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Biomedical subjects

X L Liu

Publications and source records attributed to X L Liu.

At least 55 records · Page 3Linked to original sources
PubMed1996

Identification of a novel serine protease-like gene, the expression of which is down-regulated during breast cancer progression.

In an effort to isolate genes with down-regulated expression at the mRNA level during oncogenic transformation of human mammary epithelial cells (MECs), we performed subtractive hybridization between normal MEC strain 76N and its radiation-transformed tumorigenic derivative 76R-30. Here, we report the isolation of cDNA clones corresponding to a 1.4-kb mRNA species that is abundantly expressed in 76N cells but is drastically reduced in 76R-30 cells. Based on its selective expression in MECs compared with fibroblasts, the corresponding gene is designated NES1 (normal epithelial cell-specific 1). Sequence analysis of the full-length NES1 cDNA clones revealed it to be a novel gene with a predicted polypeptide of 30.14 kilodaltons; in vitro transcription and translation confirmed this prediction. Database searches revealed a 50-63% similarity and 34-42% identity with several families of serine proteases, in particular the trypsin-like proteases, members of the glandular kallikrein family (including prostate-specific antigen, nerve growth factor gamma, and epidermal growth factor-binding protein) and the activators for the kringle family proteins (including the human tissue plasminogen activator and human hepatocyte growth factor activator). Importantly, all of the residues known to be crucial for substrate binding, specificity, and catalysis by the serine proteases are conserved in the predicted NES1 protein, suggesting that it may be a protease. An antipeptide antibody directed against a unique region of the NES1 protein (amino acids 120-137) detected a specific 30-kilodalton polypeptide almost exclusively in the supernatant of the mRNA-positive MECs, suggesting that NES1 is a secreted protein. The 1.4-kb NES1 mRNA was expressed in several organs (thymus, prostate, testis, ovary, small intestine, colon, heart, lung, and pancreas) with highest levels in the ovary; a 1.1-kb transcript was found in the pancreas. Although expression of the NES1 mRNA was observed in all normal and immortalized nontumorigenic MECs, the majority of human breast cancer cell lines showed a drastic reduction or a complete lack of its expression. The structural similarity of NES1 to polypeptides known to regulate growth factor activity and a negative correlation of NES1 expression with breast oncogenesis suggest a direct or indirect role for this novel protease-like gene product in the suppression of tumorigenesis.

Amino Acid Sequence↗
PubMed1996

Mutant p53-induced immortalization of primary human mammary epithelial cells.

Mutations of the p53 gene are the most frequent genetic lesions in breast cancer, suggesting a critical role for p53 protein in normal mammary cell growth control. Indeed, the p53-targeting human papillomavirus oncogene E6 induces efficient immortalization of normal human mammary epithelial cells (MECs). To assess whether selective loss of p53 is sufficient for MEC immortalization, we introduced seven missense mutants and one single-amino acid deletion mutant (del239) of p53 into the 76N normal MEC strain. Although the missense mutants failed to immortalize MECs, the del239 mutant reproducibly immortalized these cells. The immortal cells were anchorage dependent and nontumorigenic, indicating a preneoplastic transformation. Gamma-irradiation of these cells failed to induce G1 cell cycle arrest and did not lead to an increase in WAF1 and mdm-2 mRNA levels, demonstrating a loss of the endogenous p53 function. These results demonstrate that selective ablation of p53 function by a dominant-negative mutant is sufficient for immortalization of MECs. Availability of an immortalizing as well as several nonimmortalizing p53 mutants should help identify functions critical for cell growth control by p53 in mammary epithelial cells.

Breast↗
PubMed1995

Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7.

Multiple mammary epithelial cell (MEC) types are observed both in mammary ducts in vivo and in primary cultures in vitro; however, the oncogenic potential of different cell types remains unknown. Here, we used human papilloma virus 16 E6 and E7 oncogenes, which target p53 and Rb tumor suppressor proteins, respectively, to immortalize MECs present in early or late passages of human mammary tissue-derived cultures or in milk. One MEC subtype was exclusively immortalized by E6; such cells predominated in late-passage cultures but were rare at early passages and apparently absent in milk. Surprisingly, a second cell type, present only in early-passage tissue-derived cultures, was fully immortalized by E7 alone. A third cell type, observed in tissue-derived cultures and in milk, showed a substantial extension of life span with E7 but eventually senesced. Finally, both E6 and E7 were required to fully immortalize milk-derived MECs and a large proportion of MECs in early-passage tissue-derived cultures, suggesting the presence of another discrete subpopulation. Identification of MECs with distinct susceptibilities to p53- and Rb-targeting human papillomavirus oncogenes raises the possibility that these cells may serve as precursors for different forms of breast cancer.

