Biomedical subjects

A S Tan

Publications and source records attributed to A S Tan.

32 records · Page 2Linked to original sources

PubMed1997

Patient education in the management of diabetes mellitus.

AIM: A patient education programme in the management of diabetes mellitus (DM) was piloted in a government polyclinic. This study aimed to evaluate the effectiveness of the education programme in improving knowledge of DM and skills in self-care in order to achieve long term control of DM. METHOD: The study was carried out on an intervention group of 183 diabetic patients who completed the education programme and a control group of 95 diabetic patients who attended the clinic during the period of the study. The patients were assessed on their knowledge of diabetes and their practice for good control of the disease (dietary practice, compliance, home monitoring) through a questionnaire. Long term control was assessed by their glycosylated haemoglobin levels. The education programme comprised individual counselling using a diabetes education guide, talks, videoshows and food displays. RESULTS: The intervention group showed a significant and greater improvement in the knowledge of the disease and self-care and in the dietary practice (taking more unpolished rice/high fibre food, reducing calories intake and cutting down oily/fatty food) when compared to the control group. Compliance with medication and the mean HbA1c levels were also improved in the intervention group. CONCLUSION: In this study the educational intervention was observed to have improved the diabetic patients' knowledge of the disease and self-care and the long term control of the disease. Patient education is thus an important component in the management of diabetes mellitus.

Adult↗

PubMed1996

CD95 (Fas/Apo-1)-induced apoptosis results in loss of glucose transporter function.

Treatment of activated human T cells with CD95 (Fas/Apo-1) ligand or Abs against CD95 results in apoptotic cell death. Although cellular responses to CD95 ligation have been described in some detail, the early molecular events that result in T cell death are only now beginning to be elucidated. Using Jurkat cells as a model of activated human T cells, we have investigated the effects of CD95 ligation on glucose transport and on glucose transporter function. We show that within minutes of CD95 activation, the ability to transport glucose across the plasma membrane is compromised and that transient exposure to Abs against CD95 for as little as 3 min results in reduced glucose transport and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) responses measured at 16 h. The effects of CD95 ligation on glucose transport are shown to be associated with loss of affinity of glucose transporters for glucose without altered maximum velocity and without changes in the cell surface expression of Glut 1, the predominant glucose transporter isotype on Jurkat cells. These results support a model of CD95 induced cell death that, at least in its early stages, does not depend on signaling to the nucleus or on macromolecular synthesis. Acute regulation of glucose transport is proposed to be an early effector mechanism in CD95-induced apoptotic cell death.

Antibodies, Monoclonal↗

PubMed1995

Interleukin-3 facilitates glucose transport in a myeloid cell line by regulating the affinity of the glucose transporter for glucose: involvement of protein phosphorylation in transporter activation.

Growth factors promote cell survival and proliferation by activating signal transduction pathways that result in progression through the cell cycle and differential gene expression. Uptake of simple sugars needed for basal cell metabolism, and for macromolecular synthesis necessary for cell growth and proliferation, is thought to follow as a consequence of signal transduction to the nucleus. However, in the presence of inhibitors of DNA synthesis and respiration, growth factors can still promote cell survival responses in the short term, raising the possibility that they may also regulate critical membrane and cytosolic processes necessary for cell survival. We have tested this hypothesis directly by investigating the role of the haemopoietic growth factor, interleukin-3 (IL-3), in the regulation of glucose transport in the bone marrow-derived cell line, 32D. We show that IL-3 promotes glucose transport by actively maintaining the affinity of the plasma membrane, glucose transporter for glucose (Km 1.35 +/- 0.15 mM, n = 4). Withdrawal of IL-3 for 1 h resulted in reduced affinity for glucose (Km 2.96 +/- 0.28 mM, n = 4) without an associated change in Vmax. Furthermore, glucose transporter molecules as the cell surface, as determined by cytochalasin B binding to isolated plasma membranes, did not differ significantly between control and IL-3-treated cells. Inhibition of DNA synthesis with mitomycin C or with the respiratory poison, sodium azide, did not affect the ability of IL-3 to promote glucose transport. In contrast, the tyrosine kinase inhibitors genistein and erbstatin extensively inhibited control and IL-3-stimulated glucose transport, some preference of IL-3-stimulated glucose transport, some preference for IL-3-stimulated responses being observed at low inhibitor concentrations. The light-activated protein kinase C inhibitor, calphostin C, also inhibited control and IL-3-stimulated glucose transport but without preference for IL-3 responses. Additionally, the tyrosine phosphatase inhibitor, orthovanadate, stimulated control and IL-3-dependent glucose transport by 50-80% while the protein kinase A inhibitor, KT5720, inhibited glucose transport by about 20% at plateau values. These results indicate that IL-3 is involved in continuous maintenance of glucose transporter activity by a mechanism that involves tyrosine kinases and protein kinase C, and demonstrate that this activation is not dependent on respiration or signal transduction to the nucleus.

