In this paper, the number of rhizosphere and non-rhizosphere microbial organisms of fusarium wilt resistant and susceptible watermelon under soil culture and soilless substrate culture was studied by traditional culture methods. The results showed that, the number of rhizoshpere microorganisms was significantly higher than non-rhizosphere, and the number was changed with the stage of watermelon grow, the number was the lowest in seedling stage and increased with the watermelon grow, and achieved highest at the flowering and fruiting stage, decreased with the watermelon ageing. The fusarium wilt resistant of watermelon was correspondence with number of rhizosphere bacteria; the number of rhizosphere bacteria of resistant watermelon was higher than that of susceptible watermelon in each stage under soil culture and soilless culture. The fusarium wilt resistant of watermelon is no correspondence with number of rhizosphere fungi and actinomycete. The number of non-rhizosphere microbial organisms was changed in a small range in the whole growing stage. The non-rhizosphere bacteria have no significant change in the whole stage under soil culture and increased quickly under soilless substrate culture and decreased at the later stage. The non-rhizosphere fungi and actinomycete reached highest at the later stage under soil culture or soilless sub-strate culture.

The traditional culture methods and the molecular biology methods were used to study the rhizosphere bacterial diversity between fusarium wilt resistant and susceptible watermelon. The results showed that the diversity and the equality of cultured rhizosphere bacteria of resistant watermelon were higher than those of the susceptible watermelon. The reason was that the cultured rhizosphere bacterial di- versity index H′ and 1/D of the resistant watermelon were higher than those of the susceptible watermelon and that the cultured rhizosphere bacterial equality index E of the resistant watermelon were higher than those of the susceptible watermelon. The dominant cultured bacterial genotypes were different between re- sistant and susceptible watermelon. The genotype 1 is the dominant genotype of resistant watermelon, con- sists 51.1%. The genotype 7 is the dominant genotype of susceptible watermelon, consists 58.7%.

Objective To detect the effect of cell density off both integrin αγβ6 expression and matrix metalloproteinase-9(MMP-9)secretion in colon cancer cells. Methods Flow cytometry was applied to analyze αγβ6 expression in human WiDr colon cancer cell lines and human HaCaT keratinocyte cells, respectively,at high-and low-cell density culture.The MMP-9 aetivity level for various coloIl cancer cell lines, WiDr and SW480 cells at high-and low-cell density culture was analyzed using Biotrak MMP-9 activity assay and Gelatin Zymography assay, respectively. Results High cell density significantly enhances integrin αγβ6 expression for WiDr cells expressing αγβ6 compared with low density,but no increase was observed for human keratinocyte HaCaT cells. Biotrak MMP-9 assay indicated that the amount of MMP-9 secreted per cell for WiDr and SW480 B6 cells at high cell density culture was(3.3±1.2)×10-7ng/cell and(27.2±3.0)× 10-7ng/cell respectively; However,at low cell density it was(1.8±0.7)× 10-7ng/cell and(10.9±2.0)×10-7 ng/cell,respectively. It was 2-3-fold higher for WiDr and SW480 β6 cells at high cell density compared with that at low cell density, but no density-dependent increase observed for SW480 wild cells lack αγβ6 expression(t=0.47,P＞0.05),MMP-9 secretion for SW480 wild cells was(3.9±1.7)× 10-7 ng/cell at hish cell density and(3.8 ±0.7)×10-7 ng/cell at low eell density(P＞0.05),respectively. Gelatin zymography assay also indicated that the level of MMP-9 in SW480 B6 cells expressing αγβ6 was evidently higher at high density than at low density, however no density-dependent increase observed for SW480 wild cells and HaCaT cells. Conclusions High cell density induces integrin αγβ6 expression and promotes MMP-9 secretion in colon cancer cells, which constitutes the basis for a self-perpetuating system of tumor infiltrating growth in colon cancer progression.

OBJECTIVETo investigate the regulation trend of Jinxin Oral Liquid (JXOL) on the expression of negative regulatory factor of TLR3 signaling pathway SOCS1 in the lung tissue of RSV infected BALB/c mice at different time points.

METHODSTotally 75 BALB/c mice were randomly divided into 5 groups, i.e., the normal control group, the model group, the ribavirin group, the high dose JXOL group, and the equivalent dose JXOL group, 15 in each group. Each group had 3 intervention ways (I, II, and III) with 5 mice treated in each group. BALB/c mice were nasally infected with respiratory syncytial virus (RSV), and treated by different intervention ways. After intervention, mice were killed and their lung tissues were sampled, mRNA expression levels of RSV-M, SOCS1, and IFN-β were detected by Real time PCR. The expression of SOCSl at the protein level was detected by Western blot.

