Membrane retrieval in the guinea pig neurohypophysis: biochemical characterization of a retrieval structure

[3H]Choline and [35S]methionine injected into the guinea pig hypothalamus in vivo were incorporated into the lipids and proteins, respectively, of secretory vesicles transported to the neural lobe. Prolonged in vivo stimulation of hormone secretion by dehydration decreased the [3H]choline content of secretory vesicles, with a concomitant increase in the [3H]choline content of a membrane fraction isolated on sucrose gradients. After stimulation of neural lobes in vitro in the presence of horseradish peroxidase, this extracellular fluid marker was found in the same membrane fraction. SDS electrophoresis of membrane proteins radiolabelled by [35S]methionine in vivo demonstrated that this fraction contained at least one major protein also present in the secretory vesicle membrane. These results suggest that we have isolated a membrane fraction containing the structure(s) involve in membrane retrieval in the neurohypophysis.     

Role of the Cell Surface of Methanosarcina mazei in Cell Aggregation

Colonial aggregates of Methanosarcina (= Methanococcus) mazei were examined with scanning and transmission electron microscopy. Cells are irregular and grouped into multicellular sarcinal colonies, which may disaggregate in older cultures. The protoplast is bounded by a typical trilaminar plasma membrane, outside of which is a matrix of loose fibrils. The presence and compactness of matrix material are responsible for the close packing of cells, and colony disaggregation seems to be the result of matrix shedding and degradation. The cell envelope contains complex hetero polysaccharides of N-acetylgalactosamine and galacturonic and glucuronic acids. Polymers extruded by M. mazei are likely quite adhesive in nature, accounting for its strong adherence to surfaces and hardiness compared with many other methanogens.     

Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid

Addition of thrombin to human platelets results in production of lysophosphatidic acid. Such synthesis of lysophosphatidic acid can be inhibited by mepacrine, an inhibitor of the phospholipase A2 which attacks phosphatidic acid to give lysophosphatidic acid. In the present study, mepacrine was used at a concentration of 2.5-20 microM, sufficient to block aggregation and lysophosphatidic acid formation induced by 0.1 U/ml thrombin. Mepacrine, at this concentration, also blocked thrombin-induced phosphorylation of platelet myosin light chain and a 47 kDa protein, thrombin-induced secretion and thrombin-induced release of arachidonic acid from platelet phospholipids. However, mepacrine also partly inhibited the formation of phosphatidic acid in response to thrombin, consistent with some simultaneous inhibition of phospholipase C. Lysophosphatidic acid (2.5-22 microM) overcame the mepacrine block in thrombin-stimulated aggregation, protein phosphorylation and secretion without stimulating the release of arachidonic acid from platelet phospholipids or the formation of lysophosphatidic acid, and only slightly increasing phosphatidic acid formation. The results suggest that lysophosphatidic acid primarily acts distal to mepacrine inhibition of phospholipase A2 and phospholipase C and are consistent with the possibility that lysophosphatidic acid might be a mediator of part of the effects of low-dose thrombin on human platelets.     

Investigation of the instability of plasmids directing the expression of Met-prochymosin in Escherichia coli

The causes of the instability of a multicopy plasmid, pCT70, which directs the expression of calf prochymosin in Escherichia coli, were investigated. Plasmid pAT153 and its derivative, pCT54, were stable for more than 90 generations in continuous culture with glucose limitation. The multicopy plasmid pCT66, which expressed very low levels of prochymosin due to poor translational efficiency, and low copy number plasmids which efficiently expressed the prochymosin gene, were also stable. These results indicated that high level translation of the recombinant gene was the cause of the instability of pCT70. The maximum specific growth rate of E. coli(pCT70) was reduced by 30% compared with E. coli(pCT66). To fulfil the requirements of a production system, a dual origin plasmid with controllable copy number was developed. Both this plasmid (pMG165) and a derivative which contained the prochymosin gene (pMG168) were stable when maintained at low copy number. When the copy number of plasmid pMG168 was increased by putting replication under the control of the lambda PR promoter and the cI857 temperature sensitive repressor, expression of prochymosin was achieved. This strategy enables large-scale production of prochymosin without the need for antibiotic selection or other methods of preventing plasmid loss.     

Sensitization to stress: the enduring effects of prior stress on amphetamine-induced rotational behavior

In rats with a unilateral 6-OHDA lesion of the substantia nigra exposure to footshock or immobilization stress produced a long-lasting enhancement in the rotational behavior evoked by a subsequent injection of amphetamine. However, the effect was dependent on the environmental context in which stress was applied. It is suggested that stress may induce enduring changes in brain and behavior similar to those produced by psychomotor stimulant drugs.     

