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M Saxena

Publications and source records attributed to M Saxena.

At least 37 records · Page 2Linked to original sources
PubMed1999

Crosstalk between cAMP-dependent kinase and MAP kinase through a protein tyrosine phosphatase.

The haematopoietic protein tyrosine phosphatase (HePTP) is a negative regulator of the MAP kinases Erk1, Erk2 and p38. HePTP binds to these kinases through a kinase-interaction motif (KIM) in its non-catalytic amino terminus and inactivates them by dephosphorylating the critical phosphorylated tyrosine residue in their activation loop. Here we show that cyclic-AMP-dependent protein kinase (PKA) phosphorylates serine residue 23 in the KIM of HePTP in vitro and in intact cells. This modification reduces binding of MAP kinases to the KIM, an effect that is prevented by mutation of serine 23 to alanine. The PKA-mediated release of MAP kinase from HePTP is sufficient to activate the kinase and to induce transcription from the c-fos promoter. Expression of a HePTP serine-23-to-alanine mutant inhibits MAP-kinase dissociation and activation and induction of transcription from the c-fos promoter. We conclude that HePTP not only controls the activity of MAP kinases, but also mediates crosstalk between the cAMP system and the MAP-kinase cascade.

Alanine↗
PubMed1998

Lymphocyte activation: the coming of the protein tyrosine phosphatases.

The molecular mechanisms of signal transduction have been at the focus of intense scientific research world-wide. As a result, our understanding of protein tyrosine kinase-mediated signaling has advanced at an unprecedented pace during the past decade. In contrast, the study of protein tyrosine phosphatases is in its infancy, but is currently gathering momentum and is predicted to become a "hot topic" in the field within the next few years. This review summarizes the current state-of-the art in our understanding of the structure, regulation and role of protein tyrosine phosphatases in lymphocyte activation.

Animals↗
PubMed1998

Negative regulation of T cell antigen receptor signal transduction by hematopoietic tyrosine phosphatase (HePTP).

The hematopoietic tyrosine phosphatase (HePTP) is predominantly expressed in thymocytes and T lymphocytes and at lower levels in other hematopoietic cells. Expression of the gene is enhanced by the T cell growth factor interleukin-2, suggesting a role for HePTP in T cell proliferation or differentiation. We report that HePTP blocks T cell antigen receptor (TCR)-induced transcriptional activation of a reporter gene driven by a nuclear factor of activated T cells(NFAT)/AP-1 element taken from the interleukin-2 gene promoter. This effect was specific to HePTP and was abolished by a mutation (C270S) that impaired its phosphatase activity. Co-expression of HePTP also reduced TCR-induced activation of the mitogen-activated protein kinase Erk2 and the TCR-induced appearance of phosphorylated Erk. In contrast, HePTP did not affect the activation of the N-terminal c-Jun kinase, Jnk. Together these findings suggest that HePTP plays an active negative role in TCR signaling by dephosphorylating one or several signaling molecules between the receptor and the mitogen-activated protein kinase pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

Modification of phosphatidylinositol 3-kinase SH2 domain binding properties by Abl- or Lck-mediated tyrosine phosphorylation at Tyr-688.

In cells expressing the oncogenic Bcr-Abl tyrosine kinase, the regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on tyrosine residues. We report that this phosphorylation event is readily catalyzed by the Abl and Lck protein-tyrosine kinases in vitro, by Bcr-Abl or a catalytically activated Lck-Y505F in co-transfected COS cells, and by endogenous kinases in transfected Jurkat T cells upon triggering of their T cell antigen receptor. Using these systems, we have mapped a major phosphorylation site to Tyr-688 in the C-terminal SH2 domain of p85. Tyrosine phosphorylation of p85 in vitro or in vivo was not associated with detectable change in the enzymatic activity of the phosphatidylinositol 3-kinase heterodimer, but correlated with a strong reduction in the binding of some, but not all, phosphoproteins to the SH2 domains of p85. This provides an additional candidate to the list of SH2 domains regulated by tyrosine phosphorylation and may explain why association of phosphatidylinositol 3-kinase with some cellular ligands is transient or of lower stoichiometry than anticipated.

