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Biomedical subjects

S Chen

Publications and source records attributed to S Chen.

At least 937 records · Page 52Linked to original sources
PubMed1996

Leukemic burden in subpopulations of CD34+ cells isolated from the mobilized peripheral blood of alpha-interferon-resistant or -intolerant patients with chronic myeloid leukemia.

We attempted to determine the frequency of normal hematopoietic stem cells (HSC) and contaminating leukemic cells in mobilized peripheral blood (MPB) collected from chronic myeloid leukemia (CML) patients, intolerant of alpha-interferon or with interferon-resistant disease. A total of 14 MPB samples, six from patients in chronic phase (CP) and eight from patients in accelerated phase or blast crisis (AP/BC) were studied. Cytogenetic analysis of MPB collected from AP/BC patients showed that 100% of the cells were Ph+, whereas cells from four of five CP MPB were Ph-. By contrast, fluorescence in situ hybridization (FISH) analysis of CP MPB showed a mean frequency of 14.7% Ph+ cells, while AP/BC MPB contained 39.2% Ph+ cells. In an attempt to purify normal HSC, subpopulations of the MPB CD34+ cells were isolated based on expression of the Thy-1 antigen (CDw90). The mean Ph+ cell frequency as determined by FISH within the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- populations from CP patients was 19.2% and 33.9%, respectively. In the AP/BC patients, levels of residual leukemic cells were significantly greater with mean Ph+ cell frequencies of 59.2% and 72.7% for the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- fractions, respectively. The frequency of cobblestone area forming cells (CAFC) was used as a means of quantitating the numbers of functional HSC within these cell subpopulations. The mean CAFC frequency was 1 of 19 for the CD34+Thy-1+Lin- cells as compared with 1 of 133 for the Thy-1-fraction indicating a higher frequency of primitive progenitor cells in the Thy-1+ subpopulation. CD34+ cell subsets from two patients were also injected into SCID-hu bone assays to determine the in vivo behavior of these cell populations. After 8 weeks, multilineage donor engraftment was observed in these grafts. FISH analysis of the donor cells within the grafts showed that 55.3% and 60.0% of the cells were Ph+. We conclude that unfractionated MPB from this patient population is not leukemia-free and that the CD34+Thy-1+Lin- cell subpopulation, although predominantly enriched for normal HSC, still contains substantial numbers of residual leukemic cells.

Adolescent↗
PubMed1996

Transient expression of lyn gene in Purkinje cells during cerebellar development.

Expression of the lyn gene, a member of sarcoma proto-oncogene family, was analyzed immunohistochemically during cerebellar development in the rat. Lyn immunoreactivity was intense in axons, dendrites and somata of Purkinje cells from gestational day 18 to postnatal day 15 and then decreased. lyn gene expression clearly followed the appearance and the maturation of dendritic arbors. A rapid decrease in Lyn protein, after the 18th postnatal day, left only a few scattered positive Purkinje cell somata in the adult. External and undifferentiated internal granule cells were weak in Lyn immunoreactivity but gradually increased during development. Clusters of positive granule cells were found along the Purkinje cell layer with parasagittal bands crossing the granular layer by 21 days. These bands persisted into adulthood. Cerebellar nuclei lacked immunoreactivity in early development but only fastigial neurons began to acquire lyn gene expression by the 15th postnatal day. The corresponding appearance of the lyn gene expression and the formation of Purkinje cell dendritic arbors suggests that Lyn protein is involved in dendrogenesis. On the other hand, the late onset of immunoreactivity in fastigial neurons and granule cells implies a role in cell maintenance.

Animals↗
PubMed1996

Temporal regulation of gene expression in adipocyte differentiation.

Cells usually become 'committed' to differentiate long before any actual morphological change is apparent. In one model commitment is a decision which corresponds to the expression of a control gene, while differentiation is the ultimate consequence of that decision. We have been studying adipocyte commitment and differentiation at the molecular level. Earlier we showed that the introduction of a specific DNA sequence into 'uncommitted' cells renders those cells committed to differentiate into adipocytes. Here we report temporal regulation of the expression of this DNA sequence; furthermore, we show that this RNA is in the non-polyA+ fraction of total cellular RNA. These data suggest that coordinate regulation of this and other genes is important in promoting differentiation.

3T3 Cells↗
PubMed1996

Cloning of rat 92-kDa type IV collagenase and expression of an active recombinant catalytic domain.

