Search PubMed⌕ Search

Biomedical subjects

W Liu

Publications and source records attributed to W Liu.

At least 649 records · Page 36Linked to original sources
PubMed1997

Cloning and expression of a gene encoding a protein obtained from earthworm secretion that is a chemoattractant for garter snakes.

The protein ES20, derived from earthworm shock secretion, is a vomeronasally mediated chemoattractant for garter snakes (Jiang, X. C., Inouchi, J., Wang, D., and Halpern, M. (1990) J. Biol. Chem. 265, 8736-8744). Based on its 15-residue N-terminal amino acid sequence, degenerative oligodeoxynucleotide probes were synthesized and used to screen a cDNA library that was constructed in sense orientation using a Uni-ZAPTM XR vector and XL1-Blue MRF' host. A gene was cloned from a polymerase chain reaction as well as from the cDNA library. A combination of the forward degenerative primer and T7 primer was used to obtain gene-specific DNA fragments, from which probes were synthesized and successfully used in screening the cDNA library. The ES20 gene is about 700 base pairs long and encodes 208 amino residues. The ES20 gene was excised from a recombinant plasmid pSK-ES20, ligated to pQE30 expression vector, and transformed into Escherichia coli strain JM109. The selected recombinant plasmids were transformed into expression host cell, E. coli M15[pREP4]. Three transformants were selected, induced with isopropyl-1-thio-beta-D-galactopyranoside for fusion gene expression and an expressed 20-kDa fusion protein purified under denaturing conditions. This protein was refolded and gave a positive reaction against ES20-specific polyclonal antibodies. The fusion protein that had not been denatured remained as an aggregate and was an active chemoattractant for garter snakes.

Amino Acid Sequence↗
PubMed1997

BDNF protection of auditory neurons from cisplatin involves changes in intracellular levels of both reactive oxygen species and glutathione.

Previous studies have shown that brain derived neurotrophic factor (BDNF) can protect auditory neurons from cisplatin toxicity in vitro. To explore the mechanism of BDNF mediated neuronal protection sequential confocal microscopic sampling of auditory neurons measured intracellular levels of reactive oxygen species (ROS) in response to withdrawal of BDNF supplementation, cisplatin exposure, and BDNF protection from cisplatin damage in normal and oxidative stress states. Additionally, we examined intraneuronal levels of the free radical scavenger glutathione (GSH) in response to withdrawal of BDNF. Withdrawal of BDNF resulted in increased production of ROS and decreased survival of auditory neurons. Levels of GSH within neurons increased after BDNF withdrawal, and this increase was shown to lag behind the production of ROS. Auditory neurons in cultures supplemented with BDNF and exposed to cisplatin showed significantly lower levels of ROS and increased survival compared to neurons in unprotected, cisplatin exposed cultures. Neurons treated with buthionine sulfoximine (an inhibitor of GSH synthesis), supplemented with BDNF, and exposed to cisplatin showed significantly higher intracellular levels of ROS compared to the neurons in BDNF supplemented cultures exposed to cisplatin. These results suggest that intracellular levels of ROS play an important role in cisplatin induced cell death of auditory neurons and that production of ROS can be ameliorated through supplementation with BDNF. GSH appears to mediate BDNF protection of these neurons from cisplatin induced ROS and subsequent damage.

Animals↗
PubMed1997

Two Sp sites are important cis elements regulating the upstream promoter region of the gene for rat type I hexokinase.

