Objective To study the relationship between hepatitis B virus(HBV)pre-S gene mutations and progression of liver disease.Methods The entire pre-S1,pre-S2 genes were amplified hy nested polymerase chain reaction(PCR)and the products were digested by NlaⅢ.The method for detecting pre-S2 start codon mutation was established based on the digested restriction fragment length polymorphism(RFLP).Pre-S gene deletion was revealed by electrophoresis on polyacrylamide gel(PAGE).Pre-C A1896 and basic core promoter T1762/A1764 mutations were identified by direct sequencing of PCR products.The 138 sera from patients with HBV-related disease,including asymp- tomatic carriers(ASC),chronic hepatitis(CH),liver cirrhosis(LC),hepatocarcinoma(HCC),were tested by these methods.Results The detection rate of pre-S deletion mutation was higher in patients with HCC(56.3%)and LC(42.9%)than those with CH(11.8%)and ASC(8.1%,P

Objective To investigate the mechanism of BVT. 2733 on insulin resistance, by using diet-induced obese (DIO) mice model. Methods After having been balanced for 3 days, the C57BL/6J mice were randomly divided into a normal diet group and a high-fat diet (HFD) group. After 20 weeks, the obese mice were further randomly divided into an obese control group, a BVT. 2733 group and a pioglltazone (PGZ) group and they were orally administered with placebo, BVT. 2733 and PGZ separately for two weeks.Adiponectin and leptin mRNA expression levels from adipose tissue were analyzed with real-time quantitative PCR. The levels of plasma glucose, serum insulin and adiponectin were measured with biochemical technology, radioimmunoassay and ELISA. Adipocyte sizes were observed with immunohistocbemistry.Results The body weight, plasma glucose and serum insulin levels raised(P<0.05)in the HFD group and the adipocyte sizes were bigger. Serum insulin levels significantly reduced (P<0.05) and adipocyte sizes reduced, while plasma adiponectin level raised (P<0.01)in the two treatment groups as compared with those in obese controls. Both the mRNA expressions of adiponectin and leptin upregulated(P<0.05)in the PGZ group, but their expressions in the BVT. 2733 group did not alter significantly. The body weight of the mice reduced significantly in the BVT. 2733 group. Conclusion BVT. 2733 can reduce body weight significantly and improve insulin resistance, but cannot influence the expression of adipocytokines.

Objective To investigate the change of HO-1 expression in adipose tissue of obese young SD rats as well as its relationship with macrophage infiltration and polarization. Methods Three-week old SD rats (n=24) were randomly divided into 2 groups, routine diet group (NC) and high fat diet group (FC). After feeding 4 weeks, triglyceride (TG), high den?sity lipoprotein (HDL-C), fasting glucose and insulin were compared between these two groups and the insulin resistance in?dex was calculated. The gene expressions of HO-1, IL-6, IL-10 and MCP-1 were assessed by quantitative PCR. Infiltration and polarization of macrophages and M2 macrophages in the visceral adipose tissue were examined by immunohistochemis?try. Results The levels of FINS, FBG and HOMA-IR in rats of FC group were higher than those of rats in NC group after 4 weeks feeding (P<0.05). The level of HO-1, IL-6, MCP-1 in rats from FC group were significantly higher while level of IL-10 were lower compared with those in rats from NC group after 4 weeks of feeding (P<0.05). In samples from FC groups, more macrophages were detected in adipose tissue by DAB staining than those from NC group. There was no significant dif?ference (P>0.05) in MOD value of F4/80 and CD206 between these two groups (P>0.05). Conclusion The infiltration of macrophage in visceral adipose tissue of obese young SD rats significantly increased while HO-1 expression was reactively increased. This insinuated that HO-1 might play an important role in anti-inflammatory mechanism through regulating polar?ization of macrophages.

