Aged ; China ; Drug Resistance, Bacterial ; Humans ; L Forms ; drug effects ; Middle Aged ; Mining ; Mutation ; Mycobacterium tuberculosis ; drug effects ; Silicotuberculosis ; microbiology

Adult ; Animals ; Asthma ; epidemiology ; etiology ; Female ; Humans ; Immunoglobulin E ; blood ; Male ; Occupational Diseases ; epidemiology ; etiology ; Pyroglyphidae ; Skin Tests

Objective To study the lysozyme activity in Oncomelania hupensis and observe its inhibitory effect on bacterial growth.Methods Soft tissues of Oncomelania hupensis were initially homogenized and immersed in Tris-HCl-TritonX-114 buffer solution for 24 hours then the supernatant was collected after centrifugation at 10 000 × g for 10 minutes.The supernatant was incubated in a 37 ℃ water bath for 15 minutes and centrifuged again at 2000 × g for 10 minutes.The precipitate was put into ultrafiltration tube (relative retention molecular mass =3000) and centrifuged at 4 ℃,5 000 × g for 30 minute to obtain concentrated enzyme.The protein content,lysozyme activity and the antibacterial effect on Micrococcus lysodeikticus,Shigella dysenteriae,Staphylococcus aureus,Escherichia coli and Candida albicans were measured with bicinchonininc acid(BCA) method,turbidimetric method and agar diffusion (K-B) method,respectively.Results The antibacterial protein lysozyme was identified in gastropod protein concentration of the concentrated enzyme was 3.428 g/L.Average activity,total activity,and specific activity were (760 ± 120) × 103 U/L,(1520 ± 240) × 103 U/L and (221.70 ± 35.00)U/mg,respectively.The enzyme had produced exclusive inhibitory effects on growth of Micrococcus lysodeikticus and Shigella dysenteriae.Average inhibitory diameters were 10-12 and 12-15 mm,respectively.No inhibition zone was observed in saline control,Staphylococcus aureus,Escherichia coli and Candida albicans.Conclusions Lysozyme can be extracted from soft tissues of Oncomelania hupensis with Tris-HCl-TritonX-114 buffer solution,and the enzyme has inhibitory effect on growth of Micrococcus lysodeikticus and Shigella dysenteriae but has no antibacterial effect on Staphylococcus aureus,Escherichia coli and Candida albicans.

Objective To investigate the infestation status and environment tolerance of ornithonyssus bacoti (O. bacoti) in stored traditional Chinese herb Semen Coicis (Coix seed). Methods 800 g of stored Semen Coicis was initially taken, and subjected to passing a 40-mesh sampling sieve. Then 80 g of Coix seed particles was sampled to directly isolate the mites that were made into slide specimen in conventional technique, and identified as the procedures described in previous literature. Results A total of 449 mites were isolated from the 80 g stored Semen Coicis particles, and were identified as O. bacoti. Conclusions O.bacoti was first detected in the stored traditional Chinese medicinal Semen Coicis with which infestation is serious. This status should call out attention to prevent the disease associated with transmission of pathogens from O.bacoti.

OBJECTIVETo clone tenecin gene, an antibacterial peptide gene, from Tenebrio molitor for its prokaryotic expression and explore the molecular mechanism for regulating the expression of antibacterial peptide in Tenebrio molitor larvae.

METHODSThe antibacterial peptide was induced from the larvae of Tenebrio molitor by intraperitoneal injection of Escherichia coli DH-5α (1×10(8)/ml). RT-PCR was performed 72 h after the injection to clone Tenecin gene followed by sequencing and bioinformatic analysis. The recombinant expression vector pET-28a(+)-Tenecin was constructed and transformed into E. coli BL21(DE3) cells and the expression of tenecin protein was observed after IPTG induction.

RESULTSTenecin expression was detected in transformed E.coli using SDS-PAGE after 1 mmol/L IPTG induction. Tenecin gene, which was about 255 bp in length, encoded Tenecin protein with a relative molecular mass of 9 kD. Incubation of E.coli with 80, 60, 40, and 20 µg/ml tenecin for 18 h resulted in a diameter of the inhibition zone of 25.1∓0.03, 20.7∓0.06, 17.2∓0.11 and 9.3∓0.04 mm, respectively.

CONCLUSIONSTenecin protein possesses strong antibacterial activity against E. coli DH-5α, which warrants further study of this protein for its potential as an antibacterial agent in clinical application.

Amino Acid Sequence ; Animals ; Anti-Bacterial Agents ; pharmacology ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; drug effects ; genetics ; metabolism ; Genetic Vectors ; genetics ; Insect Proteins ; biosynthesis ; genetics ; Larva ; chemistry ; Microbial Sensitivity Tests ; Molecular Sequence Data ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; pharmacology ; Tenebrio ; chemistry

OBJECTIVETo evaluate the biological activity of polysaccharide of Cipangopaludina chinensis (PCC) against HBV in vitro.

METHODHepG2 2. 2. 15 cells were taken as the in vitro experimental model. The cell toxicity was observed by MTT. PCC of different safe concentrations and positive control medicine 3TC were added into the cells. Cell control without medicine was set at the same time. Cultural supernatants were collected at 9 d. HBsAg and HBeAg in cultural supernatants were tested by ELISA. The content of HBV-DNA was detected by TaqMan probe fluorescence quantitative PCR.

RESULTTC0 and TC50 of PCC in HepG2 2. 2. 15 cell culture were 1 g . L-1 and >10 g . L-1, respectively, suggesting low toxicity in cells. IC50 of PCC in HepG2 2. 2. 15 cells HBsAg and HBeAg were 0. 501, 0. 401 g. L-1, with SI being >19.96 and >24. 94, respectively. PCC could effectively inhibit the secretion of HBsAg and HBeAg, and have a better effect on HBeAg than on HBsAg. PCC had a significant inhibitory effect on HBV-DNA in HepG2 2. 2. 15 cells at concentrations of 0. 1, 1 g . L-1 P <0.05).