Multiple transcriptional start sites have been identified for the gene encoding the rat Type I isozyme of hexokinase (White, J.A., Liu, W., and Wilson, J. E., Arch. Biochem. Biophys. 335, 161-172, 1996); these are clustered at positions approximately -460, -300, and -100 relative to the translational start codon (ATG, with A being +1). PC12 cells and H9c2 cells were transfected with luciferase reporter constructs containing genomic sequence between positions -3366 and -171. Marked (85%) decrease in promoter activity was associated with deletion of sequence between -742 and -516. In DNase I footprinting experiments, two regions, called P1 (-552 to -529) and P2 (-480 to -458) boxes, were protected by proteins present in nuclear extracts from PC12 cells. Mutation or deletion of the P2 box had no effect on promoter activity; protection in this region, which includes the most upstream cluster of transcriptional start sites, is attributed to binding of RNA polymerase II or associated factors. In contrast, mutations or deletions in the P1 box had markedly detrimental effects on promoter activity and on binding of proteins in PC12 cell nuclear extracts. Maintenance of a consensus Sp1 binding site centrally located in the P1 box was critical for both promoter activity and binding. A second Sp1 site (-570), just upstream from the P1 box, was also shown to be functionally important but no protection of this region was detected in footprinting experiments, presumably reflecting lower affinity at this site under the conditions used. Supershift experiments demonstrated the involvement of Sp1, Sp3, and Sp4 in formation of complexes with the P1 box region and implicate these transcription factors in regulating promoter activity associated with this region. Another series of reporter constructs, including sequence between -171 and -1, permitted detection of an additional promoter activity downstream from -364. While not yet extensively characterized, it is already evident that the cis elements influencing the downstream promoter activity are distinct from the Sp factors determined to be important in expression from the upstream promoter region.

Animals↗
PubMed1997

The tumor-derived fetal-intestinal alkaline phosphatase cDNA is identical in sequence to the adult intestinal alkaline phosphatase isozyme gene.

The alkaline phosphatase (AP) of Caco-2 cells, a cell line derived from a human adenocarcinoma of the colon, is quite similar to fetal intestinal AP in its enzymatic properties. The nucleotide sequence of a cDNA encoding AP produced in Caco-2 cells was examined. The sequence was identical to one of the three sequences of adult intestinal AP reported previously. We further investigated the entire nucleotide sequence of cDNA of intestinal-type AP produced in cancer cell lines such as HuH-7 cells, FL-amnion cells, and HuG-1 cells. The sequence of these cell APs was identical to that of Caco-2 cell AP. These results indicate that cancer cells producing intestinal-type AP have the same nucleotide sequence as that of adult intestinal AP, and suggest that the differences in electrophoretic mobilities of these cell APs compared with adult intestinal AP may be due to post-translational modifications.

Adult↗
PubMed1997

Expression of epidermal growth factor receptor and human papillomavirus E6/E7 proteins in cervical carcinoma cells.

BACKGROUND: Epidermal growth factor receptor (EGF-R) proteins are highly expressed in many tumors, including those of the cervix. We have observed previously that the introduction of a transcription unit containing an antisense sequence for the E6/E7 genes of human papillomavirus (HPV) 18, along with a transcription unit containing a sense complementary DNA sequence for the wild-type retinoblastoma (Rb) gene, decreased the growth of human cervical carcinoma HeLa cells (HPV 18 positive) both in vitro and in vivo. To clarify the regulatory mechanisms by which this reduction in cell proliferation occurred, we studied the expression of EGF-R proteins in these cells. METHODS: Western blot and northern blot techniques were used to measure EGF-R expression, and a pulse-chase immunoprecipitation assay was used to measure the stability of EGF-R protein in HeLa cells and HeLa cells that had been transfected with the antisense E6/E7 or sense Rb sequences. Cell proliferation was measured by use of a tetrazolium-based colorimetric assay for numbers of viable cells. RESULTS: The introduction of sense Rb or antisense E6/E7 transcription units or a combination of these two transcription units into HeLa cells dramatically decreased the level of EGF-R proteins in these cells; EGF-R levels were not affected at the transcriptional level but at the post-transcriptional level. Addition of the anti-EGF-R-specific monoclonal antibody 225mAb to HeLa cells caused 53% (95% confidence interval = 44%-62%) growth inhibition. CONCLUSIONS: These results suggest that HeLa cervical carcinoma cells are dependent on EGF-R for proliferation and that changes in functional levels of the E6/E7 HPV proteins and endogenous Rb proteins may alter the growth rate of cervical cancer cell lines by reducing the stability of EGF-R at the post-transcriptional level.

Blotting, Northern↗
PubMed1997

Multidirectional instability of the thoracic spine due to iatrogenic pedicle injuries during transpedicular fixation. A biomechanical investigation.

