Role of the tubulin-microtubule system in lymphocyte activation

The role of the tubulin-microtubule system was examined in human peripheral blood leukocytes after activation with phytohemagglutinin (PHA). Soluble tubulin and microtubules were measured with a [(3)H]colchicine-binding assay. It was found that the tubulin content of PHA-activated lymphocytes was consistently increased relative to total protein content after 36 h of culture. There was no increase in the proportion of total tubulin synthesis which was present as microtubules at 36 h. Nevertheless, as a result of increased tubulin synthesis, there was a two-to three-fold increase in total microtubular mass. Colchicine, which disrupts microtubles, was used to assess the role of microtubule assembly in the sequence of events which follow lymphocyte activation, namely lymphokine release, protein synthesis, RNA synthesis, and DNA synthesis. Colchicine consistently inhibited DNA synthesis but did not inhibit release of the lymphokine, osteoclast activating factor (OAF). Protein and RNA syntheses were inhibited much less than DNA synthesis. The fact that some effects of PHA on lymphocytes appear to require intact microtubules and at least one does not suggest that the microtubule dependent step in PHA-stimulated lymphocyte activation occurs at a stage after propagation of the signal from the membrane to the cell interior.     

The effect of dibromodulcitol on resting and dividing lymphoid cells

The effect of dibromodulcitol (DBD) on the incorporation of labelled precursors into DNA and RNA fractions of PHA-stimulated human lymphocytes and of P388F lymphoma cells at various stages of their growth was studied. Both cell systems showed sensitivity to the drug within the concentration range of 1–10 μg/ml.When DBD was added before phytohaemagglutinin (PHA), human lymphocytes showed a DNA labelling that was more affected than RNA. In contrast, by adding DBD after PHA, RNA labelling was much more inhibited than DNA. In the latter case, the decrease in DNA labelling occurred only 24 h after drug treatment whereas RNA labelling was decreased 1 h after treatment. Levels of DBD which normally produced 30% inhibition in plating efficiency of P388F lymphoma cells affected uridine-5-T incorporation to a different extent at different stages of growth of the culture. Enhanced RNA labelling occurred in early exponential stage while at later stages of growth, RNA synthesis was depressed.     

Involvement of polyadp-ribose polymerase in the initiation of phytohemagglutinin induced human lymphocyte proliferation

Nicotinamide (10 mM) or 3-aminobenzamide (5 mM) added at the onset of phytohemagglutinin (PHA) treated human lymphocyte cultures provoke a marked inhibition of the PHA induced DNA synthesis and cell proliferation as well as of poly(ADPR) polymerase activity. When the inhibitors of poly(ADPR) polymerase are added at a later stage of culture (48 h) no inhibition of the stimulation of DNA synthesis and cell proliferation by PHA in human lymphocyte cultures is observed. The intervention of ADP ribosylation at the initiation of DNA synthesis is suggested.     

DNA repair after gamma irradiation in lymphocytes exposed to low-frequency pulsed electromagnetic fields

The effect of exposure to extremely low-frequency pulsed electromagnetic fields (EMFs) on DNA repair capability and on cell survival in human lymphocytes damaged in vitro with gamma rays was studied by two different micromethods. In the first assay, which measures DNA repair synthesis (unscheduled DNA synthesis, UDS), lymphocyte cultures were stimulated with phytohemagglutinin (PHA) for 66 h and then treated with hydroxyurea (which blocks DNA replication), irradiated with 100 Gy of 60Co, pulsed with [3H]thymidine ([3H]TdR), and then exposed to pulsed EMFs for 6 h (the period in which cells repaired DNA damage). In the second assay, which measures cell survival after radiation or chemical damage, lymphocytes were first irradiated with graded doses of gamma rays or treated with diverse antiproliferative agents, and then stimulated with PHA, cultured for 72 h, and pulsed with [3H]TdR for the last 6 h of culture. In this case, immediately after the damage induced by either the radiation or chemicals, cultures were exposed to pulsed EMFs for 72 h, during which cell proliferation took place. Exposure to pulsed EMFs did not affect either UDS or cell survival, suggesting that this type of nonionizing radiation--to which humans may be exposed in the environment, and which is used for both diagnostic and therapeutic purposes--does not affect DNA repair mechanisms.     

