Neutral endopeptidase from nuchal ligament of fetal calves

The nuchal ligament of unborn calves contains a neutral endopeptidase that is biochemically and immunologically similar to the neutral endopeptidase (NEP), or enkephalinase, from human kidney. Enzymatic activity was inhibited more than 90% by phosphoramidon (1 microM). The specific activity in membrane fractions, as determined by hydrolysis of the dansylated substrate, DAPGN, was similar in tissue from fetuses of gestational ages ranging from 100 to 280 days. NEP activity in adult ligament tissue, however, was less than 10% of that in fetal tissue. Fibroblasts dissociated from ligament tissue by collagenase displayed less NEP activity than did preparations of intact ligament, and activity was even lower in cultured cells. By contrast, fibroblasts cultured from fetal calf lungs had NEP activity comparable to that in the ligament tissue. When ligament fibroblasts were cultured on subcellular matrices derived from fetal lung fibroblasts the NEP activity increased relative to those cultured on plastic alone. These studies confirm the presence of neutral endopeptidase (NEP) in the nuchal ligament of the fetal calf. The consistent activity through a range of gestational ages and the influence of the subcellular matrix suggest that this enzyme might be involved in growth of the ligament during fetal life.     

Characterization of the muscarinic cholinergic receptor in the opossum (Didelphis virginiana, Kerr) submandibular gland: differences in receptor density and subtype compared with higher mammalian species

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.     

Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments

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.     

Photosynthetic capacity in microalgae associated with Antarctic pack ice

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?     

Effect of rate of change of luteinizing hormone concentration on in-vitro progesterone secretion within rat corpora lutea during differentiation.

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.     

Electrophoretic resolution and fluorescence detection of N-linked glycoprotein oligosaccharides after reductive amination with 8-aminonaphthalene-1,3,6-trisulphonic acid.

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.