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Biomedical subjects

Y Gong

Publications and source records attributed to Y Gong.

At least 73 records · Page 4Linked to original sources
PubMed2001

Sequence and chromosomal assignment of a human novel cDNA: similarity to gamma-aminobutyric acid transporter.

Gamma-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the mammalian brain. Although initially thought to be confined to the central nervous system, GABAergic activity has also been described in other tissues throughout the body. In the present study, we report the cloning and localization of human GABA transporter cDNA and document its expression in various human tissues. A human liver cDNA library was initially screened by a 32P-labeled murine brain GABA transporter 3 (GAT-3) cDNA probe, and full-length cDNA was cloned by employing Marathon-Ready human kidney cDNA. The human GABA transporter cDNA encoded a 569 amino acid hydrophobic protein with 12 transmembrane domains (TMs). Search of published sequences revealed high homology with rat GAT-2, murine GAT-3 cDNA, human solute carrier family 6 member 13 (SLC6A13), and a human peripheral betaine/GABA transporter. Northern blot analyses demonstrated that the human GABA transporter is expressed strongly in the kidney and to a lesser extent in the liver and brain. The sequence was well matched with human chromosome 12p13.3, suggesting the human GABA transporter contains 14 exons. The above findings confirm the existence of and further characterize a specific GABA transporter in human tissues.

Amino Acid Sequence↗
PubMed2001

[Epidemiological effects of live attenuated hepatitis A vaccine (H(2)-strain): results of A 10-year observation].

OBJECTIVE: To assess the long-term epidemiological effects after a mass vaccination program, using live attenuated hepatitis A vaccine (H(2)-strain). METHODS: Shengsi county and Jiaojiang city in Zhejiang province were chosen where high incidence rate of hepatitis A was observed. Mass vaccination program of H(2)-strain vaccine was carried out on 1 - 15 year olds. Cohort studies included two groups were carried out in Shengsi. Cases reported program on hepatitis A was under the national standard on clinical diagnosis and partly through laboratory monitor of anti-HAV IgM and IgG, ELISA and Abbott EIA. Population size was estimated by the yearly reports of census bureau of the county and the city. Serum tests including neutralization test, immune-recall responses, and anti-HAV IgG were performed 10 years after the initial immunization. RESULTS: The morbidity of hepatitis A was significantly different between vaccinated groups and non-vaccinated groups (P < 0.01) in the cohort study. The protective efficacy in the site was 100 percent. There was a significantly negative correlation of regression between vaccine coverage and hepatitis A morbidity. In both sites, hepatitis A incidence rate showed decrease of 94.5% and 90.3% in whole population, with 96.9% and 97.9% in 1 - 15 year olds respectively. Neutralization test showed that 63.8% of the serum negative to ELISA after vaccination could still neutralize certain HAV. Immune-recall response after the booster was seen in vaccine group with 80.2% positive rate of anti HAV-IgG, 10 years after immunization among vaccinees in both sites. Not a single hepatitis A case was identified. CONCLUSION: Live attenuated hepatitis A vaccine seemed to have obviously long-term epidemiological effects.

Adolescent↗
PubMed2001

[Purification and gene cloning of a novel fibrinolytic protease from Streptomyces sp. C3662].

A novel fibrinolytic protease from Streptomyces sp. C3662 was purified by (NH4)2SO4 fractionation, DEAE-Sepharose and CM-Sepharose chromatography. The molecular weight of the protease was indicated to be 30 kD by SDS-PAGE. Using a E. coli/Streptomyces shuttle plasmid pIJ699 as the vector, shot-gun cloning was performed to clone the gene of the protease. One clone with fibrinolytic activity harboring a plasmid that contains a DNA fragment of 6 kb was obtained from 3000 clones. Sequence analysis reveals that the open reading frame of the gene of the protease is 903 bp in size, encoding a putative protein of 300 amino acids with 30 kD. The overall GC% and the third codon GC% of the ORF were 68.33% and 95.6%, respectively. Comparison of homologue showed that the purative protein is highly homologous with other proteases of Streptomyces sp.