Chemotactic responses of human polymorphonuclear leukocytes to cyclic GMP and other compounds

Some metabolic properties of small molecular weight nuclear RNA (snRNA) components have been studied in human lymphocytes cultured with PHA. Pulse-labelling experiments with ³H-uridine in 3 h-intervals around the onset of DNA synthesis showed no qualitative or quantitative differences in the snRNA labelling pattern. Long labelling experiment with ³H-methionine demonstrated the following relative degrees of methylation: tRNA (1.0), 5S RNA (0), D (0.3), 5.5S RNA (0.2), C (0.6), A (0.2), L (0) and rRNA (0.2). Chase-experiments with ³H-methionine showed that the snRNA components D, C and A are metabolically stable with half-lives of not less than 30 h. Actinomycin D (0.05 μg/ml) reduced markedly the synthesis of rRNA and 5 S RNA whereas the synthesis of D, C, A and L was unaffected or only slightly affected. Actinomycin D at a concentration of 0.25 μg/ml inhibited the synthesis of D, C and A. Cycloheximide (0.19 μg/ml) reduced the synthesis of D, C and rRNA to about 50% of control whereas 5S RNA synthesis was only slightly inhibited and tRNA synthesis was unaffected.     

The induction of the human T-cell growth factor receptor precedes the production of RNA and occurs in the presence of inhibitors of RNA synthesis

The receptor for T-cell growth factor (TCGF) is an activation antigen that is present in low amounts on a small fraction of resting T lymphocytes. The TCGF receptor on human T cells can be detected with the anti-Tac monoclonal antibody within 7-12 h of stimulating the cells with phytohemagglutinin (PHA). In the current studies, we examined human lymphocytes cultured alone, with PHA, or with PHA plus sufficient actinomycin-D to inhibit RNA synthesis. After varying intervals, aliquots of the lymphocytes were stained with acridine orange (AO) or pyronin-Y(PY) to measure RNA and/or with anti-Tac plus FITC goat anti-mouse Ig. Tac expression began to increase after 6-8 h incubation with PHA, whereas increases in PY or AO staining were not detected until 12 h or later. Furthermore, the initial increase in Tac expression was not affected by sufficient actinomycin-D to block all detectable nucleic acid synthesis. Therefore, it appears that the initial expression of TCGF receptors detected after lymphocyte activation does not require de novo production of RNA.     

Measurement of spontaneous and X-irradiation-induced 6-thioguanine-resistant human blood lymphocytes using a T-cell cloning technique

Lymphocytes separated from human peripheral blood were cultured in vitro, in the presence of 6-thioguanine (TG), to select and clone rare TG-resistant (TGr) cells present in the circulation in vivo. The incidence of such TGr cells ranged from 0.83 X 10(-5) to 2.53 X 10(-5) (mean 1.48 X 10(-5) ) in healthy individuals aged between 19 and 79 years; did not differ between males and females; but increased significantly with age at a rate of 2.4 cells/10(7) lymphocytes/year. Exposure of lymphocytes (G0) in vitro to X-ray doses of upto 200 rad resulted in a dose-dependent increase in TGr cell frequencies. The rates of increase were approximately in proportion to the square of the dose and these rates were closely similar to those obtained in cultured skin fibroblasts and suggest that the bulk of these mutations are a consequence of chromosome structural aberrations. The cloned TGr cells are considered to be HPRT- mutants and the mutation frequencies in lymphocytes determined using this cloning technique were compared with the variant frequencies obtained in earlier experiments utilising an autoradiographic technique to detect azaguanine-resistant (AGr) variant cells. Mutation frequencies with the cloning technique were 10-20-fold lower than variant frequencies with the autoradiographic method.     

Spatial redistribution of ribosomal chromatin in the fibrillar centres of human circulating lymphocytes after stimulation of transcription

We have studied the distributional changes of the completely extended ribosomal chromatin present in the fibrillar centres of resting human lymphocytes after phytohemagglutinin (PHA) treatment. In thin sections of resting lymphocytes selectively stained for DNA, the extended non-nucleosomal chromatin was located in a solitary, large agglomerate which corresponds to the solitary, large fibrillar centre observed in uranium-lead-stained sections. At 20 h after PHA stimulation the ribosomal chromatin agglomerate appeared to be fragmented into smaller agglomerates which correspond to numerous fibrillar centres surrounded by a thick rim of dense fibrillar component. The mean area of ribosomal chromatin agglomerates from resting lymphocytes was found to be 0.772 mu 2 + 0.125 SD, whereas in stimulated lymphocytes it was found to be 0.184 mu 2 + 0.052 SD. At 20 h after PHA treatment ribosomal RNA (rRNA) synthesis was 8-fold greater than the control value, whereas DNA synthesis had not started. These results indicate that ribosomal chromatin of resting lymphocyte fibrillar centres contains transcribable sequences, temporally not expressed.     

