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Biomedical subjects

T H Zhou

Publications and source records attributed to T H Zhou.

At least 19 recordsLinked to original sources
PubMed2001

Production, purification, and characterization of an intracellular aflatoxin-detoxifizyme from Armillariella tabescens (E-20).

Some Armillariella tabescens (E-20) multienzymes have previously been reported to present detoxifying activities against aflatoxins. In this paper, we describe the isolation purification of an intracellular enzyme, named aflatoxin-detoxifizyme, which exhibited detoxification activity on aflatoxin B(1) (AFB(1)). This aflatoxin-detoxifizyme exhibited a specific activity of 7.09 nmol min/mg at pH 6.0 and 28 degrees C. The apparent molecular mass was 51.8 kDa as determined by SDS-PAGE. The isoelectric point was estimated to be 5.4 and optimum activity for the enzyme was found at pH 6.8 and 35 degrees C. The activity of the purified enzyme was confirmed by Ames test.

Aflatoxin B1↗
PubMed2001

[Cloning and expression of a synthetic gene encoding magainin-melittin hybrid peptide in Escherichia coli and studies on its antibacterial activity].

A hybrid peptide gene was designed and synthesized. Its encoding peptide is constructed from residues 3-14 of magainin and residues 1-13 of melittin. The MA-E gene was cloned into plasmids pUC18 and pBV220. By DNA sequencing, the whole sequences of this gene is confirmed to be correct. The recombinant plasmid pBMA-E was expressed in E. coli DH5 alpha. A gene product band can be seen with Tricine-SDS-PAGE. The MA-E hybrid peptide was purified by immobilized metal affinity chromatography. Bioactivity assay was carried out in liquid turbidity method. The bactericide value to E. coli K12D31 is 0.182.

Antimicrobial Cationic Peptides↗
PubMed2001

[Study on the structure of epimerides of cycloclausenamide].

AIM: To study the structures of the epimerides of cycloclausenamide. METHODS: The structures of compound I, extracted from Clausena lansium (Lour.) Skeels, and synthesized compound III were determined by single crystal X-ray diffraction analysis. The stereo-structures of compound II and IV were also built up through Tripos force field based on crystal structures of compound I and III. RESULTS: The molecular formula and molecular weight were found to be C18H17O2N and 279.34 respectively. Compound I crystallized in monoclinic system, space group P2(1) with a = 0.5928(1), b = 1.5014(1), c = 1.6190(1) nm, V = 1.4410(3) nm3, Z = 4, Dx = 1.288 g.cm-3, Rf = 0.075, Rw = 0.073(w = 1/sigma 2|F|), S = 3.983; compound III crystallized in triclinic system, space group P1 with a = 0.5667(1), b = 1.2934(1), c = 2.1119 (1) nm, alpha = 102.17(1), beta = 90.25(1), gamma = 102.65(2) degrees, V = 1.4770(5) nm3, Z = 4, Dx = 1.224 g.cm-3, Rf = 0.047, Rw = 0.051(w = 1/sigma 2|F|), S = 0.467. CONCLUSION: These results showed that compound I and III both are cycloclausenamide except that the directions of the phenyl group on C6 are different. Cycloclausenamide can form 4 pairs of epimerides but the directions of the phenyl group does not affect their energy in free state.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed2001

[In vitro metabolic studies of the novel anti-anxietic drug AF-5 and its metabolites in human liver microsome incubation system].

AIM: To study the metabolism of a novel anti-anxietic drug AF-5 and its metabolites (I, II) in human liver microsome incubation system. METHODS: Human liver microsomes were prepared, the enzyme activity was determined to be 8.79 mg.mL-1 by Lowry's method. The human liver microsome incubation system consisted of: human liver microsomes 2 mg.mL-1, glucose-6-phosphate (G-6-P) 0.01 mmol.mL-1, glucose-6-phosphate dehydrogenase (G-6-PDH) 1 U.mL-1, magnesium chloride (MgCl2) 4.0 mumol.mL-1, coenzyme II in oxidized form (NADP) 0.5 mumol.mL-1, and coenzyme I in reduced form (NADH) 1.0 mumol.mL-1. Two milligrams of AF-5 solubilized by Tween 80 was then added, the mixture was diluted to 5 mL with Tris-HCl solution and the mixture was incubated in a 37 degrees C water bath with shaking. Oxygen was passed over the liquid surface for 0.5 min every 20 minutes. The incubation was carried out for 40 min and 100 min respectively. Three volumes of ethyl ether were added to stop the metabolism, and more ethyl ether was used to extract the metabolites for 3 times. The ether extracts were pooled together, dried with anhydrous sodium sulfate, then evaporated to dryness. The residue was dissolved in 0.5 mL n-hexane and analyzed by GC/MS under the following conditions: 150 degrees C (1 min)[formula: see text]180 degrees C (1 min)[formula: see text]260 degrees C (2 min), in the total ion current mode, EI: 70 eV, interface temperature: 250 degrees C, ion source temperature: 200 degrees C. RESULTS: Two major metabolites were found and identified in this incubation system, and demonstrated that the in vitro metabolic pathway was that the carbon 4 was first oxidized to hydroxyl group, then further oxidized to a carbonyl group. CONCLUSION: In human liver microsome incubation system AF-5 was completely metabolized in 100 min to the hydroxy derivative I and carbonyl derivative II, with hydroxymetabolite as the major metabolite. Metabolite I was further transformed to metabolite II, which was not metabolized any further by the human liver microsomes.

