Biomedical subjects

Lin Hou

Publications and source records attributed to Lin Hou.

28 records · Page 2Linked to original sources

PubMed2003

Molecular cloning of a novel human gene encoding histone acetyltransferase-like protein involved in transcriptional activation of hTERT.

To isolate proteins involved in hTERT transcriptional regulation, the HeLa cDNA library was screened using the hTERT promoter-based yeast one-hybrid assay. A positive clone was rescued and proved to contain an open reading frame and the upstream coding sequences were obtained by 5'-RACE. The assembled full cDNA consisted of a 2.5 kb reading frame encoding 834 amino acids, in which a conserved N-acetyltransferase domain (GNAT family) was searched out in bioinformatics, and thus named as hALP (human N-acetyltransferase-like protein, GenBank Accession No. AF 489535). The expression of native hALP was identified in HeLa cells and proved to distribute in the cellular nucleus. The binding potential of hALP to hTERT promoter was confirmed by EMSA and the interacting sequence involved to -201- to -56-nt upstream region of the promoter. On transfection assay, hALP could obviously transactivate hTERT promoter and stimulate endogenous telomerase activity of cells. The analysis on histone acetyltransferase showed that hALP could specifically acetylate free histones in vitro. The investigation suggested that hALP influences the activity of histone acetylation and could up-regulate telomerase activity through transactivation of hTERT promoter.

Amino Acid Sequence↗

PubMed2003

[Studies on the molecular mechanism of GM(2) gangliosidosis].

OBJECTIVE: To study the molecular mechanism of GM(2) gangliosidosis. METHODS: The skin fibroblasts from 4 patients with GM(2) gangliosidosis were subjected to culture. Enzyme activities assay, Western blot and immunocytochemical analysis were performed using the cultured fibroblasts. RESULTS: The hexosaminidase (Hex) activities of 4 patients with GM(2) gangliosidosis were significantly decreased. The activities were 12% 3% 15% and 6% of control values, respectively. Western blot analysis indicated that the amount of Hex mature alpha- and beta- subunits (alpha m, beta m) was decreased in cells from patients 2 and 3, but only decreased alpha m was found in patient 1 and both alpha m and beta m were normal in cells from patient 4. Immunocytochemical analysis revealed the accumulated GM(2) ganglioside in cells from patients 1-4. CONCLUSION: The pathogenesis of GM(2) gangliosidosis was associated with deficiency of Hex alpha m and beta m and GM(2) activator caused by HEXA, HEXB and GM(2)A gene mutations.

Adult↗

PubMed2003

[Expression of telomerase gene hTERT in testes of infertile male and its significance].

OBJECTIVES: To study the expression and the significance of telomerase gene hTERT in testes of infertile male. METHODS: By using in situ hybridization(ISH) techniques, the expression of telomerase gene hTERT mRNA in testes of 47 infertile male and 10 normal testicular tissues were observed. RESULTS: In male testes, there was a positive correlation between the expression of hTERT and the quantity and density of germ cells(spermatogonia, spermatocyte, spermatid). The expression of hTERT in some germinal cell of maturation arrest patients were not significantly different with those of normal. CONCLUSIONS: Our results suggest that the deficiency of telomerase might be a factor for germinal cell maturation arrest and there might be some other etiological factors in these patients. Our study provides experimental groundwork for the gene therapy of male infertility.

Humans↗

PubMed2003

[Characterization of telomerase regulation association gene TRAP and cancer biology].

OBJECTIVE: To investigate expression of TRAP in human tumors and correlativity between TRAP and hTERT and to explore the role of TRAP in tumorigenesis. METHODS: TRAP was expressed in E. coli. and polyclonal antibody was prepared by immunization of New Zealand rabbits. Expressions of TRAP gene and hTERT in human tumors were analyzed by immunohistochemistry. The eukaryotic expression vectors of TRAP were constructed and transferred stably into HeLa cells to be analysed with Northern blot, TRAP-PCR ELISA, growth curve, nude mouse transplantation. RESULTS: Polyclonal antibody was obtained from New Zealand rabbits immunized with TRAP fuse protein, which was inducibly expressed in E. coli., and its specificity was verified in Western blot. TRAP staining was detected in 213 (86.2%) of 247 malignant tumors. On the contrary, TRAP in all tissues adjacent to tumors was negative. There is a obvious difference between borderline, pre-cancerous and benign lesion in the expression of TRAP. Furthermore, hTERT staining was conducted and its result was consistent with that of TRAP. Up-regulation and down-regulation of hTERT expression and telomerase activity were observed respectively in HeLa cells transfected stably with sense and antisence TRAP recombination. Increased proliferation presented in the former and growth inhibition in the latter. Besides, in nude mice, tumorigenecity of sense TRAP-transfected HeLa cells was enhanced while tumor growth of antisense TRAP-transfected HeLa cells was retarded. CONCLUSION: The expression of TRAP, which could be detected in most of human cancers and part of pre-cancerous lesions, was correlated with expression of hTERT. The status of TRAP expression could regulate telomerase activity and present effects on growth and tumorigeneity of cancer cells. This suggests that TRAP is involved in human tumorigenesis through regulation of telermarase activity.