Amino Acid Sequence↗
PubMed2001

[The construction of recombinant plasmids of Toxoplasma gondii P30 and the immunoprotective activity of the P30 constructs].

OBJECTIVE: To identify the potential DNA vaccine candidate which can induce the protective immune response to Toxoplasma gondii by inoculating mice with plasmid DNAs encoding three different forms of P30 antigen (membranous secretory, and intracellular). METHODS: Three forms of recombinant plasmid: pcDNA3-P30Mb(contain the whole P30 gene sequence, including the gene encoding signal peptide and hydrophobic tail), pcDNA3-P30Se(contain the whole P30 gene sequence, without the gene encoding hydrophobic tail) and pcDNA3-P30In(contain the whole P30 gene sequence, without the gene encoding signal peptide) were constructed by PCR and subcloning technique. The mice were immunized with different forms of recombinant plasmids and IgG antibodies in the mice were detected by ELISA and Western blotting. RESULTS: Three forms of expression recombinant plasmid of Toxoplasma gondii P30 gene were successfully constructed. The P30 inserts were identified by restrictive enzyme digestion and sequencing. ELISA and Western blotting analysis demonstrated that specific IgG antibody could be induced in three immunized groups, but there was some difference in appearence time and intensity of IgG. CONCLUSION: Genetically immunization of mice with the recombinant plasmids could elicit specific IgG antibodies. In respect to IgG response, the immune efficiency of the three forms of recombinant plasmids was different at the beginning (2 wk), but 4 wk later approximately same.

Animals↗
PubMed2001

[Conjugation of mitoxantrone-loaded nanospheres and anti-C-erbB-2 monoclonal antibodies].

AIM: To improve the treatment efficacy of anti-tumor drug mitoxantrone, the conjugation of mitoxantrone-loaded nanospheres and anti-C-erbB-2 monoclonal antibodies were prepared. METHODS: Mitoxantrone-loaded nanospheres were prepared with emulsion-heating solidification technique. A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), was used as the crosslinker of mitoxantrone-loaded nanospheres and anti-C-erbB-2 monoclonal antibodies; pharmaceutical properties of immunonanocapsuls were studied; the conjugates of nanospheres and monoclonal antibodies was confirmed with immunological methods such as slide agglutination test, fluorescent immunossay and rosset formation test, fluorescent staining and scanning electron microscope. RESULTS: Mitoxantrone-loaded nanospheres were spherical, with smooth surface and median diameter of 0.665 micron. When stored at 3-5, 20-25 and 37 degrees C, RH 75% for three months, the appearance, morphology, size distribution, drug loading and in vitro release characteristics showed no significant change and the stability was satisfactory. The size analysis demonstrated that there was no obvious increase in the particle size of nanoparticles after conjugation. Immunological tests indicate highly selective binding of antibody-targeted nanospheres to C-erbB-2-overexpressing cells SK-BR-3. CONCLUSION: The conjugation of mitoxantrone-loaded nanospheres and anti-C-erbB-2 monoclonal antibodies can keep the activity of anti-C-erbB-2 and increase the therapeutic efficacy of anti-mammary cancer drugs.

Antibodies, Monoclonal↗
PubMed2001

[Effect of radix angelicae sinensis decoction for supplementing blood on inhibiting the increase of endothelial cell monolayer permeability induced by hypoosmotic solution].

OBJECTIVE: To study effect of radix Angelicae sinensis decoction for supplementing blood (RASDSB) on inhibiting the increase of endothelial cell monolayer permeability induced by hypoosmotic solution. METHODS: The endothelial cells isolated from newborn bovine aorta were cultured on polycarbonate microporous filter membrane to develop compact endothelial monolayer. Fluid filtration coefficient (Kf), filtration volume (Jv) and osmotic reflective coefficient (sigma) to protein of the endothelial monolayer were measured treated by hypoosmotic solution (changing concentration of serum in M199 solution from 20% to 2%) for 120 min or by hypoosmotic solution containing 10(-4) g x ml(-1) RASDSB for 120 min after perfused Hanks balanced salt solution containing 5 g x L(-1) albumin. RESULTS: Kf and Jv of the endothelial monolayer treated by hypoosmotic solution increased and sigma of that decreased. RASDSB could inhibit above-mentioned change. Morphological analysis demonstrated that RASDSB could inhibit widening of intercellular distance and enlargement of cellular area in the endothelial monolayer induced by hypoosmolality. CONCLUSION: Hypoosmotic solution could increase endothelial cell monolayer permeability and RASDSB could inhibit the increase.

