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Biomedical subjects

J Yan

Publications and source records attributed to J Yan.

At least 181 records · Page 10Linked to original sources
PubMed1999

[In situ enzyme immunoassay for detection of hepatitis A virus].

OBJECTIVE: To establish a more simple and sensitive technique for identification of hepatitis A virus (HAV). METHODS: HAV vaccine strain H2M20-K and wild-type strain He34 were used to infect KMB17 deploid cells in 96-well tissue culture plates and in situ enzyme immunoassay was used to detect viral infection with specific HA monoclonal antibody, A(OD) value of 1.8 was used as positive indicator. RESULTS: Observation of viral replication showed the peak titer of viral infectivity reached at 20-25 days after viral infection, which was 4-6 day searlier than that of ELISA endpoint assay. Comparing the viral infectivity titers of 13 individual samples, the two methods revealed no significant statistic difference (t = 1.13, P > 0.05). Our tests showed that in situ EIA could better reflect the results of neutralization and 5 HAV positive isolates were directly isolated from 12 faecal samples after the first passage culture. CONCLUSION: Although the sensitivities of the two tests were similar, the direct detection of virus/cell system with in situ EIA showed simple, easy to operate and reproducible, it may replace the conventionally used ELISA endpoint assay for identification of HAV, even for the other viruses.

Cell Line↗
PubMed1998

Electrophysiological responses to bitter stimuli in primate cortex.

Studies investigating fine details of gustatory coding in the domain of each basic taste quality have been completed for sweet, salt, and sour stimuli. In the present experiment, we used chemicals that humans describe as predominantly bitter. We recorded the activity of 50 taste neurons in insular cortex of two cynomolgus macaques. Stimuli were water, fruit juice, glucose, NaCl, HCl, and 16 bitter solutions. In a multidimensional taste space the 16 bitter stimuli formed a coherent cluster composed of three main subgroups: (1) QHCl, phenylalanine, theophylline, caffeine, propyl-thiouracil (PROP), and phenylthiocarbamide (PTC), all of which humans describe as rather purely bitter, (2) MgCl2, CaCl2, NH4Cl, and arginine, which humans describe as salty-bitter, and (3) urea, cysteine, and vitamin B1, which are described as sour-bitter. Vitamin B2, histidine and nicotine were in the center of the bitter cluster. Human descriptions of taste qualities conformed well to the presumed quality of each stimulus as inferred from its position in the multidimensional space (MDS), reinforcing the use of the macaque as a neural model for human gustation.

Animals↗
PubMed1998

Purification from bovine serum of a survival-promoting factor for cultured central neurons and its identification as selenoprotein-P.

We purified from bovine serum a glycoprotein that promotes the survival of rat embryonic neurons cultured from septum and other brain regions. A 40,000-fold purification was achieved by using a combination of ammonium sulfate precipitation, Zn2+ affinity chromatography, Cibacron blue 3-GA dye affinity chromatography, ABx ion exchange chromatography, and preparative PAGE. The active protein had an apparent molecular weight of 50-60 kDa. The concentration required for half-maximal survival (EC50) was 12 ng/ml ( approximately 200 pM) for the final fraction. Amino acid sequencing after cyanogen bromide cleavage yielded two sequences that are homologous to regions of deduced sequence of the selenoprotein-P (SPP) family in bovine, rat, and human. Antibodies against a synthetic peptide within the bovine SPP sequence immunoprecipitated and inhibited the survival-promoting activity of a partially purified serum fraction. The purified protein supported neuronal survival more effectively than inorganic selenium. These results suggest that SPP or an SPP-like protein contributes to the neuronal survival-promoting activity of serum.

Amino Acid Sequence↗
PubMed1998

Ras isoforms vary in their ability to activate Raf-1 and phosphoinositide 3-kinase.

Ha-, N-, and Ki-Ras are ubiquitously expressed in mammalian cells and can all interact with the same set of effector proteins. We show here, however, that in vivo there are marked quantitative differences in the ability of Ki- and Ha-Ras to activate Raf-1 and phosphoinositide 3-kinase. Thus, Ki-Ras both recruits Raf-1 to the plasma membrane more efficiently than Ha-Ras and is a more potent activator of membrane-recruited Raf-1 than Ha-Ras. In contrast, Ha-Ras is a more potent activator of phosphoinositide 3-kinase than Ki-Ras. Interestingly, the ability of Ha-Ras to recruit Raf-1 to the plasma membrane is significantly increased when the Ha-Ras hypervariable region is shortened so that the spacing of the Ha-Ras GTPase domains from the inner surface of the plasma membrane mimicks that of Ki-Ras. Importantly, these data show for the first time that the activation of different Ras isoforms can have distinct biochemical consequences for the cell. The mutation of specific Ras isoforms in different human tumors can, therefore, also be rationalized.