RESULTSCompared with the normal control group, the mRNA expression level of SOCS1 and IFN-β, and the protein expression level of SOCS1 increased significantly in the model group intervened by intervention I and II (all P < 0.01), but the mRNA expression level of IFN-β decreased significantly in model group intervened by intervention III (P < 0.01). Compared with the model group, the mRNA expression level of RSV-M all significantly decreased in the high dose JXOL group and the equivalent dose JXOL group intervened by 3 intervention ways (all P < 0.01). The mRNA expression level of SOCS1 significantly decreased in the high dose JXOL group intervened by intervention I and III and the equivalent dose JXOL group intervened by 3 intervention ways (all P < 0.01). The mRNA expression level of IFN-β significantly decreased in the high dose JXOL group intervened by intervention I and II and the equivalent dose JXOL group intervened by intervention I (all P < 0.01), while it significantly increased in the high dose JXOL group intervened by intervention III and the equivalent dose JXOL group intervened by intervention III (all P < 0.01). The protein expression level of SOCS1 significantly decreased in the high dose JXOL group intervened by intervention I and the equivalent dose JXOL group intervened by 3 intervention ways (all P < 0.01), while it significantly increased in the high dose JXOL group intervened by intervention III (all P < 0.01). Compared with the high dose JXOL group, the mRNA expression level of RSV-M decreased significantly in the equivalent dose JXOL group intervened by intervention I and II (P < 0.01). The mRNA expression level of SOCS1 and IFN-β decreased significantly in the equivalent dose JXOL group intervened by intervention I (P < 0.01), but the mRNA expression level of IFN-β increased significantly in the equivalent dose JXOL group intervened by intervention II and III (all P < 0.01). The protein expression level of SOCS1 decreased significantly in the equivalent dose JXOL group intervened by 3 intervention ways (all P < 0.01).

CONCLUSIONSJXOL could inhibit the expression of SOCS1 in the lung tissue of RSV infected BALB/c mice at different time points. Its regulatory effect might be associated with promoting the expression of interferon type I and further fighting against RSV.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Lung ; metabolism ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; Respiratory Syncytial Virus Infections ; drug therapy ; metabolism ; Respiratory Syncytial Viruses ; Ribavirin ; Signal Transduction ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; metabolism ; Toll-Like Receptor 3 ; metabolism

OBJECTIVETo observe the therapeutic effect of self-formulated Xiaokang Zhongzi Recipe (XZR) in treating male infertility with positive antisperm antibody (AsAb).

METHODSOne hundred and ten male infertility patients with positive AsAb were randomly assigned, according to randomized digital table, to the trial group treated with XZR, and the control group, treated with prednisone, 55 in each group. Clinical therapeutic effect and adverse reactions of the drugs were observed after 3 months of treatment.

RESULTSIn the trial group after treatment, 38 patients (69.1%) were cured, 14 (25.4%) improved and 3 treated in vain (5.5%, including 2 dropped out). While in the control group, the corresponding numbers were 12 (21.8%), 11 (20.0%) and 32 (58.2%, including 4 dropped out, 20 remained AsAb positive and 8 with AsAb reverted to positive after negative conversion). The negative conversion rate and average negative conversion time in the trial group and the control group were respectively 94.5%, (45 +/- 14) days and 41.8%, (62 +/- 21) days. Significant difference between the two groups was shown in therapeutic effectiveness and average negative conversion time (P < 0.01). Adverse reactions occurred in 3 cases (5.5%) in the trial group and 8 (14.5%) in the control group.

CONCLUSIONXZR is better than prednisone in treating male infertility with positive AsAb, and with fewer and milder adverse reactions.

Adult ; Autoantibodies ; blood ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Infertility, Male ; drug therapy ; etiology ; immunology ; Male ; Phytotherapy ; Spermatozoa ; drug effects ; immunology ; Treatment Outcome ; Young Adult

OBJECTIVETo illuminate the preliminary genotype and allele frequency distribution of D6S477 and the other four short tandem repeat(STR) loci in Chinese Han population in Qingdao area and to probe the possibility of their genetic application.

METHODSTwo hundred ACD-blood specimens were collected from the unrelated individuals in Qingdao. The DNA samples were extracted with Chelex method and were amplified by polymerase chain reaction technique. The PCR products were analyzed by polyacrylamide gel electrophoresis and displayed using silver staining.

RESULTSThe authors obtained the allele frequency distribution and preliminary genotype of D6S477, D9S1118, D18S865, D19S400 and D20S161 STR loci. No deviation from Hardy-Weinberg equilibrium was observed in the five loci.

CONCLUSIONAll the five loci have higher chance of exclusion and discriminating power, and they will be useful markers for researches in genetics.

Asian Continental Ancestry Group ; genetics ; China ; Gene Frequency ; Genotype ; Humans ; Linkage Disequilibrium ; Microsatellite Repeats ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic

OBJECTIVETo know the genotype and allele frequency distribution of D1S549, D3S1754 and D12S375 in Chinese Han population in the Qingdao area and to study the three short tandem repeat(STR) loci for genetic application.

METHODSACD-blood specimens were collected from the unrelated individuals in Qingdao. The DNA samples were extracted with the use of Chelex method and were amplified by polymerase chain reaction (PCR) technique. The PCR products were analyzed by polyacrylamide gel electrophoresis and were visualized by silver staining.

