PubMed Health⌕ Search

Biomedical subjects

L Xia

Publications and source records attributed to L Xia.

At least 19 recordsLinked to original sources
PubMed1997

CaN19 expression in benign and malignant hyperplasias of the skin and oral mucosa: evidence for a role in regenerative differentiation.

CaN19, a member of the S100 family of calcium-binding proteins, is known to be "underexpressed" in cultured breast carcinoma-derived cell lines relative to their normal counterparts. By Northern blotting, we confirm these results and find that CaN19 is also markedly "underexpressed" in several carcinoma-derived cell lines of the skin, oral mucosa, and urogenital tract. However, exceptions to the inverse correlation between CaN19 expression and malignancy have been identified, bringing into question the hypothesis that CaN19 functions as a tumor suppressor gene. Unexpectedly, CaN19 mRNA was strongly expressed in bulk specimens of basal and squamous cell carcinomas of the skin and oral cavity. However, in situ hybridization revealed only limited CaN19 expression in tumor cells themselves; the bulk of expression is localized to hyperplastic perilesional epidermis. Tumor cell expression of CaN19 was similar in primary and locally metastatic tumors, indicating that this gene is not necessarily down-regulated during tumor progression. Coordinate overexpression of CaN19 and the "hyperproliferalive" keratin K6a was observed only in tissues undergoing squamous differentiation. Taken together with other recent results from our laboratory, these findings suggest the hypothesis that CaN19 participates in an epidermal growth factor receptor-dependent pathway of regenerative squamous differentiation.

Basal Cell Carcinoma↗
PubMed1997

Identification of specific nucleotide sequences and structural elements required for intronic U14 snoRNA processing.

Vertebrate U14 snoRNAs are encoded within hsc70 pre-mRNA introns and U14 biosynthesis occurs via an intron-processing pathway. We have shown previously that essential processing signals are located in the termini of the mature U14 molecule and replacement of included boxes C or D with oligo C disrupts snoRNA synthesis. The experiments detailed here now define the specific nucleotide sequences and structures of the U14 termini that are essential for intronic snoRNA processing. Mutagenesis studies demonstrated that a 5', 3'-terminal stem of at least three contiguous base pairs is required. A specific helix sequence is not necessary and this stem may be extended to as many as 15 base pairs without affecting U14 processing. The spatial positioning of boxes C and D with respect to the terminal stem is also important. Detailed analysis of boxes C and D revealed that both consensus sequences possess essential nucleotides. Some, but not all, of these critical nucleotides correspond to those required for the stable accumulation of nonintronic yeast U14 snoRNA. The presence of box C and D consensus sequences flanking a terminal stem in many snoRNA species indicates the importance of this "terminal core motif" for snoRNA processing.

Animal Population Groups↗
PubMed1997

Construction and expression of mouse-human chimeric antibody SZ-51 specific for activated platelet P-selectin.

A murine monoclonal (mAb) SZ-51 specific for human P-selectin may be used for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi. In order to reduce the immunogenicity of the murine mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human chimeric reagents. The E. coli expression vector pHEN1-SZ51Fab/Hu was constructed by fusing the variable regions of mAb SZ-51 with human IgG gamma 1CH1 and C kappa genes. The constructs were introduced into E. coli HB2151 for expression of soluble chimeric Fab fragment. We also constructed two fusion products by joining the variable regions of mouse antibody to the appropriate constant regions of human Ig gamma 1 and kappa. These chimeras were cloned into two eukaryotic selectable expression vectors separately, which were then contransfected into a non-Ig secreting murine myeloma line SP2/0 with lipofectin reagent. Six cell lines remained positive for Ig secretion. The highest producing cell line, which showed stable integration and expression at 5 mg/l of culture, was selected for the large scale production of chimeric antibody. Immunoblotting analysis demonstrated that both of the chimeric antibodies (SZ51Fab/Hu, SZ51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P-selectin. In addition, the whole chimeric antibody can compete for binding to activated platelets with murine SZ-51. Therefore, the SZ-51 chimeric antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.

Animals↗
PubMed1996

A truncated hMSH2 transcript occurs as a common variant in the population: implications for genetic diagnosis.