Anti-Anxiety Agents↗
PubMed2001

[Role of analytical chemistry in the development of new drugs].

In this paper, the role of analytical chemistry in drug research, its development and some basic research related to developing new drugs are concisely discussed, including the establishment of quality control standards, finger prints of natural medicine products, resolution of chiral compounds and drug metabolism studies. The incorrect point of view of some people on pharmaceutical analysis is mentioned and the indispensability of this discipline in drug research and development is also emphasized.

Chemistry Techniques, Analytical↗
PubMed2000

Significant down-regulation of alpha-albumin in human hepatoma and its implication.

The potential association of alpha-albumin (ALF) with hepatocellular carcinoma (HCC) was investigated. Expression of ALF was significantly reduced in HCC tumor tissue as compared with the paired peritumor tissue from 16 patients and in four HCC cell lines as compared with normal hepatocytes. ALF mRNA was also down-expressed in circulating HCC cells compared to circulating normal hepatocytes. The proliferation of Hep3B cells was inhibited by over-expression of ALF. Taken together, ALF is significantly down-regulated in HCC, and this might facilitate the proliferation of HCC. Thus, detection of ALF mRNA, in addition to that of alpha-fetoprotein (AFP) mRNA, might help to distinguish normal or malignant hepatocytes in peripheral blood.

Albumins↗
PubMed2000

Identification of alternative splicing variants of the beta subunit of human Ca(2+)/calmodulin-dependent protein kinase II with different activities.

The beta subunit of human Ca(2+)/calmodulin-dependent protein kinase II (beta CaMKII) was identified by searching through an expressed sequence tag database and rapid amplification of cDNA 5'-ends and was assigned to chromosome 7. Reverse transcription-polymerase chain reaction and sequencing analysis identified at least five alternative splicing variants of beta CaMKII (beta, beta6, betae, beta'e, and beta7) in brain and two of them (beta6 and beta7) were first detected in any species. When expressed in HEK 293 cells, the Ca(2+)/calmodulin-dependent kinase activity of beta7, the shortest variant, was much lower than that of either beta (the longest one) or betae (the medium one), suggesting possible regulation of beta CaMKII activity by alternative splicing.

Alternative Splicing↗
PubMed2000

Identification of a human brain-specific isoform of mammalian STE20-like kinase 3 that is regulated by cAMP-dependent protein kinase.

A novel isoform of mammalian STE20-like kinase 3 (MST3) with a different 5' coding region from MST3, termed MST3b, was identified by searching through expressed sequence tag data base and obtained by rapid amplification of cDNA 5'-ends. MST3b was assigned to the long arm of human chromosome 13, D13S159-D13S280, by use of the National Center for Biotechnology Information sequence-tagged sites data base. Reverse transcription-polymerase chain reaction and Northern blot analysis with a probe derived from 5' distinct sequence of MST3b revealed that the expression of MST3b mRNA is restricted to the brain, in contrast to ubiquitous distribution of MST3 transcript. Western analysis confirmed the brain-specific expression of MST3b protein. In situ hybridization of rat brain sections with a MST3b-specific probe indicated that MST3b is widely expressed in different brain regions, with especially high expression in hippocampus and cerebral cortex. When expressed in human embryonic kidney 293 (HEK293) cells, MST3b effectively phosphorylated myelin basic protein, as well as undergoing autophosphorylation. Interestingly, expression of MST3, but not MST3b, in HEK293 cells was able to activate the endogenous p42/44 mitogen-activated protein kinase (MAPK) up to 4-fold, whereas neither isoform activated p38 MAPK under the same conditions. Further experiments demonstrated that MST3b, but not MST3, was effectively phosphorylated by activation of cyclic AMP-dependent protein kinase (PKA) in both in vivo and in vitro assays. The mutation of Thr-18 into Ala in MST3b (T18A), a putative PKA phosphorylation site that is absent in MST3, abolished its phosphorylation by PKA. Consequently, expression of the T18A mutant in HEK293 cells led to partial activation of p42/44 MAPK, indicating that MST3b is under the regulation of PKA. Taken together, our data provide evidence that the two isoforms of STE20-like kinase 3 are differentially distributed and regulated.