Angelica sinensis↗
PubMed2000

[Effect of tea polyphenols and tea pigments on inducing the activity of phase II detoxicating enzymes and on the chemoprevention of liver precancerous lesions].

In order to elucidate the chemoprevention mechanism of tea, the regulative effect of tea polyphenols and tea pigments on phase II enzyme, glutathions S-transferase (GST) activity and its isoenzymes in Wistar rat were studied. Rat liver precancerous lesions were initiated with diethylnitrosamine (DEN), promoted with partial hepatectomy and carbon tetrachloride. The rats were killed after 8 weeks of treatment. Liver cytosol GST activities were tested. The GST 1-1, 1-2, 3-3 and Pi isoenzymes were detected with Western Blot method. The results showed that the activities of GST increased greatly in the tea polyphenol and tea pigment groups, while the activities were decreased in positive control group. Both tea polyphenold and tea pigments increased the activities of classes GST alpha (GST1-1, 1-2) and GST mu(GST3-3). The increase of GST alpha was more in tea polyphenol group, and the increase of GST mu was more in tea pigment group. Both tea polyphenols and tea pigments inhibited the over expression of GST Pi in the rats treated with DEN/PH/CCl4. The study indicated that tea polyphenols and tea pigments modulated phase II enzymes effectively, thus inhibited the occurrence and development of the precancerous lesions of rat liver.

Animals↗
PubMed2000

Dissecting the nucleotide binding properties of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase with fluorescent 3'(2)'-o-anthraniloyladenosine 5'-triphosphate.

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7, 8-dihydropterin, the first reaction in the folate biosynthetic pathway. Like other enzymes in the folate pathway, HPPK is an ideal target for development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from humans and animals. Using 3'(2')-o-anthraniloyladenosine 5'-triphosphate as a fluorescent probe, a fluorometric competitive binding assay has been developed for measuring the dissociation constants of various compounds that bind to the ATP site of HPPK. The fluorometric assay has been used to determine the nucleotide specificity and dissect the energetics of the binding of MgATP. The order of affinity of various nucleoside triphosphates for HPPK is MgATP>MgGTP>MgITP>MgXTP approximately MgUTP approximately MgCTP. The affinity of MgATP for HPPK (K(d)=2.6+/-0.06 microM) is 260-fold higher than that of MgGTP and more than 1000-fold higher than those of the other nucleoside triphosphates, indicating that HPPK is highly specific with respect to the base moiety of the nucleotide. The affinity of ATP for HPPK in the presence of Mg(2+) is 15 times that in the absence of Mg(2+), indicating that the metal ion is important for the binding of the nucleotide. Removal of the gamma-phosphate from MgATP reduces its affinity for HPPK by a factor of approximately 21. The affinity of AMP for HPPK is about one third that of ADP and almost the same as that of adenosine. The result suggests that among the three phosphoryl groups of MgATP, the gamma-phosphoryl group is most critical for binding to HPPK and the alpha-phosphoryl group contributes little to the binding of the nucleotide. The affinity of MgATP is 18 times that of MgdATP, indicating that the 2'-hydroxyl group of MgATP is also important for binding. van't Hoff analysis suggests that binding of MgATP is mainly driven by enthalpy at 25 degrees C and the entropy of binding is also in favor of the formation of the HPPK.MgATP complex.

Binding, Competitive↗
PubMed2000

Solid-phase parallel synthesis of azarene pyrrolidinones as factor Xa inhibitors.