STUDY DESIGN: Pedicle fracture was simulated in an in vitro model, and its effect on multidirectional stability provided by pedicle instrumentation was quantified. OBJECTIVES: To quantify the multidirectional flexibility of padicle instrumentation due to different iatrogenic pedicle injuries. SUMMARY OF BACKGROUND DATA: Misplacement of the screw and iatrogenic pedicle fracture are the main complications of pedicle instrumentation. Despite the increasing number of clinical studies dealing with this issue, there is little reliable information concerning the biomechanical effects of an intraoperative pedicle fracture. METHODS: A burst fracture was created in 10 human cadaveric five-vertebrae spine specimens, from the middle and lower thoracic spine regions. The fracture was stabilized with a semirigid pedicle screw fixation device. To simulate an intraoperative pedicle fracture, the pedicles of the instrumented segments were resected in four steps. After each pedicle injury, three-dimensional flexibility in the form of range of motion and neutral zone of the construct was determined and compared with the intact values. RESULTS: Resection of the pedicles had little effect on the multidirectional flexion-extension stability provided by the instrumentation. There were significant increases of axial rotation in the middle thoracic spine when the lateral wall was resected (range of motion, 8.2 degrees vs. 3.6 degrees; neutral zone, 4.2 degrees vs. 1.7 degrees), whereas in the lower thoracic spine, significant increases occurred only when all the pedicles were resected (range of motion, 3.8 degrees vs. 1.4 degrees; neutral zone, 1.1 degrees vs. 0.4 degree). Lateral resection of the pedicle resulted in significant increases of range of motion and neutral zone for lateral bending in both the middle thoracic spine (range of motion, 19.0 degrees vs. 10.0 degrees; neutral zone, 2.4 degrees vs. 1.1 degrees) and the lower thoracic spine (range of motion, 4.3 degrees vs. 2.5 degrees; neutral zone, 0.9 degree vs. 0.3 degree). CONCLUSIONS: Resection of the pedicles results in a significant decrease in axial rotation and lateral bending stability provided by the instrumentation. This effect was higher in the middle than in the lower thoracic spine and may be relevant to pedicle fractures produced by pedicle screws used in these regions.

Adolescent↗
PubMed1997

Structure of cardiac muscle troponin C unexpectedly reveals a closed regulatory domain.

The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties. Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca2+-binding properties compared with skeletal TnC and features a Ca2+-binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca2+-saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a "closed" conformation even in the Ca2+-bound (the "on") state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC. This structure in the Ca2+-bound state, and its subsequent interaction with troponin I (TnI), are crucial in determining the specific regulatory mechanism for cardiac muscle contraction. Further, it will allow for an understanding of the action of calcium-sensitizing drugs, which bind to cardiac TnC and are known to enhance the ability of cardiac TnC to activate cardiac muscle contraction.

Alanine↗
PubMed1997

Impaired generation of bone marrow B lymphocytes in mice deficient in C/EBPbeta.

CAAT/enhancer binding proteins (C/EBP) are a family of transcription factors that mediates adipocyte differentiation and the regulation of genes expressed in immune responses and inflammation, such as interleukin-6 (IL-6), IL-8, and granulocyte colony-stimulating factor (G-CSF). We investigated the role of C/EBPbeta (NF-IL6) in the generation of bone marrow B lymphocytes by taking advantage of C/EBPbeta-/- mice. We found that the expansion of bone marrow (BM) B lymphocytes was impaired in long-term lymphoid cultures from C/EBPbeta-/- mice. Consistent with this finding, the number of BM B cells was decreased in C/EBPbeta-/- mice. Both the levels of IL-7 gene expression and bioactive IL-7 from BM stromal cells were decreased in C/EBPbeta-/- mice. Furthermore, the proliferative responsiveness of BM B-cell precursors to IL-7 was also reduced as compared to wild-type mice, indicating that C/EBPbeta is required for the generation of BM B cells induced by IL-7. Accordingly, IL-7 stimulates the C/EBPbeta DNA-binding activity of normal BM pre-B lymphocytes as well as of 70Z/3 pre-B cells. These results point to C/EBPbeta as a critical signaling molecule in BM B lymphopoiesis.

