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Biomedical subjects

R Chakrabarti

Publications and source records attributed to R Chakrabarti.

At least 19 recordsLinked to original sources
PubMed1995

Detection of a functional promoter/enhancer in an intron-less human gene encoding a glutamine synthetase-like enzyme.

A human genomic clone, psi GS, containing an intron-less glutamine synthetase (GS)-encoding pseudogene, was isolated by screening a human library. A sequence of 3004 bp, containing the GS coding region and both the 5' and 3' flanking sequences, was identified that exhibits all the characteristics of a processed pseudogene. The coding region shows 93% identity with the human GS cDNA (hGS) sequence and contains two frame-shifts and two termination codons. The coding sequence is flanked by a 9-bp AT repeat and a putative polyadenylation site, AATAAA, at the 3' end. Primer extension analysis and S1 nuclease mapping showed a transcription start point (tsp) 62 bp upstream from the start codon indicating a shorter untranslated region than hGS. Transfection of HeLa cells with cat constructs containing portions of the 5' flanking sequence showed the presence of a functional promoter/enhancer within 200 bp of the tsp, independent of its orientation.

Amino Acid Sequence↗
PubMed1995

T cell receptor-mediated Ca2+ signaling: release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane.

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca2+ release from the intracellular stores followed by a sustained Ca2+ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca2+ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca(2+)-ATPase inhibitor, stimulated Ca2+ entry into the cells by a mechanism other than emptying Ca2+ stores. In addition, Ca2+ entry into the Ca(2+)-depleted cells was stimulated by low basal level of cytosolic Ca2+, not by the emptying of intracellular Ca2+ stores. Both the Ca2+ release and influx were dependent on high and low concentrations of extracellular Ca2+. At low concentrations, Mn2+ entered the cell through the Ca2+ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn2+ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca2+ release was inhibited by low concentrations of Ni2+, La3+, and EGTA, while Ca2+ influx was inhibited by high concentrations. Thus, in T cells Ca2+ influx occurs independently of IP3-dependent Ca2+ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca2+ influx. Extracellular Ca2+ influenced Ca2+ release and influx through the action of two plasma membrane Ca2+ entry pathways with different pharmacological and biochemical properties.

Animals↗
PubMed1995

Modulation of signal transduction in macrophages by dietary fatty acids.

Tumor growth can be altered by the amount and type of fat in the diet. Although there are several possible mechanisms for this, recent work suggests that alterations in the immune system by dietary fat may affect tumorigenesis. The focus of recent studies has been on dietary fat modulation of macrophage function because that cell plays a pivotal role in many immune responses, including anti-tumor activity. One possible mechanism of dietary fat effects on macrophages is altered signal transduction, which, in turn, could alter gene regulation and macrophage function. Initial studies tested the effects of dietary fat on kinase activity after stimulation with interferon-gamma. Macrophages from mice fed menhaden fish oil (MFO) had slightly decreased protein kinase C activity compared with macrophages from mice fed safflower oil (SAF). No differences among the diets were observed when the activity of protein kinase A and G were tested. When calcium mobilization was tested, we found that macrophages from mice fed MFO had an increased response compared with macrophages from mice fed SAF. Dietary fat also modified the response of macrophages to platelet-activating factor with respect to the induction of Ia expression. In studies to identify genes involved in dietary fat effects on macrophage function, we screened a cDNA library of macrophages treated with prostaglandin E2 (PGE2), a lipid-based mediator that can modulate macrophage function and be altered by dietary fat. The cloned gene, BTG1, was enhanced in macrophages treated with PGE2, but the relationship with dietary fat remains to be determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Insulin-sensitive association of GLUT-4 with endocytic clathrin-coated vesicles revealed with the use of brefeldin A.