A focused library (4 x 14) prepared from 4-aminopyridine and 4-, 5-, and 6-azoindole templates was synthesized using 14 polymer supported 4-amido-2,3,5,6-tetrafluorophenyl (TFP) sulfonate esters inputs. Several compounds were identified as factor Xa inhibitors (IC50< or =0.1 microM) helping to establish the SAR among these four series of azarene pyrrolidinones.

Anticoagulants↗
PubMed2000

Amido-(propyl and allyl)-hydroxybenzamidines: development of achiral inhibitors of factor Xa.

The design, synthesis and SAR of amido-(propyl and allyl)-hydroxybenzamidine coagulation factor Xa inhibitors is described. These achiral inhibitors are selective for fXa vis a vis structurally related serine proteases and are readily prepared in 6-7 linear steps. The most potent member 9j (fXa Ki = 0.75 nM) is selective (>1000-fold) and an effective anticoagulant in mammalian plasma.

Animals↗
PubMed2000

Identification of the subcellular localization of daunorubicin in multidrug-resistant K562 cell line.

We examined the subcellular distribution of daunorubicin (DNR) in resistant K562 cell line which overexpress the P-glycoprotein by confocal laser scanning microscopy. Three fluorescent probes - Rhodamine123, neutral red, NBD-ceramide, which stain the mitochondria, lysosomes, Golgi apparatus respectively, were used to identify the nature of the subcellular compartment sequestering daunorubicin. In sensitive k562 cell line, nuclear and cytoplasmic DNR fluorescence was intense and diffuse. In contrast, resistant K562 cell line showed a different DNR distribution. A bright fluorescence signal was located in the perinuclear region and peripheral plasma, the nucleus and other cytoplasmic region appear as empty, as suggested by the distribution of fluorescent probe Rhodamine123 specifically for mitochondria. Verapamil, an effective resistance modulator in P-glycoprotein MDR cells, restored the DNR distribution closer to that in the parent cells. Golgic inhibitor brefeldin A and lysosomotropic agent chloroquine had little effect on drug sequestration. Our studies demonstrate that daunorubicin may be sequestered in mitochondrial compartment in the resistant cells and P-glycoprotein plays an important role on mediating DNR transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Effect of preoperative interventions on outcome following liver resection in a rat model of cirrhosis.

BACKGROUND/AIMS: High morbidity and mortality rates in cirrhotic patients undergoing resections for hepatocellular malignancies underscore the need for identifying a therapy that will decrease fibrosis or enhance hepatic regenerative activity in the perioperative period. Thus, in the present study, 104 carbon tetrachloride-induced cirrhotic rats received either saline (untreated cirrhotic controls) or one of the following agents that have been reported to decrease hepatic fibrosis or increase hepatic regeneration; pentoxifylline, ciprofloxacin or a traditional Chinese herbal remedy (TCHR). Twelve additional rats served as healthy, non-cirrhotic controls. METHODS: Treatments were administered daily by gavage for 4 weeks followed by a 70% partial hepatectomy. Hepatic fibrosis was documented at the time of surgery by computer-assisted quantitation of collagen content. Liver function and hepatic regenerative activity were documented 24 h post partial hepatectomy by serum bilirubin determinations and a combination of 3[H]-Thymidine incorporation into hepatic DNA and proliferating cell nuclear antigen (PCNA) quantitation, respectively. RESULTS: Compared to untreated cirrhotic controls (8.1 +/- 0.7%), fibrosis was significantly reduced in the pentoxifylline- and ciprofloxacin-treated groups (4.6 +/- 0.2%, p<0.005 and 5.5 +/- 0.6%, p<0.05) but unchanged in the TCHR-treated group (6.6 +/- 11.0%). Post-operatively, total serum bilirubin levels were lower in the pentoxifylline (1.40 +/- 0.15 mg/dl,p<0.01) and ciprofloxacin (1.87 +/- 0.25 mg/dl, p<0.05)-treated groups, but unchanged in the TCHR group (2.20 +/- 0.45 mg/dl), when compared to untreated cirrhotic controls (3.00 +/- 0.37 mg/dl). Hepatic regenerative activity was also significantly improved in the pentoxifylline-treated group (17.8 +/- 2.2 versus 9.9 +/- 1.9 DPM/microg DNA in untreated cirrhotic controls, p<0.05), but unchanged in the ciprofloxacin (16.1 +/- 1.8 DPM/microg DNA) and TCHR (10.9 +/- 1.2 DPM/microg DNA)-treated groups. PCNA protein determinations were in keeping with the 3[H]-Thymidine results CONCLUSIONS: Pre-operative pentoxifylline holds promise as a useful therapeutic intervention for patients with cirrhosis requiring hepatic resection.

