PubMed Health⌕ Search

Biomedical subjects

D R Kaplan

Publications and source records attributed to D R Kaplan.

123 records · Page 7Linked to original sources
PubMed1986

Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo.

We have constructed a recombinant murine retrovirus which efficiently transduces avian pp60c-src into murine cells and which is easily rescued from infected cells in plasmid form. To characterize the virus, several randomly selected NIH 3T3 lines were isolated after infection with recombinant retroviral stocks. All lines overproduced avian pp60c-src and appeared morphologically normal. Immunoprecipitates made from these lines with antisera specific for pp60c-src were tested for their kinase activities in vitro. We find that both autokinase and enolase kinase activities increase proportionately with the level of pp60c-src in the immunoprecipitates. To further test the authenticity of the pp60c-src encoded by the retroviral vector, these analyses were repeated in the presence of polyomavirus middle T antigen. Avian pp60c-src was activated as a protein kinase, indicating that the virally encoded pp60c-src interacts normally with middle T antigen. Interestingly, by increasing the intracellular levels of pp60c-src 15-fold over normal endogenous levels, we were unable to obtain a proportionate increase in the amount of middle-T-antigen-pp60c-src complex. Finally, using the shuttle features designed into the vector, we have isolated the first fully processed cDNA encoding functional avian pp60c-src X pp60c-src synthesized in vitro with this cDNA had intrinsic protein kinase activity and no detectable phosphatidylinositol kinase activity.

Animals↗
PubMed1986

Phosphatidylinositol kinases and cell transformation.

Products of phosphatidylinositol (PI) turnover have recently been implicated as regulators of cell growth and differentiation. Transformation of cells in culture by infection with certain viruses (Rous sarcoma virus, Kirsten sarcoma virus, and polyoma virus) or by transfection with the oncogenes carried by these viruses affect the steady-state level of intermediates in the PI turnover pathway. In addition, immunoprecipitates of the transforming gene products of Rous sarcoma virus and polyoma virus contain activities of certain enzymes in the PI turnover pathway. We have previously reported that polyoma middle T immunoprecipitates can catalyze phosphorylation of PI to phosphatidylinositol-4-phosphate (PIP). This activity is not intrinsic to middle T or pp60c-src but is due to a cellular enzyme that specifically associates with the middle T/pp60c-src complex. The PI kinase is found in immunoprecipitates of the middle t protein from polyoma viruses that are capable of cell transformation but does not associate with mutants of middle t defective in transformation, suggesting that this association may be important for transformation. Two PI kinases from fibroblasts (type I and type II) that are separable by anion exchange chromatography have been partially purified and characterized. These enzymes differ in their Km for ATP as well as their Ki for adenosine and ADP. Only the type I PI kinase specifically associates with the transformation-competent mutants of middle T.

1-Phosphatidylinositol 4-Kinase↗
PubMed1985

Large-scale production of polyoma middle T antigen by using genetically engineered tumors.

A recombinant plasmid containing a metallothionein promoter-polyoma middle T cDNA fusion was constructed and used to transfect NIH 3T3 cells. Transformed cells expressing middle T were injected into nude mice. Within 3 weeks, each mouse produced tumors containing middle T equivalent to that in 250 to 1,000 100-mm dishes of polyomavirus-infected cells. This middle T, partially purified by immunoaffinity chromatography, retained activity as measured by its ability to be phosphorylated in vitro. The combined approach of fusing strong promoters to genes of interest and utilizing nude mice to grow large quantities of cells expressing the gene provides a quick, inexpensive alternative to other expression systems.

Animals↗
PubMed1984

Influenza virus-specific human cytotoxic T cell clones: heterogeneity in antigenic specificity and restriction by class II MHC products.

Human cytotoxic T lymphocytes specific for A/JAP/57 (H2N2) influenza virus were cloned from in vitro stimulations of peripheral blood lymphocytes. Analysis of the viral specificity in cytotoxic function revealed one clone that killed all type A influenza-infected targets, another clone that was specific for the hemagglutinin subtype of the immunizing influenza virus, and the third clone that demonstrated cytotoxicity restricted to the hemagglutinin of A/JAP/57 and A/JAP/62 (H2N2) and not other type A influenza strains with the H2N2 subtypes. The phenotype of these three clones was Leu 2-, Leu 3+, Leu 4+; MHC restriction of their cytotoxic function was mapped to HLA-DR by a panel of target cells as well as by inhibition of cytotoxicity with monoclonal antibodies. Proliferation of these clones, examined in a tritiated thymidine incorporation assay, was found to be driven by antigen in the absence of exogenous lymphokines. For all three clones antigen-dependent production and secretion of lymphokines with IL-2 activity was demonstrated. The antigen specificity of proliferation and factor production was shown to be identical to the pattern that each clone revealed in its cytotoxic function.

Antigens, Viral↗
PubMed1984

Antigen-dependent regulation of interleukin 2 receptor expression on cloned human cytotoxic T lymphocytes.

