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Biomedical subjects

Y Tan

Publications and source records attributed to Y Tan.

At least 127 records · Page 7Linked to original sources
PubMed1998

[Detection of mycobacterium tuberculosis by AmpliSensor-PCR technique and its clinical application].

OBJECTIVE: To explore the value of Amplisensor-PCR method in diagnosis of tuberculosis. METHOD: The samples from 784 patients with tuberculosis, 160 patients with lung cancer were detected by using AmpliSensor-PCR, PCR, smear and culture methods, and the results were compared. RESULT: The sensitivity of AmpliSensor-PCR technique was obviously higher than that of smear and culture (P < 0.01). The specificity of AmpliSensor-PCR method was higher than that of PCR method. CONCLUSION: Amplisensor-PCR method can be determined to detect the lower limit by standard curve, through which the original amount of target-DNA in samples can be calculated. AmpliSensor-PCR method shows higher specificity and sensitivity. It is of clinical value in diagnosis of pulmonary tuberculosis and particularly of extrapulmonary tuberculosis.

DNA, Bacterial↗
PubMed1998

[Effects of simulated weightlessness and irradiation on metabolism of rat myocardial cells cultured in vitro].

To investigate the changes of the metabolism of rat myocardiac cells cultured in vitro under simulated weightlessness and gamma-irradiation, rat myocardiac cells were rotated on a horizontal clinostat, and irradiated with 2Gy 60Co gamma-ray. The results showed: (1) after irradiation, the LDH activity and SOD concentrations showed a significant increase, and the MDA concentration also increased. (2) After irradiation, LDH activity increased 60% on the 3rd day, and SOD concentration almost doubled on the first day. (2) MDA concentration also increased but it reduced to normal after the 3rd day. (3) Simple rotation on the clinostat showed no effect on the parameter, but there was a synergistic effect when it was combined with gamma-irradiation.

Animals↗
PubMed1998

Effects of simulated weightlessness and gamma-irradiation on myocardial cells and osteoblasts.

The effects of simulated weightlessness and gamma-irradiation on the structure and function of the myocardial cells and osteoblasts were investigated with a rotating clinostat and 2 Gy 60Co gamma-irradiation. The results showed: (1) Under the simulated weightlessness (SWL), the diameter of the myocardial cells decreased 40%. And the long-short diameter proportion decreased 70%, (P < 0.01); SWL affected the cell skeleton and the distribution of microfilament became ununiform, the orientation was unclear; SWL had significant effects on osteoblast disintegration and proliferation. The disintegration function decreased and the proliferation process was inhibited; (2) 2 Gy 60Co gamma-ray irradiation had significant effects on Lactate Dehydrogenase (LDH) activity and Superoxide Dismutase (SOD), Malonaldehyde (MDA) content in the myocardial cells, (P < 0.05) (5) A synergistic effect was observed between irradiation and SWL.

Animals↗
PubMed1997

In vivo gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector.

Lipoproteins are emulsion particles that consist of lipids and apolipoproteins. Their natural function is to transport lipids and/or cholesterol to different tissues. We have taken advantage of the hydrophobic interior of these natural emulsions to solubilize DNA. Negatively charged DNA was first complexed with cationic lipids containing a quaternary amine head group. The resulting hydrophobic complex was extracted by chloroform and then incorporated into reconstituted chylomicron remnant particles ( approximately 100 nm in diameter) with an efficiency approximately 65%. When injected into the portal vein of mice, there were approximately 5 ng of a transgene product (luciferase) produced per mg of liver protein per 100 microg injected DNA. This level of transgene expression was approximately 100-fold higher than that of mice injected with naked DNA. However, such a high expression was not found after tail vein injection. Histochemical examination revealed that a large number of parenchymal cells and other types of cells in the liver expressed the transgene. Gene expression in the liver increased with increasing injected dose, and was nearly saturated with 50 microg DNA. At this dose, the expression was kept at high level in the liver for 2 days and then gradually reduced and almost disappeared by 7 days. However, by additional injection at day 7, gene expression in the liver was completely restored. By injection of plasmid DNA encoding human alpha1-antitrypsin, significant concentrations of hAAT were detected in the serum of injected animals. This is the first nonviral vector that resembles a natural lipoprotein carrier.