Adolescent ; Adult ; Aged ; Child ; DNA, Circular ; blood ; isolation & purification ; DNA, Viral ; blood ; isolation & purification ; Female ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Serologic Tests ; Young Adult

Objective To investigate the clinical significance of measuring resting energy expenditure (REE) for guiding an accurate nutritional support in elderly bedridden patients with nasal feeding.Methods The REE of 32 elderly bedridden patients with nasal feeding was assessed by using the Cosmed K4b2 portable telemetric gas analysis system.The waist-hip ratio, serum levels of albumin, transferrin, prealbumin and retinol-binding protein were determined to assess comprehensive nutrition status.The energy intakes were calculated, and the correlation of REE and the difference between the energy intakes and consumption with nutritional index were analyzed.Results The resting energy expendture was lower in the patients with waist-hip ratio≥0.95 than in patients with waist-hip ratio ＜0.95 (t=3.622, P＜0.01).The waist-hip ratio was reduced and serum albumin and transferrin levels were decreased along with the increase of REE in elderly patients (r=-0.55,-0.36 and-0.593, respectively, P=0.001, 0.043, ＜0.001).The difference between the energy intake and expenditure was higher in patients with waist-hip ratio≥0.95 than those with waist-hip ratio＜0.95 (t =5.643, P＜ 0.001).Serum albumin, prealbumin, transferrin and retinol-binding protein levels were increased along with the increase of the difference between the energy intake and expenditure, which showed the positive correlations (r=0.525, 0.409, 0.624, 0.414, respectively,P=0.002, 0.02, ＜0.001, 0.019).Conclusions Precise determination of REE and energy intake guided by REE are the important guarantees for the reasonable nutrition support in the elderly.

OBJECTIVETo establish a mismatched polymerase chain reaction restricted fragment length polymorphism (mPCR-RFLP) method for detection of hepatitis B virus (HBV) mutation in precore A1896, and compare with direct sequencing for evaluating its applicability.

METHODSAccording to the principle of mPCR, 194bp gene fragments in HBV precore region was amplified. The products of PCR were digested by Bsu36I and subjected to agarose gel electrophoresis. A method for detecting procore A1896 mutation was established by restricted fragment length polymorphism. Totally 134 sera were analyzed by both mPCR-RFLP and direct sequencing methods. Two sera which were identified having mixed infection with precore wild and mutant strains by mPCR-RFLP also were analyzed by cloning and sequencing.

RESULTSFrom 134 sera, 117 could be analyzed for HBV precore 1896 situation by mPCR-RFLP method, 109 could be analyzed by sequencing. In 101 sera which could be analyzed by the two methods, 54 were mutant strains and 47 were wild strains. The results of both methods were completely compatible. There was no significant difference in detective rate of HBV precore A1896 mutation between the two methods. The sequences of five clones from one serum which was identified precore mutant by mPCR-RFLP were all A1896 mutant strains. Another serum was identified as mixed infection by mPCR-RFLP, one clone was A1896 mutant strain and four were G1896 wild strains. The results of mPCR-RFLP were verified by cloning.

CONCLUSIONCompared with sequencing, the mPCR-RFLP method is simple, accurate and can be used in large-scale surveys and clinical research.

Adult ; Aged ; DNA, Viral ; blood ; genetics ; isolation & purification ; Female ; Genetic Heterogeneity ; Hepatitis B ; blood ; virology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B virus ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Young Adult

The proteins encoded by the Herpesviridae β-gene play a critical role in the replication stage of the virus. In this paper, phylogenetic analyses provided evidence that someβ-gene products, such as UL2 and UL23 from HSV1, have their homologous genes in its family, and also exist in prokaryotic organisms, indicating that these viruses appear to have been assembled over evolutionary time by numerous independent events of horizontal gene transfer.

Taking guizhi fuling capsule (GZFL) for instance, a new method about reference Chinese medicine preparation which was used as standard substance for the quality evaluation of complex Chinese medicine preparation by the fingerprint of reference preparation instead of standard fingerprint was proposed. It could eliminate the errors from different instruments, chromatographic columns and solve the problem of similarity matching in the absence of standard fingerprint. The qualification of reference GZFL was evaluated according to the quality control method of GZFL from Chinese Pharmacopoeia. Then multiple batches of GZFL were estimated, taking fingerprint of reference preparation and standard fingerprint as references, respectively, at different instruments and chromatographic columns. Finally, the packaging and expiration date for reference GZFL were confirmed according to the results of stability investigation. The results indicated that the fingerprint of reference GZFL could be used to assess the quality of GZFL better than standard fingerprint. The data of accelerated stability and long-term stability test demonstrated that reference GZFL was stable in the conditions of double blister package. Therefore, reference GZFL can be used as standard substance in quality control of GZFL.
Capsules ; Drug Stability ; Drugs, Chinese Herbal ; chemistry ; standards ; Reference Standards

OBJECTIVETo study the relationships between hepatitis B virus (HBV ) pre-S region mutations and their genotypes and the stages of liver disease of the patients.

