Surface antigen changes occurring in short-term cultures of activated human T lymphocytes: analysis by flow cytometry

Surface antigens of activated and cultured human T cells were studied using peripheral blood lymphocytes activated with conditioned medium from phytohemagglutinin-activated leukocytes and maintained in liquid culture for 2 weeks with conditioned medium containing Interleukin 2. The ensuing cell population was tested for kinetic changes in cell size and for the expression of surface antigens by immunofluorescence staining with a panel of monoclonal antibodies and analysis by flow cytometry. Upon activation, the cell population progressively increased in size to large blasts, with the rapid appearance on all of the large dividing cells of the antigen recognized by OKT9, the transferrin receptor. Cells within the population continued to express the common peripheral T-cell antigens bound by OKT3 and UCHT1, and also the antigen bound by 3A1, but never the antigen bound by OKT6, a thymic cell marker. From the time of activation an increasing proportion of the T cells, up to 80%, expressed the antigen detected with OKIa and FMC4, which recognise nonpolymorphic Ia determinants. This sequence of events was followed by a general decrease in size of the cell population, a process accompanied by further phenotypic changes. The percentage of cells expressing Ia antigens decreased, but most striking was the rapid change in the OKT4:OKT8 ratio of cells within the population, from 60:40 to 40:60. Thereafter the proportions of OKT4⁺ to OKT8⁺ cells within the cultures remained relatively stable and it is suggested that these data provide evidence for a possible change in phenotype of cultured human T lymphoblasts, from OKT4 to OKT8.     

Temperature dependence of the transition enthalpy of core particle DNA and of long DNA

The denaturation of short (145 base pairs) and long (about 8000 base pairs) DNA moelucules has been studied by adiabitic differential microcalorimetry in solutions with different NaCl content. It is found that the enthalpy of denaturation of short DNA is more sensitive to changes in T_m than that of long DNA. A comparison with other data is also given.     

A micromethod for the quantitative determination of the viability of Candida albicans hyphae

A micromethod for the quantitative determination of the viability of Candida albicans hypae was devised which takes advantage of the dimorphic nature of C. albicans which grows exclusively in the yeast form when incubated aerobically on Sabouraud dextrose agar at 30°C. When tested by thisd method, all viable, C. albicans hyphae were recognized as microcolonies consisting of one hypha surrounded by several yeast form progeny. In contrast to this, no yeast form progeny emerged from nonviable hypae. By counting appropriate total numbers (200–400) of microcolony-forming hypae and infertile hyphae, it was possible to determine the ratio of viable to nonviable cells in a given hyphal suspension. This micromethod may be used for quantitative assessment of the candidacidal effects of various antimycotic agents or phagocytes C. albicans hyphae whose viability could not have been determined by the conventional plating technique because of the species' high propensity to clump.     

The identification of dimethylphenylarsine as a microbial metabolite using a simple method of chemofocusing

Candida humicola acts on benzenearsonic acid to produce dimethylphenylarsine, which was identified by mass spectroscopy following the chemofocusing of the volatile metabolite onto a mercuric chloride impregnated filter. The same technique established that trimethylarsine is the volatile metabolic product obtained from C. humicola treated with 4-NH₂-2-OHC₆H₃AsO(OH)₂ and (CH₃)₃AsO. Arsanilic acid, 4-NH₂C₆H₄AsO(OH)₂, is not metabolized to a volatile arsine.     

Effect of food-deprivation on rat pancreatic islet cells

Somatostatin, insulin and glucagon concentrations in rat pancreas were measured following various intervals of food-deprivation. Tissue concentrations, as measured by radioimmunoassay, were correlated with A-, B-, and D-cell number and size using a scanning integrating image analyzer (Quantimet 720). Alterations in total islet hormone content were not correlated to changes in size or distribution of cells. This implies that changes in tissue content reflect changes in turnover of peptides rather than changes in cell size or number.     

Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient.

The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.     

Development of a radiometric metabolic viability testing method for human and porcine skin

A radiometric viability assay based upon the conversion of [¹⁴C]glucose into ¹⁴CO₂ by the viable cells on the dermal side of whole skin has been developed. The assay proved to be sensitive, reproducible, and practical, and was based upon the use of a microbiological growth detection system commonly used in many hospitals and laboratories. Relatively small samples of skin (0.25–1.00 g) were used in the test, and it was found that microbiological contamination did not interfere with the assay under normal conditions. The linear proportionality of the assay with both time and amount of skin assayed precluded the difficulties of nonlinear proportionality in other systems, allowing direct comparisons to be made between skin samples of different sizes and different incubation times. The assay could also detect ¹⁴CO₂ released from many radiolabeled substrates, including glucose, aspartate, glutamate, ornithine, orotic acid, and glycerol. Thus, the method could be used to test a number of cellular functions necessary for viability, including glycolysis, the functioning of the citric acid cycle and the pentose phosphate pathway, sugar and amino acid metabolism, pyrimidine biosynthesis, and cryopreservative agent metabolism. Since any of these tests could be performed in 4 hr, a viability assay based upon glycolysis alone, or in combination with any of the other tested substrates, could be carried out after allograft skin procurement before a decision needed to be made on skin cryopreservation.     

Developmental appearance of myosin heavy and light chain isoforms in vivo and in vitro in chicken skeletal muscle

Three myosin heavy chain isoforms with unique peptide maps appear sequentially in the development of the chicken pectoralis major muscle. An embryonic isoform is expressed early and throughout development in the embryo. A second isoform appears just after hatching and predominates by 10 days ex ovo. A third isoform, indistinguishable from adult myosin heavy chain, predominates by 8 weeks after hatching. This sequence of myosin isoform change does not, however, appear during myogenesis in vitro. In cultures prepared from embryonic myoblasts only embryonic myosin heavy chain is expressed. This is true even in cultures maintained for 30 days. Myosin light chain expression also changes in vivo with a progressive increase in fast light chain 3 accumulation. In vitro, however, this shift to increasing fast light chain 3 accumulation does not occur. The results indicate that the myosin heavy chain and light chain pattern observed in vitro is identical to that of the embryonic muscle and that the conditions necessary for the shift in expression to a more mature myosin phenotype are not present in myogenic cultures. These cultures are therefore potentially of great value in probing further the neural and humoral determinants of muscle fiber maturation and growth.