Animals↗
PubMed1997

Governing step of metastasis visualized in vitro.

Metastasis is the ultimate life-threatening stage of cancer. The lack of accurate model systems thwarted studies of the metastatic cell's basic biology. To follow continuously the succeeding stages of metastatic colony growth, we heritably labeled cells from the human lung adenocarcinoma cell line ANIP 973 with green fluorescent protein (GFP) by transfection with GFP cDNA. Labeled cells were then injected intravenously into nude mice, where, by 7 days, they formed brilliantly fluorescing metastatic colonies on mouse lung [Chishima, T., Miyagi, Y., Wang, X., Yang, M., Tan, Y., Shimada, H., Moossa, A. R. & Hoffman, R. M. (1997) Clin. Exp. Metastasis 15, 547-552]. The seeded lung tissue was then excised and incubated in the three-dimensional sponge-gel-matrix-supported histoculture that maintained the critical features of progressive in vivo tumor colonization while allowing continuous access for measurement and manipulation. Tumor progression was continuously visualized by GFP fluorescence in the same individual cultures over a 52-day period, during which the tumors spread throughout the lung. Histoculture tumor colonization was selective for lung cancer cells to grow on lung tissue, because no growth occurred on histocultured mouse liver tissue, which was also observed in vivo. The ability to support selective organ colonization in histoculture and visualize tumor progression by GFP fluorescence allows the in vitro study of the governing processes of metastasis [Kuo, T.-H., Kubota, T., Watanbe, M., Furukawa, T., Teramoto, T., Ishibiki, K., Kitajima, M., Moossa, A. R., Penman, S. & Hoffman, R. M. (1995) Proc. Natl. Acad. Sci. USA 92, 12085-12089]. The results presented here provide significant, new opportunities to understand and to develop treatments that prevent and possibly reverse metastasis.

Adenocarcinoma↗
PubMed1997

Comparison of androgen-independent growth and androgen-dependent growth in BPH and cancer tissue from the same radical prostatectomies in sponge-gel matrix histoculture.

BACKGROUND: In order to determine androgen sensitivities of prostate cancer and benign prostatic hypertrophy (BPH) tissues from the same patient in vitro, we used a histoculture technique to measure androgen-independent and androgen-dependent growth and compared them in paired specimens of BPH and prostate cancer from 23 radical prostatectomies. Both androgen-independent growth and androgen-dependent growth are measures of important biological characteristics of benign and malignant prostate tissue. METHODS: The effect of hydroxyflutamide and antiandrogens on dihydrotestosterone (DHT)-stimulated incorporation of 3H-thymidine into both paired specimens of BPH and cancer was utilized to measure androgen-independent and androgen-dependent growth. The percentage decrease in 3H-thymidine incorporation/microgram protein in the flutamide-treated specimen compared to the DHT-treated specimen represented androgen-dependent growth. Residual 3H-thymidine incorporation/microgram protein during hydroxyflutamide administration represented androgen-independent growth. RESULTS: Androgen-independent growth was significantly greater (P = 0.015) in the BPH compared to the cancer paired tissue. Androgen-dependent growth was significantly higher in 23 paired specimens of cancer compared to BPH (P < 0.03). CONCLUSIONS: In paired specimens of BPH and prostate cancer from the same radical prostatectomy specimen, androgen-independent growth appeared greater in BPH compared to cancer specimens; androgen-dependent growth, however, was greater in prostate cancer than in BPH. There was no correlation of either growth parameter with Gleason tumor grade. Future clinical correlations will indicate whether either growth parameter represents an important prognostic factor for prostate cancer aggressiveness stimulated 3H-thymidine incorporation into DNA.

Androgens↗
PubMed1997

Competitive interactions between cytochromes P450 2A6 and 2E1 for NADPH-cytochrome P450 oxidoreductase in the microsomal membranes produced by a baculovirus expression system.