Fusion Proteins, bcr-abl↗
PubMed1997

Insulin down-regulates the inducible nitric oxide synthase pathway: nitric oxide as cause and effect of diabetes?

Evidence in this paper indicates that insulin can down-regulate the inducible nitric oxide synthase (iNOS) pathway in vivo. The iNOS pathway is up-regulated in diabetes-prone rats and mice and is associated with an autoimmune process. However, the results presented here indicate that macrophage nitric oxide (NO) production and iNOS mRNA expression are also elevated in rats or mice made diabetic by streptozotocin injection in which there is no primary autoimmune component. Insulin administration reduces NO production in autoimmune-prone and streptozotocin-induced diabetic rodents. Finally, insulin decreases macrophage NO production in normal hosts. These results indicate that the autoimmune paradigm is inadequate to explain increased NO in diabetes. As a potential mechanism to explain insulin-mediated regulation of NO production, TGF-1 may be involved because 1) macrophages from diabetic mice produce less TGF-beta1 than macrophages from normal hosts; 2) the circulating TGF-beta1 level is lower in diabetic mice; and 3) insulin administration increases circulating TGF-beta1 in normal mice. Together, these results provide evidence that increased NO in diabetes is not only a cause but also an effect of beta-cell destruction and results in part from a heretofore unrecognized immunomodulatory activity of insulin.

Animals↗
PubMed1997

Multiple routes and regulation by tyrosine phosphorylation characterize the ATP-dependent transport of 2,4-dinitrophenyl S-glutathione in inside-out vesicles from human erythrocytes.

ATP-dependent efflux routes for 2,4-dinitrophenyl S-glutathione (DNP-SG) were investigated using inside-out vesicles from human erythrocytes. Nonlinear double-reciprocal plots of transport at substrate concentrations ranging from 0.07 to 500 micro;m indicated that multiple transport routes were operative. Sensitivity to naphthyl glucuronide separated [3H]DNP-SG transport into two low-affinity components which by computer analysis exhibited Km values of 330 and 1400 micro;m, respectively. At low substrate concentrations, two high-affinity routes were observed. The predominant activity (hMOAT3a) exhibited a Km of 0.18 micro;m (Vmax = 22 pmol/min/mg protein), whereas the second activity (MOAT3b) had a Km of 0.58 micro;m (Vmax = 16 pmol/min/mg protein). High-affinity transport of DNP-SG increased substantially (2.5-fold) in vesicles preincubated with genistein or other tyrosine kinase inhibitors. Kinetic analyses in vesicles pretreated with 50 micro;m genistein showed that increased transport resulted from the appearance of a new activity (hMOAT3c) with a Km of 0.85 micro;m and a substantially elevated Vmax (80 pmol/min/mg protein). At varying concentrations of genistein, a progression was observed that was consistent with the conversion of hMOAT3b to hMOAT3a and hMOAT3a to hMOAT3c. Phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, produced an opposite progression. Specificity studies showed that hMOAT3a exhibited the highest affinity for various anionic conjugates, and had a notable binding preference for glutathione disulfide. The relative effectiveness of the various inhibitors was similar for hMOAT3a, hMOAT3b, and hMOAT3c, as well as for a corresponding mMOAT3 activity from L1210 mouse cells. The results show that human erythrocytes contain multiple ATP-dependent efflux systems for DNP-SG and that separation of these systems can be achieved on the basis of substrate Km value and inhibitor and activator specificity. High-affinity transport can proceed via three activities which appear to be subforms of a single system with differing levels of tyrosine phosphorylation. Multiple hMOAT3 subforms provide flexibility for extruding various anionic conjugates and may have evolved in erythrocytes to expedite the efflux of GS-SG.

Adenosine Triphosphatases↗
PubMed1997

Suspension model for blood flow through stenotic arteries with a cell-free plasma layer.