Germline mutations of the hMSH2 gene are responsible for many cases of hereditary nonpolyposis colorectal cancer. While screening for hMSH2 gene mutations in hereditary nonpolyposis colorectal cancer kindreds, we observed that a previously reported germline mutation is in fact a common, alternatively spliced variant in the population. Using RT-PCR and the protein truncation test, the hMSH2 exon 13 deletion variant was found in more than 90% of individuals. The exon 13 deletion transcript was only present in lymphocyte RNA, no abnormalities were detected in genomic DNA flanking exon 13, and the deletion transcript is apparently not translated. These findings highlight further that caution should be exercised in providing genetic risk assessment on the basis of currently used germline mutation detection strategies.

Colorectal Neoplasms↗
PubMed1996

Site-directed mutagenesis of Cys324 and Cys331 in human cytosolic phospholipase A2: locus of action of thiol modification reagents leading to inactiviation of cPLA2.

Human cytosolic phospholipase A2 contains two cysteines, cyS324 and cyS331, chemical modification of which using thiol modifying reagents abolishes the activity of the enzyme [Li et al. (1994) Biochemistry 33, 8594-8603]. To verify the functional importance of the two cysteine residues, site-directed mutagenesis has been used to create six mutations at positions 324 and 331. The mutant enzymes include C324A, C331A, C324Q, C331Q, C324R, and C331S. Complete loss of activity is observed for C331Q, whereas the other mutants have retained varying degrees of activity. These results show that neither CyS324 nor CyS311 is catalytically essential for the enzyme activity. Further chemical modification studies of the mutant enzymes by thiol-specific reagents suggest that modification of Cys331 is responsible for the complete loss of the enzyme activity. The possible roles of Cys324 and Cys331 are discussed.

Amino Acid Sequence↗
PubMed1996

Preparation of an antifibrin thrombus-specific murine/human chimeric monoclonal antibody Fab fragment in Escherichia coli.

We have successfully developed a murine antifibrin monoclonal antibody designated SZ-63 with property of binding specifically with thrombus both in vitro and in vivo. In order to reduce its immunogenicity and molecular weight, a murine/human chimeric Fab fragment of the antibody was prepared. mRNA was selected on oligo (dT) cellulose from total RNA isolated from SZ-63 hybridoma cells. cDNAs coding for heavy and light variable regions were amplified by reverse transcriptase polymerase chain reaction. The amplified fragments were cloned and sequenced. The nucleotides of SZ-63 VH and VL are 354 and 321 respectively. The variable genes were then linked with human IgG gamma1 CH and kappa CL genes. Expression vector pHEN1-63 Fab/Hu was thereby constructed and chimeric Fab fragment was expressed in E. coli HB2151 cells in soluble form. Western blot and ELISA results showed that it remained the same capability of binding with cross-linked fibrin as the murine SZ-63 antibody, the content in culture is about 125 ug/L.

Animals↗
PubMed1996

Lineage-specific CaN19 expression in human skin: lack of expression in normal melanocytes.

Although expression of CaN19 is reduced in cell lines derived from tumors of diverse origin, its expression in their normal counterparts has not been studied in detail. We find that CaN19 mRNA is expressed at very low to undetectable levels in normal melanocytes as well as malignant melanoma cell lines, confirming that CaN19 expression in normal cells is lineage-dependent.

Cells, Cultured↗
PubMed1996

Effects of 2-deoxy-d-glucose on the functional state of the rat myoblast GLUT 1 transporter.