A full-length cDNA for rat 92-kDa type IV collagenase was isolated and sequenced. RNase protection assay revealed tissue specific differential expression of the 92-kDa type IV collagenase in the rat during development. Natural and modified forms of the 92-kDa type IV collagenase were expressed. One active protein, 92-CD, contained only the putative catalytic domain. Large quantities of the 92-CD were expressed in Escherichia coli, extracted from inclusion bodies, purified, and refolded to an active form. This recombinant protein was able to cleave denatured and native collagen and was inactivated by known MMP inhibitors.

Amino Acid Sequence↗
PubMed1996

G-protein-gated inward rectifier K+ channel proteins (GIRK1) are present in the soma and dendrites as well as in nerve terminals of specific neurons in the brain.

G-protein-gated inward rectifier potassium (GIRK) channels are coupled to numerous neurotransmitter receptors in the brain and can play important roles in modulating neuronal function, depending on their localization in a given neuron. Site-directed antibodies to the extreme C terminus of GIRK1 (or KGA1), a recently cloned component of GIRK channels, have been used to determine the relative expression levels and distribution of the protein in different regions of the rat brain by immunoblot and immunohistochemical techniques. We report that the GIRK1 protein is expressed prominently in the olfactory bulb, hippocampus, dentate gyrus, neocortex, thalamus, cerebellar cortex, and several brain stem nuclei. In addition to the expected localization in somas and dendrites, where GIRK channels may mediate postsynaptic inhibition, GIRK1 proteins were also found in axons and their terminal fields, suggesting that GIRK channels can also modulate presynaptic events. Furthermore, the distribution of the protein to either somatodendritic or axonal-terminal regions of neurons varied in different brain regions, which would imply distinct functions of these channels in different neuronal populations. Particularly prominent staining of the cortical barrels of layer IV of the neocortex, and the absence of this staining with unilateral kainate lesions of the thalamus, suggest that the GIRK1 protein is expressed in thalamocortical nerve terminals in which GIRK channels may mediate the actions of mu opiate receptors.

Amino Acid Sequence↗
PubMed1996

Inhibition of thromboxane A2 activity during warm ischemia of the liver.

To determine the role of thromboxane A2 (TxA2) in ischemic damage of the rat liver, we examined the effects of a TxA2 synthetase inhibitor (OKY 046) and a TxA2 receptor antagonist (ONO 3708). Rats were divided into three groups. In group I, a portion of the liver was subjected to 100 min of warm ischemia and the remaining liver resected. In group II, OKY 046 (30 mg/kg, intravenously) was given 5 min before the same procedure. In group III, ONO 3708 (10 mg/kg, intravenous) was given 5 min before ischemia. We then assessed survival, serum biochemistry, extent of histologic necrosis, and the levels of prostaglandin E2 (PGE2), TxB2, and 6-keto-PGF1-alpha. Pretreatment with OKY 046 and ONO 3708 significantly improved survival, decreased the tissue water content, and lowered the levels of serum transaminases and the extent of histological liver necrosis compared with the control group. OKY 046 markedly suppressed the level of TxB2, but not the levels of PGE2 or 6-keto-PGF1-alpha. ONO 3708 did not change the levels of PGE2, TxB2, or 6-keto-PGE1-alpha. In a liver perfusion model, OKY 046 and ONO 3708 did not suppress the uptake of trypan blue in hepatocytes. Our results demonstrate that either a TxA2 synthetase inhibitor or a TxA2 receptor antagonist can protect the liver from an ischemic insult. The effects of these drugs were due to inhibition of TxA2 synthesis and TxA2 blockade at the receptor, without modulating PGI2 or PGE1. Our results in a perfused rat liver model suggest that these drugs work during reperfusion and prevent postischemic tissue edema.

Animals↗
PubMed1996

A monoclonal antibody recognizing CD43 (leukosialin) initiates apoptosis of human hematopoietic progenitor cells but not stem cells.

CD43 (the major sialoglycoprotein of leukocytes) is an adhesion molecule broadly expressed on hematopoietic cells. A monoclonal antibody recognizing this molecule induces apoptosis of lineage marker-negative bone marrow hematopoietic progenitor cells (HPCs) that express CD34 at a high density (CD34hiLIN-). However, not all cells within this population undergo apoptosis on stimulation via CD43. Dividing progenitor cells are most highly affected, whereas more primitive quiescent cells survive anti-CD43 monoclonal antibody treatment. These surviving cells (1) are enriched for cobblestone area-forming cells, (2) repopulate fragments for human fetal bone implanted into C.B-17 scid/scid mice, (3) have a potential to differentiate in vivo to myeloid and lymphoid cells, and (4) have a high proliferative potential in long-term stromal cell-free liquid culture. These data indicate that cells with hematopoietic stem cell characteristics are relatively resistant to CD43-mediated apoptosis as compared with HPCs. Thus, CD43 may be specifically involved in the regulation of HPC proliferation.

