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Biomedical subjects

C Su

Publications and source records attributed to C Su.

At least 37 records · Page 2Linked to original sources
PubMed2000

[CDKN2/p16 gene cloning and its probe preparation and application in lung cancer].

OBJECTIVES: To clone CDKN2/p16(INK4a) gene, prepare its probe, and to study the change of CDKN2/p16(INK4a) gene in lung cancers. METHODS: Total RNA of normal lung tissue was extracted, CDKN2/p16(INK4a) gene cDNA synthesized, and CDKN2/p16(INK4a) gene recombinant vector, constructed. Southern blot was used to study CDKN2/p16(INK4a) gene in 46 cases of lung cancers, 3 cases of normal lung tissues, 6 cases of lung tissues near cancer, and 3 cases of lymph nodes with lung cancer metastasis. RESULTS: Cloned CDKN2/p16(INK4a) cDNA was proved by enzyme digestion and sequencing. Southern blot showed 4.3 kb band in normal lung tissues and lung tissues near cancers, and deletion of CDKN2/p16(INK4a) gene in cancer tissues and lymph nodes with lung cancer metastasis, with a deletion rate of 17.4% (8/46). CONCLUSION: CDKN2/p16(INK4a) gene may play a role to some extent in progression of lung cancers.

Blotting, Southern↗
PubMed2000

Relations of tumor suppressor gene p16 and RB to early diagnosis of lung cancer.

OBJECTIVES: To study the incidence and mechanism of the tumor suppressor gene p16 and RB inactivation in lung cancer, and to investigate their relations to biological characteristics, clinical pathological diagnosis and gene classification. METHODS: Immunohistochemistry, double in situ hybridization, PCR, PCR-SSCP, and sequencing were used to study the changes in the p16 and RB genes in 106 patients with lung cancer. The specimen of normal lung tissue from the patients with lung cancer and specimens of the 23 patients with benign lung diseases were studied in contrast to those of the same patients. RESULTS: The total expression rate of the p16 and RB genes in the lung cancer tissues of the 106 patients was lower than that in normal lung tissues and tissues of benign lung diseases and was specially related to histological type, metastasis, and clinical stage of lung cancer. In stage I and II lung cancer, the obvious inactivation rate of the tumor suppressor gene p16 or RB was 32.6% or 28.3%. The p16 inactivation appeared largely in non-small cell lung cancer (50.1%), and the RB inactivation largely in small cell lung cancer (88.2%). Homozygous deletion, methylation, and mutation were three mechanisms for the p16 gene inactivation. The deletion rate of the p16 exon 1 and/or exon 2 was 25.8% in the patients with non-small cell lung cancer. Fifteen patients (16.9%) showed methylation at the SmaI sites of CpG island of the p16 gene. PCR-SSCP and sequencing revealed p16 gene mutation in 9 of the 106 patients with lung cancer. CONCLUSIONS: The p16 and RB genes may play an important role in genesis and progression of lung cancer. The inactivation of the p16 or RB gene may be an early sign of lung canceration, that is important for the early diagnosis. A new gene classification model for lung cancer diagnosis would be set up based on the research of the p16 and RB genes.

DNA Methylation↗
PubMed1999

Cloning of linoleate diol synthase reveals homology with prostaglandin H synthases.