Amino Acid Sequence↗
PubMed1998

Human ULK1, a novel serine/threonine kinase related to UNC-51 kinase of Caenorhabditis elegans: cDNA cloning, expression, and chromosomal assignment.

The unc-51 gene, isolated from mutants of Caenorhabditis elegans exhibiting abnormal axonal extension and growth, encodes a novel serine/threonine kinase (K. Ogura, et al., 1994, Genes Dev. 8: 2389-2400). Here we report the molecular cloning and characterization of the human homologue of UNC-51, designated ULK1, for UNC-51 (C. elegans)-like kinase 1. Sequence analysis of the human ULK1 cDNA showed that an open reading frame is composed of 1050 amino acids with a calculated MW of 112.6 kDa and a pI of 8.80. Homology search analysis showed that ULK1 has 41% overall similarity to UNC-51 and 29% similarity to Apg1p of Saccharomyces cerevisiae. Phylogenetic analysis of ULK1, UNC-51, and Apglp suggested that they constitute a novel subfamily of serine/threonine kinases. Southern blot analyses suggested that the ULK1 gene spans 30-40 kb in the human genome as a single-copy gene. Zoo blot analysis indicated that ULK1 kinase is conserved among vertebrates including mammals, birds, reptiles, amphibians, and fish. Northern blot analysis revealed that ULK1 is ubiquitously expressed in adult human tissues such as skeletal muscle, heart, pancreas, brain, placenta, liver, kidney, and lung, whereas UNC-51 is specifically detected in the nervous system of C. elegans. Both FISH and RH mapping confirmed the regional localization of ULK1 to human chromosome 12q24.3.

Amino Acid Sequence↗
PubMed1998

Identification of mouse ULK1, a novel protein kinase structurally related to C. elegans UNC-51.

A novel protein kinase related to the C. elegans serine/threonine kinase UNC-51 was cloned from mouse. The UNC-51-Like Kinase (ULK)1 is encoded by a cDNA of 1051 amino acids with calculated MW of 113 kDa. Comparison of the ULK1 and UNC-51 shows the highest conservation in the amino-terminal kinase domain, which is followed by a proline/serine-rich (PS) domain and a conserved carboxyl-terminal (C) domain. ULK1 mRNA is expressed in various tissues, and is mapped to mouse chromosome 5F and rat chromosome 12q16.3, by fluorescent in situ hybridization. HA-tagged ULK1 is expressed as a protein of approximately 150 kDa in COS7 cells and is auto-phosphorylated in vitro in its PS domain. We propose that ULK1, UNC-51 and a yeast protein kinase Apg1p comprise a novel subfamily of protein kinase, which is structurally conserved among eukaryotes.

Amino Acid Sequence↗
PubMed1998

Characterization of the regional intestinal kinetics of drug efflux in rat and human intestine and in Caco-2 cells.

