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Biomedical subjects

Y Xie

Publications and source records attributed to Y Xie.

At least 343 records · Page 19Linked to original sources
PubMed1995

Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis.

Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner. Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes. Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program. Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator. Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes.

1-Methyl-3-isobutylxanthine↗
PubMed1995

The DNA binding activity of C/EBP transcription factor is regulated in the G1 phase of the hepatocyte cell cycle.

We have isolated the promoter of the rat C/EBP alpha gene and find a high degree of homology with the mouse gene, particularly in putative regulatory domains. Transactivation of this promoter by ectopic expression of rat C/EBP beta occurs through a C/EBP regulatory domain at position -170 to -195. An oligonucleotide corresponding to this domain binds to complexes expressed in rat liver that comprise C/EBP alpha-C/EBP beta heterodimers (alpha beta) as well as C/EBP beta complexed with itself and/or other unidentified nuclear factors (beta 1, beta 2, and beta 3). The DNA binding activity of these complexes changes both qualitatively and quantitatively following partial hepatectomy. Within 2-5 h postsurgery, the binding activity of the alpha beta complexes drops severalfold, reaching a nadir by 20 h. During the ensuing 3-8 days, as regeneration nears completion, this activity slowly returns to normal quiescent liver levels. Western blot analysis shows 3 major C/EBP alpha polypeptide species (42, 40, and 30 kDa), whose abundance in general parallels the decrease and recovery in DNA binding activity. In contrast to C/EBP alpha behavior, the DNA binding activity of the beta complexes is transiently induced severalfold during the early G1 period between 2 and 6 h posthepatectomy. The major C/EBP beta polypeptide is the 32-kDa LAP protein, whereas the LIP protein (21 kDa) is weakly expressed. Both remain essentially constant throughout the course of regeneration, suggesting that changes in DNA binding activity may reflect changes in the complexed proteins rather than the C/EBP beta polypeptides themselves. In primary hepatocyte cultures, under growth supporting conditions, in the absence of growth factors proliferation is negligible; C/EBP alpha is abundantly expressed at the outset, but is then extensively down-regulated. Epidermal growth factor causes further decay of C/EBP alpha polypeptides and DNA binding activity, and down-regulates C/EBP beta DNA binding activity as well. Addition of transforming growth factor beta completely antagonizes the effects of epidermal growth factor on C/EBP beta activity, and partially overcomes the effect on C/EBP alpha. These results demonstrate that the DNA binding activity of C/EBP alpha and C/EBP beta complexes is regulated in the regenerating liver, and in hepatocyte cultures responding to growth factors that regulate their proliferation.

Animals↗
PubMed1995

Identification of an upstream region that controls the transcription of the human autocrine motility factor receptor.

We have isolated from a human placenta cosmid library a 0.7 kb genomic clone that contains the 5' terminal portion of the autocrine motility factor receptor (hAMFR) coding region. Chloramphenicol Acetyl Transferase (CAT) reporter gene assays have identified this region as the promoter of the hAMFR gene. A single transcription initiation site (+1) has been mapped to 129 bp upstream of the ATG start codon by primer extension. DNA sequence analysis and CAT assay revealed a TATA element at the position -485/-468 which was able to conduct only a marginal transcription (less than 5% of the total activity). The majority of the hAMFR promoter's activity is contributed by a transcription initiator (Inr) element overlapping the initiation site (+1) which independently controls the transcription of the hAMFR gene. Gel mobility shift assays showed that DNA-binding proteins in HeLa cells nuclear extract can bind specifically to both promoter's elements. DNA-binding proteins were found to be differentially expressed by sparse and dense cultured normal fibroblasts. The nuclear-binding protein expressed by sparse NIH-3T3 cells induced a DNA mobility shift similarly to the nuclear protein of HeLa cells, while a different DNA-protein complex size was observed with nuclear proteins extracted from dense cultured NIH-3T3 cells. Also CAT-reporter gene analysis revealed a significant lower activity in dense NIH-3T3 cells as compared with the sparse-cultured counterparts. These results help to explain the previously observed cell-cell contact regulation of AMFR expression in normal cells and its consecutive expression in tumor cells.

3T3 Cells↗
PubMed1995

Tyrosine phosphorylation of Shc proteins and formation of Shc/Grb2 complex correlate to the transformation of NIH3T3 cells mediated by the point-mutation activated neu.