Linoleate diol synthase is a homotetrameric ferric hemeprotein, which catalyzes dioxygenation of linoleic acid to (8R)-hydroperoxylinoleate and isomerization of the hydroperoxide to (7S,8S)-dihydroxylinoleate. Ferryl intermediates and a tyrosyl radical are formed in the reaction. Linoleate diol synthase was digested with endoproteinase Lys-C, and internal peptides were sequenced. The sequence information was used for reverse transcription-polymerase chain reaction analysis, and a cDNA probe was obtained. Northern blot analysis of linoleate diol synthase suggested a 3.7-kilobase pair (kb) mRNA. A full-length clone of the linoleate diol synthase gene was obtained by screening of a genomic lambda-ZAP II library of the fungus Gaeumannomyces graminis. The 5'-untranslated region contained CAAT- and TATA-like boxes. The gene contained three short introns and spanned over 3.2-kb. The deduced open reading frame consisted of 2.9-kb, which corresponded to 978 amino acids and a molecular subunit mass of 108,000. Data base analysis with the gapped BLAST algorithm showed that 391 residues of linoleate diol synthase was 23-24% identical and 36-37% positive with the catalytic domain of mammalian prostaglandin H (PGH) synthase-2. Based on homology with PGH synthases, the proximal heme ligand of linoleate diol synthase was tentatively identified as His-379 and the important tyrosine for catalysis as residue 376 (apparent consensus EFNXXXYXWH). The distal heme ligand was tentatively identified as His-203 (apparent consensus THXXFXT). We conclude from catalytic and structural similarities that linoleate diol synthase and PGH synthases likely share common ancestry and may belong to a gene family of fatty acid heme dioxygenases.

Amino Acid Sequence↗
PubMed1999

Determination of ginsenoside Rg3 in plasma by solid-phase extraction and high-performance liquid chromatography for pharmacokinetic study.

A method using high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) is described for the determination of ginsenoside Rg3 in human plasma. A 2.5-ml volume of plasma was mixed with 2.5 ml 60% methanol aqueous solution, and centrifuged at 1100 g for 10 min, the supernatant fluid was further purified by SPE with 200 mg/5 ml 40 microns octadecyl silica and separation was obtained using a reversed-phase column under isocratic conditions with ultraviolet absorbance detection. The intra- and inter-day precision, determined as relative standard deviations, were less than 5.0%, and method recovery was more than 97%. The lower limit of quantitation, based on standards with acceptable RSDs, was 2.5 ng/ml. No endogenous compounds were found to interfere with analyte. A good linear relationship with a regression coefficient of 0.9999 in the range of 2.5 to 200 ng/ml was observed. This method has been demonstrated to be suitable for pharmacokinetic studies in humans. Method development for determination of drug with low UV absorption by SPE and HPLC is also discussed.

Calibration↗
PubMed1999

Fifty-million-year-old polymorphism at an immunoglobulin variable region gene locus in the rabbit evolutionary lineage.

The Ig heavy chain variable region (V(H)) genes encode the antigen-binding regions of antibodies. The rabbit genome contains more than 100 V(H) genes, but only one (V(H)1) is preferentially used in the VDJ gene rearrangement. Three highly divergent alleles occur at this V(H)1 locus in most rabbit populations. These three V(H) alleles are also present in snowshoe hare populations, indicating that the polymorphism of the V(H)1 alleles is trans-specific. Here we report the results of a phylogenetic analysis of rabbit Ig germ-line V(H) genes (alleles) together with V(H) genes from humans and mice. We have found that all rabbit V(H) genes belong to one mammalian V(H) group (group C), which also includes various human and mouse V(H) genes. Using the rate of nucleotide substitution obtained from human and mouse V(H) sequences, we have estimated that the V(H)1 polymorphism in the rabbit lineage has been maintained for about 50 million years. This extremely long persistence of V(H)1 polymorphism is apparently caused by overdominant selection, though the real mechanism is unclear.

Animals↗
PubMed1999

Diversity and evolution of T-cell receptor variable region genes in mammals and birds.

The receptor of a T lymphocyte (TCR) recognizes nonself antigens in the company of major histocompatibility complex (MHC) molecules presented to it by the antigen-presenting cell. The variable region of TCR is encoded by either a concatenation of variable region (TCR-V), diversity region (TCR-D), and joining region (TCR-J) genes, or a concatenation of TCR-V and TCR-J genes. The TCR-V genes exist as a multigene family in vertebrate species. Here we study the evolutionary relationships of TCR-V genes from humans, sheep, cattle, rabbits, mice, and chicken. These six species can be classified into two groups according to the frequency of gamma(delta) T-cells in their peripheral T-cell populations. The "gamma(delta) low" group of species includes humans and mice, in which gamma(delta)T-cells constitute very limited portion of the T-cell population. The "gamma(delta) high" group includes sheep, cattle, rabbits, and chicken, in which gamma(delta) T-cells comprise up to 60% of the T-cell population. Here, we compiled TCR-V sequences from the six species and conducted a phylogenetic analysis. We identified various TCR-V gene subgroups based on the analysis. We found that humans and mice have representatives from nearly all of the subgroups identified, while other species have lost subgroups to different extent. Therefore, the gamma(delta) low species have a high degree of diversity of TCR-V genes, while gamma(delta) high species all have limited diversity of TCR-V genes. This pattern is similar to that found for immunoglobulin variable region (IGV) genes.