Diphenylhydantoin inhibits cortisol-induced lysis of thymocytes

Diphenylhydantoin (DPH) shares two features with cortisol: immunosuppression and cleft palate formation. We tested the hypothesis that DPH would have effects on lymphocytes in vitro similar to those induced by cortisol, and the corollary that DPH would inhibit those cortisol effects. We found that DPH lysed rat thymocytes, although at higher concentrations than cortisol. When combined, DPH inhibited cortisol lysis of thymocytes. Neither drug lysed human phytohemagglutinin (PHA)-stimulated cells, but both drugs depressed DNA and RNA syntheses in PHA cells. DPH augmented cortisol inhibition of DNA and RNA syntheses in PHA cells and DNA synthesis in rat thymocytes. It had no effect on cortisol inhibition of RNA synthesis in rat thymocytes. It appears that DPH has a cortisol-like action (lysis of rat thymocytes). The actions of this drug enable us to show that cortisol lysis and the inhibition of DNA or RNA synthesis can be associated. These phenomena may explain some immunosuppressive effects of DPH in the human.     

Evidence that T cell activation is required for HIV-1 entry in CD4+ lymphocytes

The relationship of T cell activation to HIV entry and generation of viral DNA intermediates was studied in freshly isolated CD4+ T lymphocytes. Unstimulated cells exposed to infectious virus for up to 48 h did not synthesize any detectable unintegrated HIV DNA duplex forms or integrated genomic provirus. However, activation of these cells with either PHA or OKT3 (anti-CD3) mAb before viral exposure resulted in the generation of unintegrated HIV DNA after 6 h and integrated copies after 24 h. Cell-to-cell fusion studies showed significantly attenuated fusion between freshly isolated resting T cells and T cells constitutively expressing high levels of HIV envelope glycoprotein (HXB/gpt) compared with T cells first stimulated with either PHA or OKT3 mAb. The baseline fusion observed with resting T cells is believed to be a consequence of allogeneic stimulation by the HXB/gpt cell line. These results provide evidence that HIV entry and HIV envelope-dependent cell-to-cell fusion require T cell activation.     

Levels of hypoxanthine phosphoribosyltransferase RNA in human cells

The gene for the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) is expressed at a low level in many cells. As is the case with several other “housekeeping genes,” thorough studies of hprt gene regulation have been hampered by the low levels of its mRNA. We have used RNA/RNA hybridization in solution to determine the concentration of hprt-RNA in human cells. The sensitivity and specificity of the method have been validated, and it is shown that hprt-RNA can be accurately determined at a level of a few mRNA molecules per cell. As expected for a housekeeping gene, low and relatively constant hprt-RNA levels (0.3–0.8 pg/μg DNA) were found in primary cultures of normal amnion cells and fibroblasts, EBV-transformed lymphoblastoid cell lines, neuroblastoma, glioblastoma, and melanoma cell cultures. While resting lymphocytes were found to contain very low amounts of hprt-RNA, lymphocytes stimulated with phytohemagglutinin (PHA) showed a 10-fold increase to about 0.8–1.2 pg/μg DNA, which corresponds to 6–10 hprt-RNA molecules per cell. The level started to increase about 20 h after PHA stimulation, 5–10 h before the onset of DNA synthesis, and a steady-state level was reached after 2–3 days in culture. In PHA-stimulated lymphocytes from two brothers with inherited HPRT deficiency (LeschNyhans syndrome), the hprt-RNA level in PHA-stimulated lymphocytes was only about 25% of that in normal subjects. In T-cells selected for HPRT deficiency by growth in 6-thioguanine medium, the levels of hprt-RNA were either normal or very low, which probably reflects the different nature of the mutations involved. These results demonstrate the sensitivity of this method for determinations of low levels of RNA and clearly show induction of hprt-RNA after mitogenic stimulation of human lymphocytes.     