Animals↗

PubMed1995

Evidence that cell survival is controlled by interleukin-3 independently of cell proliferation.

Hemopoietic cell proliferation is controlled by a set of polypeptide growth factors and regulatory molecules that bind to cell surface receptors inducing cellular responses. Maintenance of a viable state, cell growth, DNA synthesis and mitosis are basic properties of proliferating cells, but links between growth factor receptors and each of these cellular outcomes are poorly understood. Most studies have monitored DNA synthesis as a measure of progression through the cell cycle or directly measured viable cell numbers, but cell survival per se as an output of receptor activation by ligand, has received little attention. In this study we have used a bone marrow-derived murine cell line that is dependent on interleukin-3 for growth, to investigate the relationship between DNA synthesis and a biochemical marker of cell survival, reduction of the tetrazolium salt, MTT. We show that at times up to 6 hr, continued DNA synthesis, RNA synthesis, protein synthesis, and mitochondrial respiration are not necessary for background or IL-3-stimulated MTT reduction. Furthermore, dibutyryl cyclic AMP promoted background and IL-3-dependent MTT reduction while simultaneously inhibiting DNA synthesis. These results provide evidence that IL-3 controls events involved in MTT reduction and cell survival independently of DNA synthesis.

Animals↗

PubMed1993

Isolation and characterization of two immunochemically distinct alkaline phosphatases from Pseudomonas aeruginosa.

We have isolated two alkaline phosphatases (H-AP and L-AP, for high and low molecular mass, respectively) from Pseudomonas aeruginosa PA01. These two enzymes were found to differ in mobility on sodium dodecyl sulphate polyacrylamide gels (H-AP, M(r) = 51,000 and L-AP, M(r) = 39,500), amino-terminal amino acid sequence and did not cross-react. Both enzymes were active as phosphomonoesterases while only L-AP demonstrated any phosphodiesterase activity. Both enzymes were purified from P. aeruginosa grown in phosphate limiting conditions using the same protocol and were identified in both periplasmic and extracellular locations. A low level of H-AP was produced constitutively whereas L-AP was produced only after induction by reduced phosphate concentration in the growth medium. An L-AP-like enzyme has been previously described, however, this is the first report of a second P. aeruginosa alkaline phosphatase.

Alkaline Phosphatase↗

PubMed1993

Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction.

The MTT assay, which is widely used to measure cell proliferation and to screen for anticancer drugs, is based on reduction of the tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by actively growing cells to produce a blue formazan product. Despite broad acceptance of this assay, neither the subcellular localization, nor the biochemical events involved in MTT reduction are known. Mitochondrial involvement in MTT reduction has been inferred from studies with respiratory inhibitors using succinate as a substrate, but the contribution of this activity to overall cellular MTT reduction is unknown. Using the bone marrow-derived cell line, 32D, we investigated the subcellular localization of MTT reduction using succinate, NADH, and NADPH as substrates. At optimum substrate concentrations, MTT reduction by whole cell homogenates was greatest with NADH and least with succinate, which accounted for less than 10% of the combined activities. Using succinate, 96% of recoverable MTT reducing activity was in particulate fractions of the cell and 77% in the mitochondrial and light mitochondrial/lysosomal fractions. When NADH and NADPH were used as substrates, increased amounts of MTT reducing activity were associated with soluble fractions of the cell and association with mitochondrial fractions was less pronounced. To further characterize MTT reduction by the mitochondrial fraction, respiratory chain inhibitors were used to explore involvement of electron transport in MTT reduction. Succinate-dependent mitochondrial MTT reduction was inhibited by 80% with chlorpromazine, 70% by antimycin A, and 85-90% by thenoyltrifluoracetone (TTFA), but inhibition was not observed with rotenone at < or = 2 microM, Amytal, or azide. These results suggest that when succinate is used as an electron donor, 70-80% of mitochondrial MTT reduction occurs subsequent to transfer of electrons from cytochrome c to cytochrome oxidase, but prior to the point of azide inhibition. In contrast to succinate, NADPH-dependent mitochondrial MTT reduction was not affected by any of the respiratory inhibitors tested, and NADH-dependent reduction was only inhibited by chlorpromazine (40-50% at plateau concentrations). These results suggest that most cellular MTT reduction occurs outside the mitochondrial inner membrane and involves NADH and NADPH-dependent mechanisms that are insensitive to respiratory chain inhibitors. This interpretation is supported by whole cell studies in which rotenone failed to affect basal and interleukin-3-stimulated MTT reduction at times up to 4 h but strongly inhibited DNA synthesis. We conclude that most cellular reduction of MTT occurs extramitochondrially and probably involves the pyridine nucleotide cofactors NADH and NADPH.