The present study investigated the interactions between cytochrome P450 (P450) enzymes and the NADPH:cytochrome oxidoreductase (OR) in the microsomal membrane. Microsomes containing human cytochrome P450 2A6 (h2A6) coexpressed with human OR (hOR) via a baculovirus expression system displayed coumarin hydroxylase activity with apparent Km and Vmax values of 0.41 microM and 4.05 nmol/min/nmol P450, respectively. Incorporation of purified rat liver cytochrome b5 (b5) into the microsomes increased the Vmax 2.5-fold, but did not affect the Km. The N-nitrosodimethylamine (NDMA) demethylase activity of human cytochrome P450 2E1 (h2E1) coexpressed similarly was characterized previously. Coumarin was shown not to be a substrate nor an inhibitor of h2E1, and NDMA was not a substrate nor an inhibitor of h2A6. In microsomes containing h2A6, h2E1, and hOR (M-h2A6-h2E1-hOR) obtained from a triple expression system, the two P450 enzymes were shown to compete with each other for interaction with hOR. In incubations with M-h2A6-h2E1-hOR, the presence of a h2A6 substrate (coumarin) decreased NDMA demethylase activity by a maximum of 47%, and the presence of a h2E1 substrate (NDMA) decreased coumarin hydroxylase activity by a maximum of 19%. This substrate-induced competition between h2A6 and h2E1 was decreased by the addition of purified b5. In the absence of a substrate, the NADPH-dependent H2O2 formation was high in both M-h2A6-h2E1-hOR and M-h2E1-hOR, but low in M-h2A6-hOR. The addition of NDMA had little effect on the H2O2 formation in M-h2A6-h2E1-hOR and M-h2E1-hOR. The addition of coumarin, however, slightly decreased H2O2 formation in M-h2A6-h2E1-hOR, but drastically increased H2O2 formation in M-h2A6-hOR. These results suggest that the presence of a h2A6 substrate decreased the electron flow to h2E1 in M-h2A6-h2E1-hOR. The activities of coumarin hydroxylase and NDMA demethylase of M-h2A6-h2E1-hOR were decreased and increased, respectively, by an increase in ionic strength. The ionic strength, however, did not drastically change the substrate-induced competition between h2A6 and h2E1 for hOR. The results demonstrate the usefulness of the coexpression system for mechanistic studies and illustrate that the interaction of monooxygenase enzymes in the microsomal membrane is regulated by the presence of substrates and b5.

Animals↗
PubMed1997

Calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine eye: high calmodulin affinity isozyme immunologically related to the brain 60-kDa isozyme.

Calmodulin-dependent cyclic nucleotide phosphodiesterase was identified in and purified to apparent homogeneity from the total calmodulin-binding protein fraction of bovine eye in a single step by immunoaffinity chromatography. The bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase is immunologically similar to the bovine brain 60-kDa isozyme. The purified enzyme had higher affinity for calmodulin than the 60-kDa phosphodiesterase isozyme from bovine brain, but similar affinity to that of the heart isozyme. When the Ca(2+)-dependence of the eye enzyme was compared to cardiac calmodulin-dependent cyclic nucleotide phosphodiesterase at an identical concentration of calmodulin, the bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase was activated at the same Ca2+ concentration as the bovine heart calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme.

Animals↗
PubMed1997

Overexpression and large-scale production of recombinant L-methionine-alpha-deamino-gamma-mercaptomethane-lyase for novel anticancer therapy.

The goal of the next generation of cancer chemotherapy is effective tumor-selectivity. A tumor-selective target with high therapeutic potential is the elevated methionine requirement of tumor cells relative to normal cells. We have termed the elevated requirement for methionine in tumors methionine dependence. To selectively target the methionine dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine alpha-deamino-gamma-mercaptomethane-lyase (methioninase, METase) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). The METase gene was then ligated into the pT7-7 overexpression plasmid containing the T7 RNA polymerase promoter and recloned in E. coli strain BL21(DE3). The pAC-1 clone was isolated by its yellow-orange color which is due to high enrichment of the pyridoxal phosphate-containing recombinant methioninase (rMETase) and distinguished rMETase-overproducer from rMETase-negative colonies. A scale-up production protocol which contained a heat step, two DEAE Sepharose FF ion-exchange, and one ActiClean Etox endotoxin-affinity chromatography columns has been established. The pAC-1 clone produces rMETase at approximately 10% of the total soluble protein and up to 1 g/liter in shake-flask culture. The protocol can produce therapeutic rMETase at the multi-gram level per batch with high yield (> 60%), high purity (> 98%), high stability, and low endotoxin. Purified rMETase is stable to lyophilization. The t1/2 of rMETase was 2 h when rMETase was administered by i.v. injection in mice. Studies of the antitumor efficacy of rMETase in vitro and in vivo on human tumors xenografted in nude mice demonstrated that all types of human tumors tested including those from lung, colon, kidney, brain, prostate, and melanoma were sensitive to rMETase. In contrast, normal cells were insensitive to rMETase in vitro and correspondingly, no toxicity was detected in vivo at the effective doses. In conclusion, the overexpression clone and large-scale production protocols for rMETase have enabled rMETase to be used as a tumor-selective therapeutic with broad indication and high promise for effective, low-toxicity human cancer therapy.