Breast↗
PubMed1994

Tumor cell-specific loss of p53 protein in a unique in vitro model of human breast tumor progression.

Mutations of the p53 gene are the most frequent genetic lesion in breast cancer. Here, we examined p53 expression in a unique in vitro model of tumor progression derived from a single breast cancer patient (21T series). While the normal mammary epithelial, fibroblast and mesothelial cells derived from this patient expressed easily detectable functional p53 protein, the primary as well as metastatic tumor cell lines demonstrated a lack of p53 protein synthesis. 21T tumor cells failed to exhibit G1 cell cycle arrest upon exposure to gamma-irradiation, and their growth was suppressed by transfection of a normal p53 cDNA, demonstrating a lack of p53-mediated function in these cells. No p53 gene deletion or rearrangements were detectable. PCR and sequence analysis of the entire coding region of p53 gene revealed a novel mutation, an insertion of a single T within codon 33, which resulted in a frame-shift and early termination. The same mutation was observed in all 21T tumor cell lines. These results demonstrate a tumor cell-specific loss of p53 protein due to a frame-shift mutation, and suggest that p53 loss may occur at a relatively early step in breast tumorigenesis before metastatic seeding or emergence of tumor heterogeneity. In addition, the availability of normal and tumor-derived epithelial cells with known p53 sequences from a single breast cancer patient should facilitate understanding of the p53 regulation in mammary cells.

Breast↗
PubMed1994

Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells.

Animals↗
PubMed1994

[Application of SRK II formula and measurement of A constant of KM intraocular lens].

The characteristics and distribution of the axial lengths and the results of keratometric measurements of 757 eyes with senile cataracts were reported and their relationship was discussed. The range of the axial lengths was 19.53-33.74 mm, the mean value being 23.81 +/- 2.26 mm (mean +/- SD), the eyes with longer axial lengths accounting for 21.8% and the eyes with axial lengths over 27 mm, 8.59%. The range of the degrees of the corneal curvatures was 39.75-50.50 D, the mean value being 44.47 +/- 0.78 D. The corneal curvatures of the eyes with shorter axial lengths are increased markedly with the shortening of the axial lengths. However, the corneal curvatures of the eyes with longer axial lengths are not decreased significantly with the lengthening of the axes. The A constant of KM intraocular lens we measured is 116.86 and the post-operative mean anterior chamber depth is 3.63 +/- 0.28 mm. If the C value is + 1.5 in eyes with 21-22 mm axial lengths, 0 in eyes with 22-24.5 mm and -0.5 in eyes with > 24.5, > 27, > 28.5 mm, the predictive error seen post-operatively is the smallest.

Aged↗
PubMed1994

[Phage typing of Salmonella typhimurium in 20 provinces, autonomous regions and municipalities of China].

Thirty strains of Salmonella bacteriophages were isolated from hospital sewage. Eleven strains selected from them, in combination with Felix Salmonella Phage O-I, were used as a phage typing set for serotyping Salmonella. Thirty-one identified types and 20 undefined types were detected in 2348 strains of Salmonella typhimurium collected from 20 provinces, autonomous regions and municipalities in China. The nine types with higher frequencies were 7774 (accounted for 48.0%), 0774 (17.1%), 6774 (7.7%), 4774 (7.3%), 5774 (3.3%). 3774 (2.2%), 4000(2.1%), 0000 (1.8%) and 7000 (1.7%). Most outbreaks of nosocomial infection and food poisoning were caused by phage types 7774 and 4774. Phage types of Salmonella typhimurium in domestic ducks, pigs, rats, snails, and in sewages were analyzed, and their significance in epidemics was discussed.

Bacteriophage Typing↗
PubMed1993

Cloning, sequencing and expression of the gene encoding NADH oxidase from the extreme anaerobic thermophile Thermoanaerobium brockii.

