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Biomedical subjects

K Yang

Publications and source records attributed to K Yang.

At least 109 records · Page 6Linked to original sources
PubMed2000

[Expression and characterization of t-PA in insect cells].

With the RT-PCR technique, human tissue-type plasminogen activator (t-PA) cDNA was amplified from human melanoma cells. The sequence was proved to be the same as those reported by foreign researchers. The t-PA cDNA containing whole reading frame was inserted into the transfer vector pBacPAK8. The constructed pBac-tPA and the linear BacPAK6 virus DNA were cotransfected with Tn-5B-1 insect cells by lipofectin-mediated transfection method. Eleven pure recombinant viruses were isolated by plaque assay, and one of them, nominated BactPA3, was selected by PCR identification and biological activity comparison. The t-PA activity expressed in serum-containing media reach the highest level at 72 h postinfection. The activity was 3.04 x 10(3) IU/ml, e.g. 1.8 x 10(4) IU/10(6) cells. The highest expression level in serum-free media was almost the same, but needed more time (at 132 h postinfection). SDS-PAGE fibrin autography showed that the molecular weight of the expressed t-PA was about 68 kDa. Its stimulation by fibrinogen, affinity with fibrin and inactivation in plasma were almost the same as the native t-PA purified from human melanoma cell culture. The half-life of t-PA expressed in serum-free media was seven minutes.

Animals↗
PubMed2000

[Infrared study on molecular structure and orientation in Langmuir-Blodgett films of chiral LCs M1].

The LB films of chiral LCs M1 were investigated by FTIR transmission and reflection absorption spectra. CaF2 and vacuum deposition gold plate were used as solid substrate. The LB films can be transferred onto the solid substrate successfully. The orientation of LB films was analysis by comparing the FTIR transmission and reflection absorbance spectroscopy. The aromatic cores part which including the C-O bond was almost perpendicular the substrate surface, the alkyl chain and C=O part has a certain angle titled onto the substrate.

English Abstract↗
PubMed1999

DNA immunization: effect of secretion of DNA-expressed hemagglutinins on antibody responses.

DNA vaccines expressing plasma membrane and secreted forms of the influenza and measles virus hemagglutinins (HAs) have been used to evaluate the effect of secretion on DNA-raised antibody responses. At low doses of DNA, the plasma membrane form of the influenza virus HA raised higher titers of antibody than the secreted form. The isotype of the DNA-raised antibodies depended on both the method of DNA delivery and the form of the expressed antigen. Following intramuscular injections, DNAs expressing membrane bound forms of the influenza and measles HAs raised predominantly IgG2a. By contrast, DNAs expressing the secreted from of the two HAs as well as another secreted protein, human growth hormone, raised predominantly IgG1. Gene gun delivery resulted in predominantly IgG1 antibody responses for both secreted and membrane bound forms of the hemagglutinins. The raising of predominantly IgG1 by i.m. delivery of the secreted form of the influenza hemagglutinin was IL-4 dependent suggesting that a T-helper 2-biased immune response had been raised.

Animals↗
PubMed1999

Short-chain fatty acid metabolism, apoptosis, and Apc-initiated tumorigenesis in the mouse gastrointestinal mucosa.

Short-chain fatty acids (SCFAs) are physiological regulators of growth and differentiation in the gastrointestinal tract, and we have previously shown that apoptosis induced in colonic cell lines by these compounds is dependent on their metabolism by B-oxidation in the mitochondria (B. G. Heerdt et al., J. Biol. Chem., 266: 19120-19126, 1991; Cancer Res., 54: 3288-3293, 1994). Because tumors initiated by an inherited Apc mutation have been reported to be linked to decreases in apoptosis in the flat mucosa of the gastrointestinal tract, the aims were to determine whether elimination of efficient metabolism of SCFAs affected apoptosis in the gastrointestinal mucosa of the mouse, and whether this altered tumorigenesis initiated by an inherited Apc mutation. We, therefore, generated mice that have a chain-terminating mutation in the Apc gene and that were either wild-type for SCFA metabolism, or deficient, due to homozygous deletion of the gene (Scad) that encodes the enzyme short-chain acyl dehydrogenase, which catalyzes the first step in SCFA B-oxidation. Scad+/+ mice maintained on a wheat bran-fiber-supplemented diet gained significantly more weight than mice maintained on AIN76A, but this was eliminated by the Scad mutation, demonstrating that uptake and metabolism of SCFAs in the gastrointestinal tract can be a significant energy source. As predicted, on either AIN76A or wheat bran diet, the Scad mutation almost completely eliminated apoptosis in the flat mucosa of the proximal colon and reduced apoptosis by 50% in the distal colon compared with littermates that were wild-type for Scad. The mutation also reduced apoptosis by approximately 50% in the duodenum in AIN76A-fed mice. These reductions in apoptosis had no effect on incidence, frequency, or site specificity of tumors initiated by the Apc mutation. Therefore, the metabolism of SCFAs by the gastrointestinal mucosa plays a role in modulating apoptosis, but a general decrease in apoptosis in the mucosa of the gastrointestinal tract is not linked to gastrointestinal tumorigenesis initiated by an inherited Apc mutation.