Animals↗
PubMed1999

Coagulase gene polymorphism of Staphylococcus aureus isolates from dairy cattle in different geographical areas.

The objectives of this study were to investigate the coagulase gene polymorphism of Staphylococcus aureus isolates obtained from bovine mastitic milk and to determine the resistance of predominant and rare coagulase genotypes to bovine blood neutrophil bactericidal activities. A total of 453 isolates were collected from four countries: the Czech Republic, France, Korea and the United States. The isolates were subtyped into 40 types by restriction fragment length polymorphism (RFLP) of the coagulase gene. Twenty-three strains from predominant and rare genotypes were evaluated for their ability to resist neutrophil bactericidal activities. There were significant (P < 0.01) differences in the average percent neutrophil killing of the predominant (16.7%) and rare (39.7%) genotypes when bacteria were opsonized with antiserum. The results indicate that the profiles of coagulase genotype differ among geographic locations, and only a few genotypes prevail in each location. In addition, the predominant genotypes were more resistant to neutrophil bactericidal activities than rare genotypes.

Animals↗
PubMed1999

A random coefficient degradation model with random sample size.

In testing product reliability, there is often a critical cutoff level that determines whether a specimen is classified as "failed." One consequence is that the number of degradation data collected varies from specimen to specimen. The information of random sample size should be included in the model, and our study shows that it can be influential in estimating model parameters. Two-stage least squares (LS) and maximum modified likelihood (MML) estimation, which both assume fixed sample sizes, are commonly used for estimating parameters in the repeated measurements models typically applied to degradation data. However, the LS estimate is not consistent in the case of random sample sizes. This article derives the likelihood for the random sample size model and suggests using maximum likelihood (ML) for parameter estimation. Our simulation studies show that ML estimates have smaller biases and variances compared to the LS and MML estimates. All estimation methods can be greatly improved if the number of specimens increases from 5 to 10. A data set from a semiconductor application is used to illustrate our methods.

Computer Simulation↗
PubMed1999

The proinflammatory cytokine, interleukin-1alpha, reduces glucocorticoid receptor translocation and function.

Proinflammatory cytokines have been shown to influence the expression and function of the glucocorticoid receptor (GR). Specifically, several studies have found that cytokines induce a decrease in GR function, as evidenced by reduced sensitivity to glucocorticoid effects on functional end points. To investigate the potential mechanism(s) involved, we examined the impact of the proinflammatory cytokine, interleukin-1alpha (IL-1alpha), on 1) GR translocation from cytoplasm to nucleus using GR immunostaining, 2) cytosolic radioligand GR binding, and 3) GR-mediated gene transcription in L929 cells stably transfected with the mouse mammary tumor virus-cholamphenicol acetyltransferase reporter gene. L929 cells were treated with IL-1alpha (100 and 1000 U/ml) for 24 h in the presence or absence of dexamethasone (Dex; 10 nM to 1 microM). IL-1alpha inhibited Dex-induced GR translocation and alone induced GR up-regulation. Pretreatment with IL-1alpha followed by Dex treatment for 1.5 h led to about 20% inhibition of Dex-induced GR-mediated gene transcription, whereas coincubation of IL-1alpha plus Dex for 24 h inhibited Dex-induced GR-mediated gene activity up to 42%. The latter effect was reversed by the IL-1 receptor antagonist. These results suggest that cytokines produced during an inflammatory response may induce GR resistance in relevant cell types by direct effects on the GR, thereby providing an additional pathway by which the immune system can influence the hypothalamic-pituitary-adrenal axis.