Animals↗
PubMed1997

Androgen and glucocorticoid receptor heterodimer formation. A possible mechanism for mutual inhibition of transcriptional activity.

The androgen and glucocorticoid hormones elicit divergent and often opposing effects in cells, tissues, and animals. A wide range of physiological and molecular biological evidence suggests that the receptors that mediate these effects, the androgen and glucocorticoid receptors (AR and GR, respectively), influence each other's transcriptional activity. We now show that coexpressed AR and GR indeed do interact at the transcriptional level and that this interaction is correlated with their ability to form heterodimers at a common DNA site, in vitro and in vivo. Furthermore, mutants that cannot heterodimerize do not inhibit each other's activity. These observations provide the first evidence that the opposing physiological effects of the androgen and glucocorticoid hormones are due to the direct physical interaction between their receptors at the transcriptional level.

Androgens↗
PubMed1997

Evidence of genetic diversity underlying Rh D-, weak D (Du), and partial D phenotypes as determined by multiplex polymerase chain reaction analysis of the RHD gene.

The human blood group Rh antigens are expressed by proteins encoded by a pair of highly homologous genes located at chromosome 1p34-36. One of the genes (RHCE) encodes Rh CcEe antigens, while the other (RHD) the D antigen. Point mutations in the RHCE gene generate the C/c and E/e polymorphisms, while it has been shown that an RHD gene deletion can generate the D-negative phenotype. We have analyzed intron 4 of the RHCE and RHD genes and have defined the site of an RHD-specific deletion located in this intron. Using a multiplex RHD typing assay, which combines a reverse polymerase chain reaction (PCR) primer, which straddles this RHD-specific sequence, and a pair of primers located in exon 10 of the RHD gene, we have analyzed 357 different genomic DNA samples derived from individuals expressing D+, D-, weak D, and partial D phenotypes. Of these, we have noted a significant discordance with our multiplex PCR assay in the D- phenotypes dCcee and dccEe (which have been previously described) and weak D phenotypes. Our results suggest that in five serologically D- individuals we have identified an apparently intact RHD gene. Sequence analysis of transcripts obtained from one of these individuals (of phenotype dCCee) illustrates the presence of full-length RHD transcripts, which have a point mutation at nucleotide 121 (C --> T), which generates an in-frame stop codon (Gln41Stop). Thus, we describe a different molecular basis for generating the D- phenotype to the complete RHD gene deletion described previously. We also show that there are discordances with serotype and the multiplex assay in weak D and partial D phenotypes, indicating that the underlying molecular basis can be heterogeneous. Existing Rh D PCR assays assume the complete absence of the RHD gene in D- phenotypes. We describe a different molecular basis for generating the D- phenotype to the complete RHD gene deletion described previously.

Amino Acid Sequence↗
PubMed1997

Molecular analysis of Rh transcripts and polypeptides from individuals expressing the DVI variant phenotype: an RHD gene deletion event does not generate All DVIccEe phenotypes.

The D antigen is a mosaic comprising at least 30 epitopes. Partial Rh D phenotypes occur when there is absence of one or more of these epitopes, with the remainder expressed. The DVI phenotype is the most common of the partial D phenotypes, lacking most D antigen epitopes (ep D) (epD1, 2, 5-8 using the 9-epitope model or epD 1-4, 7-22, 26-29 using the 30-epitope model). DVI mothers may become immunized by transfusion with D-positive blood (if typed as D-positive using polyclonal typing reagents) or by fetuses which have all of the D antigen. This situation can give rise to severe hemolytic disease of the newborn (HDN). The molecular basis of the DVI phenotype has previously been proposed to occur by two different genetic mechanisms, one (in individuals of DVICcee phenotype) where a gene conversion event generates a hybrid RHD-RHCE-RHD gene; the second (in individuals of DVIccEe phenotype) was proposed to be caused by a partial RHD gene deletion. We present evidence that in four DVICcee phenotypes studied, this phenotype is not generated by a partial RHD gene deletion, but occurs by a similar mechanism to the DVICcee phenotypes. In two individuals we have found hybrid RHD-RHCE-RHD transcripts in both DVICe and DVIcE haplotypes. These differ in that the DVICe transcripts are derived from an RHD gene where exons 4-6 have been replaced with RHCE equivalents (encoding Ala226); the DVIcE transcripts are derived from an RHD gene where exons 4 and 5 are replaced by RHCE equivalents (encoding Pro226). We provide direct evidence that Rh DVI polypeptides are expressed at the erythrocyte surface as full-length polypeptide products. We have used immunoprecipitation experiments using anti-D reactive with DVI erythrocytes followed by immunoblotting the immune complexes with rabbit sera immunoreactive to the fourth external and C-terminal domains of all Rh polypeptides. Our results illustrate that these domains are present on all Rh DVI proteins studied, and suggest that Rh DVI polypeptide species studied here exist as full-length Rh proteins.

