Fluidity of a retrovirus genome.

Comparison of the genomic sequences of the Friend spleen focus-forming virus with other murine retroviral sequences indicated that the spleen focus-forming virus was derived from at least three retroviruses. The 5' end of the virus, from the primer binding site through most of gag, was derived from AKV. The rest of gag and all of pol were of uncertain origin, but were probably derived from the same xenotropic virus that gave rise to the 5' half of env. The remainder was derived from Friend murine leukemia virus. The positions of a 585-base deletion, a 6-base duplication, and a point insertion that leads to a frame shift and premature protein termination in the ecotropic 3' end of env were invariant between three spleen focus-forming virus strains, indicating that they had a single common ancestor. However, the point of crossover between xenotropic viral sequences and Friend murine leukemia virus was different in each strain, and two strains were much more closely related to each other than to the third in the xenotropic region, indicating that these strains had diverged by multiple recombinations. Furthermore, a different nucleotide comprised the single point insertion near the 3' end of env, suggesting that these viruses have an extremely high transition and transversion rate.     

The effects of a settled industrial–domestic sewage works effluent from percolating filters on the survival and growth of various stages of rainbow trout, Salmo gairdneri (Richardson)

Comparisons were made of the mortality, growth and condition factor over a 50-day period of eleutheroembryos, alevins, 2-month fry, 4-month fry and 1 + rainbow trout, Salmo gairdneri , in water of three qualities–Birmingham tap-water, 50% and 100% settled effluent from percolating filters (Minworth Effluent Treatment Works) when dissolved oxygen levels were maintained near 100% ASV.
There was no significant mortality among life stages in control tap-water but the LT50 values of life stages in both 50% effluent and 100% effluent revealed significant differences. Inter-treatment comparisons showed similar mortalities of eleutheroembryos, of 2-month fry and of 1 + fish in the three treatments but LT50 values of 4-month fry showed significant differences. Growth rates of 2-month and 4-month fry were suppressed in both effluents after 7 days and there was a significant deterioration in the K factor of these life stages after 14 days. Predicted toxicities (the sum of the proportions of the 48-h LC50 of the individual poisons to rainbow trout) were compared with the observed toxicity in 50% and 100% effluent. The relevance of in vivo testing in comparison to predictive techniques is compared.     

Synaptosomal Calcium Metabolism During Hypoxia and 3,4-Diaminopyridine Treatment

A decline in the calcium-dependent release of neurotransmitters appears to underlie the decreased neuronal function that accompanies reduced oxygen tensions (hypoxia). To determine if alterations in calcium uptake are primary to these changes, synaptosomal calcium uptake was measured in the presence of 100%, 2.5%, or 0% oxygen. Calcium uptake declined 60.2 +/- 0.1 and 82.4 +/- 2.5% with 2.5% and 0% when compared with 100% oxygen, respectively. 3,4-Diaminopyridine stimulated calcium uptake by synaptosomes when they were incubated in low-potassium media. It also diminished the hypoxic-induced decline in calcium uptake to 30.6 +/- 3.1 and 33.5 +/- 3.1% with 2.5% and 0% oxygen, respectively. External binding to the synaptosomal plasma membrane declined to 29.2 +/- 0.3 or 11.8 +/- 0.9% when the oxygen tension was reduced to 2.5% or 0% oxygen. 3,4-Diaminopyridine increased this superficial binding from 111.7 +/- 0.3 to 86.5 +/- 0.9 or 23.4 +/- 0.9% with 100%, 2.5%, or 0% oxygen when compared with 100% oxygen without 3,4-diaminopyridine, respectively. Thus, the decline in neuronal processing that accompanies acute hypoxia may be due to altered calcium homeostasis, which diminishes neurotransmitter release.     

Epitope mapping of the human surface suppressor/cytotoxic T cell molecule Tp32

The complexity of the suppressor/cytotoxic subset marker of human T lymphocytes was demonstrated by biochemical analysis, cross-blocking experiments, phylogenetic comparisons, and functional studies. At the biochemical level, the antigen was shown to be a heteromultimer of at least three polypeptide chains covalently associated into four different higher m.w. species. Sixteen different murine monoclonal antibodies were used to map epitopes of this heteromultimeric complex. Cross-blocking experiments undertaken with six directly labeled reference antibodies identified at least seven spacially distinct epitopes. Flow microfluorometric analysis of peripheral blood lymphocytes showed two distinct subpopulations of bright and dull-stained cells that differed approximately 10-fold in antigen density. The distribution of epitopes on bright and dull cells was not uniform because in several combinations, blocking was observed on bright cells only. Studies with nonhuman primate T cells demonstrated a high degree of phylogenetic heterogeneity in the antigen. The combined cross-blocking and primate data divided the 17 antibodies into 15 groups. Each of the antibodies was capable of blocking lysis by alloreactive cytotoxic T lymphocytes, indicating that the mechanism of inhibition may not necessarily involve hindrance of an active site.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

A cortical granule-specific enzyme,B-1,3-glucanase,in sea urchin eggs

The ultrastructural localization of B-1,3-glucanase in three species of sea urchin eggs was determined using a monospecific antibody in an electronmicroscopic immunogold procedure. In all three species, Lytechinus variegatus, Strongylocentrotus purpuratus, and Arbacia punctulata, B-1,3-glucanase was localized specifically to the cortical granules. No other organelle within the egg contained significant label. During the fertilization reaction, B-1,3-glucanase was released from cortical granules into the perivitelline space and became associated with the hyaline layer. No significant label was found in association with the fertilization envelope.     