The interaction of the adipocyte/skeletal muscle glucose transporter (GLUT-4) with clathrin lattices may be important in maintaining its intracellular distribution. To better understand the role of clathrin lattices in the sorting of GLUT-4, we have attempted to determine the cellular origin of clathrin-coated vesicles (CCVs) that contain this transporter. The fungal toxin brefeldin A (BFA) causes the selective disassembly of clathrin lattices at the trans-Golgi network (TGN), but not at the plasma membrane (PM), thus providing a way of estimating the proportion of GLUT-4 in PM- versus TGN-derived clathrin lattices. Exposure of 3T3-L1 adipocytes to BFA resulted in a rapid disassembly of clathrin lattices at the TGN, observed by optical sectioning microscopy, and to a pronounced decrease in the yield of CCVs purified from these cells. Thus, CCVs isolated from BFA-treated cells are likely to be derived from the PM. Immunoblotting experiments revealed the presence of GLUT-4 in such CCVs, suggesting that under basal conditions the transporter is continually retrieved from the PM through the CCV pathway. Exposure of both BFA-treated or non-treated cells to insulin resulted in a 4-6-fold increase in the concentration of GLUT-4 at the PM. In parallel, the concentration of GLUT-4 in PM-derived CCVs decreased by 60%. These results suggest (a) that the effect of insulin to increase the cell surface concentration of GLUT-4 is not inhibited by BFA, and (b) that a decreased association of GLUT-4 with endocytic CCVs may be important in facilitating its increased cell surface concentration in response to the hormone.

3T3 Cells↗
PubMed1994

Changes in glucose transport and transporter isoforms during the activation of human peripheral blood lymphocytes by phytohemagglutinin.

We have explored the mechanism of stimulation of glucose transport during PHA stimulation of human peripheral blood lymphocytes (HPBT) enriched in T cells. Equilibrium exchange flux of 3-O-methyl glucose (3-O-MG) was stimulated two- and fourfold at 24 and 48 h after PHA stimulation, respectively. The increase was transient in that the flux rate returned to control (unstimulated) levels by 96 h. Immunoblotting and immunoprecipitation using specific Abs revealed that resting HPBT expresses glucose transporter isoforms GLUT-2 and GLUT-3 but not GLUT-1. After PHA stimulation, GLUT-1 expression was induced predominantly in the plasma membrane, whereas GLUT-3 expression was simultaneously down-regulated. GLUT-1 expression was detectable at 24 h, peaked at 48 h, and disappeared at 96 h. The total number of glucose transporters per cell measured as the total capacity of D-glucose displaceable cytochalasin B binding did not change significantly at any time after PHA stimulation. PHA stimulation also caused expression of high affinity IL-2R and secretion of IL-2. The IL-2 secretion was transient, which peaked at 24 h, slightly preceding the GLUT-1 expression peak and disappeared at 72 h. In PHA-activated HPBT cells synchronized at G0-G1, GLUT-1 was not expressed but was rapidly induced by exposure to IL-2. This induction did not occur in the presence of cyclosporin A, which inhibits IL-2 secretion. Based on these observations, we conclude that PHA stimulation increases glucose transport partly by inducing the expression of GLUT-1 instead of GLUT-3 and that GLUT-1 expression is induced by signals generated by IL-2 binding to its high affinity receptors.

Cells, Cultured↗
PubMed1994

Glucocorticoids regulate intestinal glutamine synthetase gene expression in endotoxemia.

PURPOSE: Although glutamine is required to maintain gut mucosal metabolism and function, intestinal glutamine uptake from the gut lumen and from the bloodstream is decreased during sepsis. We hypothesized that endogenous mucosal glutamine biosynthesis is increased during endotoxemia, and we attempted to define the "stress" mediators that regulate the activity of small intestinal glutamine synthetase (GS), the principal enzyme of de novo glutamine biosynthesis in the gut. METHODS: Adult rats received Escherichia coli lipopolysaccharide (LPS) (7.5 mg/kg intraperitoneally), RU 38486 (a glucocorticoid antagonist; 10 mg/kg by gavage) 2 hours prior to LPS administration, antibody to tumor necrosis factor (TNF) (4 mg/kg intraperitoneally) prior to LPS administration, or ketorolac tromethamine (a prostaglandin synthesis inhibitor; 1 mg/kg intraperitoneally) followed by LPS administration. Mucosal GS activity was assayed 12 hours after LPS administration. In a separate set of studies, cultured intestinal mucosal cells (Caco-2) were exposed to LPS, interleukin 1 (IL-1), IL-6, TNF-alpha, interferon-gamma, prostaglandin E2, or dexamethasone. Twelve hours later, GS activity was assayed and messenger RNA was extracted. The GS transcripts were labeled with a GS complementary DNA probe radiolabeled with phosphorus 32, were quantitated by phosphoimaging, and were normalized to beta-actin. RESULTS: In vivo LPS treatment increased mucosal GS activity by 250%. Pretreatment with antibody to TNF or ketorolac did not inhibit the LPS-induced increase in mucosal GS, whereas pretreatment with RU 38486 attenuated the increase in gut GS activity by 60%. Lipopolysaccharide, IL-1, IL-6, TNF-alpha, gamma-interferon, and prostaglandin E2 did not increase GS activity in Caco-2 cells, whereas dexamethasone increased GS activity and messenger RNA 2.5-fold and threefold, respectively. These data indicate that cytokines and prostaglandins (prostaglandin E2) do not regulate mucosal GS expression during endotoxemia. Glucocorticoids, however, stimulate GS gene expression directly. CONCLUSIONS: This hormonally mediated response may support de novo mucosal GS during septic states when uptake of glutamine from the lumen and blood is decreased.