The effects of the red cell concentration, the shape of the stenosis and a peripheral layer on blood flow characteristics due to the presence of a mild stenosis, are investigated. To account for the red cell concentration and the peripheral layer, blood is represented by a two-fluid model of particle-fluid suspension, and to estimate the effect of the stenosis shape, a suitable geometry has been considered such that the axial shape of the stenosis can be changed easily just by varying a parameter (referred to as the shape parameter). It is shown that the flow resistance increases with the cell concentration but decreases with increasing shape parameter. The existence of the peripheral layer causes significant reduction in the flow resistance. The wall shear stress distribution in the stenotic region and its magnitude at the maximum height of the stenosis (i.e., at stenosis throat) possess the variations similar to the resistance to flow with respect to any parameter except the shape parameter. The latter is independent of the shape whereas the former decreases in the converging zone as the shape parameter increases while it increases in the diverging zone in a similar situation. To discuss the physiological relevance, the analytical results are used to estimate the blood flow characteristics for different diseases using the experimental data and the present theoretical approach.

Anemia, Sickle Cell↗
PubMed1996

MOAT4, a novel multispecific organic-anion transporter for glucuronides and mercapturates in mouse L1210 cells and human erythrocytes.

Glucuronides and mercapturates were examined as possible high-affinity substrates for a low-affinity ATP-dependent transport system for 2,4-dinitrophenyl S-glutathione (DNP-SG) in mouse L1210 cells. Initial inhibitor studies with inside-out vesicles revealed that the low-affinity transport of [3H]DNP-SG (Km 450 microM) exhibits a high sensitivity to N-acetyl 2,4-dinitrophenyl cysteine (NAc-DNP-Cys) (Ki 5.0 microM) and alpha-naphthyl beta-D-glucuronide (naphthyl glucuronide) (Ki 8.5 microM). Direct transport measurements showed the presence of ATP-dependent uptake activities for NAc-DNP-[35S]Cys and naphthyl [14C] glucuronide, and Km values for half-maximal transport were comparable to the Ki values of these compounds for inhibition of [3H]DNP-SG transport. Transport of [3H]DNP-SG, NAc-DNP-[35S]Cys and naphthyl [14C]glucuronide each showed the same sensitivity to various anions and anion conjugates. Inhibition was competitive and was most potent for bilirubin ditaurate, indoprofen, 4-biphenylacetic acid, 4-acridine 4 beta-D-glucuronide, N-acetyl leukotriene E4, 17 beta-oestradiol 3 beta-D-glucuronide and taurolithocholate 3-sulphate. Inside-out vesicles from human erythrocytes contain a comparable ATP-dependent transport system. These results show that NAc-DNP-Cys and naphthyl glucuronide are high-affinity substrates for a single system identified previously as a low-affinity transporter of DNP-SG. Substrate and inhibitor studies identify this system as a novel multispecific organic-anion transport system (MOAT4) that accommodates glucuronides and mercapturates and is distinct from other MOAT transporters. Human erythrocytes contain an additional ATP-dependent system for NAc-DNP-Cys (Km 33 microM) that does not transport monoglucuronides.

Acetylcysteine↗
PubMed1996

Nitric oxide mediates early dysfunction of rat and mouse islets after transplantation.

Evidence presented in this paper indicates that nitric oxide (NO), generated by a nonspecific "wound"-type of inflammation, is an important mediator of the early dysfunction of transplanted islets in rodents. Although allogeneic islets stimulate NO production to a greater degree than syngeneic islets, the amounts of NO produced after either are significantly elevated above baseline. Inhibition of NO production by N(G)-monomethyl-L-arginine (NMA), markedly decreases the time needed to restore euglycemia after intraportal transplantation of syngeneic islets in diabetic rats. The dose of NMA used was not observably toxic, with no significant changes in blood pressure, hepatic artery blood flow, serum hepatic enzyme levels, or in weight compared with control animals. In rat recipients of intraportal syngeneic transplants, evidence that NO is produced at the site of implantation includes (1) an early and transient increase in posttransplant hepatic vein nitrate levels (pretransplant, 90 microM; 24 hr, 230 microM; 48 hr, 250 microM; 72 hr, 170 microM; and 96 hr, 140 microM), (2) concurrent appearance of inducible NO synthase mRNA in liver extracts, and (3) immunohistochemical localization of inducible NO synthase within the transplanted islets. Suppression of NO production or inhibition of NO activity is a potential strategy to increase the early function and engraftment transplanted islets in the clinical setting.