Acid Phosphatase↗

PubMed2003

[Preclinical research of a new therapy for Gaucher's disease with F213I mutation].

OBJECTIVE: To design and make trial of a new therapy for Gaucher disease. METHODS: A substrate analogue of beta-Glc (glucocerebroside analogue, GCA) was used as a molecular chaperon. Normal and mutant skin fibroblasts were cultured with or without GCA. The activity of beta-Glc was assayed by fluorescent enzymologic techniques. The amount of beta-Glc was determined using Western blot. The beta -Glc was localized by double cell stain experiment. The degradation of glucocerebroside was assessed by thin layer chromatography (TLC) experiment using 14C-Serine. RESULTS: It was found that GCA could enhance the activity and amount of beta-Glc with F213I mutation. It also promoted the beta-Glc with F213I mutation to the lysosome and accelerated the degradation of glucocerebroside. CONCLUSION: The low molecular compound analogous to beta-Glc substrate (GCA ) may be a new therapeutic strategy for Gaucher's disease with F213I mutation.

Amino Acid Substitution↗

PubMed2003

[Expression of telomerase and p53 in ameloblastoma].

OBJECTIVE: To detect the expression of p53 protein, hTR, and hTERT mRNA in ameloblastoma (AB) and to investigate the clinical biological characteristics of AB. METHODS: We detected hTR and hTERT Mrna in 54 cases of AB by in situ hybridization, and p53 protein by S-P method. RESULTS: The positive ratio of hTR, hTERT mRNA was 2/7 and 1/7 in normal oral mucosa, 81.2%(13/16) and 87.4% (14/16) in odontogenic keratocyst (OKC), 94.4%(51/54) in AB, respectively. There was a significant difference in these three groups (P < 0.001). A strong correlation between hTR and hTERT mRNA was found (rs = 1.000, P < 0.001). p53 protein was expressed in 87.5% (42/49) AB, 44%(11/25) OKC, 33.3%(1/3) normal oral mucosa, respectively. A strong correlation between hTERT mRNA and p53 protein was found (rs = 0.848, P < 0.001). A moderate correlation between hTR mRNA and p53 protein was found (rs = 0.536, P < 0.001). CONCLUSION: The positive ratios of hTR and hTERT were higher than p53 protein in AB. Telomerase activity and p53 expression play an important role in genesis and development of AB.

Adolescent↗

PubMed2002

Development of a monoclonal antibody for neutralization of human telomerase activity.

To develop monoclonal antibodies (MAbs) to neutralize human telomerase, an epitope (hTERT(7)) in reverse transcriptase domain of hTERT was synthesized and was used to immunize BALB/c mice. Hybridomas were generated and screened by enzyme-linked immunoadsorbent assay (ELISA) for specific MAbs. One hybridoma M2 clone, isotyped IgG(1), was established. The competitive assay confirmed that the M2 antibody was hTERT(7) specific, and the affinity constant was about 1 x 10(6) M(-1). M2 could recognize cell extracts from HeLa cancer cells but not those of normal 2BS cells in ELISA assay. For in situ staining immunohistochemically, the positive staining presented in the nuclear compartment of HeLa, while 2BS was nonreactive. In TRAP-PCR ELISA, M2 markedly decreased the activities of human telomerase in HeLa cells. The sequencing of M2 heavy chain variable region proved its mouse origin. The results demonstrated that the developed mouse MAb should be hTERT specific and could not only recognize native cellular hTERT in ELISA and immunohistochemistry, but also neutralize telomerase activities. Thus, it could be hoped that the antibody could be used in clinical diagnosis and treatment of cancers.