Production of alpha-lymphotoxin by human T-cell subsets

Human T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed-lymphocyte culture. These sensitized T cells were fractionated on the basis of their possession of Fc receptors for IgG (TG+) or IgM (TM+), or the absence of both IgG and IgM receptors (TG-M-). When restimulated with alloantigen of the same derivation, TG+, TM+, and TG-M- cells yielded almost equal amounts of cytotoxin. Anti-alpha-lymphotoxin serum neutralized most of this cytotoxic activity indicating that alpha-lymphotoxin (alpha-LT) constituted most of this activity. Although TG-M- cells function as effectors in allogeneic cytotoxicity, TG+ cells lyse IgG-coated targets in an antibody-dependent cell-mediated cytotoxic (ADCC) reaction, which has been shown to be mediated in part by alpha-LT. Whether TM+ cells can be cytotoxic is not clear. In addition, freshly isolated human T-cell subsets were stimulated with phytohemagglutinin-P (PHA-P). After PHA stimulation, TG+, TM+, and TG-M- cells produced similar amounts of soluble cytotoxin, which was largely neutralized by anti-alpha-LT. The TG+ cells incorporated less thymidine than the TM+ or TG-M- cells. Likewise, OKT4+ and OKT8+ subsets, isolated with the aid of monoclonal OKT8 or OKT4 antibody and complement, yielded lymphotoxin after stimulation with PHA. It is shown that all T-cell subsets, as defined here, can produce lymphotoxin. Furthermore, depending on the assay system, cytotoxicity can be clearly demonstrated in all of these subsets, except in TM+ cells, where positive and negative results have been reported.     

Microtubules, colchicine, and lymphocyte blastogenesis.

We have studied the time course of disassembly of microtubules of resting and stimulated mouse lymphocytes caused by the drug colchicine, as well as the effect of this compound on DNA and RNA synthesis of human and mouse lymphocytes. Fine-structure studies with the electron microscope showed a great increase in number of microtubules resulting from stimulation of mouse lymphocytes by the mitogenic lectin Con A. The presence of a network of microtubules was demonstrated in resting lymphocytes by use of the technique of immunofluorescence; this technique was not effective for the study of the microtubules of stimulated lymphocytes in the blast stage. The disappearance of microtubular networks in some cells (approximately 25%) was caused by the protocol of colchicine treatment used in many laboratories (30 min at 10(6) M); a 6- to 8-h treatment was required to cause all cells to lose their microtubules. It is indicated in these findings that there is need for extreme caution in implicating microtubule disruption as the cause of certain colchicine effects, such as that on the Con A-induced inhibition of receptor-ligand migration. The addition of colchicine to stimulated cells at varying times of culture caused marked inhibition of DNA synthesis provided that sufficient time (approximately 20 h for maximum inhibition) elapsed between addition of the drug to the stimulated culture and assay of DNA synthesis. Our data on the time course of inhibition of DNA synthesis by alpha-methyl mannoside (alpha MM) and by colchicine do not exclude the possibility that the latter compound may act partially by affecting the commitment of stimulated lymphocytes to DNA synthesis but they show that it can inhibit well after commitment is complete. The later the time of assay of thymidine incorporation, the more disparate were the curves relating the effects of alpha MM and colchicine to DNA synthesis of human cells. In the case of mouse splenic lymphocytes, there was no resemblance between the time course of the alpha MM and of the colchicine effects. Synthesis of RNA after 12 h of culture of stimulated human lymphocytes was also sensitive to colchicine.     

Regulation of interleukin 2 receptor expression: effects of phorbol diester, phospholipase C, and reexposure to lectin or antigen

Anti-Tac monoclonal antibody identifies the receptor for interleukin 2 (IL 2, or T cell growth factor) present on activated human T lymphocytes. By using tritiated anti-Tac, we now report a sensitive and specific binding assay to evaluate cell surface IL 2 receptor expression. IL 2 receptors on human peripheral blood lymphocytes can be detected within 6 hr after PHA stimulation. PHA-induced receptor expression is inhibited by actinomycin D and cycloheximide, but not by mitomycin C, suggesting a requirement for de novo RNA and protein synthesis, but not DNA synthesis. Scatchard analysis of [3H]-anti-Tac binding to lymphocytes stimulated with PHA for 3 days revealed from 20,000 to 60,000 molecules of antibody bound per cell, and a Kd of 1 to 3 x 10(-10) mol/l. Sequential binding studies of activated human lymphocytes maintained in long-term culture with IL 2 demonstrated a progressive decline in receptor number correlating with diminished growth rate. Restimulation with lectin or antigen increased the number of IL 2 receptors, suggesting that IL 2 dependent immune responses may be regulated, at least in part, by IL 2 receptor expression. Receptor number was also increased by PMA. Moreover, similar effects were produced by incubation with phospholipase C but not interleukin 1. Because both PMA and phospholipase C result in activation of protein kinase C, these data suggest the possibility that activation of protein kinase C may induce IL 2 receptor expression.     