Animals↗
PubMed1999

Ligand-receptor binding measured by laser-scanning imaging.

This report describes the integration of laser-scanning fluorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-bound, cyanine dye-labeled ligands, [Cy]ligands, were discriminated from those free in solution by measuring the accumulated fluorescence associated with a receptor-containing particle. To illustrate the various binding formats accommodated by this technique, saturation- and competition-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have successfully applied this technique to the analysis of G protein-coupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laser-scanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step fluorescent immunoassays.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Mouse models for colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers in the Western world. Much has been learned about colorectal cancer from human inherited syndromes, such as familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC). Mouse models for CRC were generated by introducing mutations into the mouse genes, whose human counterparts were implicated in the onset and progression of CRC. Central among these are mice carrying mutations in the Adenomatous polyposis coli (Apc) gene. Although most of these Apc mutations share some common phenotypes as homozygous embryonic lethality and tumor predisposition, the severity of the tumor predisposition is variable. Mice with mutations in the mismatch repair genes, Msh2 and Mlh1, exhibit a mismatch repair defect and are predisposed to developing gastrointestinal cancer, lymphomas and tumors of other organ systems. Mice carrying a mutation in the Pms2 gene are predisposed to lymphomas and other tumors. Mice with a mutation in the Msh6 gene have a defect in base mismatch repair and show a tumor predisposition phenotype. Mice with mutations in Mlh1, Pms2 and Msh5 have defects in meiosis suggesting unique roles for these genes in gametogenesis.

Adenomatous Polyposis Coli↗
PubMed1999

Novel somatic mutations in the VHL gene in Swedish archived sporadic renal cell carcinomas.

Frequent loss-of-function somatic mutations of the VHL gene have been detected in sporadic renal cell carcinoma (RCC), indicating that inactivation of the VHL gene plays a critical role in RCC. In this study, we collected 35 archived Swedish sporadic RCCs identified from an epidemiological study on occupational exposure and kidney cancer to test how well stored pathological specimens could be retrieved and analyzed for VHL mutations. Thirty specimens were successfully analyzed with PCR-SSCP and sequencing. Aberrant SSCP bands were detected in 16 out of the 30 samples (53%). Sequencing analysis of the aberrant bands revealed seven deletions, one insertion, one base substitution on a splicing site, six missense mutations, one silent mutation and several base substitutions in the 5' non-coding region and intron 1. Most were novel somatic mutations that have not been reported in sporadic RCC. The mutations were found in three types of non-papillary RCC cases, i.e. 14 clear cells, one granular chromophilic and one sarcomatoid RCC. Interesting multiple mutations were found in three cases (5, 3, 2 mutations, respectively).

Adult↗
PubMed1999

Action of capsaicin on dorsal root-evoked synaptic transmission to substantia gelatinosa neurons in adult rat spinal cord slices.

An action of capsaicin was investigated on dorsal root-evoked synaptic transmission to substantia gelatinosa (SG) neurons in adult rat spinal cord slices by use of the whole-cell voltage-clamp technique. In 79% of neurons examined, superfusing capsaicin (1 microM) for 30 s depressed a C-fiber-evoked excitatory synaptic current in a manner sensitive to a capsaicin-receptor antagonist, capsazepine (10 microM). On the contrary, Adelta-fiber-evoked excitatory and inhibitory synaptic currents were unaffected by capsaicin in all of cells tested. It is concluded that capsaicin specifically acts on C-afferents, resulting in an inhibition of evoked excitatory transmission to the SG; this may contribute to, at least in part, an acute analgesic action of capsaicin.

Animals↗
PubMed1999

Oxidative refolding of recombinant prochymosin.