The gene encoding the enzyme NADH oxidase from the extreme thermophile Thermoanaerobium brockii has been isolated from a recombinant library of genomic DNA and sequenced. An open reading frame corresponds to the 651 amino acids of the enzyme's subunit, which include characteristic FAD- and NADH-binding sequences, as well as cysteines which are involved in the FeS cluster present in the enzyme. The enzyme is expressed either from its own promoter or from vector promoters in Escherichia coli. After heat-treating the recombinant extracts at 70 degrees C, most of the host proteins are denatured, leaving the NADH oxidase 5- to 10-fold enriched.

Amino Acid Sequence↗
PubMed1993

Growth of an androgen-sensitive human prostate cancer cell line, LNCaP, in nude mice.

This study was undertaken to establish an androgen-sensitive model of human prostatic carcinoma in nude mice. The androgen-sensitive prostatic carcinoma cell line, LNCaP, was suspended in reconstituted basement membrane (Matrigel) and injected subcutaneously into nude mice. The LNCaP cell line was chosen for the study, because the cell line is androgen-sensitive and secretes prostate specific antigen (PSA) into culture media. Following injection of 1 x 10(6) LNCaP cells with 0.25 ml of Matrigel, 88% of mice exhibited palpable tumor burdens after 12 weeks of observation. In addition, significant levels of PSA were observed in the serum of LNCaP-bearing mice. Bilateral orchiectomy of mice resulted in tumor regression and stabilization of serum PSA levels, compared to testis-intact controls. A significant correlation of PSA to tumor volume and weight was observed. The castrate level of testosterone was confirmed by radioimmunoassay and was similar to testosterone levels in female nude mice. Matrigel allows for a conducive environment to propagate LNCaP cells in nude mice. Furthermore, the growth can be manipulated by castration, leading to involution of tumor and stabilization of serum PSA level. This in vivo model of hormone-sensitive human prostate cancer cell line will serve as a model for the study of prostate tumor biology and treatment.

Animals↗
PubMed1993

Characteristics of the epitope of protein-reactive anti-peptide antibodies.

The specificity of hen egg-white lysozyme (HEL)-reactive rabbit antibodies induced by the peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) of HEL was clarified by analyzing their cross-reactions with various avian lysozymes and their reaction with synthetic peptides (sequences 59-82) in which alanine was substituted for the amino acid at certain positions. The Arg-68 residue of HEL plays a dominant role in the binding, while Gly-71, Ser-72, Arg-73, and Pro-79 also contribute to the binding of two anti-Ploop I.II antibodies (rabbit number 125 and 126). These residues, although remote in sequence, are grouped together in the crystal structure of HEL and may form an area of contact with the antibody. Contributions by Trp-63, Ile-78, and Asn-77 to the binding of the two antibodies to HEL were excluded. These results support the idea that the anti-Ploop I.II antibodies recognize a conformational type of epitope which is similar to that of native HEL. The immunogenicity of the reduced and alkylated form of Ploop I.II was also tested, but it failed to induce an HEL-reactive antibody.

Alanine↗
PubMed1993

[Histochemical study of intrinsic laryngeal muscles in neonates].

The motor end-plates and muscle fiber types in muscles of cricothyroid, posterior cricoarytenoid, thyroarytenoid were in five neonates and two adults studied by staining with acetylcholinesterase (AChE) and succinic dehydrogenase (SDH). The muscle fibers in neonate could be classified into three types: red, white and intermediate. The diameters of muscle fibers were different, with the white muscle fiber widest and the red ones narrowest. Each of the laryngeal muscles was composed of three muscle types in proportion. The motor end-plate showed a concentrated distribution band.

Acetylcholinesterase↗
PubMed1993

[Effects of stimulation of SI cortex and cerebral peduncle on the nociceptive responses of spinal dorsal horn neurons in the rat].

Single spinal dorsal horn neurons were recorded extracellularly with glass micropipettes, and the effect of stimulation of SI cortex or cerebral peduncle (CP) was observed on the long latency discharges (C-fiber evoked response) of the wide dynamic range (WDR) neurons induced by intense electrical skin stimulation. Inhibition was the predominant effect of SI cortex stimulation on the C-fiber evoked responses of WDR neurons. The effect of CP stimulation was similar to that of SI cortex stimulation, except that the former was much more effective than the latter. The inhibition lasted a period of time varying from 400 ms to more than 10 min after CP stimulation in different neurons. Systemic naloxone had no significant effect on the inhibition produced by CP stimulation. Systemic methysergide reduced or eliminated the inhibition in a part of the neurons tested, suggesting that 5-HT, but not opioid peptides, is involved partially in mediation of the descending inhibitory effect originating from the cortex.