Animals↗

PubMed1993

Cyclic adenosine monophosphate promotes cell survival and retards apoptosis in a factor-dependent bone marrow-derived cell line.

Hemopoietic growth factors promote cell survival, proliferation and differentiation, but whether these processes, which often occur in concert, are mediated through the same or different receptor signaling mechanisms is not known. Using the bone marrow-derived IL-3-dependent cell line, 32D, we show that dibutyryl cyclic adenosine monophosphate (dbcAMP) retards the rapid loss of viable cells seen in the absence of IL-3. This effect is shown to be concentration-dependent and detectable within 16 hours of culture and is not associated with cell differentiation. At earlier times (2 to 7 hours), when no significant changes in cell numbers were observed, dbcAMP stimulated the reduction of dimethylthiazoldiphenyl tetrazolium bromide (MTT), and this effect was indistinguishable from that seen with IL-3. In contrast, control cells deprived of growth factor showed a decline in MTT response over this period. The effect of dbcAMP in maintaining cell viability and MTT responsiveness was associated with a concentration-dependent inhibition of 3H-thymidine incorporation into DNA, and retardation of the intranucleosomal cleavage of DNA that is associated with apoptosis. These results suggest that in 32D cells, cAMP can act to promote cell survival and retard apoptosis, quite independently of cell proliferation, by stimulating the activity of mitochondrial enzymes involved in MTT reduction.

Adenosine Triphosphate↗

PubMed1992

The protein kinase C inhibitor, calphostin C, inhibits succinate-dependent mitochondrial reduction of MTT by a mechanism that does not involve protein kinase C.

The light-activated protein kinase C inhibitor, calphostin C, is shown to inhibit the ability of IL-3-dependent 32D cells to reduce the tetrazolium salt, MTT. To determine whether this inhibition was mediated through mitochondria which have been implicated in MTT reduction, isolated mitochondria were treated with calphostin C in the presence of various substrates for mitochondrial electron transport and EDTA (to exclude PKC involvement). Calphostin C extensively inhibited succinate-dependent MTT reduction (IC50 = 110nM) but had little effect on either NADH- or NADPH-dependent MTT reduction. An alternative protein kinase C inhibitor, H7, did not affect succinate-dependent mitochondrial MTT reduction, and the protein kinase A inhibitor, KT5720, had little effect on either cellular or mitochondrial MTT reduction. These results show that in addition to its role as a PKC inhibitor, calphostin C is also a potent inhibitor of succinate-dependent mitochondrial electron transport.

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

PubMed1989

Expression of specific high-affinity binding sites for erythropoietin on rat and mouse megakaryocytes.

Considerable experimental and clinical evidence suggests a relationship between erythropoiesis and thrombopoiesis. This is supported by observations that erythropoietin (Epo), the primary regulator of erythropoiesis, can affect platelet production when injected into animals. In this study we provide experimental evidence for a direct effect of Epo on thrombopoiesis by demonstrating that 125I-labeled recombinant human Epo binds to rat and mouse bone marrow megakaryocytes. Thus, autoradiographic analysis using cold competition to measure specific binding has been used to demonstrate that Epo binding to megakaryocytes increases with megakaryocyte maturation. When corrected for cell size, Epo binding sites per unit surface area increase from Stage I megakaryoblasts to Stage II megakaryocytes, and then remain approximately constant throughout further megakaryocyte maturation. Receptor density on megakaryocytes is similar to that on pronormoblasts in the rat, and in mice is 60% that on pronormoblasts. No binding of Epo to platelets or to naked megakaryocyte nuclei was detected. Equilibrium binding studies with partially purified rat megakaryocytes (20%-40% pure), where megakaryocytes are the only significant Epo binding cell population, showed a single class of saturable, high-affinity binding sites present on average at 6500 binding sites per megakaryocyte with a KD of 287 pM. Binding of [125I]Epo to rat megakaryocytes was inhibited with an antiserum against murine erythroblasts. These results suggest that the effects of Epo on thrombopoiesis may be directly mediated through specific, high-affinity binding sites for Epo on the surface of maturing megakaryocytes.