Animals↗
PubMed1999

Sensitization to the cytotoxicity of adriamycin by verapamil and heat in multidrug-resistant Chinese hamster ovary cells.

Development of multidrug resistance to anticancer agents is a major limitation for the success of cancer chemotherapy. The chemosensitizer verapamil increases intracellular accumulation of drugs such as adriamycin in certain multidrug-resistant cell lines. When combined with verapamil, hyperthermia should be able to alter membrane permeability to adriamycin and to enhance the cytotoxicity of the drug. Verapamil increased the cytotoxicity of adriamycin in multidrug-resistant Chinese hamster ovary cells (CH(R)C5) but not in drug-sensitive cells (AuxB1). Hyperthermia (42 degrees C) alone clearly increased the cytotoxicity of adriamycin in AuxB1 cells. There was also a small increase in CH(R)C5 cells at 42 and 43 degrees C. In drug-resistant cells, the cytotoxicity of adriamycin increased considerably when verapamil was combined with heat. This effect was dependent on temperature and increased with time of incubation. At 37 degrees C, verapamil increased the uptake of adriamycin in CH(R)C5 cells, while drug efflux decreased. When verapamil was combined with hyperthermia, drug efflux decreased even further. These results led to an overall increase in intracellular accumulation of the drug. In drug-sensitive cells, hyperthermia increased both the uptake and efflux of adriamycin, but verapamil had no effect. Verapamil plus heat increased the cytotoxicity of adriamycin in drug-resistant cells, and this was accompanied by altered permeability of the membrane to the drug. Hyperthermia combined with verapamil could be beneficial by increasing the effectiveness of adriamycin in the elimination of multidrug-resistant cells in a localized target region.

Animals↗
PubMed1999

[Homozygous deletion of CDKN2/p16 gene in lung cancers].

OBJECTIVE: To study the relationship between CDKN2/p16 gene homozygous deletion and lung cancer progression. METHODS: Improved multiplex PCR technique was applied to detect deletion of CDKN2/p16 gene exon1 and exon2 in 89 cases of lung cancers. RESULTS: Gene deletion rate was increased by the improved PCR technique. Exon 1 and exon 2 deletion rates were 19.1% (17/89) and 22.5% (20/89) respectively, with total rate of exon1 and/or exon2 deletion 25.8% (23/89). Deletion of CDKN2/p16 gene occurred in non-small cell lung carcinoma (NSCLC) and was related to metastasis and progressive stage. CONCLUSION: Abnormality of CDKN2/p16 gene is a genetic factor for NSCLC susceptibility, and may play a role to some extent in NSCLC malignant progression.

Aged↗
PubMed1999

[Experimental study of interleukin-4 gene therapy for glioma].

OBJECTIVE: To study IL-4 gene therapy for glioma. METHODS: Using recombinant retrovirus vector, mIL-4 was transfected into psi 2 packaging cells and rat C6 glioma cells, respectively by lipofectamine. The IL-4 secretting cell clones psi 2 IL-4 and C6IL-4 were studied in vitro and in vivo. RESULTS: Apart from a decrease in cloning efficiency, no difference was observed between the transfected and the wild type glioma cells in proliferation and cell cycle in vitro. Tumorigenicity of subcutaneously and intracranially inoculated C6IL-4 was reduced. Tumor growth could be suppressed by perineoplastic inoculation of psi 2 IL-4 cells. CONCLUSION: IL-4 gene-modified C6 glioma has decreased tumorigenicity. Local production of IL-4 by psi 2 IL-4 cells can lead to regression of established glioma.

Animals↗
PubMed1998

A protein radical and ferryl intermediates are generated by linoleate diol synthase, a ferric hemeprotein with dioxygenase and hydroperoxide isomerase activities.