6-Thioguanine resistant T-lymphocyte determination as a possible indicator of human ionizing radiation exposure

We used the autoradiographic assay to assess human in vivo somatic cell gene mutation at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in T-lymphocytes. Cells able to incorporate tritiated thymidine in vitro in 6-thioguanine containing short-term cultures were enumerated in order to determine 6-thioguanine resistant (TGr) variant frequencies in cryopreserved lymphocytes from control individuals and 3 persons suspected of 60Co exposure from an accident in Cd. Juárez, México. The data indicate that the lymphocyte TGr variant frequency assay may be potentially usefull for human population monitoring following accidental exposures to ionizing radiation.     

Bromodeoxyuridine labelling as an alternative method to identify 6-thioguanine-resistant mutant lymphocytes in humans

6-Thioguanine-resistant (TG^R) mutant lymphocytes in human blood are usually enumerated by the cloning assay which allows the molecular characterisation of the HPRT mutations to be detected. A “short-term” alternative approach is provided by the anti-bromodeoxyuridine (anti-BrdU) technique in which TG^R lymphocytes are identified immunocytochemically by their ability to synthesise DNA in the presence of 6-thioguanine (TG). We have evaluated the influence of various experimental factors that could affect the frequency of TG^R lymphocytes. A standard protocol is proposed, based on 24-h cold storage of isolated lymphocytes at 4°C and 40-h culture with and without TG, the last 16 h with BrdU. The harvested cells are treated with hypotonic (0.075 M) KCl, fixed with methanol:acetic acid (3:1) and put on microscopic slides. For the TG cultures, all cells are prepared on the slides, while slides from the control cultures are made by a 1/50 dilution. DNA is denatured by formamide, and the BrdU label is identified by anti-BrdU antibody detected by immunoperoxidase staining using a peroxidase-conjugated secondary antibody with diaminobenzidine as substrate. In 10 donors, the frequency of TG^R lymphocytes (variant frequency, V_f) detected by this protocol ranged from 69.65×10⁻⁶ to 83.45×10⁻⁶, and split measurements showed a relatively small intra-assay variation in V_f values of each donor. BrdU in DNA was also detected by immunofluorescence using a fluorescein-conjugated anti-BrdU monoclonal antibody. This method, facilitating easy identification of positive cells and rapid microscopic scoring, may serve as a basis for an automated analysis of TG^R lymphocytes. V_f values detected by the anti-BrdU assay are higher than mutant frequencies obtained by the cloning assay, which has been assigned to the presence of non-mutant phenocopies considered to represent spontaneously cycling lymphocytes. Although the anti-BrdU assay is rapid and easy and has been shown to respond to genotoxic exposures, its true value could be evaluated only when it can be ascertained that phenocopies do not significantly contribute to the V_f values obtained.     

Enumeration of 6-thioguanine-resistant peripheral blood lymphocytes in man as a potential test for somatic cell mutations arising in vivo.

An autoradiographic method to enumerate variant 6-thiogunanine-resistant (TGr) peripheral blood lymphocytes (PBLs) that occur in vivo in man is described. Variant cells are detected in PBL cultures stimulated to tritiated thymidine (3HTdr) incorporation in vitro with phytohemagglutinin (PHA) in the presence of TG. Cells with the naturally-occurring Lesch--Nyhan (LN) mutation served as prototype-variant cells. PBLs from a LN hemizygous male were found to be resistant to TG inhibition of PHA-stimulated 3HTdr in corporation in vitro while a LN heterozygous female was found to be a mosaic with 2/1000 PBLs resistant to 2 X 10(-4) M TG. Experiments with artificial mixtures of LN and normal PBLs showed that the LN cells were virtually all detectable even when present in low frequency (10(-5)). TGr PBLs were found in healthy non-LN individuals at median frequencies of 1.0 X 10(-4) and 1.1 X 10(-4) when determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG respectively. Their frequencies were not age-related. TGr PBL-variant frequencies (Vf's) were determined in 47 cancer patients who were being treated with cytotoxic agents that are known to be mutagens. The median TGr PBL Vf determined at 2 X 10(-3) M TG in cancer patients was 2.2 X 10(-4) while, when determined at 2 X 10(-4) M TG, it was 8.5 X 10(-4). The distribution of Vf's for the treated cancer-patient group differed from that for the normal control group in that more than half of the treated cancer patients had TGr PBL Vf's greater than the highest seen for controls. Unlike those of the normal controls, the TGr PBL Vf's of treated cancer patients differed if determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG, a behavior that suggested partial resistance and mimicked that seen with LN TGr PBLs. PBLs resistant to 2,6-diaminopurine (DAPr) were not found in two individuals, although the TGr PBL Vf was elevated in one. TGr PBL Vf's were greatly elevated under conditions of in vivo selection in patients receiving purine-analogue immunosuppressive therapy. The TGr PBL enumerative assay system is presented as one of potential value to detect somatic cell mutations occurring in vivo in man.     