Animals↗

PubMed1989

Monoclonal antibodies that bind to hemopoietic stem cells: characterization and immunohistochemical localization of cells expressing gp50-65.

The cell surface of hemopoietic stem cells has been shown to express several antigens in common with more mature hemopoietic cells. One set of stem cell antigens is defined by a group of three monoclonal antibodies (13C6, 1C10, and 1A9), selected on the basis of binding to subpopulations of spleen colony-forming stem cells (CFU-S). These antibodies are shown to recognize cell surface glycoproteins of 50-65 kd (gp50-65) that occur widely on hemopoietic cells. Each cell type investigated shows a distinctive pattern of expression of these glycoproteins. To further investigate the presence of gp50-65 on stem cells, low-density bone marrow cells were labeled with 1C10 and sorted according to fluorescence intensity. Most (73%) of the stem cells (CFU-S10) were recovered in the two most highly fluorescent fractions containing 2.6% of starting marrow cells. Immunohistochemistry of frozen sections of normal spleen and spleens during repopulation after lethal irradiation and bone marrow transplantation showed that the most strongly 1C10-labeled cells occurred under the splenic capsule and along trabeculae. Although many of these cells were also alpha-naphthylacetate esterase positive and Mac-1 positive, indicating cells of the monocyte-macrophage lineage, a distinctive population of singular cells were stained with 1C10 alone. These cells were negative for surface Ig and closely corresponded with a small population of cells in a similar location that were doubly labeled with 1C10 and anti-Thy-1. These results show that stem cells express high levels of gp50-65 and suggest that stem cells can be identified by immunohistochemical methods using dual labeling procedures.

Animals↗

PubMed1987

Polymorphic glycoprotein-1 on mouse platelets: possible role of Pgp-1 and LFA-1 in antibody-dependent platelet cytotoxicity involving complement.

The presence of the Pgp-1 glycoprotein on mouse platelets is demonstrated by antibody-binding techniques, by immunoprecipitation, and by transblotting using the monoclonal antibody (MoAb) C71/26 against Pgp-1. C71/26 immunoprecipitates as a broad band of mol wt 87,000 to 100,000 as determined by radioiodination of the platelet cell surface and by the 3H-sodium borohydride labeling technique. Immunoblotting showed Pgp-1 expression on platelets to be quantitatively similar to its presence on macrophages and resolved platelet Pgp-1 into two bands of mol wt 87,000 and 97,000 whereas Pgp-1 on parasite-elicited peritoneal macrophages showed 82,000 and 87,000 mol wt species. Platelets and monocyte/macrophage cells from either peripheral blood or from the peritoneal cavity showed homogeneous binding of Pgp-1 antibody to greater than 97% of cells by flow cytometry. In contrast, lymphocytes from peripheral blood or from the spleen showed a heterogeneous binding pattern with 20% to 30% of cells being negative, and the majority weakly positive. In functional studies, MoAbs against CR1 and CR3 substantially inhibited platelet immune adherence, whereas C71/26 showed only marginal inhibitor. In contrast, C71/26 and other MoAbs against Pgp-1 inhibited platelet-dependent cytotoxicity of antibody-coated sheep erythrocytes in the presence of C5-deficient mouse plasma whereas M1/70 against CR3 showed no effect. In this assay, MoAbs against the alpha- and beta-subunits of leukocyte functional molecule LFA-1 also inhibited platelet cytotoxicity. These results show that the platelet cell surface moieties Pgp-1 and LFA-1 are involved in or closely associated with antibody-dependent cellular cytotoxicity by platelets.

Animals↗

PubMed1985

Cell-lineage antigens of the stem cell-megakaryocyte-platelet lineage are associated with the platelet IIb-IIIa glycoprotein complex.