RESULTSEight alleles were found at D1S549 locus, eight alleles at D3S1754 locus and five alleles at D12S375 locus, and 22, 19 and 14 genotypes were identified respectively. No deviation from Hardy-Weinberg equilibrium was observed in the three loci. The heterozygosities expected of them were 0.7988, 0.7087 and 0.75 respectively. The exclusion probability was calculated as 0.6592 for D1S549, and 0.5605 for D3S1754, and 0.5864 for D12S375. The discriminating power of the three loci were 0.9143, 0.8382 and 0.8861. Comparison of the allelic frequencies in Qingdao area with those in Hans of Chengdu area by chi-square test showed a difference statistically significant at D1S549 locus but no difference at D3S1754 and D12S375 loci.

CONCLUSIONThis study reveals the structure of the three loci and the obtained data are beneficial to understanding the population genetics in Chinese Han population. All of the three loci have higher chance of exclusion and higher discriminating power, and they will be useful markers for individual identification, paternity test and genetics purposes.

China ; ethnology ; Gene Frequency ; Humans ; Polymorphism, Genetic ; Tandem Repeat Sequences

Metabolic engineering is the analysis of metabolic pathway and designing rational genetic modification to optimize cellular properties by using principle of molecular biology. Aromatic metabolites such as tryptophan, phenylalanine, and tyrosine are essential amino acids for human and animals. In addition, phenylalanine is used in aspartame production. Escherichia coli and many other microoganism synthesize aromatic amino acids through the condensation reaction between phospho-enolpyruvate (PEP) and erythrose-4-phosphate(E4P) to form 3-deoxy-D-arabinoheptulosonate 7-phosphate(DAHP). But many enzymes compete for intracellular PEP, especially the phosphotransferase system which is responsible for glucose transport in E. coli. This system uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. To channel more carbon flux into the aromatic pathway, one has to overcome pathways competing for PEP. ppsA and tktA are the key genes in central metabolism of aromatic amino acids biosynthesis. ppsA encoding phosphoenolpyrucate synthetase A (PpsA) which catalyzes pyruvate into PEP; tktA encoding transketolase A which plays a major role in erythrose-4-phosphate (E4P) production of pentose pathway. We amplified ppsA and tktA from E. coli K-12 by PCR and constructed recombinant plasmids of them in pBV220 vector containing P(R)P(L) promoter. Because of each gene carrying P(L) promoter, four productions of ligation were obtained. The monoclonal host containing recombinant plasmids was routinely grown in Luria-Bertani (LB) medium added Ampicillin at 37 degrees C overnight, and then inoculated in LB (Apr) medium by 3%-5% in flasks on a rotary shaker at 30 degres C, induced at 42 degrees C for 4.5 hours when OD600 = 0.4, cells were obtained by centrifugation at 10,000 r/min at 4 degrees C. The results of SDS-PAGE demonstrated that the bands at 84kD and 73kD were more intensive than the same ones of the controls. The specific activity of PpsA in crude extracts was increased by 10.8-fold, and TktA, by 3.9-fold. When both genes were co-expressed in E. coli, the activity of PpsA varied from 2.1-9.1 fold comparing to control, but the activity of TktA was relatively stable(3.9-4.5 fold). Whatever the two genes were expressed respectively or cooperatively, both could promote the production of DAHP, the first intermediate of the common aromatic pathway, but co-expression was more effective on forming DAHP. The results demonstrate that co-expression of ppsA and tktA can improve the production of DAHP to near theoretical yield. This report details a different strategy based on co-expression of two genes in one vector in vivo to release the burden and paves the way for construction of genetic engineering bacteria for further research.
Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; enzymology ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Pyruvate Synthase ; genetics ; metabolism ; Transketolase ; genetics ; metabolism

Aged ; Biopsy, Needle ; methods ; Coal Mining ; Humans ; Lung ; pathology ; Lung Neoplasms ; complications ; diagnosis ; Middle Aged ; Pneumoconiosis ; complications

The key and crucial step of metabolic engineering during quinic acid biosynthesize using shikimic acid pathway is high expression of quinate 5-dehydrogenase. The gene qa-3 which code quinate 5-dehydrogenase from Neurospora crassa doesn't express in Escherichia coli. By contrast with codon usage in Escherichia coli, there are 27 rare codons in qa-3, including eight AGG/AGA (Arg) and nine GGG (Gly). Two AGG are joined together (called box R) and some GGG codons are relative concentrate (called box G). Along with the secondary structure of mRNA analysed in computer, the free energy of mRNA changes a lot from -374.3 kJ/mol to least -80.5 kJ/mol when some bases in the end of qa-3 were transformed, and moreover, the change of free energy is quite small when only some bases in the box G and box R transformed. After the change of rare codon and optimization of some bases in the end, qa-3 was expression in E. coli and also the enzyme activity of quinate 5-dehydrogenase can be surveyed accurately. All the work above benefit the further research on producing quinic acid engineering bacterium.
Alcohol Oxidoreductases ; biosynthesis ; genetics ; Base Sequence ; Codon ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Hydro-Lyases ; genetics ; Molecular Sequence Data ; Neurospora crassa ; enzymology ; genetics ; RNA, Messenger ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Shikimic Acid ; metabolism ; Transformation, Bacterial