Animals↗
PubMed1994

Dexamethasone increases jejunal glutamine synthetase expression via translational regulation.

Glutamine provides energy and precursors for nucleotide biosynthesis for the gut mucosa, and it is essential for intestinal metabolism and function. During stress states, glutamine uptake of circulating and luminal glutamine may be diminished, but the ability of the gut mucosa to synthesize glutamine de novo in response to this decreased delivery remains undefined. Since the glucocorticoids play an important role in regulating interorgan glutamine metabolism during catabolic states, we hypothesized that these hormones induce the expression of gut mucosal glutamine synthetase (GS), the enzyme that catalyzes the intracellular biosynthesis of glutamine. Adult rats were treated with dexamethasone (DEX, 0.5 mg/kg intraperitoneally) or saline (controls). At various times after treatment (4, 12, 24, 48, and 72 hours), jejunal mucosal GS-specific activity was assayed, and total RNA was extracted. GS transcripts were detected by Northern blot analysis, using a radiolabeled rat GS cDNA probe. Transcripts were quantitated by phospho-imaging and normalized to beta-actin. An anti-GS polyclonal antibody was used to quantitate GS protein concentrations by Western blot analysis. The relative quantities of GS translated were measured using a cell-free protein-synthesizing system (reticulocyte lysate assay). Data were analyzed using analysis of variance and were considered statistically significant for p < 0.05. DEX increased GS activity by 45% 12 hours after administration. Western blot analysis revealed an increase in the concentration of the GS protein in the jejunum of DEX-treated animals. Northern blot analysis demonstrated no significant change in GS mRNA levels after DEX treatment, indicating the possibility of post-transcriptional regulation. In vitro translational experiments demonstrated that the quantity of GS translated was increased by 25% after the administration of DEX. These data suggest that glucocorticoids may increase jejunal mucosal GS levels by accelerating protein translation. This adaptive response could provide glutamine for the gut mucosa during stress, when exogenous glutamine supplies may be rate limiting.

Animals↗
PubMed1994

A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal.

The unique COOH-terminal 30-amino acid region of the adipocyte/skeletal muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test whether an underlying mechanism of this domain's function involves glucose transporter endocytosis rates, transfected cells were generated expressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUT1) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incubation of COS-7 or CHO cells expressing the HA-tagged chimera with anti-HA antibody at 37 degrees resulted in an increased rate of antibody internalization compared to cells expressing similar levels of HA-tagged GLUT1, which displays a cell surface disposition. Colocalization of the internalized anti-HA antibody in vesicular structures with internalized transferrin and with total transporters was established by digital imaging microscopy, suggesting the total cellular pool of transporters are continuously recycling through the coated pit endocytosis pathway. Mutation of the unique double leucines 489 and 490 in the rat GLUT4 COOH-terminal domain to alanines caused the HA-tagged chimera to revert to the slow endocytosis rate and steady-state cell surface display characteristic of GLUT1. These results support the hypothesis that the double leucine motif in the GLUT4 COOH terminus operates as a rapid endocytosis and retention signal in the GLUT4 transporter, causing its localization to intracellular compartments in the absence of insulin.

Amino Acid Sequence↗
PubMed1994

Cytokines decrease glutaminase expression in human fibroblasts.