Hyperbaric hyperoxia reversibly inhibits erythrocyte phospholipid fatty acid turnover

The present study is one component of a comprehensive investigation of oxygen tolerance of tissues and organs in normal human subjects. The focus of this study was the acylation of membrane phospholipid in situ by erythrocytes. Activation of exogenous [9,10-3H]oleic acid to acyl thioester and transesterification of the acyl thioester into phospholipid by intact human erythrocytes incubated in vitro decreased 30% after exposure of 10 human subjects to hyperbaric hyperoxia (100% O2, 3 ATA, 3.5 h). Partial recovery of activity could be detected when additional cells were obtained from these subjects and assayed in vitro 24 h after cessation of exposure. No significant change in membrane phospholipid fatty acid composition was detected under these conditions. The reduced glutathione content of intact erythrocytes increased by 15% after hyperbaric hyperoxia and remained elevated 24 h after exposure. In isolated membranes prepared from the same cells activation of [9,10-3H]oleic acid to acyl thioester and its transesterification into phospholipid did not change after hyperoxia. Since the ability of intact cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be necessary for the maintenance of membrane integrity during exposure to oxidative stress, the decrease in [9,10-3H]oleic acid incorporation by human erythrocytes detected in vitro after hyperbaric hyperoxia in vivo may reflect an early event in the pathogenesis of oxygen-induced cellular injury and may be a useful index for assessment of the tolerance of tissues to hyperoxia.     

Age-related augmentation of phosphorylase b kinase in hepatic tissue from the glycogen-storage-disease (gsd/gsd) rat.

The effects of food deprivation on body weight, liver weight, hepatic glycogen content, glycogenolytic enzymes and blood metabolites were compared in young and old phosphorylase b kinase-deficient (gsd/gsd) rats. Although the concentration of glycogen in liver from 9-week-old female gsd/gsd rats (730 mumol of glucose equivalents/g wet wt.) was increased by 7-8% during starvation, total hepatic glycogen was decreased by 12% after 24 h without food. In 12-month-old male gsd/gsd rats the concentration of liver glycogen (585 mumol of glucose equiv./g wet wt.) was decreased by 16% and total hepatic glycogen by nearly 40% after food deprivation for 24 h. Phosphorylase b kinase and phosphorylase a were present at approx. 10% of the control activities in 9-week-old gsd/gsd rats, but both enzyme activities were increased more than 3-fold in 12-month-old affected rodents. It is concluded that the age-related ability to mobilize hepatic glycogen appears to result from the augmentation of phosphorylase b kinase during maturation of the gsd/gsd rat.     

Compartmentation and regulation of acetylcholine synthesis at the synapse.

Acetylcholine and choline release was measured by using an automated and modified version of the chemiluminescence technique of Israel & Lesbats [(1981) Neurochem. Int. 3, 81-90]. A comparison of acetylcholine and choline release from synaptosomes demonstrated that acetylcholine release was K+-stimulated and inhibited by the Ca2+ ionophore A23187 and cyanide. Choline release, however, did not vary markedly under different conditions, suggesting that it is not associated with acetylcholine release at the nerve ending. Total acetylcholine synthesis in synaptosomal preparations was measured concurrently with the incorporation of [14C]acetyl and [3H]choline moieties by using the chemiluminescence method. Under sub-optimal glucose concentrations or in the absence of treatment of the synaptosomes with the acetylcholinesterase inhibitor phospholine, the incorporation of radioactivity exceeded total synthesis, indicating that cycling between acetylcholine and its precursors may occur. After treatment with phospholine, acetyl-group incorporation from D-[U-14C]glucose occurred without dilution of the precursor at optimal (1.0 mM) and low (0.1 mM) glucose concentrations; however, at very low (0.01 mM) glucose concentrations, dilution by a small endogenous pool occurred. [14C]Acetyl incorporation into acetylcholine was compared with various metabolic parameters. A closer correlation was observed between [14C]acetyl-group incorporation into acetylcholine and the calculated acetyl-carrier efflux from the mitochondria than with the calculated pyruvate-dehydrogenase-complex flux. The results are discussed with respect to the regulation of acetylcholine concentrations at the synapse and the mechanism whereby turnover occurs.