The disulphide-coupled refolding of recombinant prochymosin from Escherichia coli inclusion bodies was investigated. Prochymosin solubilized from inclusion bodies is endowed with free thiol groups and disulphide bonds. This partially reduced form undergoes renaturation more efficiently than the fully reduced form, suggesting that some native structural elements existing in inclusion bodies and remaining after denaturation function as nuclei to initiate correct refolding. This assumption is supported by the finding that in the solubilized prochymosin molecule the cysteine residues located in the N-terminal domain of the protein are not incorrectly paired with the other cysteines in the C-terminal domain. Addition of GSH/GSSG into the refolding system facilitates disulphide rearrangement and thus enhances renaturation, especially for the fully reduced prochymosin. Based on the results described in this and previous papers [Tang, Zhang and Yang (1994) Biochem. J. 301, 17-20], a model to depict the refolding process of prochymosin is proposed. Briefly, the refolding process of prochymosin consists of two stages: the formation and rearrangement of disulphide bonds occurs at the first stage in a pH11 buffer, whereas the formation and adjustment of tertiary structure leading to the native conformation takes place at the second stage at pH8. The pH11 conditions help polypeptides to refold in such a way as to favour the formation of native disulphide bonds. Disulphide rearrangement, the rate-limiting step during refolding, can be achieved by thiol/disulphide exchange initiated by free thiol groups present in the prochymosin polypeptide, GSH/GSSG or protein disulphide isomerase.

Air↗
PubMed1999

Tumorigenesis in Mlh1 and Mlh1/Apc1638N mutant mice.

An3 1 KAL I MutL homologue 1 (MLH1) is a member of the family of proteins required for DNA mismatch repair. Germ-line mutations in MLH1 lead to the cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). We generated mice carrying a null mutation in the Mlh1 gene. We showed that mice heterozygous and homozygous for the Mlh1 gene are predisposed to developing tumors of the gastrointestinal (GI) tract, lymphomas, and a number of other tumor types. We also examined the role of adenomatous polyposis coli gene (Apc) gene mutations in the GI tumors of Mlh1 mutant mice by different methods and showed that the GI tumors in Mlh1 mice express little or no adenomatous polyposis coli protein. When an Apc gene mutation was bred into the Mlh1 mutant mice, the GI tumor incidence increased 40-100-fold. The wild-type Apc allele in these tumors was found to contain mutations. Together, these results show that we have developed two mouse models for human HNPCC and that the mechanisms of tumor development in the GI tract of these mice involve loss of Apc gene function in a manner very similar to that seen in the GI tumors of HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed1999

International study of expert judgment on therapeutic use of benzodiazepines and other psychotherapeutic medications: VI. Trends in recommendations for the pharmacotherapy of anxiety disorders, 1992-1997.

OBJECTIVE: To assemble expert clinical experience and judgment regarding the treatment of anxiety disorders in a systematic, quantitative manner, particularly with respect to changes during the preceding five years. METHOD: A panel of 73 internationally recognized experts in the pharmacotherapy of anxiety and depression was constituted by multistage peer nomination. Sixty-six completed a questionnaire in 1992, and 51 of those completed a follow-up questionnaire in 1997. This report focuses on the experts' responses to questions about therapeutic options relevant to seven vignettes describing typical cases of different anxiety disorders. RESULTS: The preferred initial treatment strategy in 1992 was a combination of medication with a psychological therapy for all vignettes except simple phobia, where a psychological procedure alone was favored. There was little change in 1997, primarily some decrease in the choice of psychological therapy and some increase in the choice of medication for social phobia. Experts recommending a medication in 1992 most often chose as first-line treatment a benzodiazepine anxiolytic (BZ) for panic disorder (PD), generalized anxiety disorder (GAD), simple phobia, and adjustment disorder. They recommended a beta-blocker most often for social phobia and a tricyclic anti-depressant (TCA) for agoraphobia and obsessive-compulsive disorder (OCD). Nearly a fourth chose a combination of medications, usually a TCA plus a BZ. In 1997, the expert panel's most frequent recommendation for agoraphobia, PD, and OCD changed to a specific serotonin reuptake inhibitor (SSRI); and they also recommended these compounds more often for GAD, social phobia, and simple phobia. Fewer experts chose BZs or TCAs. However, in 1997 many again chose a combination of medications, often a BZ plus a SSRI, so that, overall, there was only a small decline in recommendations for BZs. As second-line medications (1997 only), the experts recommended SSRIs most often for most vignettes, but a TCA for PD and GAD. Recommendations for a combination of medications rose substantially for most vignettes, usually a BZ plus an antidepressant. CONCLUSIONS: Combined cognitive-behavioral therapy plus medication was highly favored by the experts as the initial treatment strategy for anxiety disorders. During the preceding five years, SSRIs displaced older antidepressants as the experts' first-line choices for the pharmacotherapy of anxiety disorders. In case of an unsatisfactory response, the experts' second-line choices more often were an older antidepressant or a combination of an antidepressant plus a BZ. According to the experts' judgements, the BZs, especially combined with an antidepressant, remain mainstays of pharmacotherapy for anxiety disorders.

