Diversity of Microflora in Colonic Mucus from Severe Ulcerative Colitis Patients Analyzed by Terminal Restriction Fragment Length Polymorphism and Clone Libraries of Bacterial 16S rRNA Gene Sequences

Although the gut microflora is thought to be an essential factor in the development of ulcerative colitis (UC), the entire gut microflora occurring in UC remains unknown. Most studies use feces to represent the microflora distribution;however, here we analyzed the bacterial diversity in colonic mucus from UC patients receiving colectomy surgery and control patients. The diversity of microflora was investigated using a combination of terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of the 16S rRNA gene sequences. In the T-RFLP analysis, the number of terminal restriction fragments (T-RFs) decreased significantly in UC patients when compared to control samples. Also in the clone library analysis, the number of operational taxonomic units (OTU) and the Shannon diversity index were reduced significantly in UC patients. These molecular analyses reveal an overall dysbiosis in UC patients. No specific pathogen was found, and a strong negative correlation in relative abundance of bacterial populations was observed between the phyla Bacteroidetes and Firmicutes in the UC patients. This is the first report showing a significant correlation between these two phyla, which may be important characteristics in the pathogenesis of UC.

Antibiotic-Resistant Bacterial Group in Field Soil Evaluated by a Newly Developed Method Based on Restriction Fragment Length Polymorphism Analysis

Spreading of antibiotic resistant bacteria into environment is becoming a major public health problem, implicating affair of the indirect transmission of antibiotic resistant bacteria to human through drinking water, or vegetables, or daily products. Until now, the risk of nosocomial infection of antibiotic resistant bacteria has mainly been evaluated using clinical isolates by phenotypic method. To evaluate a risk of community-acquired infection of antibiotic resistant bacteria, a new method has been developed based on PCR-RFLP without isolation. By comparing restriction fragment lengths of the 16S rDNA gene from bacterial mixture grown under antibiotic treatment to those simulated from the DNA sequence, bacterial taxonomies were elucidated using the method of Okuda and Watanabe [1] [2]. In this study, taxonomies of polymyxin B resistant bacteria group in field soils, paddy field with organic manure and upland field without organic manure were estimated without isolation. In the both field soils, the major bacteria grown under the antibiotic were B. cereus group, which had natural resistance to this antibiotic. In field applied with organic manure, Prevotella spp., and the other Cytophagales, which were suggested to be of feces origin and to acquire resistance to the antibiotic, were detected. When numbers of each bacterial group were roughly estimated by the most probable number method, B. cereus group was enumerated to be 3.30 × 106 MPN/g dry soil in paddy field soil and 1.32 × 106 MPN/g dry soil in upland filed. Prevotella spp. and the other Cytophagales in paddy field were enumerated to be 1.31 × 106 MPN, and 1.07 × 106 MPN·g-1 dry soil.

Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS ASSOCIATED WITH FACTOR Ⅷ CARRIER DETECTION USING DNA RFLPs IN CHINESE

RFLPs for XbaI, BelI and BglI sites of human FⅧwere informative for 48%, 41% and 15% of females studied, respectively. BglI RFLP is different from that reported by Chan et al, a fact suggests Yangtze River region population of China would be at variance with the Southern Chinese population in certain RFLP distribution. TaqI allelic system Ⅰin the DXS52 region also shows the same variance among them, but heterozygous rate 0f 71% for system Ⅰ(alleles 1 to 8) and 49% for system Ⅱ(αand βalleles) were very similar. Using the Bell/XbaI RFLPs, accurate information could be obtained from this study for 56% of women who were at risk for hemophilia A (HA) carriership. The carrier of the remaining 44% could be determined by utilizing the TaqI RFLP. In addition, we report a new intergenie polymorphism (9%) at DXS115 as a marker for detection of heterozygotes in families at risk for HA. The advantage of using the XbaI/KpnI RFLP is that both the intragemie RFLP and the new intergenie RFLP can be evaluated on the same blot at the same time.