Animals↗
PubMed1992

A thermostable NADH oxidase from anaerobic extreme thermophiles.

A high-abundance NADH-oxidizing enzyme (NADH: acceptor oxidoreductase, EC 1.6.99.3) has been identified and isolated from a range of anaerobic extreme thermophiles, including strains of Clostridium thermohydrosulfuricum and Thermoanaerobium brockii. By use of a pseudo-affinity salt-promoted adsorbent, a nearly pure sample was obtained in one step; remaining impurities were separated by ion-exchange. The fully active purified enzyme contains FAD (two molecules per subunit of 75-78 kDa) and iron-sulphur, and is hexameric in its most active form. The reaction with oxygen is a one- or two-electron transfer to produce superoxide radical and H2O2; other acceptors include tetrazolium salts, dichlorophenol-indophenol, menadione and ferricyanide. The role of the enzyme is not clear; it was found not to be NAD:ferredoxin oxidoreductase, which is a major NADH-utilizing enzyme in these organisms.

Chromatography, Gel↗
PubMed1992

The weaver gene encodes a nonautonomous signal for CNS neuronal differentiation.

In the neurological mutant mouse weaver, CNS precursor cells in the external germinal layer (EGL) of the cerebellar cortex proliferate normally, but fail to differentiate and die in the proliferative zone. To examine the autonomy of expression of the weaver gene, we carried out cell-mixing experiments in vitro. In homotypic, reaggregate cultures, weaver EGL precursor cells expressed the general neuronal markers N-CAM, L1, and MAP2, but failed to express the late neuronal antigens TAG-1 and astrotactin, to extend neurites or to migrate on glial fibers. After reaggregation with wild-type EGL precursor cells, weaver precursor cells extended neurites equivalent in length to wild-type cells, migrated along astroglial fibers, and expressed TAG-1 and astrotactin. Rescue of neurite production was also achieved by the addition of membranes from, but not by medium conditioned by wild-type cells. These findings suggest that the weaver gene acts non-autonomously, encoding a membrane-associated ligand that induces EGL neuronal differentiation.

Animals↗
PubMed1992

Changes in serum osmolality and osmolar discrepancy in burned patients.

Forty-three patients with burns of various depth and burn surface area were studied to determine the relationship between burn severity and changes of serum osmolality. The results showed that the osmolality in four patients who died were significantly higher than the osmolalities of survivors with very serious burns, whose osmolalities decreased gradually to normal level. Hyperosmolality and obvious osmolar discrepancy often co-exist. It is suggested that the changes of osmolality and osmolar discrepancy can reflect the burn severity and prognosis of the patients.

Adolescent↗
PubMed1992

Cloning, sequencing, and expression of the Zymomonas mobilis fructokinase gene and structural comparison of the enzyme with other hexose kinases.

The frk gene encoding the enzyme fructokinase (fructose 6-phosphotransferase [EC 2.7.1.4]) from Zymomonas mobilis has been isolated on a partial TaqI digest fragment of the genome and sequenced. An open reading frame of 906 bp corresponding to 302 amino acids was identified on a 3-kbp TaqI fragment. The deduced amino acid sequence corresponds to the first 20 amino acids (including an N-terminal methionine) determined by amino acid sequencing of the purified protein. The 118 bp preceding the methionine codon on this fragment does not appear to contain a promoter sequence. There was weak expression of the active enzyme in the recombinant Escherichia coli clone under control of the lac promoter on the pUC plasmid. Comparison of the amino acid sequence with that of the glucokinase enzyme (EC 2.7.1.2) from Z. mobilis reveals relatively little homology, despite the fact that fructokinase also binds glucose and has kinetic and structural properties similar to those of glucokinase. Also, there is little homology with hexose kinases that have been sequenced from other organisms. Northern (RNA) blot analysis showed that the frk transcript is 1.2 kb long. Fructokinase activity is elevated up to twofold when Z. mobilis was grown on fructose instead of glucose, and there was a parallel increase in frk mRNA levels. Differential mRNA stability was not a factor, since the half-lives of the frk transcript were 6.2 min for glucose-grown cells and 6.6 min for fructose-grown cells.

Amino Acid Sequence↗