BACKGROUND: Glutamine metabolism in fibroblasts is essential for energy production, nucleotide biosynthesis, and growth during wound healing. Because cytokines can impair fibroblast proliferation, we tested the hypothesis that cytokines impair glutamine metabolism. We studied the influence of several cytokines on the expression of glutaminase, the major enzyme of intracellular glutamine metabolism in fibroblasts. METHODS: Human foreskin fibroblasts were incubated for 6 and 12 hours with varying doses (10, 100, or 1000 units/ml) of interleukin (IL)-1, IL-6, tumor necrosis factor-alpha, or gamma-interferon. Cell lysates were assayed for glutaminase-specific activity, and glutaminase protein content was measured by Western blotting with a polyclonal antibody. Total cellular RNA was extracted, and relative glutaminase messenger RNA levels were determined by Northern blotting with a 32P-labeled glutaminase complement DNA-derived probe. These mRNA levels were normalized by blotting with a beta-actin cDNA-derived probe as control. Cell nuclei were isolated, and nuclear run-ons were used to determine relative glutaminase mRNA transcription rates. RESULTS: IL-1, IL-6, tumor necrosis factor-alpha, and gamma-interferon decreased glutaminase activity and protein concentration after a 12-hour incubation in a dose-independent fashion. No difference was noted at 6 hours. Western blot analysis showed a 30% to 60% reduction in glutaminase protein in treated cells. These cytokines also decreased glutaminase mRNA levels, consistent with transcriptional regulation. This was confirmed by nuclear run-on assays that showed a decrease in the number of glutaminase transcripts. CONCLUSIONS: A variety of different pro-inflammatory cytokines decrease glutaminase expression in cultured human fibroblasts. This cytokine-mediated inhibition of glutamine metabolism may limit the availability of key glutamine-derived intermediates and impair fibroblast proliferation in certain patients.

Cells, Cultured↗
PubMed1993

Fibrinolytic activity, clotting factors, and long-term incidence of ischaemic heart disease in the Northwick Park Heart Study.

Fibrinolytic activity (FA) was measured by dilute blood clot lysis time at entry to the Northwick Park Heart Study in 1382 white men aged 40-64, of whom 179 subsequently experienced episodes of ischaemic heart disease during a mean follow-up period of 16.1 years. There was a significant interaction between age and low FA (p = 0.02) with respect to ischaemic heart disease: a difference of one standard deviation in FA was associated with a difference of about 40% in ischaemic heart disease risk (p = 0.002) in those aged 40-54 at entry. The FA association remained after adjusting for plasma fibrinogen. High fibrinogen concentrations themselves were also associated with ischaemic heart disease, as was high factor VII activity with fatal events. Low FA in younger men may exert a long-term influence by impairing the removal of fibrin deposits that contribute to atherogenesis. Low FA appears to be a leading determinant of ischaemic heart disease in younger men and methods of enhancing fibrinolytic activity, whether by life-style changes or pharmacologically, should be considered.

Adult↗
PubMed1993

Interaction of the eucaryotic peptide chain initiation factor eIF-4A with the specific elements at the 5'-untranslated sequence of human asparagine synthetase mRNA.

The resistance of certain tumor cells to the chemotherapeutic agent L-asparaginase has often been found to be associated with the presence of asparagine synthetase activity. In an attempt to study the translational regulation of the asparagine synthetase gene, the 5'-untranslated region of human asparagine synthetase cDNA was mapped by antisense oligonucleotide-mediated hybrid arrest translation in reticulocyte lysate. Three consecutive cis-acting regulatory elements, spanning from -60 to -120 bases from the initiation codon, in the 5'-untranslated region of the asparagine synthetase gene, were identified. T1 RNase footprinting analysis showed that those regulatory elements can be protected from T1 digestion when incubated with reticulocyte lysate. A 46-kDa trans-acting protein factor that interacts with the cis-acting regulatory element of asparagine synthetase mRNA was detected. This 46-kDa protein factor is most likely to be the eucaryotic peptide chain initiation factor eIF-4A as determined by immunoprecipitation experiments using a monoclonal antibody raised against reticulocyte eIF-4A.

Aspartate-Ammonia Ligase↗
PubMed1992

Transport function and subcellular distribution of purified human erythrocyte glucose transporter reconstituted into rat adipocytes.