Based on the rationale that internalized 2-deoxy-D-glucose (dGlc) is toxic to cells, glucose transport (GLUT) defective myoblast mutants have been isolated by their ability to grow in glucose-free medium containing dGlc. Recent studies revealed that the GLUT 1 transport process was activated when GLUT 3-GLUT 4-mutants were grown in glucose-free medium. It was therefore puzzling why these GLUT3-GLUT4-myoblasts could survive in the presence of dGlc during the mutant selection process. The present study revealed that GLUT 1 transport affinity in dGlc-grown cells was at least four folds lower than that in control cells. This loss of GLUT 1 transport activity was apparent only after exposure to the toxic sugar analogues for more than 10 hrs. This dGlc-mediated effect was not due to competitive inhibition by the residual dGlc carried over from growth medium, changes in glycolytic enzymes, nor accumulation of the negatively charged dGlc-6-PO4. In fact, GLUT 1 transcript level was elevated in dGlc-treated cells. Both immunoprecipitation and immunoblotting studies indicated that the size of the GLUT 1 transporter in dGlc-grown myoblasts was reduced from 52 kDa to that of the unglycosylated form (38 kDa). These findings suggest that growth in the presence of dGlc inhibits glycosylation of the GLUT 1 transporter, thus reducing its transport affinity. This inability of the GLUT 1 transporter to take up dGlc may therefore explain why GLUT 3-GLUT 4-mutants are able to grow in the presence of the toxic dGlc during the mutant selection procedure.

3-O-Methylglucose↗
PubMed1996

Interleukin-1 stimulates the central release of corticotropin-releasing hormone in the primate.

The cytokine interleukin-1 (IL-1) is present in the brain and is known to cause a variety of neuroendocrine and immune effects in the rodent; the neuropeptide corticotropin-releasing hormone (CRH) plays a critical role in mediating many of these effects. Little is known about these neuropeptide interactions in the primate. We have therefore examined the effects of IL-1 alpha on the release of CRH in the ovariectomized rhesus monkey in vitro and in vivo. In 3 animals, the effect of IL-1 alpha on CRH release from the superfused hypothalamus was studied in vitro. The hypothalamus was divided in half and fragments from each half were superfused separately. Mean CRH release was 262 +/- (SE) 46 pg/20 min and increased to 1,340 +/- 470 pg/20 min after exposure to IL-1 alpha (p < 0.05). The effect of IL-1 alpha on CRH release into cerebrospinal fluid (CSF) in vivo was studied in 8 animals with chronic cannulas implanted in the lateral ventricle for IL-1 infusion; indwelling catheters were also placed via lumbar puncture and threaded into the cervical area for CSF collection. CSF was collected at a rate of 800 microliters/h during a 4-hour baseline period and for 4-8 h after intracerebroventricular infusion of 4.2 micrograms of IL-1 alpha. CRH increased significantly over time in CSF after IL-1 alpha infusion; the mean concentration of CRH increased from 83 +/- 17 pg/ml during the baseline period to 203 +/- 40 pg/ml after IL-1 alpha infusion (p < 0.01). We conclude that IL-1 stimulates central CRH release in the primate and that the effects of cytokines on the release of this important neuromodulator can be monitored in chronically cannulated animals in vivo.

Animals↗
PubMed1996

Elements essential for processing intronic U14 snoRNA are located at the termini of the mature snoRNA sequence and include conserved nucleotide boxes C and D.

Essential elements for intronic U14 processing have been analyzed by microinjecting various mutant hsc70/Ul4 pre-mRNA precursors into Xenopus oocyte nuclei. Initial truncation experiments revealed that elements sufficient for U14 processing are located within the mature snoRNA sequence itself. Subsequent deletions within the U14 coding region demonstrated that only the terminal regions of the folded U14 molecule containing con- served nucleotide boxes C and D are required for processing. Mutagenesis of either box C or box D completely blocked U14 processing. The importance of boxes C and D was confirmed with the excision of appropriately sized U3 and U8 fragments containing boxes C and D from an hsc7O pre-mRNA intron. Competition studies indicate that a trans-acting factor (protein?) is binding this terminal motif and is essential for U14 processing. Competition studies also revealed that this factor is common to both intronic and non-intronic snoRNAs possessing nucleotide boxes C and D. Immunoprecipitation of full-length and internally deleted U14 snoRNA molecules demonstrated that the terminal region containing boxes C and D does not bind fibrillarin. Collectively, our results indicate that a trans-acting factor (different from fibrillarin) binds to the box C- and D-containing terminal motif of U14 snoRNA, thereby stabilizing the intronic snoRNA sequence in an RNP complex during processing.