PLC-gamma, ras-GAP and Shc have been proposed to be in vivo substrates for the neu-encoded p185neu receptor tyrosine kinases. We compared the tyrosine phosphorylation levels of PLC-gamma, ras-GAP and Shc in two NIH3T3 derived cell lines, transformed B104-1-1 and non-transformed DHFR/G8 cells in which point-mutation activated and normal rat neu genes were transfected and expressed, respectively. We found that tyrosine phosphorylation of Shc and formation of Shc/Grb2 complex were more significant in B104-1-1 cells than in DHFR/G8 cells, while no obvious difference could be detected for the tyrosine phosphorylation levels of ras-GAP and PLC-gamma between these two cell lines. Furthermore, we observed that association with Shc was severely impaired by deletion of most of the major autophosphorylation sites of the point-mutated neu. The truncated neu product, however, fully retained its ability to transform NIH3T3 cells, induce Shc tyrosine phosphorylation and Shc/Grb2 complex formation. Our results suggest that tyrosine phosphorylation of Shc which allows formation of Shc/Grb2 complex may play an important role for cell transformation induced by the point mutation-activated neu, and that stable binding to mutant p185neu may not be necessary for Shc to mediate this signaling pathway.

3T3 Cells↗
PubMed1995

Four hydrophobic segments in the NH2-terminal third (H1-H4) of Na,K-ATPase alpha subunit alternately initiate and halt membrane translocation of the newly synthesized polypeptide.

Transmembrane disposition of the NH2-terminal third of the Na,K-ATPase alpha subunit was studied using an experimental approach that involved in vitro endoplasmic reticulum membrane insertion of chimeras. These chimeras consisted of four truncated amino-terminal segments of the alpha subunit linked at amino acid residues 126, 179, 313, and 439 to chloramphenicol acetyltransferase (CAT), a reporter protein, that contains a consensus sequence for N-linked glycosylation. The fusion sites were located after one of the four hydrophobic segments (H1-H4). The results showed that the chimeras in which the alpha subunit was truncated at positions 126 and 313 were glycosylated, and the glycosylated peptides were protected by membranes from proteolysis. However, the other two chimeras were not glycosylated and the inserted peptides were digested by protease into fragments which did not immunoprecipitate with anti-CAT. These results clearly demonstrate that hydrophobic segments H1 and H3 function as signal/anchor type II, and H2 and H4 function as halt transfer signals. Furthermore, membrane insertion of the NH2-terminal third of Na,K-ATPase alpha subunit is achieved by a series of alternate signal/anchor type II and halt transfer sequences.

Amino Acid Sequence↗
PubMed1995

Control of aggregation in protein refolding: a variety of surfactants promote renaturation of carbonic anhydrase II.

The denaturation and renaturation of carbonic anhydrase II (CAII) has been studied in several laboratories. Both thermodynamic and kinetic evidence support the existence of at least two intermediates between denatured and native protein. Previous studies have shown that on rapid dilution of a CAII solution from 5 M to 1 M guanidinium chloride, aggregation strongly competes with renaturation at higher protein concentrations, suggesting an upper limit for [CAII] of approximately 0.1%. Our experiments show 60% renaturation at 0.4% [CAII] and that aggregate formation is partially reversible. This yield can be substantially increased by several surfactant additives, including simple alkanols as well as micelle-forming surfactants. Effective surfactants (promoters) act by suppressing initial aggregate formation, not by dissolving aggregates. Promoters act on either the first folding intermediate (I1) or oligomers thereof. Eight of the 18 surfactants examined showed promoter activity, and no correlation was evident between promoter activity and chemical structure or surface tension lowering. These results indicate discrimination (molecular recognition) by I1 and/or its oligomers.

Animals↗
PubMed1995

Sister-chromatid exchanges in lymphocytes from methimazole-induced hypothyroid mice.

The inhibitory effect of an antithyroid drug on mouse T lymphocytes was investigated. Inbred C57BL/6 mice were provided with an antithyroid drug, methimazole, for 2, 4 and 6 weeks and the in vitro responses of the lymphocytes were studied. The proliferative responses of T lymphocytes from the spleen of methimazole (MMI)-treated mice significantly (p < 0.05) decreased following concanavalin A stimulation, and the inhibitory effect became prominent with the increased duration of MMI treatment. A concomitant increase in the frequency of induced sister-chromatid exchanges was also observed in these T lymphocytes. When the splenocytes were stimulated with concanavalin A for 24 h, their ability to produce interleukin-2 (IL-2) was significantly decreased (p < 0.05). The results indicated that methimazole interfered with the normal proliferation of T lymphocytes by suppressing the production of IL-2, a cytokine also known as T cell growth factor, as well as inducing a higher incidence of sister-chromatid exchange during cell division.

Animals↗
PubMed1995

Ion channel involvement in anoxic depolarization induced by cardiac arrest in rat brain.