CONCLUSIONPCC has the effect against HBV activity in vitro to some extent, with low toxicity, thereby having a good prospect for application.

Animals ; Antiviral Agents ; adverse effects ; pharmacology ; DNA, Viral ; metabolism ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; drug effects ; metabolism ; Humans ; Polysaccharides ; adverse effects ; pharmacology ; Snails ; chemistry

OBJECTIVETo evaluate the replication and encapsidation of HBV mutants with the truncated C gene.

METHODSThe HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro. The replication and encapsidation of HBV mutants were investigated by Southern blotting, PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome.

RESULTSThe C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication defective, however, all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay.

CONCLUSIONThe C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely, however, the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-transfected with the helper construct which lacks part of 5 prime-proximal HBV RNA packaging signal Epsilon.

Cell Line, Tumor ; Hepatitis B Core Antigens ; genetics ; Hepatitis B virus ; genetics ; physiology ; Humans ; Mutation ; Plasmids ; genetics ; Transfection ; Virus Replication

OBJECTIVE: To investigate the feasibility of applying multiple displacement amplification (MDA) to DNA typing in forensic pathological section. METHODS: Ninety-eight pieces of pathological sections were prepared in terms of 3 factors as the period of preservation, tissue types and death ages, and randomized into groups by Latin square by double 7-order design. Silicon bead method was used to extract the DNA template. Compared with the PCR amplification performed directly by AmpFlSTR Identifiler kit in the control group, MDA was performed before amplification in the experimental group. Based on the samples from fresh autopsies as the standard genotypes, the number of detection and the detection rate were analyzed and compared between the experimental group and the control group. RESULTS: Between the control group and the experimental group, there was significantly statistical difference regarding the rate of DNA typing in each period of the tissue sections preserved (P<0.01). The detection rate of the 16 loci in the experimental group was more than 95% when the period of the tissue sections were preserved within 360d. There was significant difference in different tissue types (P<0.01). But there was no significant difference in different death ages (P>0.01). CONCLUSION MDA is efficacious in DNA typing of forensic pathological sections, for it can improve the DNA template quantification through abating the inhibiting factor's concentration of PCR and reducing the rate of allele drop out (ADO). However, the period of the sections preserved and tissue types would affect the results of genotyping by MDA.
Adolescent ; Adult ; Age Factors ; Brain Chemistry ; Cadaver ; Child ; Child, Preschool ; DNA/genetics* ; DNA Fingerprinting/methods* ; Feasibility Studies ; Forensic Pathology/methods* ; Frozen Sections ; Genetic Loci ; Genotype ; Humans ; Infant ; Kidney ; Liver ; Loss of Heterozygosity/genetics* ; Middle Aged ; Nucleic Acid Amplification Techniques/methods* ; Polymerase Chain Reaction/methods* ; Preservation, Biological/methods* ; Time Factors ; Young Adult

Objective To study the drug-resistant characteristics genetic mutation of rpoB in Mycobacterium tuberculosis L-forms among patients of pneumoconiosis complicated with pulmonary tuberculosis. Methods A total of 42 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected, including 31 drug-resistant strains. Their genomes DNA were extracted and target genes amplified by PCR. Hot regions in the rpoB gene were analyzed by automated DNA sequenator. Results No mutation of rpoB was identified in 11 rifampicin-sensitive strains while conformation changes were fotmd in 31 rifampicin-resistant strains. The mutation rate was 93.55% (29/31) in resistant strains, mainly concentrated in codon 531 (51.6%, 16/31) and 526 (32.26%, 10/31), happened base substitutions, including 27 unit point mutation and 2 two point mutation. The newly found mutation of codon 516 had not been reported by internal or overseas scholars. Conclusion The substitution of highly conserved amino acids encoded by rpoB gene resulted in the molecular mechanism was responsible for RFP resistance in Mycobacterium tuberculosis L-forms. It also proved that rpoB gene was in diversiform.

OBJECTIVETo study the characteristics of drug-resistant genetic mutation of rpoB in multiple drugs resistant bacillus tuberculosis (MDR-TB) among patients of pneumoconiosis complicated with pulmonary tuberculosis.

METHODSA total of 114 clinical isolated strains of Mycobacterium tuberculosis were collected, MDR-TB were identified by conventional antimicrobial susceptibility test (AST). Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator.

RESULTSThe results by AST showed that there were 31 strains of MDR-TB in the 114 clinical isolated strains, the rate of drug resistance was 27.19% (31/114). No mutation of rpoB was identified in 10 rifampicin-sensitive strains that randomly chosen, while conformation changes were found in MDR-TB strains, and the mutation rate of rpoB was 93.55% (29/31) in resistant strains, mainly concentrated in codon 531 (45.16%, 14/31) and 526 (29.03%, 9/31), happened base substitutions, including 27 unit point mutation and 2 two point mutation.

CONCLUSIONSThe substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for RFP resistance in MDR-TB among patients of pneumoconiosis complicated with pulmonary tuberculosis. It also proves that rpoB gene is diversiform.

Adult ; Aged ; Bacterial Proteins ; genetics ; DNA-Directed RNA Polymerases ; Drug Resistance, Multiple, Bacterial ; genetics ; Genes, Bacterial ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Mining ; Mutation Rate ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Pneumoconiosis ; microbiology ; Sequence Analysis ; Tuberculosis, Pulmonary ; microbiology