IL 2 receptor expression as a function of time after antigenic stimulation was examined on antigen-dependent human CTL clones specific for type A influenza virus. The anti-Tac monoclonal antibody was used to follow IL 2 receptor levels on the cloned cells. Shortly after antigenic stimulation, IL 2 receptor expression was maximal; by 1 wk, however, levels had decayed considerably, and by 2 wk only background expression remained. Reexpression of IL 2 receptors could be induced by exposure of quiescent clones to antigen or lectin. IL 2-driven proliferation of the cytotoxic clones was also examined, and it decayed with the same kinetics as IL 2 receptor levels. Proliferation of quiescent cells could also be obtained by antigen-specific stimulation. Thus, IL 2 receptor expression by human CTL clones at least in part regulates the antigen-specific proliferation of these cells.

Antibodies, Monoclonal↗
PubMed1983

Insulin as a surface marker on isolated cells from rat pancreatic islets.

Immunoreactive insulin was shown to exist as a surface molecule in the plasma membrane of dispersed rat pancreatic islet cells. The intact cells were stained by immunofluorescence with a guinea pig antisera specific for insulin. The hormone on the cell surface could not be accounted for by insulin bound to specific receptors or nonspecifically absorbed to cells. Thus, surface insulin was demonstrated to be a specific membrane antigen for islet cells. Furthermore, the proportion of islet cells with insulin on the cell surface was directly correlated with insulin secretion in several different settings. This correspondence was demonstrated by varying the glucose concentration in the medium, by withholding Ca2+, which inhibits secretion, and by adding theophylline, which potentiates secretion. Consequently, these results suggested that insulin as a membrane protein was a marker for cells that actively secreted the hormone and may have been derived in the fusion process of secretory granules with the plasma membrane.

Animals↗
PubMed1979

Platelet alpha granules contain a growth factor for fibroblasts.

Platelets contain a polypeptide growth factor that stimulates the replication of normal connective tissue cells; this platelet-derived growth factor (PDGF) is released during the clotting process. Human platelets from normal volunteers were disrupted by nitrogen cavitation, and the subcellular organelles were fractionated by ultracentrifugation through a 30%--60% sucrose gradient. Electron microscopy revealed that fraction 7 (density 1.23 g/liter) contained the largest number of alpha granules. The specific activity of platelet fibrinogen, an alpha-granule marker, was also highest in this fraction. The subcellular fractions were assay for the presence of PDGF and for beta-thromboglobulin. PDGF was assayed quantitatively by the stimulation of DNA synthesis in confluent growth-arrested BALB/c-3T3 cells, whereas the concentration of beta-thromboglobulin was determined by radioimmunoassay. The highest concentrations of both PDGF and beta-thromboglobulin were found in the alpha-granule fraction. In contrast, beta-glucuronidase, a lysosomal enzyme, was more diffusely distributed and had its highest specific activity in fractions of lower density than those for PDGS, beta-thromboglobulin, or fibrinogen. The data demonstrate that the alpha granules of platelets provide a unique delivery system for PDGF, a polypeptide hormone with growth-promoting activity for connective tissue cells.

Animals↗
PubMed1978

Alteration of clonal profile. I. Effect of sublethal irradiation on the responses to phosphorylcholine in BALB/c mice.

BALB/c mice exhibit greater than 90% H8 clonal dominance in the immune response to phosphorylcholine. Adult mice exposed to 500 rads were initally unable to produce a humoral immune response to both phosphorylcholine and trinitrophenol antigens, and the direct plaque-forming cell response was slowly regained over several weeks. Clonotypic analysis wity antisera directed against the H8 idiotype showed that the H8 clone initially dominated the recovery of the response to phosphorycholine but that 60 days after the irradiation significant numbers of non-H8 clones could be detected. This same pattern could be seen in mice irradiated with 100 rads, a dose that does not completely abrogate the H8 response to phosphorylcholine. Sublethal irradiation of neonates before they had acquired responsiveness to phosphorylcholine could also eventually lead to the emergence of non-H8 idiotypes. Thus, a radiosensitive element regulates the expression of clonal dominance in anti-phosphorylcholine responses of BALB/c mice.

Animals↗
PubMed1978

Clonal dominance: loss and restoration in adoptive transfer.

An adoptive transfer system was used to study the mechanism responsible for clonal dominance of the anti-phosphorylcholine response in BALB/c mice. The adult spleen contains phosphorylcholine-specific precursor cells that are capable of developing into antibody-producing cells after transfer into lethally irradiated animals. The neonatal liver of the BALB/c mouse lacks precursor cells specific for phosphorylcholine but contains immature cells that differentiate into specific precursors during the normal course of ontogeny. The transfer of fetal or neonatal liver cells into lethally irradiated recipients prevents the appearance of the dominant H8 clone which constitutes the majority of the clones responding to phosphorylcholine in adult BALB/c mice. However, if these cells are transferred into neonatally suppressed recipients that lack the H8 idiotype, dominance of the H8 clone can develop. The conversion of the committed immature progenitor cell into a responsive B lymphocyte precursor is a regulated event. Regulation at the level of progenitor cells determines the eventual clonal profile of the immune response to phosphorylcholine. It is suggested that selection of the dominant clone occurs at this level.

