br-cAMP). On Day 0, the granulosa cells of the preovulatory follicles were small (53 +/- 0.5 microns2) with a cytoplasmic to nuclear (Cy:Nu) ratio less than or equal to 1.5. By Day 2, corpora lutea (CL) were present and composed of 95% small luteal cells (diameter less than 125 microns2, Cy:Nu greater than or equal to 3.0) and 5% large luteal cells (diameter greater than 125 microns2, Cy:Nu ratio greater than or equal to 3.0). The percentage of large luteal cells increased to 36 +/- 7% by Day 5, suggesting that they are derived from a select population of small luteal cells. Basal progesterone secretion increased from 38 +/- 5 on Day 0 to 1010 +/- 48 pg/mg/ml on Day 8. The rate of 5 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8; 30 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8, but not on Day 5; 8-br-cAMP stimulated progesterone secretion on both Days 2 and 8. These data demonstrate that once granulosa cells are induced to luteinize they lose their capacity to secrete progesterone in response to 5 ng LH/min and do not regain their responsiveness to LH rate until they completely differentiate. The loss of this LH responsiveness appears to be due to an inability to stimulate sufficient intracellular cAMP concentrations, since cAMP stimulates progesterone secretion on both Days 2 and 8. 相似文献
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The relative mobilities of various N-linked oligosaccharides reductively aminated to the charged fluorophore 8-amino-naphthalene-1,3,6-trisulphonic acid (ANTS) were determined by electrophoresis on high-density polyacrylamide slab gels. Each ANTS-derivatized oligosaccharide was assigned a relative migration index (RMI) expressed in terms of glucose equivalents, which was conveniently estimated by reference to a homologous series of ANTS--maltooligosaccharides run on each gel as oligosaccharide size standards. High-mannose-, complex- and hybrid-type structures were generally well resolved and easily visualized at picomole levels by simple UV light excitation. Application of these methods for the qualitative analysis of the oligosaccharides released from bovine fetuin and bovine asialofetuin by peptide-N-glycosidase F illustrates the usefulness of these techniques as fast, simple, and inexpensive tools for the characterization of N-linked oligosaccharides attached to glycoproteins. 相似文献
40.
R G Painter R Dukes J Sullivan R Carter E G Erd?s A R Johnson 《The Journal of biological chemistry》1988,263(19):9456-9461
Intact human neutrophils hydrolyzed N-formyl-Met-Leu-[3H]Phe (fMLP) and released Leu-[3H]Phe, cleaving 45-50% of the peptide within 20 min at 37 degrees C. The dipeptide after its release was then hydrolyzed to free amino acids by a dipeptidase (EC 3.4.13.11). This activity, present in plasma membrane-enriched fractions of neutrophil lysates, was also inhibited over 90% by phosphoramidon, an inhibitor of neutral endopeptidase (NEP, EC 3.4.24.11). Dithiothreitol and EDTA inhibited the activity to a comparable degree, suggesting the requirement for a heavy metal cofactor. Bestatin and amastatin, inhibitors of aminopeptidases (but not human kidney NEP), did not inhibit the rate of fMLP degradation but prevented the production of free phenylalanine and enhanced the accumulation of Leu-Phe. Of other inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin slightly enhanced the rate of fMLP hydrolysis by neutrophils, and others tested were ineffective. Rabbit antiserum to homogeneous human kidney NEP reacted specifically with a 100-kDa protein present in sodium dodecyl sulfate-solubilized neutrophils. The Mr of this protein was slightly larger than that of the kidney enzyme in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum incubated with intact cells specifically inhibited the degradation of fMLP over 70%. First, we confirm that NEP present on the plasma membrane cleaves fMLP at the Met-Leu bond; then the dipeptide Leu-Phe is cleaved by a dipeptidase. Finally, inhibition of NEP completely blocks fMLP-mediated chemotaxis. Thus, the enzyme may play an important role in modulating chemotactic responses. 相似文献