DNA analysis and sorting of rat testis cells using two-parameter flow cytometry

By use of two-parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell compartments could be distinguished without pre-enrichment of the samples. Cells in these compartments were identified by sorting and subsequent microscopic examination.     

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.     

The role of environmental sodium chloride relative to calcium in gill morphology of freshwater salmonid fish

Summary Ultrastructure, distribution and abundance of cell types were examined in the gills of two freshwater salmonid species, Salmo fario and Salmo gairdneri, in media of selected ion content. In plain hard water (PW) with high concentrations of Ca²⁺, Na⁺, and Cl^-, gill chloride cells (CC) were confined to trailing edges and interlamellar regions of filaments whereas in mountain soft water (MW) with low concentrations of Ca²⁺, Na⁺, and Cl^-, CC were more numerous on filaments and covered lamellae, particularly along trailing edges. CC also appeared on lamellae of PW trout acclimated to soft water in a pond. This proliferation was not alleviated when ambient Ca²⁺ levels were raised (MW + Ca²⁺) but regressed in elevated NaCl media (MW + NaCl). The regression process involved an initial covering of CC by pavement cells followed by cytolysis and then eventual disappearance of CC. In MW, mucous cells were distributed mainly on trailing edges and, to a lesser extent, leading edges of filaments; they were absent from lamellae regardless of external ion levels.The results of this study shed some light on the functional significance of CC in freshwater fish. It is suggested that proliferation of CC is an adaptive response to dilute freshwater (i.e. [NaCl]<0.1 mequiv·1^-1).     

Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II).

Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer. Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms. All three protein products of the uvrA, uvrB, and uvrC genes were required for incision. End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's. DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments. The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.     

Effect of visual cues from female on the postejaculatory operant behavior of male rat

Bar-press response reinforced by the contact with estrous female were investigated in male rats in two experimental situations, when the visual communication with female was prevented or when it was permitted. After the instrumental responses directly preceded by ejaculations, depending on the increasing duration of response latency, three categories of male behavior were observed: avoiding of the contact with female, precopulatory behavior, or copulatory behavior. The enabling the visual communication with a female caused the significant increase of occurrence of copulatory behavior and significant decrease of contact-avoiding behavior, whereas the precopulatory behavior showed unsignificant increase. It is suggested that when prevented to see the female male rats can perform the instrumental response even when the sexual arousal is to low for precopulatory behavior. Such unrewarded responses lead to the appearance of frustration. On the contrary, when the female is visible the instrumental response is delayed up to the moment when the visual cues coming from female can evoke at least precopulatory behavior.     

Uptake of l-Ascorbate by Intact Spinach Chloroplasts

Uptake of l-[1-¹⁴C]ascorbate by intact ascorbate-free spinach (Spinacia oleracea L. cv Vital^r) chloroplasts has been investigated using the technique of silicone oil filtering. Rates greater than 100 micromoles per milligram chlorophyll per hour (external concentration, 10 millimolar) of ascorbate transport were observed. Ascorbate uptake into the sorbitol-impermeable space (stroma) followed the Michaelis-Menten-type characteristic for substrate saturation. A K_m of 18 to 40 millimolar was determined. Transport of ascorbate across the chloroplast envelope resulted in an equilibrium of the ascorbate concentrations between stroma and medium. A pH optimum of 7.0 to 7.5 and the lack of alkalization of the medium upon ascorbate uptake suggest that only the monovalent ascorbate anion is able to cross the chloroplast envelope. The activation energy of ascorbate uptake was determined to be 65.8 kilojoules (16 kilocalories) per mole (8 to 20°C). Interference of ascorbate transport with substrates of the phosphate or dicarboxylate translocator could not be detected, but didehydroascorbate was a competitive inhibitor. Preloading of chloroplasts with didehydroascorbate resulted in an increase of V_max but did not change the K_m for ascorbate. Millimolar concentrations of the sulfhydryl reagent p-chloromercuriphenyl sulfonate inhibited ascorbate uptake. The data are interpreted in terms of ascorbate uptake into chloroplasts by the mechanism of facilitated diffusion mediated by a specific translocator.     

Tissue uptake of circulating hyaluronic acid

Previous work in the rabbit has shown that there is a significant flux of plasma hyaluronic acid (HA) which is taken up and degraded mainly in the liver but also concentrated in the spleen. Purified 14C-labelled HA of high average molecular wt prepared by biosynthesis from D-[U-14C] glucose was injected i.v. in mice and its tissue distribution was determined by whole-body autoradiography during the next 24 h. As blood levels declined, radioactivity was concentrated in the liver and spleen as found in the rabbit, and also in bone marrow and lymph nodes. Distribution was uniform in liver tissue, concentrated and relatively persistent in the periphery of lymph nodes, and distinctly nodular within the spleen. Analysis of an aqueous liver extract taken 4 h after injection identified 14C in HA, in a macromolecular fraction resistant to fungal hyaluronidase, and in metabolites of low molecular wt. These findings confirm and extend observations based on tissue extraction in rabbits. The pattern of distribution through the body and the restricted localization within spleen and lymph nodes further suggest that HA is absorbed from plasma and tissue fluids by elements of the reticuloendothelial system.     

Differential size variations between transcriptionally active and inactive telomeres of Trypanosoma brucei

We have studied the genes coding for the variant-specific surface antigen (VSA) in a series of seven trypanosome clones derived from AnTat 1.1: 1.1 leads to 1.3 leads to 1.6 leads to 1.16 leads to 1.1C leads to 1.3B leads to 1.18 These genes are all telomeric (1-5), and their surrounding, although sometimes similar, differs in each case. The length between these antigen genes and the corresponding DNA end appears to increase at each antigenic switch, with however occasional sharp size reductions, often linked to the involvement of the telomere in gene expression. This increase is due to a constant "growth" of the telomeres, at a rate of about 28 bp per day in at least four cases and probably linked to chromosome duplication. The telomere harbouring the transcribed VSA gene is growing slightly faster (about 36 bp per day), and it is the only one whose size reduction is progressive, leading to a terminal length heterogeneity within a clone. As a result, the active VSA gene is found in a population of telomeres which, as the trypanosomes divide, becomes increasingly heterogeneous, with however a preferred discrete size class about 1.4 kb smaller. The fact that the "active" telomere is the only one in a chromatin conformation highly sensitive to DNAaseI (1-4, 6), suggests that chromatin structure influences the rate and extent of both size increase and shortening of telomeres.