Hirsutinolides,glaucolides and sesquiterpene lactone from Vernonia species

The investigation of nine Vernonia species afforded in addition to known sesquiterpenes 28 new ones. The structures were elucidated by high field ¹H NMR spectroscopy and the configurations were determined by NOE difference spectroscopy and, in one case, by X-ray analysis. The results indicated that configurations of several previously reported sesquiterpene lactones have had to be revised. In addition to known types two new ones, the jalcaguaianolides and the vernojalcanolides, are described. Furthermore some unusual reaction products are presented which, in part, led to some natural occurring lactones.     

Effects of CO(2) Enrichment and Carbohydrate Content on the Dark Respiration of Soybeans

During the period of most active leaf expansion, the foliar dark respiration rate of soybeans (Glycine max cv Williams), grown for 2 weeks in 1000 microliters CO₂ per liter air, was 1.45 milligrams CO₂ evolved per hour leaf density thickness, and this was twice the rate displayed by leaves of control plants (350 microliters CO₂ per liter air). There was a higher foliar nonstructural carbohydrate level (e.g. sucrose and starch) in the CO₂ enriched compared with CO₂ normal plants. For example, leaves of enriched plants displayed levels of nonstructural carbohydrate equivalent to 174 milligrams glucose per gram dry weight compared to the 84 milligrams glucose per gram dry weight found in control plant leaves. As the leaves of CO₂ enriched plants approached full expansion, both the foliar respiration rate and carbohydrate content of the CO₂ enriched leaves decreased until they were equivalent with those same parameters in the leaves of control plants. A strong positive correlation between respiration rate and carbohydrate content was seen in high CO₂ adapted plants, but not in the control plants.

Mitochondria, isolated simultaneously from the leaves of CO₂ enriched and control plants, showed no difference in NADH or malate-glutamate dependent O₂ uptake, and there were no observed differences in the specific activities of NAD⁺ linked isocitrate dehydrogenase and cytochrome c oxidase. Since the mitochondrial O₂ uptake and total enzyme activities were not greater in young enriched leaves, the increase in leaf respiration rate was not caused by metabolic adaptations in the leaf mitochondria as a response to long term CO₂ enrichment. It was concluded, that the higher respiration rate in the enriched plant's foliage was attributable, in part, to a higher carbohydrate status.

     

The protective effect of dietary fish oil on murine lupus

Dietary marine lipids markedly reduce the severity of glomerulonephritis and its associated mortality in inbred strains of mice developing autoimmune disease, a model for human systemic lupus erythematosus. We report here the influence of varying the dose of menhaden oil and the timing of its administration on the mortality of female (NZB x NZW) F1 mice. After ingesting 25 wt% menhaden oil (MO) for periods of 1.5 weeks to 12 months, there was a stable content of tissue n-3 fatty acids, with total n-3 fatty acids of 28% and 35% in spleen and liver, respectively. The extent of protection from mortality was dependent on the dose of MO with marked protection at doses of 11 to 25%, marginal protection at 5.5% and no protection at 2.5% MO. Delay in the institution of MO until ages 5 or 7 months still resulted in large reductions of mortality. Conversely, institution of a MO diet from 6 weeks until ages 5 to 7 months followed by a change to beef tallow resulted in little protection. Serum levels of 4 cyclooxygenase products were reduced ranging from 26 to 76% in mice fed MO diets, compared to mice fed beef tallow, based on radioimmunoassay. The degree of reduction of mortality on different doses of MO was correlated best with tissue levels of C22:5, and levels of C20:5 and C22:6 were similar at high and low doses of MO, suggesting that levels of 22:5 may be related to the protective effects of marine lipids on autoimmune disease.     

Localization of C4 genes within the HLA complex by molecular genotyping

Segregation of the complement component, C4, was analyzed in six families that each included an individual who inherited an HLA haplotype where a crossover event had occurred in the region between HLA-B and HLA-DR. Two cDNA clones corresponding to the C4 gene were utilized as probes in Southern blot analysis of DNA from members of each family. Restriction fragment length polymorphisms (RFLP) were observed and were assigned to haplotypes. In one family RFLP, hybridizing with the C4 probes, segregrated with HLA-B, and in four families RFLP segregated with HLA-DR; one family was not informative in this respect. These analyses have made it possible to localize the genes for C4 between HLA-B and HLA-DR by molecular genotyping and to characterize three different genomic configurations of C4 genes by limited restriction mapping.Abbreviations RFLP restriction fragment length polymorphisms - LCL lymphoblastoid cell lines