Amino Acid Sequence↗
PubMed1997

Role of cytochromes P450 in the metabolism of methyl tert-butyl ether in human livers.

Methyl tert-butyl ether (MTBE) is widely used as a gasoline oxygenate for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health is a major public concern. However, information on the metabolism of MTBE in human tissues is lacking. The present study demonstrates that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and a marker for exposure to MTBE. The activity is localized in the microsomal fraction (125 +/- 11 pmol TBA/ min per mg protein, n = 8) but not in the cytosol. This activity level in human liver microsomes is approximately one-half of the value in rat and mouse liver microsomes. Formation of TBA in human liver microsomes is NADPH-dependent, and is significantly inhibited by carbon monoxide (CO), an inhibitor of cytochrome P450 (CYP) enzymes, suggesting that CYP enzymes play a critical role in the metabolism of MTBE in human livers. Both CYP2A6 and 2E1 are known to be constitutively expressed in human livers. To examine their involvement in MTBE metabolism, human CYP2A6 and 2E1 cDNAs were individually co-expressed with human cytochrome P450 reductase by a baculovirus expression system and the expressed enzymes were used for MTBE metabolism. The turnover number for CYP2A6 and 2E1 was 6.1 and 0.7 nmol TBA/min per nmol P450, respectively. The heterologously expressed human CYP2A6 was also more active than 2E1 in the metabolism of two other gasoline ethers, ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME). Although the contributions of other human CYP forms to MTBE metabolism remain to be determined, these results strongly suggest that CYP enzymes play an important role in the metabolism of MTBE in human livers.

Adult↗
PubMed1997

Cymbidium mosaic potexvirus RNA: complete nucleotide sequence and phylogenetic analysis.

The complete nucleotide sequence of the genomic RNA of cymbidium mosaic potexvirus (CymMV) was determined to be 6227 nucleotides in length, excluding the poly (A) tail at the 3' terminus. Similar to other potexviruses, its genome organisation is comprised of five major open reading frames (ORFs 1 to 5), encoding a Mr 160 KDa putative RNA-dependent RNA polymerase (RdRp); a Mr 26KDa/13KDa/10KDa triple-gene-block (TGB) and a Mr 24 KDa coat protein. The CymMV encoded proteins shared a high degree of homology to their corresponding proteins of other members of the potexvirus group. The nucleotide sequence of the 5' noncoding region (NCR) of CymMV and all other potexviruses initiates with GAAAA. CymMV possesses the shortest 5' NCR among all potexviruses. Based on phylogenetic comparisons of RdRp and coat protein, CymMV shares a close relationship to PAMV, NMV, WClMV and SMYEaV. This is believed to be the first record of the complete nucleotide sequence of CymMV.

Amino Acid Sequence↗
PubMed1997

A novel method for diabetes diagnosis based on electronic nose.

A novel non-invasive method for diabetes diagnosis based on an electronic nose is proposed in this paper. The principles of the method and results of clinical experiments are presented. These results show the convenience, painlessness and non-invasiveness of the method.

Algorithms↗
PubMed1997

Expression of human phenylalanine hydroxylase activity in T lymphocytes of classical phenylketonuria children by retroviral-mediated gene transfer.