In order to delineate the insulin-independent (constitutive) and insulin-dependent regulations of the plasma membrane glucose transporter concentrations in rat adipocytes, we introduced purified human erythrocyte GLUT-1 (HEGT) into rat adipocytes by poly(ethylene glycol)-induced vesicle-cell fusion and its transport function and subcellular distribution in the host cell were measured. HEGT in adipocytes catalysed 3-O-methylglucose equilibrium exchange with a turnover number that is indistinguishable from that of the basal adipocyte transporters. However, insulin did not stimulate significantly the HEGT function in adipocytes where it stimulated the native transporter function by 7-8-fold. The steady state distribution and the transmembrane orientation assays revealed that more than 85% of the HEGT that were inserted in the physiological, cytoplasmic side-in orientation at the adipocytes plasma membrane were moved into low-density microsomes (LDM), while 90% of the HEGT that were inserted in the wrong, cytoplasmic side-out orientation were retained in the plasma membrane. Furthermore, more than 70% of the LDM-associated HEGT were found in a small subset of LDM that also contained 80% of the LDM-associated GLUT-4, the insulin-regulatable, native adipocyte glucose transporter. However, insulin did not cause redistribution of HEGT from LDM to the plasma membrane under the condition where it recruited GLUT-4 from LDM to increase the plasma membrane GLUT-4 content 4-5-fold. These results demonstrate that the erythrocyte GLUT-1 introduced in adipocytes transports glucose with an intrinsic activity similar to that of the adipocyte GLUT-1 and/or GLUT-4, and enters the constitutive GLUT-4 translocation pathway of the host cell provided it is in physiological transmembrane orientation, but fails to enter the insulin-dependent GLUT-4 recruitment pathway. We suggested that the adipocyte plasma membrane glucose transporter concentration is constitutively kept low by a mechanism where a cell-specific constituent interacts with a cytoplasmic domain common to GLUT-1 and GLUT-4, while the insulin-dependent recruitment requires a cytoplasmic domain specific to GLUT-4.

Adipose Tissue↗
PubMed1992

Demonstration of an insulin-insensitive storage pool of glucose transporters in rat hepatocytes and HepG2 cells.

The subcellular distribution of glucose transporters in rat hepatocytes and HepG2 cells was studied in the absence and in the presence of insulin. Glucose transporters were quantitated by measuring glucose-sensitive cytochalasin B binding and by protein immunoblotting using isoform-specific antibodies. Plasma membrane contamination into subcellular fractions was assessed by measuring distribution of 5'-nucleotidase and cell surface carbohydrate label. In hepatocytes, GLUT-2 occurred in a low-density microsomal (LDM) fraction at a significant concentration, and as much as 15% of cellular GLUT-2 was found intracellularly that cannot be accounted for by plasma membrane contamination. In HepG2 cells which express GLUT-1 and GLUT-2, the two isoforms showed distinct subcellular distribution patterns: GLUT-2 was highly concentrated in LDM while very little GLUT-1 was found in this fraction, indicating that a large portion of GLUT-2 occurs in intracellular organelles. Insulin treatment did not change the subcellular distribution patterns of glucose transporters in both cell types. Our results suggest that rat hepatocytes and HepG2 cells possess an intracellular storage pool for GLUT-2, but lack the insulin-responsive glucose transporter translocation mechanism.

Adipose Tissue↗
PubMed1992

Adaptive regulation in skeletal muscle glutamine metabolism in endotoxin-treated rats.

The effects of a single dose of endotoxin (7.5 mg/kg BW) on skeletal muscle glutamine metabolism were studied in vivo in rats to gain further understanding of the altered glutamine metabolism that characterizes sepsis and other catabolic diseases. In endotoxin-treated animals the arterial glutamine concentration fell early initially and then increased compared with control values. Twelve hours after treatment, the arteriovenous concentration difference for glutamine across the hindquarter doubled, resulting in a significant increase in net muscle glutamine release in endotoxin-treated rats. As a consequence, the muscle glutamine concentration fell in the endotoxin-treated animals by 25%-40%, an event that was apparent as early as two hours after endotoxin treatment. Skeletal muscle glutaminase activity, the major enzyme of glutamine breakdown, was unchanged by endotoxemia, but expression of glutamine synthetase mRNA and glutamine synthetase specific activity increased in a time-dependent fashion. The glutamine depletion that develops in skeletal muscle during endotoxemia is caused by accelerated muscle glutamine release rather than an increase in intracellular degradation or a fall in intracellular biosynthesis. The adaptive increase in glutamine synthetase expression that occurs requires de novo RNA and protein synthesis and may be designed to prevent complete depletion of the intracellular glutamine pool.

Adaptation, Physiological↗
PubMed1991

Interrelationships between mevalonate metabolism and the mitogenic signaling pathway in T lymphocyte proliferation.

