Biomedical subjects

T Yi

Publications and source records attributed to T Yi.

69 records · Page 4Linked to original sources

PubMed1993

Hck tyrosine kinase activity modulates tumor necrosis factor production by murine macrophages.

The hematopoietic cell kinase (hck) is a member of the src family of tyrosine kinases, and is primarily expressed in myeloid cells. Hck expression increases with terminal differentiation in both monocyte/macrophages and granulocytes and is further augmented during macrophage activation. Recent evidence has implicated src-related tyrosine kinases in critical signaling pathways in other hematopoietic lineages. Herein we demonstrate that manipulation of the level of hck expression in the murine macrophage cell line BAC1.2F5 alters the responsiveness of these cells to activation by bacterial lipopolysaccharide (LPS) but does not affect survival or proliferation. Overexpression of an activated mutant of hck in BAC1.2F5 cells augments tumor necrosis factor (TNF) production in response to LPS, whereas inhibition of endogenous hck expression, by antisense oligonucleotides, interferes with LPS-mediated TNF synthesis. Together, these observations suggest that hck is an important component of the signal transduction pathways in activated macrophages.

Base Sequence↗

PubMed1993

JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells through interaction with its receptor (EPOR). Although EPOR is a member of the cytokine receptor superfamily and lacks a kinase domain, EPO induces tyrosine phosphorylation, which is correlated with gene transcription and mitogenesis. Here we demonstrate that EPO induces tyrosine phosphorylation of JAK2 kinase and activates its in vitro autophosphorylation. Using EPOR mutants, phosphorylation and activation of kinase activity correlate with the induction of mitogenesis. Furthermore, JAK2 physically associates with a membrane-proximal region of the EPOR cytoplasmic domain that is required for biological activity. The results support the hypothesis that JAK2 is the kinase that couples EPO binding to tyrosine phosphorylation and mitogenesis.

3T3 Cells↗

PubMed1993

Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene.

Mice homozygous for the recessive allelic mutation motheaten (me) or viable motheaten (mev) on chromosome 6 develop severe defects in hematopoiesis. In this paper we present the findings that the me and mev mutations are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. High resolution mapping localized me to an area tightly linked to Hcph on chromosome 6. Abnormalities of the Hcph protein product were demonstrated by Western blot analysis and by activity assays in both me/me and mev/mev mice. Molecular analysis of the Hcph cDNA identified abnormal transcripts in both mutants. DNA sequence analyses of cDNA and genomic clones revealed that both the me and mev mutations are point mutations that result in aberrant splicing of the Hcph transcript. These findings provide the first available animal models for a specific protein-tyrosine phosphatase deficiency, thus facilitating determination of the precise role of this signaling molecule in hematopoiesis.

Animals↗

PubMed1993

Determination of nutritional requirements in rats: variation with time of weight gain responses to indispensable amino acids.

The Saturation Kinetics Model (SKM) can be used to describe physiological responses as functions of a limiting dietary nutrient. Physiological responses also vary with time, and, by graphing each parameter of the SKM as a function of time, the model equation can be used to produce a three-dimensional response surface, allowing the investigator to predict requirement as a function of both dietary nutrient concentration and time. To test this hypothesis, rats were fed diets containing graded levels of indispensable amino acids (0-10 g/100 g). The inhibition form of the SKM was able to predict the complete response range of weight gain, food intake, weight-specific weight gain and weight-specific food intake for each amino acid on a day-by-day basis. With a complete response range established, nutrient requirements (defined as maximum responses) could be determined explicitly by the equation: Requirement = (K0.5.KS)0.5, where K0.5 and KS are parameters of the SKM. No arbitrary decisions (such as break points, slopes, percentages of maximum, etc.) were necessary to accomplish this. This approach allows one to set nutritional requirements and optimize responses in a dynamic system without causing inhibiting and/or toxic responses. In each case (valine, methionine, threonine and histidine), the dietary amino acid concentration required for maximal weight gain exceeded current NRC recommendations, which may be low because they fall in the more time-dependent portion of the response curve whereas maximum responses do not.