Animals↗
PubMed2000

Increased GABAergic activity inhibits alpha-fetoprotein mRNA expression and the proliferative activity of the HepG2 human hepatocellular carcinoma cell line.

BACKGROUND/AIMS: Gamma aminobutyric acid (GABA) is a potent inhibitory neurotransmitter with growth regulatory properties. Recent data indicate that increased GABAergic activity inhibits hepatocyte proliferation in regenerating livers. In the present study, we aimed to investigate whether GABA inhibits the growth of malignant hepatocytes. METHODS: Increasing concentrations of muscimol (0.05-50 microM), a specific GABA(A) receptor agonist, were added to HepG2 human hepatocellular carcinoma cells and alpha-fetoprotein (AFP) and albumin mRNA expression were determined for varying periods of time (maximum 24 h) thereafter. Cell proliferation was also documented after 48 h of exposure to muscimol. RESULTS: Muscimol significantly (p<0.0001) decreased AFP mRNA expression (maximum decrease: 65% below baseline values) without affecting albumin mRNA expression. However, the effect on AFP mRNA was transient (maximum duration: 3-6 h) and not associated with changes in cell proliferation. Because preliminary data indicate that GABA(A) receptor activity is markedly downregulated in malignant hepatocytes, transfection studies were performed wherein HepG2 cells were cotransfected with GABA(A) receptor beta2 and beta2 subunit genes in a pCDM8 expression vector or vector alone followed by re-exposure to either muscimol (5 betaM) or saline. In this series of experiments, in addition to AFP mRNA inhibition being as extensive and more prolonged (maximum duration: 6-12 h) in muscimol-treated, GABA(A) receptor-transfected cells, proliferative activity was also significantly inhibited when compared to saline-treated GABA(A) receptor-transfected controls (p<0.01) and muscimol-treated cells transfected with vector alone (p<0.005). CONCLUSION: The results of this study indicate that increased GABAergic activity inhibits AFP mRNA expression and cell proliferation in this malignant hepatocyte cell line.

Albumins↗
PubMed2000

Spectroscopic study of trypsin, heat and triton X-100-induced denaturation of the chlorophyll-binding protein CP43.

Trypsin-, heat- and Triton X-100-induced denaturation of CP43, the core antenna complex of photosystem II purified from spinach, has been investigated using absorption, fluorescence and circular dichroism spectroscopy. Triton X-100 was found to bring about considerable dissolution of pigments from the protein to the monomeric state in solution and destruction of the interactions among the chlorophyll, carotene and protein. Heat induced significant unfolding of the protein secondary structure and loss of excitonic interactions of the pigments, but no apparent dissolution of the pigments from CP43. Trypsin caused structural changes in the extrinsic part of the protein but no change of the native state of the pigments. Trypsin, heat and Triton X-100 treatments increased the light sensitivity of chlorophyll in CP43 to different extents. The results suggest that the protein and beta-carotene can protect the chlorophyll from light-induced destruction in CP43.

Binding Sites↗
PubMed2000

Effect of freezing on the activity of catalase in apple flesh tissue.