Linoleate diol synthase (LDS) was isolated as a hemeprotein from the fungus Gaeumannomyces graminis. LDS converts linoleate sequentially to 8R-hydroperoxylinoleate (8-HPODE) through an 8-dioxygenase by insertion of molecular oxygen and to 7S,8S-dihydroxylinoleate through a hydroperoxide isomerase by intramolecular oxygen transfer. Light absorption and EPR spectra of LDS indicated that the heme iron was ferric and mainly high spin. Oxygen consumption during catalysis started after a short time lag which was reduced by 8-HPODE. Catalysis declined due to suicide inactivation. Stopped flow studies with LDS and 8-HPODE at 13 degreesC showed a rapid decrease in light absorption at 406 nm within 35 ms with a first order rate constant of 90-120 s-1. Light absorption at 406 nm then increased at a rate of approximately 4 s-1, whereas the absorption at 421 nm increased after a lag time of approximately 5 ms at a rate of approximately 70 s-1. EPR spectra at 77 K of LDS both with linoleic acid and 8-HPODE showed a transient doublet when quenched after incubation on ice for 3 s (major hyperfine splitting 2.3 millitesla; g = 2.005), indicating a protein radical. The relaxation properties of the protein radical suggested interaction with a metal center. 8-HPODE generated about twice as much radical as linoleic acid, and the 8-HPODE-induced radical appeared to be stable. Our results suggest that LDS may form, in analogy with prostaglandin H synthases, ferryl intermediates and a protein radical during catalysis.

Electron Spin Resonance Spectroscopy↗
PubMed1998

Retinoblastoma, a tumor suppressor, is a coactivator for the androgen receptor in human prostate cancer DU145 cells.

The retinoblastoma protein may function as a tumor suppressor by controlling the progression of the normal cell cycle. Inactivation of Rb has been regarded as an important event in prostate carcinogenesis. However, the detailed mechanism of how Rb is linked to androgen-androgen receptor (A-AR), the major factor in promotion of prostate tumor growth, remains unclear. Using GST-Rb pull down assay and mammalian two-hybrid system, we report here that Rb can bind specifically to AR in an androgen-independent manner. Transient transfection assay demonstrates that cotransfection of AR and Rb can further induce AR transcriptional activity 4-fold in the presence of 1 nM dihydrotestosterone in DU145 cells. Interestingly, cotransfection of Rb and ARA70, the first identified AR coactivator, with AR can additively induce AR transcriptional activity 13-fold (from 5-fold to 64-fold). In conclusion, our discovery that Rb can function as a coactivator to induce AR transcriptional activity in prostate cells may represent the first data to link a negative growth regulatory protein function in a positive manner, by inducing the transcriptional activity of AR.

Cell Cycle↗
PubMed1998

Manganese lipoxygenase. Purification and characterization.

A linoleic acid (13R)-lipoxygenase was purified to homogeneity from the culture medium of Gäumannomyces graminis, the take-all fungus, by hydrophobic interaction, cation exchange, lectin affinity, and size-exclusion chromatography. The purified dioxygenase lacked light absorption between 300 and 700 nm. Gel filtration indicated an apparent molecular mass of approximately 135 kDa in 6 M urea and approximately 160 kDa in buffer. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was heterogeneous in size and consisted of diffuse protein bands of 100-140 kDa. Treatment with glycosidases for N- and O-linked oligosaccharides yielded a distinct protein of approximately 73 kDa on SDS-PAGE. Atomic emission spectroscopy indicated 0.5-1.0 manganese atom/enzyme molecule. The isoelectric point was approximately 9.7, and the enzyme was active between pH 5 and 11 with optimum activity at pH 7. 0. For molecular oxygen, Km was 30 microM and Vmax 10 micromol mg-1min-1; for linoleic acid, Km was 4.4 micromol, Vmax 8.2 micromol mg-1min-1, and the turnover number 1100 min-1. The enzyme oxidized linolenic acid twice as fast as linoleic acid. The main products were identified by mass spectrometry as 13-hydroperoxy-(9Z,11E, 15Z)-octadecatrienoic and 13-hydroperoxy-(9Z,11E)-octadecadienoic acids, respectively. After reduction of the hydroperoxide, steric analysis of methyl 13-hydroxyoctadecadienoate by chiral high performance liquid chromatography yielded one enantiomer (>95%), which co-eluted with the R-stereoisomer of methyl (13R, 13S)-hydroxyoctadecadienoate. Arachidonic and dihomogammalinolenic acids were not substrates, while oxygen consumption, UV analysis, and mass spectrometric analysis indicated that gamma-linolenic acid was oxygenated both at C-11 and C-13. The enzyme was active at 60 degreesC and after treatment with 6 M urea. It was strongly inhibited by 10-50 microM concentrations of eicosatetraynoic acid and a lipoxygenase inhibitor (N-(3-phenoxycinnamyl)acetohydroxamic acid), but many other lipoxygenase inhibitors (100 microM) were without effect. We conclude that, after deglycosylation, the enzyme has the same size on SDS-PAGE as mammalian and marine lipoxygenases, but it differs from all previously described lipoxygenases in three ways. It is secreted, it forms (13R)-hydroperoxy-(9Z, 11E)-octadecadienoic acid, and it contains manganese.