Adult↗
PubMed1996

NMR determination of the structure of Julibroside J1.

Julibroside J1 is a new triterpenoid saponin which contains one triterpene, two monoterpenes and nine sugar residues. The structure has been determined almost exclusively by high-resolution NMR methods. The 1H and 13C NMR spectra of Julibroside J1 C5D5N have been assigned by homonuclear and heteronuclear correlation experiments, such as COSY, CH-COSY, TOCSY, HMBC, HMQC-COSY, HMQC-TOCSY and NOESY. Anomeric configurations were obtained by combined use of 1JCH and 3JH1,H2 and NOESY data. The particular sugar residues were identified by utilizing 3JHH values obtained from TOCSY cross-peaks, NOE difference spectra, and several 1D-TOCSY spectra, and three-bond intra-ring cross-peaks from a HMBC spectrum. Linkage assignments were made using the HMBC spectrum, and supplemented by NOE data from the NOESY spectrum. The structure of Julibroside J1 was characterized as 3-O-[beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6) -beta-D- glucopyranosyl]-21-O-((6S)-2-trans-2-hydroxymethyl-6-methyl-6-O-[4-O-((6 S)- 2-trans-2,6-dimethyl-6-O-(6-deoxy-beta-D-glucopyranosyl)-2,7-octadien oyl)-6- deoxy-beta-D-glucopyranosyl]-2,7-octadienoyl)-acacic acid 28-O-(beta-D-glucopyranosyl-(1-->3)-[alpha-L-arabinofuranosyl-(1-->4)]- alpha-L-rhamnopyranosyl-(1-->2))-beta-D-glucopyranosyl ester.

Carbohydrate Conformation↗
PubMed1996

RER, an evolutionarily conserved sequence upstream of the rhodopsin gene, has enhancer activity.

Previous transgenic mouse experiments localized the mammalian rhodopsin gene promoter to a region just upstream of the mRNA start site, and also suggested the existence of a second more distal regulatory region. A highly conserved 100-base pair (bp) sequence which is homologous to the red and green opsin locus control region is located 1.5-2 kilobases upstream of the rhodopsin gene (depending on the species). In order to test the activity of this 100-bp region, transgenic mice were generated with bovine rhodopsin promoter/lacZ constructs which differed only by the presence or absence of the sequence. Of 11 lines generated, all demonstrated photoreceptor-specific expression of the transgene, but the lines with the putative regulatory region showed significantly higher expression. Additional transgenic lines in which the region was fused to a minimal heterologous promoter did not show transgene expression in the retina. Gel mobility shift and DNase I footprint assays demonstrated that bovine retinal nuclear extracts contain retina-specific as well as ubiquitously expressed factors that interact with the putative regulatory region in a sequence-specific manner. These results indicate that the 100-bp sequence can indeed function in vivo as a rhodopsin enhancer region.

Animals↗
PubMed1996

Clonal stability of blood cell lineages indicated by X-chromosomal transcriptional polymorphism.

The idea that stem cells oscillate between a state of activity and dormancy, thereby giving rise to differentiating progeny either randomly or in orderly clonal succession, has important implications for understanding normal hematopoiesis and blood cell dyscrasias. The degree of clonal stability in individuals also has practical implications for the evaluation of clonal lymphomyeloproliferative diseases. To evaluate the clonality pattern of the different types of blood cells as a function of time we have validated the applicability, sensitivity, and reproducibility of a thermostable ligase reaction to detect transcripts of the G6PD allele on the active X-chromosome in normal heterozygous females. While the ratio of the two X-chromosome-derived allelic transcripts varied widely among hemopoietic tissues in a given individual, this allelic ratio was virtually identical in all types of mature myeloid and lymphoid cells. Longitudinal studies indicated constancy of the G6PD allelic ratio in blood cells over a 912-d period of observation in healthy females. The individual variability observed in this allelic ratio suggests that the progeny of a relatively small number of original embryonic hemopoietic stem cells, approximately eight, contribute to the sustained production of all types of blood cells in healthy individuals.