Amino Acid Sequence↗

PubMed2002

[Monoclonal antibodies against human telomerase reverse transcriptase: preparation, characterization, and application].

OBJECTIVE: To develop monoclonal antibodies against the catalytic subunit of human telomerase hTERT for its expression detection of human tumors. METHODS: A dominant epitope in hTERT (peptide hTERT(9))was automatically synthesized based on Fmoc method, and was used to immunize BALB/c mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization of which were performed by Western blotting and immunohistochemical staining. RESULTS: Antigenic peptide hTERT(9) was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis. Three hybridoma cell lines secreting anti-hTERT(9) antibodies designated as H4, G8 and A11 were established after primary screening and consequent three rounds of limited dilution. Both of H4 and G8 were IgM, while A11 was IgG1 in isotyping. The competitive assay showed that the antibodies were hTERT(9) specific, and the affinity of G8 was stronger than that of H4 and A11 assayed by affinity ranking. However, in Western blotting, both of H4 and G8 stained an about 123 000 protein band with HeLa and 293 cell extracts but not with normal 2BS cells. Besides, positive staining presented in the nucleus of HeLa, while 2BS was non-reactive immunohistochemically. The sections from paraffin-embedded blocks of 127 cases of human cancer, 40 of precancerous and 19 of benign tumors were in situ stained by G8 antibody, the results showed that the human cancer tissues were 80.31% (102/127) positive in specific nuclear reaction, on the contrary, only a minority of precancerous lesions present weak positive (17.5%, 7/40), and negative in benign tumors (0/19). CONCLUSIONS: The monoclonal antibodies developed against synthetic peptide were hTERT-specific and could recognize both the native and the denatured form. Thus their use in immunoblotting or immunohistochemistry for detecting the telomerase hTERT expression of cancer cell and tissues was promising.

Animals↗

PubMed2002

Single domain antibody to human telomerase catalytic subunit: preparation and characterization.

OBJECTIVE: To develop a recombinant single domain antibody against hTERT, human telomerase catalytic subunit. METHODS: A previously prepared His-tagged hTERT fusion protein was used as the antigen, and the variable regions in heavy chain (VH) of immunized mice were RT-PCR amplified and cloned into the pCANTAB 5E, a phagemid vector. By transfection, the display library of mouse VH was developed. The candidate clones were selected by affinity panning, and soluble VH were obtained after expression in E. coli, HB2151. The resultant single VH antibodies were characterized on their binding potentials by western blotting. RESULTS: An about 350 bp VH fragment was amplified from spleen cells of mice immunized by His-tagged hTERT and expressed by phage displayed as VH library. The size of the library was 8 x 10(4). After three rounds of affinity panning, 4 independent clones were chosen and consequently expressed as soluble single domain antibodies (Mr = 16 000). In Western blot analysis, the single domain antibody from 2 of 4 clones proved to react with the His-tagged hTERT fusion protein (Mr = 167 000) without dependence of His-tags and also detect the native hTERT (Mr = 127 000) extracted from the human HeLa cancer cell line. DNA sequencing showed both of the single domain antibodies were encoded by the heavy chain variable region of the mouse. CONCLUSIONS: The single domain antibodies developed were hTERT recognizable and hTERT specific, thus providing a basis for application of recombinant single domain antibody in inhibition of telomerase activity and anticancer therapy.

Amino Acid Sequence↗

PubMed1998

Protection of Mitochondria from Free Hydroxyls by Zn(7)- and Cd(7)- Metallothioneins.

Zn(7)- and Cd(7)-metallothioneins were prepared. Mitochondria were isolated from rat hearts. Mitochondria lipid fluidity, protein conformation and mobility were determined by using ESR spin label. Ca(2+)-Mg(2+)-ATPase activity and (45)Ca uptake activity were assayed. It was found that, after being damaged by hydroxyl radicals, mitochondria lipid fluidity and protein mobility decreased, protein conformation changed and mitochondria Ca(2+)-Mg(2+)-ATPase activity, (45)Ca uptake activity were inhibited. Both Zn(7)- and Cd(7)-metallothioneins could antagonize hydroxyl radicals and protect myocardial mitochondria. However, this effect of Zn(7)-metallothionein was stronger than that of Cd(7)-metallothionein, because the apo-metallothionein has about 10 000 fold higher affinity for Cd than for Zn, so, Zn(7)-metallothionein would release more Zn(2+). More reduced thiol groups would be revealed.

Journal Article↗