Anoxic depolarization (AD) and failure of ion homeostasis play an important role in ischemia-induced neuronal injury. In the present study, different drugs with known ion-channel-modulating properties were examined for their ability to interfere with cardiac-arrest-elicited AD and with the changes in the extracellular ion activity in rat brain. Our results indicate that only drugs primarily blocking membrane Na+ permeability (NBQX, R56865, and flunarizine) delayed the occurrence of AD, while compounds affecting cellular Ca2+ load (MK-801 and nimodipine) did not influence the latency time. The ischemia-induced [Na+]e reduction was attenuated by R56865. Blockade of the ATP-sensitive K+ channels with glibenclamide reduced the [K+]e increase upon ischemia, indicating an involvement of the KATP channels in ischemia-induced K+ efflux. The KATP channel opener cromakalim did not affect the AD or the [K+]e concentration. The ischemia-induced rapid decline of extracellular calcium was attenuated by receptor-operated Ca2+ channel blockers MK-801 and NBQX, but not by the voltage-operated Ca2+ channel blocker nimodipine, R56865, and flunarizine.

Adenosine Triphosphate↗
PubMed1995

Postnatal changes in size and actomyosin content of rabbit gastric myocytes.

Maximal tension generated by gastric muscle is three to four times greater in weanlings than in newborn rabbits. To determine if this functional maturation is accompanied by structural changes, we compared length-tension relationships, myocyte number and size, and actomyosin content in muscle from the gastric body of newborn (1 day) and weanling (12 weeks) rabbits. Passive tension at optimal length (Lo) was six times greater in circular smooth muscle strips from weanling rabbits than from newborn rabbits. Active tension at Lo in weanling rabbits was three times greater than in newborn rabbits. For morphometry, muscle cross-sections stretched to Lo in the circular axis were photographed with electron microscopy (5300x). Cell number/unit area was counted in circular muscle layers from newborns and weanlings. Cross-sectional area of each cell was measured by computerized planimetry. There were 2.5 times more cells per unit area in newborn than in weanling tissue, P < 0.001. However, the mean cell area in newborns (5.4 +/- 4.6 microns2) was less than that in weanlings (13.5 +/- 11.7 microns2). Consequently, the muscle cells occupied similar total areas in newborns and weanlings. We measured actin and myosin heavy chain in full-thickness muscle homogenates using SDS gel electrophoresis and densitometric scanning. Actin and myosin concentrations were lower in newborns (9.6 +/- 1.3 micrograms g-1 wet weight and 5.6 +/- 0.7 micrograms g-1 wet wt, respectively) than in weanlings (17.7 +/- 3.0 micrograms g-1 wet wt and 8.2 +/- 1.6 micrograms g-1 wet wt respectively), each P < 0.01. The proportion of myosin heavy chain isozymes did not change with age. We conclude that there are postnatal increases in cell size and the quantity of actin and myosin in rabbit gastric muscle. The increase in quantity of contractile protein may be in part responsible for age-dependent increases in maximal tension.

Actomyosin↗
PubMed1995

Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr.

We investigated the fate of human immunodeficiency virus type 1 (HIV-1) viral DNA in infected peripheral blood lymphocytes and immortalized T-cell lines by using a replication-defective HIV-1. We observed that integrated HIV-1 DNA and viral gene expression decrease over time. A frameshift mutation in vpr resulted in maintenance of the HIV-1 provirus and stable persistence of viral expression. Transfection of vpr together with the neomycin resistance gene in the absence of other viral genes decreased the formation of geneticin-resistant colonies, indicating either a cytotoxic or a cytostatic effect upon cells. Therefore, maintenance of HIV-1 infection within an infected proliferating population is due to two competing processes, the rate of viral spread and the degree of cell growth inhibition and/or death induced by Vpr.

Animals↗
PubMed1995

Functional changes in dorsal root ganglion cells after chronic nerve constriction in the rat.