Animals↗
PubMed1995

Intronic U14 snoRNAs of Xenopus laevis are located in two different parent genes and can be processed from their introns during early oogenesis.

U14 is a member of the rapidly growing family of intronic small nucleolar RNAs (snoRNAs) that are involved in pre-rRNA processing and ribosome biogenesis. These snoRNA species are encoded within introns of eukaryotic protein coding genes and are synthesized via an intron processing pathway. Characterization of Xenopus laevis U14 snoRNA genes has revealed that in addition to the anticipated location of U14 within introns of the amphibian hsc70 gene (introns 4, 5 and 7), additional intronic U14 snoRNAs are also found in the ribosomal protein S13 gene (introns 3 and 4). U14 is thus far a unique intronic snoRNA in that it is encoded within two different parent genes of a single organism. Northern blot analysis revealed that U14 snoRNAs accumulate during early oocyte development and are rapidly expressed after the mid-blastula transition of developing embryos. Microinjection of hsc70 pre-mRNAs into developing oocytes demonstrated that oocytes as early as stages II and III are capable of processing U14 snoRNA from the pre-mRNA precursor. The ability of immature oocytes to process intronic snoRNAs is consistent with the observed accumulation of U14 during oocyte maturation and the developmentally regulated synthesis of rRNA during oogenesis.

Animals↗
PubMed1995

Evidence for a novel exon in the coding region of the adenomatous polyposis coli (APC) gene.

Germline mutations of the tumor suppressor gene APC cause familial adenomatous polyposis. Somatic APC alterations are involved in several sporadic neoplasms, including colorectal, duodenal, gastric, and esophageal carcinoma. The APC mRNA is encoded by 15 exons. Additional transcripts have been reported, due to alternative splicing of coding as well as noncoding regions. Two mRNA isoforms occur due to a deletion of exon 7 or a partial deletion of exon 9. We have identified a novel exon, flanked by APC exons 10 and 11, which is expressed as an alternatively transcribed product of the gene. Further, we have shown that the novel exon consists of a heptad repeat motif and is conserved across species.

Adenomatous Polyposis Coli↗
PubMed1995

Use of hexose transport mutants to examine the expression and properties of the rat myoblast GLUT 1 transport process.

Rat L6 myoblasts were recently shown to possess the GLUT 1, 3 and 4 transporters, and not the GLUT 2 isoform [1]. This investigation examined the expression and properties of the GLUT 1 isoform. GLUT 1 transcript level was significantly reduced in cells grown at high densities and during myogenic differentiation. A comparison of the GLUT 1 and 4 transcript levels in myogenesis-competent and impaired cells revealed an inverse relationship between these two isoforms. This relationship was confirmed by studies using two independent spontaneous GLUT 3- GLUT 4- mutants, M1 and M3. These mutants possessed very high level of the GLUT 1 isoform, but negligible amount of the GLUT 3 and 4 isoforms. GLUT 1 expression was also subject to positive regulation. Glucose starvation was found to increase not only the levels of the GLUT 1 transcript and transporter, but also the intrinsic activity of the GLUT 1 transporter. Studies with M1 and M3 mutants revealed that the GLUT 1 transporter was not functional in glucose-grown cells, even though it was present at a very high level in the plasma membrane. This transporter became functional when cells were starved for glucose. The functional GLUT 1 transporter had an apparent Km value of around 0.9 mM, and was sensitive to cytochalasin B, phloretin, phlorizin and pCMBS.

3-O-Methylglucose↗
PubMed1995

CCAAT-box contributions to human thymidine kinase mRNA expression.

In order to examine the role of two inverted CCAAT boxes near the start of transcription of the human thymidine kinase (TK) gene, a series of constructs were prepared in which one or both CCAAT boxes were deleted or mutated. These altered promoters (1.2 kb of 5'-flanking sequence) were used to express a TK minigene containing the first two exons and introns followed by the remainder of the cDNA. RNA blots were prepared from stable cell lines of ts13 cells containing these constructs under three conditions: 1) serum deprived cells, 2) serum stimulated cells, and 3) cells that had been stimulated with serum, but were arrested in the G1 phase of the cell cycle by the temperature sensitive mutation carried by these cells. TK mRNA expression from each construct was suppressed by the temperature sensitive block to cell cycle progression. Measurement of protein expression from the various altered TK promoters indicated that both CCAAT boxes contribute to promoter strength. These experiments also suggested that the two CCAAT boxes were not equivalent and that the distal CCAAT could substitute for the proximal CCAAT, but the converse was not true.