Animals↗
PubMed1996

Identification of efflux systems for large anions and anionic conjugates as the mediators of methotrexate efflux in L1210 Cells.

Two ATP-dependent efflux systems for methotrexate have been identified in inside-out vesicles from an L1210 mouse cell variant with a defective influx carrier for methotrexate. Transport at 40 muM [3H]methotrexate was separated by inhibitors into two components comprising 62 and 38% of total transport activity. The predominant route was inhibited by low concentrations of indoprofen (Ki=2.5 muM, 4-biphenylacetic acid (Ki=5.3 muM), and flurbiprofen (Ki=5.2 muM, whereas the second component showed a high sensitivity to the glutathione conjugates of bromosulfophthalein (Ki=0.08 muM), ethacrynic acid (Ki=0.52 muM, and 1-chloro-2,4-dinitrobenzene (Ki=0.77 muM). Bilirubin ditaurate was a potent inhibitor of both transport components (Ki=1.5 and 0.17 muM, respectively). Separation of transport activities without interference from the other route was achieved by adding an excess (100 muM) of either the glutathione conjugate of ethacrynic acid or biphenylacetic acid. Double-reciprocal plots of transport at various substrate concentrations gave Km values of 170 and 250 muM for methotrexate transport via the anion-sensitive and conjugate-sensitive routes, respectively. A comparison of inhibitor specificities indicated that the anion-sensitive transport activity in vesicles represents efflux system II for methotrexate in intact cells and is the same system identified previously in vesicles as an anion/anion conjugate pump. The conjugate-sensitive activity corresponds to efflux system I for methotrexate in intact cells and is the same system identified in vesicles as the high-affinity glutathione conjugate pump.

Adenosine Triphosphate↗
PubMed1996

Efficacy of antimicrobial treatment in non-dysenteric persistent diarrhoea in a community setting.

OBJECTIVES: To determine the efficacy of antimicrobial treatment in non-dysenteric persistent diarrhoea in a community setting. METHODS: In this double-blind field trial, 156 children aged 4-36 months with persistent diarrhoea not associated with Giardia lamblia infestation seeking treatment in a community outpatient clinic, were randomized to receive a combination of nalidixic acid and metronidazole, metronidazole alone, or placebo for 7 days. RESULTS: In comparison with placebo, metronidazole treatment did not result in a significant reduction in the mean post-enrollment diarrhoeal duration and stool frequency, increase in the proportion of patients recovered by days 3, 5 and 7 of treatment, and increase in weight gain at days 7 and 14. Comparing the combination of nalidixic acid and metronidazole with metronidazole alone, 17.5% more children treated with the combination recovered by day 3 of treatment (p = 0.08) and the mean stool frequency ascertained on day 7 for the previous 24 h was 26.8% less in them (p = 0.05). The weight gains at days 7 and 14 were similar in the two groups. CONCLUSIONS: These findings indicate that metronidazole offers no therapeutic benefit in persistent diarrhoea not associated with Giardia lamblia and nalidixic acid has only a modest clinical benefit, which is not substantial enough to warrant its routine use.

Anti-Infective Agents↗
PubMed1995

ATP-dependent efflux of 2,4-dinitrophenyl-S-glutathione. Properties of two distinct transport systems in inside-out vesicles from L1210 cells and a variant subline with altered efflux of methotrexate and cholate.