Amino Acids↗

PubMed1993

Hematopoietic cell phosphatase associates with the interleukin-3 (IL-3) receptor beta chain and down-regulates IL-3-induced tyrosine phosphorylation and mitogenesis.

Hematopoietic cell phosphatase (HCP) is a tyrosine phosphatase with two Src homology 2 (SH2) domains that is predominantly expressed in hematopoietic cells, including cells whose growth is regulated by interleukin-3 (IL-3). The potential effects of HCP on IL-3-induced protein tyrosine phosphorylation and growth regulation were examined to assess the role of HCP in hematopoiesis. Our studies demonstrate that, following ligand binding, HCP specifically associates with the beta chain of the IL-3 receptor through the amino-terminal SH2 domain of HCP, both in vivo and in vitro, and can dephosphorylate the receptor chain in vitro. The effects of increasing or decreasing HCP levels in IL-3-dependent cells were assessed with dexamethasone-inducible constructs containing an HCP cDNA in sense and antisense orientations. Increased HCP levels were found to reduce the levels of IL-3-induced tyrosine phosphorylation of the receptor and to dramatically suppress cell growth. Conversely, decreasing the levels of HCP increased IL-3-induced tyrosine phosphorylation of the receptor and marginally increased growth rate. These results support a role for HCP in the regulation of hematopoietic cell growth and begin to provide a mechanistic explanation for the dramatic effects that the genetic loss of HCP, which occurs in motheaten (me) and viable motheaten (mev) mice, has on hematopoiesis.

Animals↗

PubMed1993

Association of hematopoietic cell phosphatase with c-Kit after stimulation with c-Kit ligand.

Protein tyrosine phosphorylation and dephosphorylation have been implicated in the growth and functional responses of hematopoietic cells. Recent studies have identified a novel protein tyrosine phosphatase, termed hematopoietic cell phosphatase (HCP) or PTP1C, that is predominantly expressed in hematopoietic cells. HCP encodes a cytoplasmic phosphatase that contains two src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules with activated growth factor receptors containing intrinsic protein kinase activity, we assessed the association of HCP with two hematopoietic growth factor receptors, c-Kit and c-Fms. The results demonstrate that HCP transiently associates with ligand-activated c-Kit but not c-Fms and that this association occurs through the SH2 domains. In both colony-stimulating factor 1- and stem cell factor-stimulated cells, there is a marginal increase in tyrosine phosphorylation of HCP. Lastly, HCP can dephosphorylate autophosphorylated c-Kit and c-Fms in in vitro reactions. The potential role of HCP in stem cell factor signal transduction is discussed.

Amino Acid Sequence↗

PubMed1993

Expression of a novel form of Tec kinase in hematopoietic cells and mapping of the gene to chromosome 5 near Kit.

The Tec kinase was initially identified as a novel cytoplasmic protein tyrosine kinase that is preferentially expressed in the liver and is highly homologous to the Drosophila Dsrc28C src-related tyrosine kinase. In screening of interleukin 3 (IL-3)-dependent myeloid leukemia cells for protein tyrosine kinases, we observed that all cell lines examined expressed high levels of Tec transcripts. However, characterization of Tec cDNAs indicated that they differed significantly from the published sequence. Most strikingly, an insertion of 41 bp in the 5' region affects the initiation codon and results in replacing the published 13 amino acid amino-terminal sequences with 94 amino acids. Using polymerase chain reaction (PCR) analysis, only the form containing the insertion was detected in hematopoietic cells. In addition, we found an in-frame insertion of 66 bp that introduces an additional 22 amino acids into the SH3 domain. This insertion restores conserved SH3 sequences that are found in the src gene family and in the Dsrc28C gene. By PCR analysis, approximately equal levels of Tec transcripts containing the intact SH3 domain and containing the 22 amino acid deletion were found in hematopoietic cells. Lastly, by interspecies backcross analysis, we show that the Tec gene is tightly linked to the c-Kit gene on mouse chromosome 5.

