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.     

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.     

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.     

Neural coding of difference frequencies in the midbrain of the electric fishEigenmannia: Reading the sense of rotation in an amplitude-phase plane

Eigenmannia is able to discriminate the sign of the difference, Df, between the frequency of a neighbor's electric organ discharge (EOD) and that of its own EOD. This discrimination can be demonstrated at the level of individual neurons of the midbrain. Intracellular and extracellular recordings of such sign-selective cells revealed the following: Units preferring positive Dfs and units preferring negative Dfs were found with equal frequency. The degree of selectivity was also similar for these two classes of neurons. All sign-selective units were sensitive to the magnitude of the frequency difference, i.e. the beat rate. Most units responded best to beat rates in the 4-8 Hz range. Sign-selectivity was observed only when the jamming signal (S2) was presented through electrodes other than those used to deliver the mimic (S1) of the fish's EOD, i.e. only when amplitude modulations were accompanied by modulations of differential phase. Intracellular studies suggest that most sign-selective neurons of the tectum are large, multipolar cells in the stratum album centrale. These cells send projections to the reticular formation, to lamina 9 of the torus semicircularis and to the N. electrosensorius.     

Predator-mediated habitat use: some consequences for species interactions

Synopsis Behavioral responses to predators can have a major impact on a fishes' diet and habitat choice. Studies with the bluegill sunfish, Lepomis macrochirus, demonstrate that bluegills undergo pronounced shifts in diet and habitat use as they grow in response to changes in their vulnerability to predators. Other species of fish exhibit similar habitat shifts with body size, presumably also in response to changing predation risks and/or foraging gains. An important but little appreciated consequence of this type of predator-mediated habitat use is that predation risk, by structuring size and/or age-specific resource use, may also indirectly affect species interactions. This paper discusses some of the ways in which behavioral responses to predators may affect intra- and interspecific competition in fish. Observational and experimental studies with sunfish (Centrarchidae) provide most of the examples. These studies suggest that the nonlethal effects of predators may be as important as the actual killing of prey.     

Multiple conformations of amino acid residues in ribonuclease A

The highly refined 1.26 A structure (R = 0.15) of phosphate-free bovine pancreatic ribonuclease A was modeled with 13 residues having discrete multiple conformations of side chains. These residues are widely distributed over the protein surface, but only one of them, Lys 61, is involved in crystal packing interactions. The discrete conformers have no unusual torsion angles, and their interactions with the solvent and with other atoms of the protein are similar to those residues modeled with a single conformation. For three of the residues--Val 43, Asp 83, and Arg 85--two correlated conformations are found. The observed multiple conformations on the protein surfaces will be of significance in analyzing structure-function relationships and in performing protein engineering.     

Structural requirements for anion substrates of the methotrexate transport system in L1210 cells

A broad spectrum of structurally diverse anions reversibly inhibits the influx of methotrexate in L1210 cells. Several of the more effective anions and their respective inhibition constants (K_i values) were: 5-methyltetrahydrofolate (0.3 μm), bromosulfophthalein (2 μm), thiamine pyrophosphate (3 μm), 8-anilino-1-naphthalene sulfonate (7 μm), phthalate (20 μm), and AMP (50 μm). Moderate inhibition was observed with P_i (K_i = 400 μm) and other divalent inorganic anions, while small monovalent anions such as Cl^? (K_i = 30 mm) were the least effective. When these same anions were tested for an effect on methotrexate efflux, stimulation was observed with some anions, while others had no effect. Enhancement was produced by folate compounds and p-aminobenzoylglutamate, small monovalent (e.g., Cl^?, acetate, and lactate) and divalent (e.g., phosphate and succinate) anions, a few nucleotides (e.g., AMP), and thiamine pyrophosphate, while little or no effect was associated with trivalent anions (e.g., citrate), most nucleotides, and large organic anions (e.g., bromosulfophthalein, NAD, and NADP). Anions with the ability to promote methotrexate efflux in control cells lost this capacity upon exposure of the cells to an irreversible inhibitor of methotrexate influx. These results support the hypothesis that methotrexate transport proceeds via an anion-exchange mechanism and moreover provide evidence that anion substrates for this system can be identified by their ability to promote methotrexate efflux. Anions which appear most likely to participate in this exchange cycle in vivo are P_i and AMP.     

Inhibition of estrogen-activated sexual behavior by androgens

Experiments to determine the potential of androgen to inhibit estrogen-activated female sexual behavior in rats were conducted. Treatment with either testosterone propionate (0.8 or 1.6 mg/day) or dihydrotestosterone propionate (0.2, 0.4, or 0.8 mg/day) significantly reduced the incidence of lordosis in ovariectomized females receiving estradiol benzoate (1 microgram/day). A similar suppression of estrogen-activated lordosis by testosterone was observed in castrated male rats. Flutamide, an androgen-receptor blocker, prevented the inhibition of lordosis by testosterone in females, indicating that the interaction of testosterone or a metabolite with an androgen receptor may be an important feature of this inhibition. Furthermore, the ability of dihydrotestosterone to inhibit lordosis at lower doses than testosterone suggests that the conversion of testosterone to dihydrotestosterone may also be necessary. These experiments demonstrate the potential of testosterone to inhibit the occurrence of female sexual behavior in rats, in contrast to its established facilitative effect on this behavior.     

THE LIPID COMPOSITION OF THORACOSPHAERA HEIMII: EVIDENCE FOR INCLUSION IN THE DINOPHYCEAE1

The fatty acid, sterol and chlorophyll composition of the calcified, unicellular alga Thoracosphaera heimii (Lohmann) Kamptner are reported. The presence of 4,23,24-termethyl-5α-cholest-22E-en-3β-ol (dinosterol), 4,23,24-trimethyl-5α-cholest-22E-en-3-one (dinosterone) and the predominance of C₁₈, C₂₀ and C₂₂ unsaturated fatty acids, including the acid 18:5ω3, indicates that T. heimii is a dinoflagellate. The fatty acid: sterol ratio (1.3), is typical of dinoflagellates. The geochemical significance of dinosterone, the high relative concentration of 4-desmethyl-5α-stanols and the role of 23-methyl-5α-cholest-22E-en-3β-ol in the biosynthesis of dinosterol in T. heimii are also discussed.