PURPOSE: The aim of the present study was to investigate the transport kinetics of intestinal secretory processes in the jejunum, ileum and colon of rats and humans and in Caco-2 cells, in vitro. METHODS: Etoposide, vinblastine sulphate and verapamil hydrochloride were chosen as model substrates since they have been reported to undergo efflux in various other tissues. The concentration dependence, inhibition, directionality, temperature dependence, proton/sodium dependence, and ATP dependence of efflux were studied using side-by-side diffusion chambers and brush border membrane vesicles (BBMVs). Intestinal tissue from rats and humans and Caco-2 cells (passage no. 26) were used. Directional steady state effective permeabilities were calculated from drug appearance in the apical (AP) or basolateral (BL) chambers. Kinetic studies were carried out by investigating substrate efflux at concentrations ranging from 0.2 microns to 1000 microns. Since substrate efflux may be a result of more than one transporter, the hybrid efflux Km (Michaelis-constant), Pc (carrier-mediated permeability), and Pm (passive permeability) were determined as a function of intestinal region. Inhibitor studies were performed using quinidine (0.2mM), a mixed inhibitor of P-glycoprotein (Pgp) and Multidrug Resistance-Associated Protein (MRP), and Leukotriene C4 (100 nM), an inhibitor of MRP and the canalicular multispecific organic anion transporter (cMOAT). Temperature dependent efflux was determined by investigating the BL to AP transport at temperatures ranging from 3 degrees C to 37 degrees C. Energies of activation (Ea) were determined from an Arrhenius analysis. Sodium, proton, and ATP dependence were determined using BBMVs. Immunoquantitation of Pgp, MRP and Lung Resistance Protein (LRP) in Caco-2 cells were carried out using Western blot analysis. RESULTS: Active efflux of all substrates was observed in all regions of rat and human intestine and in Caco-2 cells. Directionality was observed with BL to AP transport exceeding AP to BL transport. The BL to AP/AP to BL permeability ratio, the efflux ratio, ranged from 1.4 to 19.8. Ileal efflux was significantly higher (p < 0.001) than in other regions. Kinetic studies revealed that hybrid efflux Km values ranged from 4 to 350 microns. In some cases, efflux was not saturable due to the solubility limits of the compounds utilized in this study. In presence of inhibitors, efflux ratios approached 1. BL to AP transport was temperature dependent in rat ileum for all substrates. Each of the intestinal efflux was found to be 11.6, 8.3, and 15.8 kcal/mole for etoposide, vinblastine and verapamil, respectively, suggesting an active, energy-dependent efflux mechanism. Substrate efflux was not sodium or proton dependent but was dependent on ATP. Using Western blot analysis the presence of Pgp, MRP, and LRP was demonstrated in Caco-2 cells and the amount of each transport protein varied as a function of passage number. CONCLUSIONS: Using multiple putative efflux substrates, the current results demonstrate that intestinal efflux was regionally dependent, mediated by multiple efflux transporters, the Km's were in the micro-molar range, and involved an energy dependent mechanism(s).

Adenosine Triphosphate↗
PubMed1998

Molecular dissection of developmental behavior of plant height in rice (Oryza sativa L.).

A doubled haploid population of 123 lines from IR64/Azucena was used to dissect the developmental behavior and genotype by environment interaction for plant height by conditional and unconditional quantitative trait loci (QTL) mapping methods in rice. It was shown that the number of QTL detected was different at various measuring stages. Some QTL could be detected at all stages and some only at one or several stages. More QTL could be found on the basis of time-dependent measures of different stages. By conditional QTL mapping of time-dependent measures, it is possible to reveal dynamic gene expression for quantitative traits. Mapping QTL for genetic main effects and GE interaction effects could help us in understanding the nature of QTL x environment interaction for the development of quantitative traits.

Adaptation, Physiological↗
PubMed1998

The P2 protein of rice dwarf phytoreovirus is required for adsorption of the virus to cells of the insect vector.

Intact particles of rice dwarf phytoreovirus adsorbed to and entered monolayer-cultured cells of the insect vector Nephotettix cincticeps and multiplied within the cells. Particles that lacked the P2 protein neither attached to nor infected such cells. Furthermore, P2-free particles obtained from a transmission-competent isolate of the virus were unable to infect insect vectors that had been allowed to feed on these virus particles through a membrane. However, when such virus particles were injected into insects via a glass capillary tube they successfully infected the insects, which became able to transmit the virus. These results support the hypothesis that, while P2-free particles can neither interact with nor infect cells in the intestinal tract of the insect vector, they do retain the ability to infect such cells when physically introduced into the hemolymph by injection.

Adsorption↗
PubMed1998

14-3-3 facilitates Ras-dependent Raf-1 activation in vitro and in vivo.

14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. We show here that 14-3-3 is bound to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol. A model that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation is proposed.

14-3-3 Proteins↗
PubMed1998

[A study on the risk factors of none-insulin-dependent type diabetes mellitus].

A 1:1 matched case-control study was conducted to investigate the risk factors for NIDDM in 1995. A total of 100 pairs of case and controls matched on sex, age and hopital of admission. Bivariate analysis revealed 7 factors significantly associated with NIDDM. Conditional logistic regression analysis showed that 3 of the 7 fectors: obesity, greasy and family history of diabetes were risk factors for NIDDM. That might play important roles in the etiology of NIDDM.

Aged↗
PubMed1998

CD 80(B7-1) expression on human tumor cell lines and its costimulatory signals for T cell proliferation and cytokine production.