Anti-Anxiety Agents↗
PubMed1999

Trends in recommendations for the pharmacotherapy of anxiety disorders by an international expert panel, 1992-1997.

A follow-up survey in 1997 to a 1992 study of the recommendations of an international expert panel on the use of benzodiazepines (BZDs) and other psychotherapeutic medications in the treatment of anxiety disorders suggests that the BZDs remain a mainstay of pharmacotherapy for most of these conditions. BZDs were mentioned more often than any other class of drugs as preferred first-line therapy for anxiety disorders, except obsessive compulsive disorder. The introduction of the selective serotonin reuptake inhibitors (SSRIs) did not significantly affect the experts' recommendations for the use of BZDs as first-line pharmacotherapy. Rather, the SSRIs displaced the tricyclic antidepressants. Some implications of the continuing recommendations for the use of BZDs in anxiety disorders are discussed.

Anti-Anxiety Agents↗
PubMed1999

IGF-I gene transfer in thermally injured rats.

Exogenous insulin-like growth factor-I (IGF-I) is known to improve the pathophysiology of a thermal injury, however, deleterious side-effects have limited its utility. Cholesterol-containing cationic liposomes that encapsulate complementary DNA (cDNA) are nonviral carriers used for in vivo gene transfection. We propose that liposome IGF-I gene transfer will accelerate wound healing in burned rats and attenuate deleterious side-effects associated with high levels of IGF-I. To test this hypothesis IGF-I gene constructs, encapsulated in liposomes, were studied for their efficacy in modulating the thermal injury response. Thirty adult male Sprague-Dawley rats were given a 60% TBSA scald burn and randomly divided into three groups to receive weekly subcutaneous injections of liposomes plus the lacZ gene coding for beta-galactosidase, liposomes plus cDNA for IGF-I and beta-galactosidase or liposomes plus the rhIGF-I protein. Body weights and wound healing were measured. Muscle and liver dry/wet weights and IGF-I concentrations in serum, skin and liver were measured by radioimmunoassay. Transfection was confirmed by histochemical staining for beta-galactosidase. Rats receiving the IGF-I cDNA constructs exhibited the most rapid wound re-epithelialization and greatest increase in body weight and gastrocnemius muscle protein content (P < 0.05). Local IGF-I protein concentrations in the skin were higher when compared to liposomes containing only the lacZ gene (P < 0.05) Transfection was apparent in the cytoplasm of myofibroblasts, endothelial cells and macrophages of the granulation tissue. Liposomes containing the IGF-I gene constructs proved effective in preventing muscle protein wasting and preserving total body weight after a severe thermal injury.

Animals↗
PubMed1999

Liposome-mediated NGF gene transfection following neuronal injury: potential therapeutic applications.

We have systematically investigated the therapeutic potential of cationic liposome-mediated neurotrophic gene transfer for treatment of CNS injury. Following determination of optimal transfection conditions, we examined the effects of dimethylaminoethane-carbamoyl-cholesterol (DC-Chol) liposome-mediated NGF cDNA transfection in injured and uninjured primary septo-hippocampal cell cultures and rat brains. In in vitro studies, we detected an increase of NGF mRNA in cultures 1 day after transfection. Subsequent ELISA and PC12 cell biological assays confirmed that cultured cells secreted soluble active NGF into the media from day 2 after gene transfection. Further experiments showed that such NGF gene transfection reduced the loss of chol- ine acetyltransferase (ChAT) activity in cultures following calcium-dependent depolarization injury. In in vivo studies, following intraventricular injections of NGF cDNA complexed with DC-Chol liposomes, ELISA detected nine- to 12-fold increases of NGF in rat CSF. Further studies showed that liposome/NGF cDNA complexes could attenuate the loss of cholinergic neuronal immunostaining in the rat septum after traumatic brain injury (TBI). Since deficits in cholinergic neurotransmission are a major consequence of TBI, our studies demonstrate for the first time that DC-Chol liposome-mediated NGF gene transfection may have therapeutic potential for treatment of brain injury.

Animals↗
PubMed1999

Interconversion of cortisol and cortisone by 11beta-hydroxysteroid dehydrogenases type 1 and 2 in the perfused human placenta.