Animals↗
PubMed1974

Clonal depletion in neonatal tolerance.

Specific unresponsiveness can be induced in neonatal and adult BALB/c mice by antibody against antigen-specific receptor (antireceptor antibody). When heterologous antireceptor antibody is used in the indirect fluorescence technique, the number of fluorescent cells in these animals is significantly lower than in normal animals. Fluorescent cells appear after a relatively brief incubation of cells from adult-suppressed animals, whereas no fluorescent cells are detected when cells from neonatally treated animals are incubated briefly. Evidently, treating neonatal mice with antireceptor antibody specifically depletes the antigen-responsive clone. In contrast, antireceptor antibody causes reversible blockade of responsive cells in adult-suppressed animals.

Animals↗
PubMedDate not supplied

Phase separation analysis of recombinant interleukin 2.

Phase separation in the detergent Triton X-114 has been used as a physical characteristic to distinguish secreted proteins from amphipathic proteins. Since recombinant interleukin 2 is not secreted by bacteria, but is instead obtained from bacterial lysates, we have analysed several different recombinant interleukin 2 molecules as well as the naturally synthesized cytokine by phase separation in Triton X-114. Although naturally synthesized interleukin 2 and recombinant interleukin 2 from R&D Systems partitioned into the aqueous phase as expected for secreted molecules, recombinant interleukin 2 from Cetus separated into the detergent phase, indicating a high degree of amphipathicity. A recombinant AMGEN interleukin 2 mutated protein (mutein) exhibited intermediate behavior. Cetus and AMGEN interleukin 2 differ from the R&D Systems recombinant molecule and from the native lymphokine by a change in amino acid position 125. Spontaneous dimerization of the Cetus and AMGEN interleukin 2 muteins to a 31 kD form has also been observed whereas multimeric structures have not been found in the other interleukin 2 preparations. These distinct biochemical differences between the two recombinant molecules appear to be related to small changes in the primary structure, and they may be relevant to the therapeutic use of interleukin 2.

Blotting, Western↗
PubMedDate not supplied

Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation.

Polyoma middle-T antigen is required for viral transformation of cultured cells and for tumorigenesis in animals. Like many other transforming gene products, middle-T is bound to the membrane and has an associated tyrosine kinase activity in vitro. This activity seems to result from the interaction of middle-T with pp60c-src, the cellular homologue of the transforming gene product of the Rous sarcoma virus, pp60v-src (refs 3-5). Both pp60v-src (ref. 6) and another retrovirus transforming gene product, pp68v-ros (ref. 7) were shown recently to have an associated phosphatidylinositol (PI) kinase activity in vitro and to increase PI turnover in vivo. These results suggest that viral transformation may be directly connected to a complex network of second messengers generated from PI turnover. Here, we assayed for PI kinase activity in immunoprecipitates made with middle-T- or pp60c-src-specific antisera of cells infected with polyoma virus. A PI kinase activity was detected in those immunoprecipitates which contained middle-T. Studies of mutants of middle-T defective in transformation indicate a close correlation between PI kinase activity and transformation.

1-Phosphatidylinositol 4-Kinase↗
PubMedDate not supplied

Activity-dependent activation of TrkB neurotrophin receptors in the adult CNS.

In this paper we have investigated the hypothesis that neural activity causes rapid activation of TrkB neurotrophin receptors in the adult mammalian CNS. These studies demonstrate that kainic acid-induced seizures led to a rapid and transient activation of TrkB receptors in the cortex. Subcellular fractionation demonstrated that these activated Trk receptors were preferentially enriched in the synaptosomal membrane fraction that also contained postsynaptic glutamate receptors. The fast activation of synaptic TrkB receptors could be duplicated in isolated cortical synaptosomes with KCl, presumably as a consequence of depolarization-induced BDNF release. Importantly, TrkB activation was also observed following pharmacological activation of brain-stem noradrenergic neurons, which synthesize and anterogradely transport BDNF; treatment with yohimbine led to activation of cortical TrkB receptors within 30 min. Pharmacological blockade of the postsynaptic alpha1-adrenergic receptors with prazosin only partially inhibited this effect, suggesting that the TrkB activation was partially due to a direct effect on postsynaptic cortical neurons. Together, these data support the hypothesis that activity causes release of BDNF from presynaptic terminals, resulting in a rapid activation of postsynaptic TrkB receptors. This activity-dependent TrkB activation could play a major role in morphological growth and remodelling in both the developing and mature nervous systems.

Animals↗