The transport of 2,4-dinitrophenyl-S-glutathione (DNP-SG) into inside-out vesicles from L1210 cells was employed to identify and characterize ATP-dependent efflux routes for DNP-SG. Measurements of ATP-dependent uptake at varying concentrations of [3H]DNP-SG revealed the presence of two distinct transport systems. Transport at low substrate concentrations occurred predominantly via a high affinity system (Km = 0.63 microM), whereas a low affinity system (Km = 450 microM) predominated at high concentrations of substrate. The high affinity system was characterized by a potent inhibition by the glutathione conjugates of bromosulfophthalein (Ki = 0.09 microM) and ethacrynic acid (Ki = 0.44 microM), leukotriene C4 (Ki = 0.20 microM), and the taurate diconjugate of bilirubin (Ki = 0.10 microM). The low affinity transport system for DNP-SG exhibited a high affinity for bilirubin ditaurate (Ki = 1.8 microM), indoprofen (Ki = 3.0 microM), and biphenylacetic acid (Ki = 5.9 microM). Different results were obtained with an L1210/C7 variant which has a defect in the efflux of methotrexate and cholate. Vesicles from the latter cells contain the same low affinity transport activity as parental cells, but the high affinity route is absent and has been replaced by a system with an intermediate affinity for DNP-SG (Km = 4.5 microM). These results indicate that L1210 cells contain two unidirectional efflux pumps for DNP-SG with substantial differences in inhibitor sensitivity. The high affinity system shows a binding preference for glutathione conjugates but can also accommodate large anionic conjugates, whereas the low affinity system has a binding preference for large organic anions. Results with the variant cells support the hypothesis that the high affinity transport system for DNP-SG also mediates the unidirectional efflux of methotrexate and cholate in intact L1210 cells.

Adenosine Triphosphate↗
PubMed1995

Distinct systems mediate the unidirectional efflux of methotrexate and cholate in human CCRF-CEM cells.

Human CCRF-CEM cells were shown to contain energy-dependent and unidirectional efflux systems for the extrusion of methotrexate and cholate. Efflux activity was sensitive to temperature and to energy deprivation by treatment with antimycin A and to various other compounds which do not affect energy metabolism. A comparison of inhibitor sensitivities revealed that methotrexate and cholate efflux exhibit substantial differences in half-maximal inhibition (IC50) by indomethacin, reserpine, ethacrynic acid, ketoprofen, probenecid, and bromosulfophthalein, although these systems could not be distinguished by their responses to prostaglandin A1, meclofenamic acid, indoprofen, and biphenylacetic acid. Comparisons between cell lines showed that methotrexate efflux in CCRF-CEM cells exhibits an inhibitor response comparable to a secondary efflux system for methotrexate in L1210 cells (system II), whereas the inhibitor response of cholate efflux in CCRF-CEM cells resembles efflux system I in L1210 cells, the primary efflux route for both methotrexate and cholate. These results indicate that CCRF-CEM cells contain similar but separate systems for the efflux of methotrexate and cholate. CCRF-CEM cells thus differ from L1210 cells in that the latter mediate the efflux of methotrexate and cholate primarily via a single system.

Biological Transport↗
PubMed1994

Functional implications from the effects of 1-chloro-2,4-dinitrobenzene and ethacrynic acid on efflux routes for methotrexate and cholate in L1210 cells.

1-Chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid were examined for the ability to inhibit unidirectional efflux routes in L1210 cells that extrude both methotrexate and cholate (system I) and methotrexate alone (system II). These electrophiles were selected for study because of their known ability to undergo rapid intracellular conversion to glutathione conjugates. CDNB produced typical inhibitor kinetics and was a moderate inhibitor of both system I (IC50 = 4.8 microM) and system II (IC50 = 7.5 microM) with methotrexate as the substrate. However, a complex response was observed when cholate was employed as an alternative substrate for system I. Cholate efflux was stimulated initially at low levels of CDNB, but then slowed to a net inhibition as CDNB concentrations exceeded 10 microM. The latter characteristics for CDNB were not observed with ethacrynic acid, which produced a comparable inhibition of efflux system I regardless of the substrate employed (IC50 = 4.6 microM). Efflux measurements in an L1210/C7 variant which lacks system I confirmed that CDNB stimulates the activity of a substantial and unique efflux activity for cholate (system III). The inhibition of system I and II by CDNB and ethacrynic acid was not reversed by a wash step but required inhibitor removal and subsequent incubation at 37 degrees C. This slow reversal was attributed to a time-dependent clearance of inhibitory glutathione conjugates. A correlation between efflux systems for anions and anionic glutathione conjugates was demonstrated further by the ability of prostaglandin A1 and indomethacin, two potent inhibitors of methotrexate and cholate efflux, to inhibit the efflux of 2,4-dinitrophenyl-S-glutathione. These results support the hypothesis that efflux systems for methotrexate and cholate in L1210 cells are part of a family of efflux pumps which function in vivo to extrude various anions and anionic glutathione conjugates.

Adenosine Triphosphate↗