Animals↗
PubMed2000

[The function of T7 promoter as cis-acting elements for polymerase II in eukaryotic cell].

Using the chlorampheniol acetyltransterase gene as reporter, the function of phage T7 promoter in mammalian cells was studied by inhibition of transcription with alpha-amanitin. The experiment proved that the reporter under T7 promoter was transcribed by RNA polymerase II. Competitive electropho retic mobility shift assay (CEMSA) with TATA box, CAAT box, GC box and octamer showed that the TATA box was competitive molecular for synthetic T7 promoter. It is possible that T7 promoter is bound with TF II D transcription factor. The TATA box and octamer were inserted into Pvu II site upstream from the T7 promoter of pT7CAT. Two recombinant plasmids, pT7TATACAT and pT7OCTCAT, were constructed and transfected into CHO cells. CAT-activity test showed that T7 promoter strength was increased by octamer factor, not by TATA box. These results suggested that T7 promoter functions as cis-acting elements of RNA polymerase II transcriptional system in eucaryotic cells.

Bacteriophage T7↗
PubMed2000

[A simple method for preparing reversed-phase electrochromatographic column and its evaluation].

The packed column was prepared with a simple method by combination of vacuum filling and pressure compressing. The reproducibility of efficiency and retention time was evaluated. The mixture of thiourea and three aromatic compounds was used. RSD values of retention time were smaller than 2.2% and RSD values of efficiency were smaller than 7.5%. The influence of voltage, phosphate concentration, acetonitrile volume fraction and column temperature on the electroosmotic flow and capacity factor of neutral compounds was investigated. Acetonitrile concentration had significant effect on the electroosmotic flow and capacity factor of neutral compounds. High concentration of acetonitrile improved the elution ability. Low concentration of acetonitrile improved the resolution. Voltage, phosphate concentration, and column temperature also had effects on the electroosmotic flow and capacity factor of neutral compounds, but the effects were smaller than the concentration of acetonitrile. These results gave a guide to select separation conditions in capillary reversed-phase electrochromatography.

Benzene↗
PubMed2000

[Preparation and evaluation of polyvinyl pyrrolidone-coated columns based on sol-gel chemistry for capillary electrophoresis].

Polyvinyl pyrrolidone(PVP) coating techniques based on sol-gel chemistry was investigated in this article. Multiple-step coating and single-step coating methods were developed and the coating conditions(molar ratios of MAPS to 0.1 mol/L HCl, reaction times and concentration of PVP) were selected. Electroosmosis mobility and column efficiency were used as the evaluating parameters for coated capillary columns. For multiple-step coating, the optimal sublayer sol solution was obtained by adding 0.1 mol/L hydrochloric acid to MAPS(molar ratio, 1:1) and reacted at room temperature for 2 hours. The best PVP mass concentration was 40 g/L in alcohol. For single-step coating, the sol-gel solution was obtained by mixing the following ingredients: (a)MAPS, (b) 8% PVP alcohol solution and (c) 0.1 mol/L hydrochloric acid in 1:1 molar ratio to MAPS. The mixture was kept at room temperature for 2 hours. Proper amounts of 100 g/L ammonium persulfate and 10% (V/V) N, N, N', N'-tetramethylethylenediamine were then added. The mixture was mixed ultrasonically, then centrifuged at 4,000 r/min for 5 min. The upper clean sol-gel solution was transferred into another clean vial for coating capillary column. The reproducibility of migration times and efficiencies of single-step-coated capillary was better than that of multiple-step-coated capillary. Single-step-coating method was very effective and easy. It can fit the needs in experimental work. Coating techniques based on sol-gel chemistry are prospective but still need to be further developed.

Electrophoresis, Capillary↗
PubMed1999

[The sequence analysis of the 3' end of intron 15 from human LDL receptor gene and its application in the study of gene diagnosis for familial hypercholesterolaemia].

To understand intron 15 of human LDL receptor gene, the DNA fragments from exon 15 to exon 16 and the 3' end of intron 15 were amplified with long chain PCR and anchored PCR. The 3' end of intron 15 was sequenced with Dynalbeads-Streptavidin Solid Phase technique. The sequence analysis showed that the 3' end of intron 15 contained the 3' splicing site and the branch site at 31 nucleotides upstream of the 3' end. Besides the authentic branch site, it is possible that the 3' end of intron 15 contains a cryptic site (GCCTCAC) at 20 nucleotides upstream of the 3' end. The sequences suggest that the PvuII polymorphism at Intron 15 is caused by the T-C substitution. According to the sequences of the 3' end, the new PCR-RFLP protocol for detection of PvuII polymorphism at intron 15 was developed. Using this protocol a representative familial hypercholesterolaemia family was identified with linkage analysis of PvuII polymorphism at intron 15.