Cell-mediated immunity to influenza virus antigen and mitogen in guinea pigs: measurement with a semimicro protein synthesis assay

A rapid and precise method for the assay of cell-mediated immune response basing on protein synthesis stimulation of mitogen-activated guinea pig lymphocytes is modified in a way that enables the study of virus-immunological problems. When used as a micromethod it has the following advantages over conventional methods: short-term cell culture, need of low quantities of cells and rapid preparation of great numbers of samples for radioactivity measurements. In this study we report the results of comparative experiments on measuring lymphocyte stimulation after addition of PHA and stimulation of sensitized lymphocytes following contact with homologous influenza virus antigen in vitro. The most important reaction parameters are as follows: 5-6 . 10(5) spleen lymphocytes/microculture in microtiter plates, use of Eagles's MEM cell culture medium without leucine, supplemented with HEPES buffer and 10% autologous guinea pig serum; optimum lymphocyte stimulation by addition of 0.5 microliter PHA or 0.1-1.0 microgram virus protein/ml; immuno-stimulation by PHA can be measured in vitro already after 6 h and by influenzavirus antigen already after 24 h.     

Effects of lectin activation on sialyltransferase activities in human lymphocytes

The effects of phytohemagglutinin (PHA) stimulation on the activities of sialyltransferase 1 (SAT-1), and sialyltransferase 3 (SAT-3), in human lymphocytes were investigated in vitro. For SAT-1 and SAT-3, respectively, the apparent Km values with variable CMP-NeuAc concentrations were 0.19 and 0.015 mM and with variable LacCer were 0.075 and 0.17 mM. Progressive increases in the activities of SAT-1 and SAT-3 were detected in lymphocytes stimulated with PHA, whereas no increase was observed in control lymphocytes incubated in culture medium alone. These increased activities occurred within 18-36 h of incubation and preceded optimum lymphocyte proliferation. Intact lymphocytes were needed for the lectin-stimulated increase of sialyltransferase activities because neither concanavalin A nor phytohemagglutinin added to the broken cell preparation modulated SAT-1 activity. The glycolipid products formed as a result of these enzymatic reactions in the presence of endogenous and exogenous acceptors were tentatively identified by thin-layer chromatography and autofluorography. The addition of exogenous LacCer to the SAT-1 assay resulted in the radiolabeling of a small amount of ganglioside GM1b (3.4%), but GM3 was the major labeled product (96%). When GgOse4Cer was added to the SAT-3 assay, 32% GM3 and 24.6% GM1b were detected while 44% consisted of glycolipids not labeled in assays performed without exogenous acceptors. Of the radioactivity transferred to endogenous acceptors, 81.3% was in GM3 and 14.6% in GM1b. These results demonstrate that the modulation of sialyltransferase activity occurs earlier than cellular activation.     

Presence of a reversible inhibitor for human lymphoid cell DNA synthesisin the extracts of human peripheral lymphocytes.

An endogenous inhibitor of human lymphocyte DNA synthesis contained in extracts of purified human peripheral lymphocytes is described. It was found that the peripheral lymphocyte extract inhibits the DNA synthesis of phytohemagglutinin (PHA) stimulated human peripheral lymphocytes, lymphocytes in mixed lymphocyte culture, and human lymphoid cells in a long-term culture (PGLC-33H). This extract did not inhibit the DNA synthesis of nonlymphoid cells including HeLa and human embryonic lung. The effects of the inhibitor were reversible and noncytotoxic. Initial characterization showed the inhibitor to be thermolabile, DNase resistant, trypsin sensitive, and stable in a pH range 5.4–8.4. It appears that the inhibitor contained in the purified human peripheral lymphocyte extract is similar to a previously described inhibitor extracted from a human lymphoid cell line (PGLC-33H). Quantitation of the inhibitor in various lymphoid cell populations showed the amount of inhibitor per cell to be higher in resting peripheral lymphocytes than in PHA stimulated peripheral lymphocytes or human lymphoid cells in long-term culture (PGLC-33H). This data suggest that the inhibitor described may play a regulatory role in lymphocyte metabolism.