OBJECTIVE: To investigate CD 80 expression on human tumor cell lines and establish stable CD 80 expression transfectants to illustrate CD 80 costimulation on the T cell proliferation and cytokine production. METHODS: Raji, MDA-453, MCF-7, Hela, 3AO, MKN-45 and EBV transformed B cell were detected for CD 80 expression by RT-PCR. CD 80 cDNA subcloned to retrovirus vector pLXSN, with them stable CD 80 transfectanants were established. With specific mAb BB1 and FACS assay, the expression of CD 80 were detected. Anti-CD3 induced T lymphocyte proliferation and IL-2, IFN-r production were performed to evaluate CD 80 costimulatory activity in the presence of calcium ionophore A23187 and phorbol ester PMA. RESULTS: CD 80 (B7-1) expression of MDA-453, MCF-7, Hela, 3AO, MKN-45 was negative, and that of Raji and EBV transformed B cell was positive by RT-PCR and FACS methods. Cocultured with CD 80 transfected human breast carcinoma cell line MDA-453, anti-CD3 induced T cells proliferation and cytokine production were significantly increased in the presence of A23187 and PMA, but not with parental MDA-453. CONCLUSIONS: CD 80 expression is absent on most human tumor cell line except for Raji and EBV transformed B cell. Full activation of T cell needs CD 80 costimulation. Influence of CD 80 costimulation on cytokine production is mainly regulated via IL-2 and IFN-r.

B7-1 Antigen↗
PubMed1998

[Experimental study of effects of anti-ovarian antibodies on ovarian histology and function].

OBJECTIVE: To investigate the effects of anti-ovarian antibodies (AOA) on ovarian histology and functions. METHODS: Rabbit antibodies against mice ovarian tissues were obtained by immunizing with mice ovarian extracts and purified. The effects of AOA on mice ovarian histology were examined under light and electronic microscope. In addition, changes of ovarian functions, including natural pregnant rate and pregnant mare serum gonadotropin (PMSG) + hCG induced ovulation rate and pregnant rate, were also observed. RESULTS: After treating with different doses of AOA pathological changes occurred in a variety of ovarian components, especially in zona pellucida and granulosa cells, which is more serious in high AOA level group than that in low AOA level group. The natural pregnant rate decreased to zero in both AOA groups. The ovulation and pregnant rate induced by PMSG-hCG were significant lower in both AOA groups than that in the control group especially in the high AOA level group. CONCLUSION: AOA may damage the ovarian tissues and reduce the ovulation and pregnant rate. The higher the AOA level, the more serious pathological changes and the poorer curative effects of PMSG-hCG may occur.

Animals↗
PubMed1998

[Cloning and expression of leptospiral protective antigen gene OmpL1 in BCG].

This study was intended to produce a new living vaccine against leptospirosis using BCG as vector. Leptospiral outer envelop antigen gene OmpL1 was amplified from the genome of pathogenic leptopira serova Lai 017 by PCR, and cloned in E. coli-BCG shuttle plasmid pY6002. Recombinant plasmids were isolated by dot blotting with Digoxigeninlabeled OmpL1 gene. After transforming the recombinant plasmids in BCG (Shanghai strain) by electroporation, the genomic DNA of all 21 transformants were prepared and hybridized with OmpL1. It showed that 6 of the 21 transformants were recombinants in which the OmpL1 gene had been integrated into the genome of BCG. By immunoblotting with OmpL1 infected rabbit antiserum, which was preabsorbed to remove antibody against E. coli and SPA-HRP, three recombinants, pLI1, pLI2 and pLI3, were detected to express OmpL1 protein. The ability of expression is in the order of pLI2 > pLI1 >> plI3. These studies provide the possibility of further research on the development of highly efficient recombinant vaccines against leptospirosis.

Cloning, Molecular↗
PubMed1998

[Determination of cellular and humoral immunity in mice of autoimmune ovarian failure].

OBJECTIVE: To determine cellular and humoral immunity in mice of autoimmune ovarian failure. METHODS: A mice model of autoimmune ovarian failure was established. The percentages of help T cell (CD4+) and suppressor T cell (CD8+), the ratio of CD4/CD8 and anti-ovarian antibody (AOA) were determined. RESULTS: No significant changes were found in CD4+ fraction, but the percentage of CD8+ was reduced and the ratio of CD4/CD8 increased. AOA was positive which was probably the cause of damage of the ovarian tissues, reduction of sex hormone secretion and impaired fertility. CONCLUSION: Autoimmune ovarian failure is correlated with the abnormal change of CD4/CD8 and the increase of B-lymphocytes function.

Animals↗