The stem cell-platelet lineage is uniquely defined by platelet cell-lineage antigens. These antigens are present on all stem cells measured by the spleen colony assay and become restricted to the platelet cell lineage as differentiation proceeds. In this study, anti-platelet serum (APS) has been used to identify cells in the bone marrow that express platelet cell-lineage antigens and to identify platelet cell surface molecules expressing these antigens. Anti-platelet IgG extensively absorbed with brain, thymus, and peritoneal cells bound selectively to stem cells, megakaryocyte progenitor cells (Mk-CFC), and megakaryocytes in CBA mouse bone marrow and to blood platelets. No other hemopoietic cell type, tissue, cell line, or tumor cell bound significant amounts of antibody against platelet cell-lineage antigens as determined by ability to absorb the anti-stem cell activity in APS. Studies with lactoperoxidase-labeled platelets showed that two major iodinated proteins of Mr = 114,000 and 138,000 were immunoprecipitated with APS and with antiserum that had been extensively absorbed. These proteins correspond to the platelet IIb-IIIa glycoprotein complex, which is known to express receptors for collagen and fibrinogen, molecules known to influence hemopoietic cell proliferation and tumor cell growth. A panel of six monoclonal antibodies against human IIb-IIIa inhibited spleen colony formation by 17% to 100%, J15 and A5.15 also being cytotoxic for granulocyte-macrophage progenitor cells and Mk-CFC. Other platelet monoclonal antibodies did not inhibit spleen colony formation. Although APS inhibited fibrinogen binding to platelets and platelet aggregation, these activities were greatly reduced with absorbed antiserum. Furthermore, fibrinogen treatment of bone marrow did not block the anti-stem cell activity in APS. Thus the evidence is consistent with expression of platelet cell-lineage antigens on the platelet IIb-IIIa glycoprotein complex at a site removed from the fibrinogen binding site.

Animals↗

PubMed1984

Changes in cell surface antigens during stem cell ontogeny.

A panel of monoclonal antibodies that bind to the murine pluripotential stem cell CFU-s was used to examine the antigenic profile of the stem cell during ontogeny. The results show that the stem cell surface changes dramatically during development. One group of three independently derived monoclonal antibodies binds to subpopulations (50%-70%) of stem cells at plateau values, and these populations increase marginally during development. A second group of four monoclonal antibodies, including anti-H-2Kk (11-4.1), define stem cell antigens that increase from low levels in the fetal liver to high levels in adult bone marrow. The presence of these two classes of antigens on adult splenic stem cells was in general similar to that observed on adult bone marrow. Antigens defined by the first group of monoclonal antibodies were present in similar amounts on CBA, C57B1/6, and Balb/c bone marrow stem cells, whereas antigens of the second group showed mouse strain variations. Quantitative absorption analysis was used to distinguish H-2Kk (11-4.1) from 9F6, which showed a similar developmental profile. Monoclonal antibodies recognizing subpopulations of stem cells were shown to be distinct by complementation studies and recognized antigens not present on brain tissue.

Animals↗

PubMed1984

Binding of monoclonal antibodies that inhibit spleen colony formation to leukemic cell lines.

Clonogenic tumor cells and normal stem cells share the property of extensive proliferative potential. Normal stem cells are under stringent growth restraint and respond to appropriate differentiation signals, whereas tumor stem cells have lost the ability to respond normally to these controls. In an attempt to define cell surface molecules involved in the control of hemopoietic cell proliferation and differentiation, we have produced 5 monoclonal antibodies against antigens held in common between hemopoietic stem cells and the Abelson virus-induced pre-B-lymphoma cells from which they were derived. Four of these monoclonal antibodies produced greater than 90% reduction of spleen colony-forming cells, whereas the other bound to a subpopulation (60 to 70%) of spleen colony-forming cells at plateau values. The expression of antigens recognized by these and two other anti-stem cell monoclonal antibodies has been shown to correlate with the differentiation status of a panel of tumor cell lines, with greater expression being observed on cells more closely resembling the pluripotent stem cell than mature hemopoietic cells. Immunoperoxidase staining of bone marrow showed that these antigens are mainly expressed by monocytes and blast cells. Treatment of bone marrow cells with those antibodies which extensively inhibited spleen colony formation and with rabbit complement abolished the ability of progenitor cells to form colonies in soft agar. Quantitative absorption studies distinguished the antigens recognized by two of the anti-stem cell monoclonal antibodies from those detected by anti-H-2k 11-4.1 monoclonal antibody. These observations suggest that the antigens involved may play a role in the regulation of growth and differentiation of stem cells and undifferentiated leukemic cells.

Animals↗