Amino Acid Sequence↗

PubMed1993

Use of stroma-supported cultures of leukemic cells to assess antileukemic drugs. I. Cytotoxicity of interferon alpha in acute lymphoblastic leukemia.

Leukemic cells from most cases of acute lymphoblastic leukemia (ALL) rapidly die by apoptosis in vitro, unless they are cultured onto bone marrow-derived stromal layers. We have recently established a stroma-supported tissue culture technique that allows long-term culture of leukemic lymphoblasts. In this study, we used this technique to examine interferon alpha (IFN alpha) cytotoxicity to ALL blasts. In 16 ALL cases tested (14 B-lineage ALL, 2 T-ALL), the number of cells recovered after 7 days of culture on stromal feeder layers was 60-178% (median, 108%) of those originally seeded. The percentage of lymphoblasts killed by 2000 U/ml IFN alpha 2b after 7 days of culture, ranged from < 1% to 91% (median, 56%). Cytotoxicity was (i) dose-dependent, (ii) eliminated by a neutralizing antibody to IFN alpha, and (iii) accompanied by tyrosine phosphorylation of a 135 kDa protein, which was detectable after 5 minutes of treatment. Numbers of residual normal lymphoid cells in the cultures remained low and conditioned medium prepared from IFN alpha-stimulated T, NK, and stromal cells was not cytotoxic to ALL blast cells. In contrast to results in freshly isolated ALL cells, six ALL cell lines tested were completely resistant to IFN alpha cytotoxicity. We conclude that IFN alpha is directly cytotoxic in most ALL cases but that the intensity of its effects varies widely among cases. The method used in this study may be applied to evaluate leukemic blast cell sensitivity to compounds with potential antileukemic activity, and to select patients to be entered in to clinical trials.

Adolescent↗

PubMed1992

Resource-dependent selection.

This paper presents a resource-dependent viability selection differential equation model of continuously reproducing diploid population with two alleles at one locus for a single limiting resource. This model assumes that the genotypic fitness is only a function of the limiting resource. The conditions that the interior equilibrium point of the system exists are that the heterozygote fitness is positive and the homozygote fitness is negative, or the heterozygote fitness is negative and the homozygote fitness is positive at the point. The sufficient and necessary conditions of locally asymptotical stability of the interior equilibrium point are that the heterozygote fitness is positive at the point, or the locally asymptotically stable equilibrium corresponds to the point at which the level of the limiting resource is locally minimized on the zero mean fitness curve, f = 0.

Animals↗

PubMed1992

Assignment of a novel protein tyrosine phosphatase gene (Hcph) to mouse chromosome 6.

Hematopoietic cell phosphatase (Hcph) was identified by amplification of conserved protein tyrosine phosphatase sequences from a myeloid cell line and is predominantly expressed in hematopoietic cells. Hcph is unique in containing two, tandemly repeated, src-homology 2 domains in the amino terminal region of the phosphatase. Using a genomic probe in interspecific backcross analysis, the murine Hcph gene maps to mouse Chromosome 6 and is tightly linked to the Tnfr-2 and Ly-4 genes.

Animals↗

PubMed1992

Identification of Leishmania donovani isolates from different kala-azar foci in China by kDNA hybridization.

kDNA sequence homology of Leishmania donovani isolates from three types of kala-azar foci in China were analyzed by using dot and Southern hybridization with biotin- and 32P-labelled probes. The results revealed kDNA sequence heterogeneity among Leishmania donovani isolates from the three kala-azar foci: sequence homology between isolates of hill and desert foci was higher than that between hill and plain foci isolates. The kDNA hybridization technique was also found to be specific and sensitive for direct identification of Leishmania in animal tissues. In a preliminary survey, kDNA hybridization of cutaneous tissue blots of 71 dogs from endemic regions showed a positive rate of 40.8%, and the rate of double positive cases (touch blot hybridization and bone marrow smear) reached 91.3%. The direct identification of Leishmania in tissues by kDNA hybridization seems to be a useful and convenient method for epidemiological study and clinical diagnosis, especially for species/strain characterization.