The human placenta contains two types of 11beta-hydroxysteroid dehydrogenase (11beta-HSD). The exclusive oxidase 11beta-HSD2 has been suggested to protect the fetus from high levels of maternal glucocorticoids by converting cortisol to inactive cortisone. Perfused term human placenta was used to examine the activity of the oxoreductase 11beta-HSD1 and to determine the regulation of cortisol effects on placental vascular tone and corticotropin-releasing hormone (CRH) output by 11beta-HSD. Radioimmunoassay showed that there was substantial cortisol (295+/-57 nM) detected in the fetal vein upon perfusion of cortisol (2 microM; perfusion rate, 12 ml/min) into the maternal intervillous space. Output of cortisol increased to 559+/-22 nM on the fetal side (P<0.05) with concurrent perfusion of carbenoxolone (CBX; 1 microM), a non-specific 11beta-HSD inhibitor. Cortisol formation increased in a dose-dependent manner with infusion of cortisone (0.1-2 microM) into the maternal intervillous space reaching 15 and 23 nM in fetal and maternal venous outflows respectively at 2 microM cortisone perfusion. There was no significant effect of cortisol either alone or in combination with CBX on the fetal arterial perfusion pressure, but cortisol perfusion increased CRH output into the fetal vein. It is concluded that activities of both 11beta-HSD1 and -2 are demonstrable in perfused human placenta in vitro, and these enzymes affect transplacental glucocorticoid transfer. These activities may provide a precise mechanism to control the passage of maternal glucocorticoids to the fetal circulation, and to regulate glucocorticoid effects within the placenta.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1999

Hprt mutant frequency and aromatic DNA adduct level in non-smoking and smoking lung cancer patients and population controls.

T cell cloning and 32P-post-labelling methods were used to study the mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus and the aromatic DNA adduct level (AL) in peripheral lymphocytes of newly diagnosed lung cancer patients (92 ever-smokers and 87 never-smokers) and matched population controls (82 ever-smokers and 79 never-smokers). Overall, the MF (total mean 20.6 x 10(-6)) and AL (4.1 x 10(-8)) were similar in cases and controls with the same smoking status, indicating that the disease has limited effect on the two endpoints. When cases and controls were combined, the AL was significantly higher in current smokers than in former or never-smokers (P = 0.0003) and the MF was significantly higher in ever-smokers than in never-smokers (P = 0.004). Age affected the MF significantly in ever-smokers (1.6%/year, 95% CI 0.6-2.5, adjusted for pack-years and years since last smoking), especially among cases (2.1%/year, 95% CI 0.5-3.7). An increase of AL with age was observed in currently smoking cases only (2.3%/year, 95% CI 0.3-4.2, adjusted for smoking dose). For currently smoking cases, there was also a more pronounced effect of smoking dose on both endpoints and a significant correlation between AL and MF (r = 0.52, P = 0.04) was observed among those with the highest dose. Our data also provide additional evidence for the different turnover times of smoking-induced DNA adducts and hprt mutations. The stronger increase of MF and AL with age and dose in currently smoking patients compared with controls is consistent with an interaction between smoking and genetic host factors.

Adenocarcinoma↗
PubMed1999

Retinoic acid stimulates the expression of 11beta-hydroxysteroid dehydrogenase type 2 in human choriocarcinoma JEG-3 cells.

The syncytiotrophoblasts of the human placenta express high levels of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the enzyme responsible for the inactivation of glucocorticoids. It has been proposed that the placental 11beta-HSD2 serves as a barrier to protect the fetus from high levels of maternal cortisol. To examine the hypothesis that nutritional signals regulate the expression of 11beta-HSD2 in placental syncytiotrophoblasts, we investigated the effects of retinoic acids (RAs), the major metabolites of vitamin A, on the expression of 11beta-HSD2 using human choriocarcinoma JEG-3 cells as a model. This trophoblast-like cell line displays a number of functional similarities to the syncytiotrophoblast. Treatment for 24 h with all-trans RA (1-1000 nM) resulted in a dose-dependent increase in 11beta-HSD2 activity with a maximal effect (increase to 3-fold) at 100 nM. The effect of all-trans RA (100 nM) was also time-dependent in that the effect was detectable at 6 h and reached its maximum by 48 h. Similar increases in 11beta-HSD2 activity were observed when the cells were treated with 9-cis RA. Results from semi-quantitative reverse transcription-polymerase chain reaction demonstrated that there was a corresponding increase in 11beta-HSD2 mRNA after RA treatment. Moreover, treatment with actinomycin D (100 ng/ml) abrogated the increase in 11beta-HSD2 mRNA induced by RA, indicating an effect on transcription. In conclusion, the present study has demonstrated for the first time that RA, at physiological concentrations, induces 11beta-HSD2 gene expression and enzyme activity in JEG-3 cells. If this occurs in vivo, the present finding suggests that high expression of 11beta-HSD2 in the human placenta may be maintained, at least in part, by dietary intake of vitamin A.

11-beta-Hydroxysteroid Dehydrogenases↗