Animals↗
PubMed1995

Protein that binds to the distal, but not to the proximal, CCAAT of the human thymidine kinase gene promoter.

Mobility shift assays were used to examine protein binding to the human TK gene CCAAT boxes. Similar protein binding patterns were observed with probes containing either the proximal or distal CCAAT. However, probes containing both CCAAT boxes in which one of the CCAAT boxes was inactivated by mutation did not demonstrate identical binding patterns. One of the complexes formed with the longer probes was only observed when the distal CCAAT was intact. This species was not formed with probes that only contained an intact proximal CCAAT, and its formation could only be competed by oligonucleotides containing the distal CCAAT motif. This observation reveals the existence of a protein that can bind to the distal, but not to the proximal, CCAAT of the human TK promoter. This protein may account for the previous observation that the two CCAAT motifs are not functionally equivalent. The protein that binds to the distal, but not to the proximal, CCAAT (DTK-CBP) was also present in two human cell lines. Significantly more DTK-CBP was present in nuclear extracts of HepG2 and WI38 cells than in TK-ts13 cells. However, this protein was not observed in three different murine cell lines and one primary culture. Its abundance in some human cell lines suggests it might modulate the expression of human TK mRNA in cells that express this protein.

3T3 Cells↗
PubMed1995

Ultrastructural changes in bovine oocytes cryopreserved by vitrification.

Oocytes recovered from abattoir-derived ovaries were exposed to a cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% fetal calf serum in tissue culture medium 199) for less than 20 s and vitrified either at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Survival was assessed by fertilization and culture in vitro to the blastocyst stage. To identify ultrastructural changes, some of the vitrified oocytes that were morphologically normal after thawing were immediately processed for transmission electron microscopy after DAP213 removal. Cleavage rates for vitrified IVM oocytes were 4.5 and 6.7% using one-step and three-step cryoprotectant dilution procedures, respectively. Four (3%) oocytes developed to the eight-cell stage with the three-step procedure, but none formed blastocysts. None of the GV oocytes cleaved, while 66.7% (78/117) of controls developed to the two-cell stage and 19.2% (15/78) of those became blastocysts. Vitrification induced profound ultrastructural modifications in microvilli, mitochondria, vesicle formation, and the ooplasm of GV oocytes, whereas these structures were generally better preserved in IVM oocytes. The integrity of cell organelles was relatively better maintained following the three-step than after the one-step procedure in both GV and IVM oocytes. Changes in the zona pellucida (ZP) of IVM oocytes due to vitrification were associated with fewer cortical granules in the ooplasm. Since previous work showed that short-term exposure to DAP213 did not cause ZP alterations in IVM oocytes, these findings suggest that ZP damage due to low temperatures may result from the premature release of cortical granules.

Acetamides↗
PubMed1995

Three-dimensional simulation of epicardial potentials using a microcomputer-based heart-torso model.

Previous cardiac simulation studies have focused on simulating the activation isochrones and subsequently the body surface potentials. Epicardial potentials, which are important for clinical applications as well as for electrocardiography inverse problem studies, however, have usually been neglected. This paper presents a procedure of simulating epicardial potentials using a microcomputer-based heart-torso model with real geometry. The heart model developed earlier which was composed of more than 60,000 cell units was used in this study. To simulate the epicardial potentials, an epicardial surface model which enclosed the whole heart was constructed. The heart model, together with the epicardial surface model, are mounted in an inhomogeneous human torso model. Electric dipoles, which are proportional to the spatial gradient of the action potential, are generated in all cell units. These dipoles give rise to a potential distribution on the epicardial surface, which is calculated by means of the boundary element method. The simulated epicardial potential maps during a normal heart beat and in patients with left bundle branch block (LBBB) are in close agreement with those reported in the literature.

Action Potentials↗