Catalase (CAT, EC 1.11.1.6) activity was measured in flesh tissue of six apple cultivars (Malus domestica Borkh. cvs. Braeburn, Gala, Jonagold, McIntosh, Red Delicious, and Spartan). Activity of CAT was determined for fresh and frozen tissue of the same fruit. Freezing resulted in reductions of 50 to 90% in CAT activity compared with the activity measured in crude extracts from fresh tissues. The rate of freezing had an impact on the level of reduction of CAT activity, with slower freezing procedures leading to greater losses in activity. Six additives to the extraction buffer were tested to evaluate their potential to reduce the inactivation of CAT from frozen tissue, but only EDTA and Tween 20 showed any benefit. However, EDTA and Tween 20 provided only partial recovery in CAT activity. In contrast, crude extracts prepared from fresh tissue showed no appreciable loss in CAT activity after frozen storage for two weeks at -80 degrees C. Gel electrophoresis and immunological analysis indicated that the loss in CAT activity from tissue freezing could be attributed to loss of both the tetrameric CAT enzyme structure and total CAT protein. The implications of using freezing to preserve apple tissue samples prior to catalase activity analysis is discussed.

Catalase↗
PubMed2000

The expression of insulin-like growth factor binding proteins in human hepatocellular carcinoma.

Insulin-like growth factors (IGF), IGF receptors and IGF binding proteins (IGFBPs) play an important role in cell growth and differentiation. The liver is the major source of IGF-1 and at least two IGFBPs (IGFBP-1 and IGFBP-3). IGFBPs most often serve to attenuate the effects of IGF at the receptor level and thereby limit IGF-induced cell growth and differentiation. Although changes in IGFBP expression have been described during controlled liver growth such as hepatic regeneration following partial hepatectomy, there is limited knowledge of IGFBPs gene expression in uncontrolled growth or hepatocellular carcinoma. In the present study, we employed Northern blotting techniques to document the expression of IGFBP-1, 3 and 4 in normal human livers, cirrhotic and hepatocellular carcinoma tissues. The results revealed no differences in IGFBP-1, 3 and 4 mRNA levels between normal and cirrhotic tissues. However, the expression of all three IGFBPs mRNA were significantly down regulated in hepatocellular carcinoma tissues. These findings are in keeping with IGFBPs playing an important inhibitory role in the development and/or growth of hepatocellular carcinoma in humans.

Blotting, Northern↗
PubMed2000

The effects of acute ethanol exposure on inhibitors of hepatic regenerative activity in the rat.

The purpose of this study was to identify the mechanism(s) whereby acute ethanol exposure inhibits hepatic regenerative activity in the rat. Adult, male, Sprague-Dawley rats (200-250 g) were randomized to receive either ethanol (1 g/kg i.p. q 4 h) or an equal volume of saline (controls) for 24 h beginning 1 h prior to a 70% partial hepatectomy (PHx). At 0, 3, 6, 12 and 24 h post-PHx, rats were sacrificed (N = 4-6/group), and the expression of the following genes associated with inhibition of hepatocyte proliferation were documented; p53, p21, transforming growth factor-beta1 (TGF-beta1) and gamma aminobutyric acid transport protein (GABA-TP). Inhibition of hepatic regenerative activity was confirmed by 3H-thymidine incorporation into hepatic DNA at 24 h post-PHx. The results of the study revealed that in ethanol-treated rats, DNA synthesis was inhibited by 37% when compared to saline-treated controls (p < 0.01). Regarding suppressor gene expression, both p21 and TGF-beta1 mRNA expression in ethanol-treated rats were similar to those obtained in saline-treated controls. Although p53 mRNA expression differed in the two groups, in the ethanol-treated group, p53 mRNA expression was decreased rather than increased (relative to controls) at 24 h post-PHx, a finding not in keeping with inhibition of DNA synthesis. GABA-TP mRNA was strongly expressed prior to PHx in both ethanol- and saline-treated rats. Following PHx, GABA-TP mRNA expression decreased in both groups but remained low in the saline-treated group while returning to pre-PHx values in ethanol-treated rats. In summary, the results of this study indicate that the inhibitory effects of ethanol on hepatic regeneration are not associated with significant or the appropriate changes in mRNA expression of the p53, p21 or TGF-beta1 suppressor genes. On the other hand, transcriptional changes in GABA-TP gene expression post-PHx are in keeping with an inhibitory effect of GABA on hepatic regeneration.

Animals↗