Minicultures of Mammalian Cells in a New Plastic Plate

A new disposable micro tissue culture plate was developed and tested for use in virological procedures. Miniature mammalian cell cultures (minicultures) were grown in these plates. Each plate contained 96 circular cultures in flat wells (7 mm in diameter). Replicate titrations of a number of viruses were performed in various tissues. Excellent reproducibility was demonstrated. Mean infectivity titers determined by miniculture methods were generally within 0.6 log₁₀/ml of macro tube titrations. Standard tissue culture assay techniques such as hemadsorption, interference titration, and microneutralization were easily carried out with this method and were very reproducible. Development of this noncytotoxic disposable micro tissue culture plate now permits the routine performance of rapid, reliable, and reproducible tissue culture tests at a very significant reduction in cost and labor.     

Increased Resistance Against Oxidative Stress Is Observed During A Short Period of Renal Reperfusion After A Temporal Ischaemia

Reperfusion of rat kidney submitted to temporal ischaemia induces a decrease in glutathione content. Lipid peroxidation is not detected in kidney homogenates but microsomes obtained after periods of reperfusion longer than 60 minutes show increased malondialdehyde values correlated with high oxygen consumption and superoxide free radical generation. Microsomes obtained from kidneys submitted to 15 or 60 minutes of reperfusion are resistant to NADPH-induced lipid peroxidation but after 120 minutes of reperfusion an increased lipid peroxidative response is observed. Although the mechanism of the protection found in microsomes against the induction of oxidative stress in the first 60 minutes of reperfusion is unknown, it is postulated that this subcellular fraction plays an important role in the oxidative stress observed after longer periods of reperfusion.     

Certain withanolides from Iochroma gesnerioides antagonize ecdysteroid action in a Drosophila melanogaster cell line

Sixteen withanolides isolated from Iochroma gesnerioides (Kunth) Miers (Solanaceae) have been assessed for their activities as ecdysteroid agonists and antagonists. None of the compounds showed any agonistic activity, but several showed significant antagonistic activity. With a 20-hydroxyecdysone concentration of 5 × 10^–8 M, the ED₅₀ values for 2,3-dihydro-3-methoxywithaferin A, 2,3-dihydro-3-methoxywithacnistine, 2,3-dihydro-3-methoxyiochromolide and 2,3-dihydro-3-hydroxywithacnistine are 3.5 × 10^-5 M, 1 × 10^–5 M, 5 × 10^–6 M and 2.5 × 10^–6 M, respectively.     

A spawning aggregation of Nassau grouperEpinephelus striatus (Pisces: Serranidae) in the Mexican Caribbean

Synopsis An annual spawning aggregation of Nassau grouper,Epinephelus striatus, observed off the southern coast of Quintana Roo, Mexico, on full moon days in December and January, was surveyed during the winters from 1991 to 1993. A fish aggregation had been appearing at one traditional site off Mahahual for more than 80 years, but currently it forms at undetermined sites far from the original site. Fish migration was northwards in the fore reef border along the 14 m isobath and isolated groups were observed. Through visual assessment fish aggregations were found to include 200 to 500 individuals. The fishing on Nassau grouper aggregations has been active since 1910 by using hook and line; however, recently fishermen used gill nets. It is necessary to continue the research on this aggregation and establish management guidelines to protect the species.     

Cell death,gliosis, and synaptic remodeling in the hippocampus of epileptic rats

Seizures set in motion complex molecular and morphological changes in vulnerable structures, such as the hippocampal complex. A number of these changes are responsible for neuronal death of CA3 and hilar cells, which involves necrotic and apoptotic mechanisms. In surviving dentate granule cells seizures induce an increased expression of tubulin subunits and microtubule-associated proteins, suggesting that an overproduction of tubulin polymers would lead to a remodeling of mossy fibers (the axons of granule cells). In fact, these fibers sprout in the dentate gyrus to innervate granule cell dendrites, creating recurrent excitatory circuits. In contrast, terminal mossy fibers do not sprout in the CA3 field. Navigation of mossy fiber's growth cones may be facilitated by astrocytes, which would exert differential effects by producing and excreting cell adhesion and substrate molecules. In the light of the results discussed here, we suggest that in adult brain activated-resident astrocytes (nonproliferating, tenascin-negative, neuronal cell-adhesion molecule-positive astrocytes) could contribute to the process of axonal outgrowth and synaptogenesis in the dentate gyrus, while proliferating astrocytes, tenascin-positive, could impede any axonal rearrangement in CA3. © 1995 John Wiley & Sons, Inc.     

Neutralization sensitivity of human immunodeficiency virus type 1 is determined in part by the cell in which the virus is propagated.

Neutralizing antibody responses to human immunodeficiency virus type 1 (HIV-1) vary widely and have not been reproducibly associated with prognosis or disease progression. We have found that both low-passage clinical isolates and laboratory-adapted strains of HIV-1 have different sensitivities to neutralization by the same antiserum, depending on the host cell in which the viral stock is prepared. One such isolate (VL069) grown in H9 cells was neutralized by 20 human sera at a geometric mean titer of 1:2,047; this same isolate prepared in peripheral blood mononuclear cell (PBMC) culture was neutralized at a mean titer of < 1:10 by the same sera. Adsorption and mixing experiments indicated that neither antibody to H9 cell components nor blocking by excess viral antigen was responsible for the differences observed. This host cell effect is rapidly reversible upon passage of the virus from PBMCs to H9 cells and back into PBMCs. In contrast, the neutralization characteristics remained remarkably stable over extended culture in PBMCs. Two laboratory strains and five clinical isolates were evaluated in expanded studies of this phenomenon. While the neutralization characteristics of most of the strains studied were affected by the host cell in which the strain was propagated, two of the strains (one clinical isolate and one laboratory strain) appeared antigenically unaffected by their cell of origin. Host cell effect was also evident in neutralization by monoclonal antibodies directed against the CD4-binding region and the V2, V3, and gp41 regions. Possible mechanisms for this host cell effect include (i) mutation during passaging; (ii) selection in different host cells of different subpopulations of the (uncloned) viral stock; and (iii) cell-specific posttranslational modifications. To explore these possibilities, the V3 through V5 region of gp120 was sequenced in preparations made by passing VL069 into H9 cells and into PBMCs; HIVMN grown in CEM-SS cells and in PBMCs was also sequenced. In both cases, a few amino acid changes outside the V3 region were found. Studies are currently under way to assess the significance of these changes.