Animals↗

PubMed1991

Identification of novel protein tyrosine phosphatases of hematopoietic cells by polymerase chain reaction amplification.

Polymerase chain reaction (PCR) conditions were used to amplify cDNAs that encode putative protein tyrosine phosphatases (PTPs) from a murine interleukin-3-dependent myeloid cell line. Primers for the reactions were based on conserved sequences of the catalytic domain that are shared among all known PTPs. Sequencing of 100 PCR-amplified cDNA clones identified seven different cDNA sequences. Two of these sequences were identical to the murine PTP genes Ly5/CD45/LCA and LRP/R-PTP-alpha. Two of the cDNA sequences were 95% identical to human PTP epsilon (HPTP epsilon) and rat brain PTP (PTP1B), respectively, and are likely to represent their murine homologs. Three of the cDNA sequences encoded novel potential PTPs. One of the putative PTPs was ubiquitously expressed while a second was predominantly expressed in brain, kidney, and liver and at much lower levels in a variety of other cell tkpes and tissues. The third novel putative phosphatase was expressed primarily in hematopoietic cells and tissues in a pattern that was comparable with Ly5/CD45/LCA. Further characterization of these novel PTPs will provide insights into the growth regulation of hematopoietic cells.

Amino Acid Sequence↗

PubMed1983

Quantitation and detection of isotypes of anti-SS-B antibodies by ELISA and Farr assays using affinity purified antigens. An approach to the investigation of Sjögren's syndrome and systemic lupus erythematosus.

Affinity purified SS-B was characterized as a protein with immunoreactive polypeptides of 40K and 29K. A modified Farr assay and an enzyme linked immunosorbent assay (ELISA) were 100- to 1,000-fold more sensitive than immunodiffusion and showed an association with the systemic manifestations of primary sicca syndrome and Sjögren's syndrome with systemic lupus erythematosus. The ELISA was sufficiently sensitive to detect class specific antibodies in saliva and lymphocyte culture supernatants.

Adult↗

PubMed1983

Relationship of precipitating antibodies to soluble cellular antigens and histologically defined renal lesions in systemic lupus erythematosus.

Antibodies to nuclear ribonucleoprotein (nRNP), Sm, SS-A, and SS-B, were studied by a quantitative immunodiffusion technique in 48 patients with systemic lupus erythematosus (SLE). There was no correlation between the presence or titre of antibodies to any of these antigens and clinically apparent renal disease. However, among 34 patients who had renal biopsies antibodies to nRNP, Sm, and/or SS-A were seen in all 8 patients with membranous glomerulonephritis. Low-titre antibodies (less than 1:4) were seen in 3 out of 7 patients with diffuse proliferative glomerulonephritis. Longitudinal studies in 4 patients who had serial biopsies indicated that the association between serological and histological findings was maintained for prolonged periods. In 2 patients whose biopsy pattern changed, antibodies to nRNP, Sm, and SS-A antedated the development of membranous nephritis.

Antigens↗

PubMedDate not supplied

Identification of a potent peptide deformylase inhibitor from a rationally designed combinatorial library.

Peptide deformylase catalyzes the removal of the N-terminal formyl group from nascent polypeptides during prokaryotic protein synthesis and maturation and is essential for bacterial survival. Its apparent absence from mammalian organisms makes it an attractive target for designing novel antibacterial agents. Based on the substrate specificity of peptide deformylase from Escherichia coli, a focused library of peptide thiols was synthesized on TentaGel resin using a disulfide linkage. Screening of the library against the purified deformylase was carried out in solution phase after the inhibitors were released from the resin with a reducing agent. A potent deformylase inhibitor was obtained from a 750-member library and was further optimized through rational modification into a low nanomolar inhibitor (KI = 15 nM against E. coli deformylase).

Amidohydrolases↗