Amino Acid Sequence↗
PubMed1997

High efficiency of site-directed mutagenesis mediated by a single PCR product.

We describe a highly efficient procedure for site-specific mutagenesis of double-stranded plasmids. The method relies on a single PCR primer which incorporates both the mutations at the selection site and the desired single base substitutions at the mutant site. This primer is annealed to the denatured plasmid and directs the synthesis of the mutant strand. After digestion with selection enzyme, the plasmid DNA is amplified into Escherichia coli strain BMH71-18 and subjected to a second digestion and amplification into the bacterial strain DH5alpha. A mutagenesis efficiency >80% was consistently achieved in the case of two unrelated plasmids.

DNA Primers↗
PubMed1997

Fluorescent probes attached to Cys 35 or Cys 84 in cardiac troponin C are differentially sensitive to Ca(2+)-dependent events in vitro and in situ.

The goal of the current study was to generate recombinant cTnC proteins with single Cys residues as sites for attachment of fluorescent probes that can distinguish between the structural effects of myosin cross bridges and direct Ca2+ binding to cTnC (cardiac and slow skeletal troponin C) in skinned fibers. We anticipated that cTnC proteins which retain the endogenous Cys 35 (cTnC(C35)) or Cys 84 (cTnC(C84)) would provide fluorescent probes with distinct microenvironments, since these residues are on opposite sides of the globular regulatory domain. In vitro experiments that showed IAANS (2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid) coupled to Cys 35 can induce unwanted structural perturbations as evidenced by a decreased affinity of site II for Ca2+ when IAANS-labeled cTnC(C35) is bound to cTnI. Important structural features involving Cys 35 in the inactive site I are suggested by a Ca(2+)-dependent increase in reactivity of Cys 35 with sulfhydryl specific reagents when cTnC(C35) is associated with cTnI. These characteristics are not seen for cTnC(C84). When incorporated in situ into skinned cardiac muscle fibers, native cTnC with IAANS bound to both Cys 35 and Cys 84 showed a pCa50 of fluorescence which preceded that of force, while the pCa50 values of both force and fluorescence were coincident for IAANS-labeled cTnC(C84). Disruption of force-producing myosin cross bridges had no effect on the pCa50 of fluorescence for IAANS-labeled cTnC(C84), but induced a rightward shift in the pCa50 of fluorescence for IAANS-labeled native cTnC. These data can be interpreted to indicate that cTnC with IAANS bound to both Cys 35 and C84 senses either myosin cross bridges or direct Ca2+ binding and myosin-induced cooperativity, while IAANS bound to Cys 84 alone senses conformations that are tightly coupled with force generation.

Amino Acid Sequence↗
PubMed1997

Identification of the histidyl residue obligatory for the catalytic activity of the human H+/peptide cotransporters PEPT1 and PEPT2.