Alleles↗
PubMed1996

Calcitonin prevents CCl4-induced hydroperoxide generation and cytotoxicity possibly through C1b receptor in rat hepatocytes.

The effects of calcitonin (CT) on oxyradical generation and cellular damage induced by carbon tetrachloride (CCl4) were investigated in rat hepatocytes. Addition of CCl4 to the cells concentration dependently increased intracellular production of hydroperoxides and release of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The hepatocytes expressed mRNA for a CT receptor, C1b. Coaddition of CT to the cells concentration dependently suppressed the CCl4-induced increase in hydroperoxide production and also decreased the release of AST and ALT. The suppressive effect of CT on hydroperoxide production was reversed by further addition of H7 or by pretreatment with phorbol 12-myristate 13-acetate for 24 h. These results suggest that CT prevents CCl4-induced oxyradical production and cellular damage through activation of protein kinase C in hepatocytes.

Animals↗
PubMed1996

Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent.

An RNA virus, designated hepatitis G virus (HGV), was identified from the plasma of a patient with chronic hepatitis. Extension from an immunoreactive complementary DNA clone yielded the entire genome (9392 nucleotides) encoding a polyprotein of 2873 amino acids. The virus is closely related to GB virus C (GBV-C) and distantly related to hepatitis C virus, GBV-A, and GBV-B. HGV was associated with acute and chronic hepatitis. Persistent viremia was detected for up to 9 years in patients with hepatitis. The virus is transfusion-transmissible. It has a global distribution and is present within the volunteer blood donor population in the United States.

Acute Disease↗
PubMed1996

Targeted overexpression of androgen receptor with a liver-specific promoter in transgenic mice.

The rodent liver displays marked age- and sex-dependent changes in androgen sensitivity due to the sexually dimorphic and temporally programmed expression of the androgen receptor (AR) gene. We have altered this normal phenotype by constitutive overexpression of the rat AR transgene in the mouse liver by targeting it via the human phenylalanine hydroxylase (hPAH) gene promoter. These transgenic animals in their heterozygous state produce an approximately 30-fold higher level of the AR in the liver as compared with the nontransgenic control. Androgen inactivation via sulfonation of the hormone by dehydroepiandrosterone sulfotransferase (DST), an androgen-repressible enzyme, also contributes to the age- and sex-dependent regulation of hepatic androgen sensitivity. DST has a broad range of substrate specificity and is responsible for the age- and sex-specific activation of certain polycyclic aromatic hepatocarcinogens as well, by converting them to electrophilic sulfonated derivatives. In the transgenic female, the hepatic expression of DST was approximately 4-fold lower than in normal females, a level comparable to that in normal males. The hPAH-AR mice will serve as a valuable model for studying the sex- and age-invariant expression of liver-specific genes, particularly those involved in the activation of environmental hepatocarcinogens such as the aromatic hydrocarbons.

Animals↗
PubMed1996

Loss of Rb and Myc activation co-operate to suppress cyclin D1 and contribute to transformation.

Cyclin D1 can bind and phosphorylate the product (pRb) of the retinoblastoma gene (RB-1) and recent evidence suggests pRb, in turn, may regulate cyclin D1 protein expression. In transformed cell lines, loss of pRb activity strongly correlates with a decrease in cyclin D1 protein expression, and conversely, introduction of pRb can induce cyclin D1 promoter activity. We show here that pRb does not regulate cyclin D1 directly as basal and serum-stimulated levels of cyclin D1 protein and kinase activity are similar in wildtype and pRb-deficient primary mouse embryonic fibroblasts (MEFs). These observations suggest that the suppression of cyclin D1 in pRb-minus tumour cell lines requires both loss of pRb and at least one additional genetic event. We have determined that constitutive, ectopic Myc expression in pRb-deficient, but not wildtype, MEFs suppresses cyclin D1 protein expression and kinase activity. Regulation is evident at either the level of RNA or protein expression. Phenotypically, pRb-deficient MEFs consistently exhibited a delayed growth response in comparison to wildtype MEFs. This growth delay is abrogated in pRb-deficient MEFs which are expressing ectopic Myc protein, coincident with the loss of cyclin D1 protein expression. Moreover, these cells exhibit an increased proliferative capacity, and they no longer show contact inhibition. Our results support a cross-regulatory mechanism between Myc, pRb and cyclin D1 and suggest a novel role for cyclin D1 in tumorigenesis.

Animals↗