METHODSThe entire HBV pre-S1 and pre-S2 genes were amplified by polymerase chain reaction (PCR). The amplified products were digested by NlaIII restriction enzyme. A detecting method for pre-S2 start codon mutation was established according to the restriction fragment length polymorphism (RFLP) analysis. Pre-S region deletion was revealed by polyacrylamide gel electrophoresis (PAGE). Fourteen sera having pre-S deletions or pre-S2 start codon mutations and wild strains were directly sequenced. HBV genotypes were determined by RFLP based on S-gene PCR products. One hundred sixty serum samples were collected from patients with HBV related diseases and they were determined by the above methods. The relationships between HBV pre-S region mutations and their genotypes and the stages of liver disease of the patients were analysed.

RESULTSOf the 160 sera, genotype B and C were identified in 81 and 79 respectively. The detected ratios of pre-S2 start codon and pre-S deletion mutations were significantly higher in genotype C than in genotype B (43.04% vs 1.23%, 36.71% vs 19.75%, P<0.05, respectively). The detection rates of pre-S2 start codon mutation were significantly different in different groups: from 50.00% (HCC), 39.47% (LC), 8.00% (CH), to ASC (0). The detection rates of pre-S deletion mutations among patients with HCC (53.13%), LC (42.11%), CH (18.00%) and ASC (7.50%) also varied significantly. The results obtained from sequencing and PCR-RFLP/PAGE were completely compatible. Multivariate analysis indicated that genotype C (OR=6.26, P<0.01) and advanced liver disease (OR=11.99, P<0.01) were significant variables for pre-S mutations development.

CONCLUSIONThe pre-S2 start codon and pre-S deletion mutations are more common in genotype C than in genotype B. These mutations are closely related to the progression of liver disease.

Adolescent ; Adult ; Aged ; China ; epidemiology ; DNA Mutational Analysis ; DNA, Viral ; genetics ; Female ; Gene Frequency ; Genotype ; Hepatitis B ; epidemiology ; virology ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B virus ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; Polymorphism, Restriction Fragment Length ; Young Adult

OBJECTIVETo establish a simple and accurate method for rapid detection of lamivudine related mutations in hepatitis B virus (HBV) polymerase gene.

METHODSHBV polymerase gene fragments of covering B and C active region were amplified by nested polymerase chain reaction (nPCR) or nested mismatched PCR. The PCR products were digested with Nde I or Nia III and subjected to electrophoresis on agarose gel, respectively. The patterns of restriction fragment length polymorphism (RFLP) were distinguished. Using this method, thirty patients with chronic hepatitis B and treated with lamivudine for at least one year were analysed for the lamivudine related mutations in polymerase gene. Sixteen cases without lamivudine therapy were used as controls. Some of the patients were also analysed by clone sequencing.

RESULTSThe nested mismatched PCR-RFLP method was simple, accurate and rapid. The whole experiments could be finished in eleven hours. The least titers of HBV DNA which could be detected was 10.3 copies/ml. The wild or mutant strains judged by RFLP were identified by clone sequencing. Mutation in the tyrosine methionine aspartic aspartic acid (YMDD) motif of HBV polymerase gene was found in eight patients and mutations of YMDD motif associated with L526M were found in another three patients. However, there were no such mutations in the control cases.

CONCLUSIONThe nested PCR-RFLP is considered as a simple and accurate method for rapid detection of lamivudine related mutations in HBV polymerase gene. It is suitable for larger number of sample detection.

Antiviral Agents ; pharmacology ; DNA-Directed DNA Polymerase ; genetics ; Drug Resistance, Viral ; genetics ; Hepatitis B virus ; drug effects ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length