1. We studied the effects of a chronic nerve constriction on the evoked responses in dorsal root fibers in the rat to norepinephrine and to thermal stimuli applied either to the dorsal root ganglion (DRG) or the site of nerve injury. We recorded a total of 59 C fibers, 15 A delta-fibers, and 46 A beta-fibers from the L5 dorsal root of the rats 11-52 days after a loose ligation of the ipsilateral sciatic nerve. Most fibers were identified by the presence of spontaneous activity (SA) that originated partially at and/or proximal to the injury site. In addition, we recorded 20 C fibers, 1 A delta-fiber, and 28 A beta-fibers from the dorsal roots of normal, uninjured neurons. 2. In nerve-injured rats, the SA of some C fibers was generally increased by cooling and decreased by heating either site. In contrast, the SA of most A beta-fibers was increased by heating either the injury site or the DRG. Cooling the DRG decreased SA in A beta-fibers, whereas cooling the injury site typically had no effect. Excitatory responses were not evoked in any fiber group when the same thermal stimuli were applied to the nerve or DRG tested in normal, uninjured rats. 3. Norepinephrine (< 0.5 mM) applied either to the injury site or the DRG increased the SA of most C fibers and A delta-fibers but only a minority of A beta-fibers in previously injured nerves. The threshold concentration for excitation of the DRG somata of C fibers was 0.01 mM. No effects were found for fibers in uninjured nerves. 4. The effect of norepinephrine was blocked by a pretreatment with yohimbine, an alpha 2-blocker, but not with prazosin, an alpha 1-blocker. 5. Stimulation of the sympathetic trunk (L2-L3) excited most C fibers and a minority of A beta-fibers. In contrast, the SA of a minority of C fibers and A beta-fibers was depressed during sympathetic stimulation. 6. After a chronic nerve constriction the DRG becomes a source of abnormal activity modulated by sympathetically released norepinephrine acting on alpha 2 receptors in DRG somata. This neuropathic activity may contribute to cutaneous pain and hyperalgesia.

Animals↗
PubMed1995

Growth hormone and colony-stimulating factor 1 share multiple response elements in the c-fos promoter.

The c-fos protooncogene is induced by GH rapidly, but transiently. Induction requires C kinase activation and the serum response element, and recent binding studies have also implicated the sis-inducible element (SIE). However, no systematic study of the promoter elements responsible for transactivation by GH has been undertaken. Here we used Chinese hamster ovary K1 cells transiently cotransfected with rabbit GH receptor and c-fos promoter-luciferase constructs to demonstrate that the major region responsible for GH induction is located between 284-396 base pairs upstream of the transcription start site. Full induction by GH requires all of the known elements located in this region to be intact, including the SIE or signal transducer and activator of transcription binding element. We also report novel negative elements located around -216 upstream of the start site that reduce induction by GH and provide gel shift evidence for factors binding in this region. Cotransfection of Chinese hamster ovary K1 cells with c-fms and c-fos promoter constructs followed by the addition of CSF-1 revealed that these same c-fos elements contribute to transactivation by c-fms. Serum also uses the same elements to induce c-fos expression, except for the SIE. These results indicate that GH receptor and c-fms tyrosine kinase operate through multiple common response elements to regulate c-fos gene expression despite their structural differences.

Animals↗
PubMed1995

[Preliminary study of a recombinant polyvalent vaccine of Plasmodium falciparum and its immunological activity].

A hybrid gene named HGFC coding three protective antigenic epitopes of Plasmodium falciparum and two exogenous T cell activating epitopes was designed and synthesized. A multicopy hybrid gene named HGF-CAC was also constructed. The two genes were cloned into expression vector pWR450-1 and the hybrid fusion proteins containing forgine antigens and beta-galactosidase were expressed in E. Coli. The molecular weights of the fusion proteins were 65KDa and 77KDa respectively. The expression rate was about 35% of total bacterial proteins. The fusion protein could react specifically with mouse and rabbit antibodies against antigens of Plasmodium falciparum. The rabbit immune serum against the purified fusion protein could specifically recognize the antigens of Plasmodium falciparum and effectively inhibit the in vitro development of the parasites. The inhibitory capacity of the immune sera to parasite invasion was enhanced as the amount of the sera increased and the incubation time of the sera with the parasites was prolonged. After 72h incubation at 20% concentration with the parasites, the serum suppressed the multiplication of parasite to a level of 82% and caused degeneration and death of the parasites. The results indicated that the recombinant hybrid antigen of Plasmodium falciparum has immunological activity and protectivity. It is probably a candidate malaria vaccine.

Amino Acid Sequence↗
PubMed1995

[Expression of chemically synthesized hybrid genes of Plasmodium falciparum and preliminary characterization of expressed products].

The chemically synthesized hybrid genes HGFC and HGFCAC were respectively recombinated with expression vector pWR450-1 and transferred into Escherichia coli. After adding IPTG to the cultural media, the bacteria harboring recombinant plasmid expressed the fusion proteins of exogenous gene products and partial amino acid of beta-galactosidase. The molecular weights of the two fusion proteins were 65 kDa and 77 kDa, respectively. The expressed fusion proteins could be recognized by antibodies of rabbit anti-RESA peptide EENVEHDA by Western blotting, indicating that the fusion proteins harbored the antigenic epitopes of Plasmodium falciparum.

Animals↗