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Non-random chromosome loss in PHA-stimulated lymphocytes from normal individuals.

31773 lymphocyte metaphase cells from 280 karyotypically normal men aged 18-46 were examined for chromosome gain or loss. Chromosome loss was much more common than chromosome gain. Frequency of chromosome loss did not conform to a binomial distribution. There is a striking non-linear, inverse relationship between likelihood of loss and chromosome length. Chromosome gain shows a near binomial distribution between cells and no clear relationship to chromosome length. These facts indicate that the hypodiploid cells mostly arose as technical artefacts during slide preparation but that hyperdiploid cells were mainly due to non-disjunctional gain.

Adolescent↗

Recovery of age-related decline of thymic endocrine activity and PHA response by lysin-arginine combination.

The frequent association of malnutrition, infectious diseases and aging has stressed the role played by some nutrients on the immune efficiency and by nutrient supplementation on the age-dependent immunological decline. In the present paper there are reported evidences that oral administration of two amino acids--lysine and arginine--recovers, in old Balb/c mice, the mitogen responsiveness, the expression of T-cell markers and the production of thymic serum factor (thymuline). The effect of the amino acids or of their combination, as present in a commercially available form (Neoiodarsolo), seems to consist mainly of the reactivation of the endocrine activity of the thymus. Similar reactivation is achieved also in old humans. These data suggest that the age-dependent decline of thymic hormonal activity is not an intrinsic and irreversible event and that some nutritional intervention, such as amino acid treatment, likely through the stimulation of neuroendocrine network, may reactivate the endogenous production of thymic hormones.

Aging↗

Inhibition of phytohemagglutinin (PHA) induced human T-cell colonies by methylprednisolone.

The mechanisms underlying the inhibition of T-cell proliferation by methylprednisolone (MPS) have been investigated using a T-cell colony assay. Cell fractionation experiments have demonstrated that MPS exerted its effects at the level of both T-cells and monocytes. Thus, monocytes treated with MPS released a soluble factor that had suppressor activity on T-cell proliferation. Moreover, MPS directly blocked the proliferative capacity of T-cells, as demonstrated by the finding that MPS-treated T-cells formed reduced numbers of colonies even under optimal culture conditions and in the presence of exogenous IL-2.

Antigen-Presenting Cells↗

Comparison of MHC antigen expression on PHA- and MLC-induced T cell lines with that on T and B lymphoblastoid cell lines by cell cycle dependency.

Although it is well known that the expression of major histocompatibility complex (MHC) antigens on the surface of lymphoblastoid cell lines are cell cycle dependent, the way in which the MHC antigen expression on activated T cells varies with cell cycle phase has not previously been described. Using 11 lymphoblastoid cell lines from malignant and nonmalignant tissues (B cells, T cells, and myeloid cells) and five activated T cell lines (two cell lines activated by phytohemagglutinin and three alloreactive T cell clones), MHC antigen expression was quantitatively studied by dual-beam flow cytometry. Correlated measurements of surface antigen quantity (immunofluorescence), DNA content (Hoechst 33342), and cell size (light scatter), uninfluenced by induction synchrony and cell fixation, were performed. The data indicate that cell surface antigen quantity and cell surface area demonstrate specific values at each phase of the cell cycle when the cells are in logarithmic growth. Examining cells in logarithmic growth, it was confirmed, for all lymphoblastoid cell lines, that the quantity of MHC antigens on G2 (S + G2 + M) cells was greater than that on G1 cells. In addition, it was found, by analyzing antigen quantity and surface area, that class I antigen density in the G2 phase is 17% less than that in the G1 phase in leukemic T cell lines, and that both class I and class II antigen densities in the G2 phase were 21% less than that in the G1 phase in lymphoblastoid B cell lines. In activated T cells, class I antigen density in the G2 phase was 11% less than that in the G1 phase, while class II antigen density in the G2 phase was 12% greater than that in the G1 phase. We describe four important observations in this report. In both G1 and G2 phases, activated T cells express: quantitatively fewer class I antigens than lymphoblastoid B cell lines; similar quantity of class I antigens as that of leukemic T cell lines; and similar quantity of class II antigens as that of lymphoblastoid B cell lines. Also, class II antigens are expressed in greater density in the G2 phase than in the G1 phase in activated T cells. In contrast, lymphoblastoid B cell lines express greater density of class II antigens in the G1 phase than in the G2 phase of the cell cycle. These findings differ from previous reports, suggesting that G1 phase cells may have a more significant role than G2 phase cells as target cells for MHC restricted cytotoxic cells.

B-Lymphocytes↗

Differential DNA cross-linking and cytotoxicity in PHA-stimulated human lymphocytes exposed to melphalan, m-L-sarcolysin and peptichemio.

DNA interstrand, as well as DNA protein cross-linking, was more efficient in phytohaemagglutinin-stimulated human lymphocytes exposed to m-L-sarcolysin as compared to melphalan at equivalent concentrations of the drug. The cellular uptake of m-L-sarcolysin was more efficient as compared to melphalan. With peptichemio, a mixture of 6 peptides containing m-L-sarcolysin, an intermediate level of DNA cross-linking was found. The differences in DNA cross-linking between the 3 drugs were parallel to differences in cytotoxicity. Peptichemio has been ascribed anti-metabolic properties in addition to the alkylating properties conferred by its m-L-sarcolysin content. In the phytohaemagglutinin-stimulated lymphocytes, however, the effect of peptichemio seems linked to its capacity for DNA cross-linking.

Cell Survival↗

Diacylglycerol production in Jurkat T-cells: differences between CD3, CD2 and PHA activation pathways.

Diacylglycerol (DAG) production induced after stimulation with either CD3 mAb, a pair of CD2 mAbs or phytohaemagglutinin has been monitored in Jurkat T-cells prelabelled to isotopic equilibrium with seven [3H]- or [14C] fatty acids. It was found that CD3 induced a high production of arachidonic acid-labelled DAG and a modest production of oleic acid-DAG. The reverse was observed when using CD2 as activator. Phytohaemagglutinin induced a high production of these two DAG subspecies and in addition induced the production of linolenic acid-labelled DAG. Whatever the activator used no changes were observed in DAG production when cellular phospholipids were prelabelled with either myristic, palmitic, stearic or linoleic acids. All together our results strongly suggest that the three activation pathways previously described in T-lymphocytes might differ either at the level of the transduction mechanism or the phospholipid pools solicited during the activation process.

Antibodies, Monoclonal↗

Unfolding and refolding of leucoagglutinin (PHA-L), an oligomeric lectin from kidney beans (Phaseolus vulgaris).

The unfolding and refolding of Phaseolus vulgaris Leucoagglutinin, a homotetrameric legume lectin, was studied at pH 2.5 and 7.2 using fluorescence, far- and near-UV circular dichroism (CD) spectroscopy, 8-anilino-1-naphthalene sulfonate (ANS) binding and FPLC techniques. This protein was found to refold even at pH 2.5 and also exhibited high refolding yield around 60% at pH 2.5 and 85% at pH 7.2. The refolding at pH 2.5 takes place with the formation of a dimeric intermediate. Although the hydrodynamic radius of the completely renatured protein and the dimer at pH 2.5 was found to be same, the ANS binding as well as far-UV CD spectra of the two were different. The denaturation kinetics at pH 2.5 followed single exponential pattern with the rate of denaturation being independent of protein concentration. The renaturation kinetics on the other hand was dependent on the protein concentration providing further evidence of an intermediate state during refolding. From these experiments the folding pathway of the protein at pH 2.5 was proposed.

Circular Dichroism↗

Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway.

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.

Acyltransferases↗