Classical phenylketonuria (PKU) is a metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. At present, T lymphocyte-directed gene therapy is the only means for which a safety record has been established. Thus, we investigated the applicability of this strategy to PKU gene therapy. We first looked for tetrahydrobiopterin (BH4) and dihydropteridine reductase (DHPR) activity, which are required for the phenylalanine hydroxylation reaction and BH4 regeneration, respectively, in T cells isolated from PKU children. We found that T cells contained a small amount of biopterin, but significant DHPR activity, and that the intracellular biopterin content could be increased by exogenous BH4 supplementation. Moreover, PKU T cells were capable of taking up phenylalanine efficiently and effluxing acquired tyrosine. Finally, a recombinant retrovirus containing the human PAH cDNA was constructed and used to transduce isolated PKU T cells. Viral-transduced T cells produced high levels of PAH activity as compared to control mock-infected T cells. These results indicate that T lymphocytes express all that is required for synthesizing/replenishing constituents of the phenylalanine hydroxylation reaction and expressing transduced phenylalanine hydroxylase cDNA.

Biopterins↗
PubMed1997

Metastatic patterns of lung cancer visualized live and in process by green fluorescence protein expression.

We demonstrate here the visualization of human lung cancer metastasis live and in process in nude mice by green fluorescent protein (GFP) expression. The human lung adenocarcinoma cell line Anip 973 stably transfected with the humanized GFP-S65T cDNA was selected for very bright green fluorescence. GFP-transfected lung cancer cells were initially inoculated subcutaneously in nude mice. Five weeks after transplantation, the resulting tumor had reached over 1 cm in diameter and had very bright GFP fluorescence. Fragments of subcutaneous tumor were implanted onto the visceral pleura of the left lung of nude mice by surgical orthotopic implantation (SOI) of histologically-intact tissue via transverse thoracotomy. The ipsilateral resulting tumor was highly fluorescent due to GFP expression. GFP expression allowed the visualization of the advancing margin of the ipsilateral tumor into the fresh normal lung tissue. Lymphogenous and direct-seeding metastases in the pulmonary hilum, cervical lymph nodes, the mediastinum and contralateral pleural cavity and contralateral lung in the SOI-treated mice were brightly visualized by GFP expression in fresh tissue. GFP-transfected and untransfected tumor had similar metastatic characteristics suggesting that GFP expression had no effect on metastasis itself. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance in the visualization and study of lung cancer metastasis in process.

Adenocarcinoma↗
PubMed1997

A hypothetical model for the active site of human cytochrome P4502E1.

1. A model for the active site structure of human cytochrome P4502E1 based on the coordinates of cytochrome P450BM-3 crystal structure and the sequence analysis information on P4502E1 was proposed. 2. The sequence alignment of mammalian P4502 family and P450BM-3 indicated a 48%, similarity and 25% identity. Secondary structural prediction displayed a similar pattern of distribution in the main frame of secondary elements, alpha-helices and beta-sheets. The locations of secondary elements also mapped well. In addition, the amino acids responsible for the conserved secondary structural regions showed the most similarity between the two proteins. In contrast, the amino acids responsible for the loop region had the least similarity in our alignment. 3. The predicted P4502E1 active site model shows that the active site is small and contains mainly hydrophobic residues. The substrate binding pocket is located on top of pyrrole rings A and D of the haen; in contrast, the access to B and C rings is partially or completely blocked by protein side chains. 4. Residues within possible contact of a representative substrate, N-nitrosodimethylamine, are He115, Ala299, Thr303, Val364 and possibly He469.

Amino Acid Sequence↗
PubMed1997

Nucleotide polymorphism in colicin E2 gene clusters: evidence for nonneutral evolution.

To explore the molecular mechanisms behind the diversification of colicin gene clusters, we examined DNA sequence polymorphism for the colicin gene clusters of 14 colicin E2 (ColE2) plasmids obtained from natural isolates of Escherichia coli. Two types of ColE2 plasmids are revealed, with type II gene clusters generated by recombination between type I ColE2 and ColE7 gene clusters. The levels and patterns of DNA polymorphism are different between the two types. Type I polymorphism is distributed evenly along the gene cluster, while type II accumulates polymorphism at an elevated rate in the 5' end of the colicin gene. These differences may be explained by recombinational origins of type II gene clusters. The pattern of divergence between the ColE2 gene cluster and its close relative ColE9 is not correlated with the pattern of polymorphism within ColE2, suggesting that this gene cluster is not evolving in a neutral fashion. A statistical test confirms significant departures from the predictions of neutrality. These data lend further support to the hypothesis that colicin gene clusters may evolve under the influence of nonneutral forces.

Base Sequence↗