Histidyl residues are known to be essential for the catalytic function of the H(+)-coupled peptide transporters expressed in the intestine and the kidney, most likely participating in the binding and translocation of H+. Three histidyl residues are conserved among the intestinal and renal peptide transporters (PEPT1 and PEPT2, respectively) from different animal species. In hPEPT1, these residues are His-57, His-121, and His-260. The corresponding residues in hPEPT2 are His-87, His-142, and His-278. We have individually mutated each of these histidyl residues in hPEPT1 and in hPEPT2 and compared the catalytic function of the mutants with that of their respective wild type transporters by expressing the transporters in Xenopus laevis oocytes and also in HeLa cells. His-57 in hPEPT1 and His-87 in hPEPT2 were found to be absolutely essential for catalytic activity because the corresponding mutants had no detectable peptide transport activity. His-121 in hPEPT1 is not essential since mutation of this residue did not impair transport function. His-142 in hPEPT2 was found to play a significant role in the maintenance of transport function but was not found to be obligatory because the mutant had appreciable transport activity. The obligatory histidyl residue (His-57 in hPEPT1 and His-87 in hPEPT2) is located in an almost identical topological position in both transporters, near the extracellular surface of the second putative transmembrane domain. The second conserved histidyl residue is located in the fourth putative transmembrane domain in hPEPT1 as well as in hPEPT2. The third conserved histidyl residue is present in the cytoplasmic loop between the transmembrane domains 6 and 7 and is unlikely to play any significant role in the binding and translocation of H+ and this was supported by the findings that mutation of this histidyl residue in hPEPT1 did not interfere with transport function. The loss of transport function of hPEPT1 and hPEPT2, when His-57 in hPEPT1 and His-87 in hPEPT2 were mutated, was not due to alterations in protein expression because the expression levels of these mutants were similar to those of the respective wild type transporters in HeLa cells as assessed by immunoblot analysis. Confocal analysis of immunofluorescence in X. laevis oocytes expressing the wild type and the three histidine mutants of hPEPT1 showed that the transporter protein is expressed exclusively in the plasma membrane and that the level of expression is comparable among the wild type and the three mutants. These site-directed mutagenesis studies clearly show that His-57 in hPEPT1 and His-87 in hPEPT2 are the most critical histidyl residues necessary for the catalytic function of these transporters.

Amino Acid Sequence↗
PubMed1997

Effect of retinoic acid on otic capsule chondrogenesis in high-density culture suggests disruption of epithelial-mesenchymal interactions.

Previous studies have shown that in utero exposure of the mouse embryo to nonphysiological levels of all-trans retinoic acid (RA) produces malformations of the epithelial-derived auditory and vestibular receptors of the inner ear and its surrounding cartilaginous capsule. In this study, we demonstrate the effects of all-trans RA in high-density cultures of the periotic mesenchyme fated to form the otic capsule. Our results demonstrate an inhibition of chondrogenesis in cultured periotic mesenchyme + otic epithelium of embryonic age E10.5 days (E10.5) in response to all-trans RA exposure. However, at later stages of development (i.e., E12, E14), when epithelial-mesenchymal interactions are no longer required for initiation of chondrogenesis, exposure to this teratogen has no effect on the chondrogenic process. Two analogues of all-trans RA, i.e., cis-RA and trans-retinol, were investigated for their biological activity in chondrogenic cultures of inner ear mesenchyme + epithelium. Moreover, we tested the inductive capability and responsiveness of in utero RA-exposed inner ear tissues when cultured with inner ear tissues that were not exposed to this teratogen. Our results support the hypothesis that all-trans RA disrupts otic capsule formation by interfering with the tissue interactions required for its normal differentiation and development.

Animals↗
PubMed1997

Effects of ligustrazine on bone marrow microvessel system in the early period of acute radiation injury in mice.

Sublethally irradiated mice were immediately treated with 250 mg/kg Ligustrazine Phosphiatis intraperitoneally twice a day for seven days, and the bone marrow microvessels of the Ligustrazine group was much greater than that of the control group. On the 7th day, the amount of the control group decreased to normal, while the ligustrazine group was still increasing, and the microvessel area was enlarged obviously.. The percentage of the hematopoietic tissue volume in bone marrow between the two groups had no significant difference in the first 7 days. On the 7th day after irradiation, the peripheral neutrophilic granulocytes increased in the Ligustrazine group. The results suggested that early use of Ligustrazine after acute radiation injury might improve the blood supply of bone marrow, and be helpful for recovery of hematopoiesis.

Animals↗