Upon stimulation with antigen or antibodies directed at the CD3.T cell receptor complex, T lymphocytes undergo a series of biochemical events that result in DNA synthesis and cellular proliferation. The purpose of the current study was to explore the role of mevalonic acid and its metabolites in this process. Stimulation of freshly isolated human T cells with immobilized anti-CD3 monoclonal antibody (mAb) results in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase message, with maximum induction occurring at 24 h of culture, approximately 12 h before the onset of DNA synthesis. Protein kinase C (PKC) probably mediates this induction, as H7, which inhibits PKC and cyclic nucleotide-dependent protein kinases, but not HA1004, which inhibits all of these protein kinases except PKC, completely abrogates the appearance of HMG-CoA reductase message. The importance of HMG-CoA reductase induction and mevalonate production in cell cycle progression was demonstrated by the observation that either 25-hydroxycholesterol, which inhibits this induction, or lovastatin, a competitive inhibitor of HMG-CoA reductase, inhibited anti-CD3-induced T cell mitogenesis in a dose-dependent manner. The presence of lovastatin during the first 24-36 h of culture results in a progressive delay of cell cycle progression, whereas this agent, when present only for the first 12 h of culture, had no effect on T cell proliferation. These results suggest that mevalonate is required for cell cycle progression from mid-G1 into late G1. Exogenous mevalonate overcomes the antiproliferative effect of lovastatin but not of 25-hydroxycholesterol. Since 25-hydroxycholesterol suppresses the metabolism of mevalonic acid at multiple points, this result suggests that one or more metabolites of mevalonate, rather than mevalonate itself, plays an essential role in cell cycle progression. One metabolite of mevalonate, farnesol pyrophosphate, may play such a role, since free farnesol suppresses anti-CD3 mAb-induced T cell proliferation in a concentration-dependent manner. In mAb is associated with PKC-dependent induction of HMG-CoA reductase which, in turn, leads to the generation of mevalonic acid and its metabolites, one or more of which play a requisite role in cell cycle progression.

Antibodies, Monoclonal↗
PubMed1991

Early activation events render T cells susceptible to HIV-1-induced syncytia formation. Role of protein kinase C.

In human immunodeficiency virus-1 (HIV-1)-infected cell cultures, cell-to-cell fusion and the formation of multinucleated giant cells (syncytia) are induced as a consequence of interactions between the viral envelope glycoprotein on infected cells and cell surface CD4 molecules on uninfected cells. Although activated CD4+ T cells rapidly form syncytia when cultured with HIV-1 envelope glycoprotein expressing (env+) cells, freshly isolated, unstimulated CD4+ T cells do so more slowly. In these studies, we sought to explore the role of T cell activation in rendering CD4+ T cells susceptible to HIV-1-mediated syncytia formation. Our results indicate that within 2 h of exposure to immunologic stimuli, CD4+ T cells acquire the ability to form syncytia with HIV-1 env+ cells. Both cholera toxin, an inhibitor of protein kinase C (PKC) through its effects on inositol triphosphate and diacylglycerol production, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a noncompetitive inhibitor (with respect to ATP) of PKC, prevented unstimulated but not previously stimulated CD4+ T cells from forming syncytia with HIV-1 env+ cells. 1-Oleoyl-2-acetyl glycerol, an analog of the PKC activator, diacylglycerol, enhanced syncytia formation whereas ionomycin, a calcium ionophore, had no effect. These results suggest that activation of PKC is essential for previously unstimulated CD4+ T cells to become fusogenic.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Transfer of monoclonal antibodies into mammalian cells by electroporation.

A simple rapid and reproducible procedure for transferring monoclonal antibodies into mammalian cells by electroporation is described. Two functionally different monoclonal antibodies (Mab 3F3 and Mab 2B4) specific for asparagine synthetase (EC 6.3.1.1) were used for electroporation into HeLa, HT-5, and L5178Y D10/R (L-asparaginase-resistant) cells. The conditions were optimized so that the viability of the electroporated cells was very high (80-90%), and 90% of the viable cells had antibody incorporated. Electropermeabilized cells were structurally intact, and the high voltage electric pulse had no inhibitory effect on overall cellular DNA and protein synthesis. Incorporated immunoglobulins showed unaltered structural integrity and were functionally active. L5178Y D10/R cells incorporated with an antibody (Mab 3F3) known to be a potent inhibitor of tumor asparagine synthetase showed increased dependence on an exogenous source of asparagine in the culture medium, while the growth of cells incorporated with a control (noninhibitory) antibody (Mab 2B4) remained unaffected. These studies demonstrate that electroporation can be employed successfully for large scale transfer of antibodies into cultured mammalian cells for the study of cellular metabolism.

Animals↗