Ascomycota↗
PubMed1998

Manganese lipoxygenase. Discovery of a bis-allylic hydroperoxide as product and intermediate in a lipoxygenase reaction.

Linoleic acid was incubated with manganese lipoxygenase (Mn-LO) from the fungus Gäumannomyces graminis. The product consisted of (13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid ((13R)-HPOD) and a new hydroperoxide, (11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD). Incubation of (11R)-[2H]- and (11S)-[2H]linoleic acids with Mn-LO led to the formation of hydroperoxides that largely retained and lost, respectively, the deuterium label. Conversion of the (11S)-deuteriolinoleic acid was accompanied by a primary isotope effect, which manifested itself in a strongly reduced rate of formation of hydroperoxides and in a time-dependent accumulation of deuterium in the unconverted substrate. These experiments indicated that the initial step catalyzed by Mn-LO consisted of abstraction of the pro-S hydrogen of linoleic acid to produce a linoleoyl radical. (11S)-HPOD was converted into (13R)-HPOD upon incubation with Mn-LO. The mechanism of this enzyme-catalyzed hydroperoxide rearrangement was studied in experiments carried out with 18O2 gas or 18O2-labeled hydroperoxides. Incubation of [11-18O2](11S)-HPOD with Mn-LO led to the formation of (13R)-HPOD, which retained 39-44% of the 18O label, whereas (11S)-HPOD incubated with Mn-LO under 18O2 produced (13R)-HPOD, which had incorporated 57% of 18O. Furthermore, analysis of the isotope content of (11S)-HPOD remaining unconverted in such incubations demonstrated that [11-18O2](11S)-HPOD suffered a time-dependent loss of 18O when exposed to Mn-LO, whereas (11S)-HPOD incorporated 18O when incubated with Mn-LO under 18O2. On the basis of these experiments, it was proposed that the conversion of (11S)-HPOD into (13R)-HPOD occurred in a non-concerted way by deoxygenation into a linoleoyl radical. Subsequent reoxygenation of this intermediate by dioxygen attack at C-13 produced (13R)-HPOD, whereas attack at C-11 regenerated (11S)-HPOD. The hydroperoxide rearrangement occurred by oxygen rebound, although, as demonstrated by the 18O experiments, the oxygen molecule released from (11S)-HPOD exchanged with surrounding molecular oxygen prior to its reincorporation.

Ascomycota↗
PubMed1998

A simple method to enrich mRNA from total prokaryotic RNA.

Isolation of prokaryotic mRNA by the poly(dT) method has been difficult, primarily due to the great instability of the poly(A) sequence in its mRNA. We developed a simple method to remove rRNA from total RNA of Staphylococcus aureus by cloning a PCR-amplified S. aureus rRNA gene fragment into a plasmid, and then synthesizing biotin-labeled antisense rRNA to subtract rRNA. By using this method, S. aureus rRNA is significantly reduced and mRNA is enriched. This method may be used to prepare prokaryotic mRNA for many molecular biology applications.

Cloning, Molecular↗