Base Sequence↗
PubMed1998

Suppression of phospholipase C blocks Gi-mediated inhibition of adenylyl cyclase activity.

The potential effect of inhibition of phospholipase C on the response of Gi-coupled receptors was investigated in neuroblastoma x glioma hybrid (NG108-15) cells. The phospholipase C specific inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H -pyrrole-2,5-dione (U73122), which did not affect basal and forskolin-stimulated adenylyl cyclase activities, time- and dose-dependently blocked delta-opioid receptor-mediated inhibition of adenylyl cyclase activity, the EC50 (0.5 microM) of which was consistent with that for inhibition of bradykinin-dependent phospholipase C activation (EC50 = 1 microM). U73122 treatment also blocked functional responses of m4 muscarinic receptor and alpha2-adrenoceptor in NG108-15 cells and three opioid receptors (mu, delta and opioid receptor-like receptor (ORL1)) in human neuroblastoma SK-N-SH cells. 1-[6-((17Beta-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2, 5-pyrrolidinedione (U73343), an inactive analog of U73122, did not show any effect, which suggests that the blockade by U73122 of Gi-coupled receptor-mediated signaling is probably mediated through inhibition of phospholipase C, although a possible direct modification of G proteins can not be excluded. Furthermore, treatment with U73122 but not U73343 blocked the GTP-induced inhibition of adenylyl cyclase, indicating blockade at the level of Gi proteins.

Adenylyl Cyclases↗
PubMed1998

Carboxyl terminal of rhodopsin kinase is required for the phosphorylation of photo-activated rhodopsin.

Human rhodopsin kinase (RK) and a carboxyl terminus-truncated mutant RK lacking the last 59 amino acids (RKC) were expressed in human embryonic kidney 293 cells to investigate the role of the carboxyl terminus of RK in recognition and phosphorylation of rhodopsin. RKC, like the wild-type RK, was detected in both plasma membranes and cytosolic fractions. The C-terminal truncated rhodopsin kinase was unable to phosphorylate photo-activated rhodopsin, but possesses kinase activity similar to the wild-type RK in phosphorylation of small peptide substrate. It suggests that the truncation did not disturb the gross structures of RK catalytic domain. Our results also show that RKC failed to translocate to photo-activated rod out segments. Taken together, our study demonstrate the carboxyl terminus of RK is required for phosphorylation of photo-activated rhodopsin and strongly indicate that carboxyl-terminus of RK may be involved in interaction with photo-activated rhodopsin.

Cell Line↗
PubMed1997

Functional expression of opioid receptor-like receptor and its endogenous specific agonist nociceptin/orphanin FQ during mouse embryogenesis.

Expression of opioid receptor-like receptor (ORL1) and its endogenous peptide agonist nociceptin/orphanin FQ (N/OFQ) during mouse embryogenesis have been investigated. Transcripts of ORL1 and N/OFQ were detected by RT-PCR in mouse brain of day 8 embryo (E8) and the expression continued afterwards. Northern blot analysis revealed abundant expression of ORL1 at postnatal day 1 (P1) and N/OFQ at E17 and P1 in the brain but none was detected in other embryonic tissues. The presence of functional ORL1 in mouse embryonic brain was also confirmed by specific binding of [3H] N/OFQ (kd = 1.3 +/- 0.5 nM and Bmax = 72 +/- 9 fmol/mg protein) as well as by N/OFQ-stimulated G protein activation.

Animals↗
PubMed1997

Retardation of pain development: a case of recovery from congenital insensitivity to pain.

Congenital analgesia is a rare genetic disorder. We report here that a 12-year-old boy was able to recover from congenital insensitivity to pain. Neurological examinations revealed that there was a 'stocking' distribution of pain decrement on the lower extremities under the patient's knee joints. Magnetic Resonance Imaging (MRI) of his brain showed gyrus thinning with sulcus widening at both sides of the parietal lobe. Southern blot hybridization probed with cDNAs of various opioid receptors did not detect any significant abnormality. Our results suggest that this rare case may not be genetically determined.

Arthropathy, Neurogenic↗
PubMed1997

[The in vitro metabolism of hainanensine derivative HH07A by rat liver microsomes].

The metabolism of hainanensine derivative HH07A has been studied in vitro with rat microsomes. A method of HPLC-DAD was developed for screening the metabolites from the microsomal incubation system. Two metabolites were found according to their UV spectra. One of them was extracted and purified with preparative HPLC and TLC, its chemical structure was identified by UV, IR, MS and NMR.

Animals↗