Bacterial Groups Concerned with Maturing Process in Manure Production Analyzed by a Method Based on Restriction Fragment Length Polymorphism Analysis

Composting is a biological aerobic decomposition process consisted from different phases. Although the Japanese Standards for manure recommended that it took at least 6 months to complete the maturing phase, there was no reliable ground. In order to find out shortening method of the maturing phase, the microorganisms concerned with a progress of the maturing was determined by using the most probable number method (MPN) and PCR-RFLP of the 16S rDNA, which was found effective to provide numbers and taxonomy of polymyxin B resistant bacterial groups in the former paper [1]. Compared to the numbers after thermophilic phase, those of Actinobacteria, δ-proteobacteria, and the other gram negative bacteria increased to 50 times, 20 times, and 105 times, respectively, after maturing phase, while those of Bacillus spp., and α and β-proteobacteria decreased to 1/10, and 1/105 after maturing phase. Numbers of the other Fumicutes, and γ-proteobacteria remained in the same revel. Actinobacteria, δ-proteobacteria, and the other gram negative bacteria might be concerned with a progress of the maturing phase, because these bacterial groups were detected and enumerated due to their proliferation ability. Although number of Acitinobacteria might be underestimated because of a PCR bias, the method was found effective for the purpose to monitor bacteria actively proliferated in culture medium.

Evaluation of the Method Based on Restriction Fragment Length Polymorphism Analysis as Simple Analysis Method of Lactic Acid Bacteria in Foods

Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g-1) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as to amplify Bifidobacterium spp., before the method will be used as an inspection method for lactic acid bacteria.

The Use of Restriction Fragment Length Polymorphism and Fluorescence in Situ Hybridization to Investigate Microbiota of Piglets after Feeding Oregano

A total of 80 piglets (7.9 ± 1.0 kg) were used in a feeding experiment with dried oregano. The diets differed in their oregano content: 0 g, 2 g, 4 g and 8 g oregano/kg feed, corresponding to 0, 23.5, 46.9 and 93.9 mg carvacrol/kg DM. After the experimental period of 5 weeks, 20 piglets of both extreme feeding groups were slaughtered: 10 animals of the control group and 10 animals of the group that received 8 g oregano/kg. Ingesta samples of jejunum, caecum and colon were collected and analyzed by FISH and PCR RFLP to compare the diversity of microbiota. The results showed no significant changes in microbiota in response to oregano. The patterns of the PCR-RFLP showed a similarity of 61.8% - 91.8% in both feeding groups. In conclusion, an effect of oregano on the in- testinal microbiota could not be shown under the methods used.

Application of restriction site amplified polymorphism (RSAP) to genetic diversity in Saccharina japonica

Restriction site amplified polymorphism (RSAP) was used, for the first time, to analyze the genetic structure and diversity of four, mainly cultivated, varieties of the brown alga, Saccharina japonica. Eighty-eight samples from varieties "Rongfu", "Fujian", "Ailunwan" and "Shengchanzhong" were used for the genetic analyses. One hundred and ninety-eight bands were obtained using eight combinations of primers. One hundred and ninety-one (96.46%) were polymorphic bands. Nei's genetic diversity was 0.360, and the coefficient of genetic differentiation was 0.357. No inbreeding-type recession was found in the four brown alga varieties and the results of the "Ailunwan" variety using samples from 2 years showed that the variety was becoming less diverse during the selection inherent in the breeding program. Genetic diversity and cluster analyses results were consistent with these genetic relationships. The results show the RSAP method is suitable for genetic analysis. Continuous inbreeding and selection could reduce the genetic diversity effectively; therefore periodical supervision is required.

Comparing the frequencies of restriction fragment length polymorphisms for dystrophin gene in Chinese with those from Japanese and Caucasian populations^1

The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subeiones of the entire 14kb eDNA for the dystrophin as hybridization probes. Allelie fragments were detected in hybridization patterns of PvuⅡ/la. TaqⅠ/2b-3, Taq Ⅰ/5b-7. and Xba Ⅰ/10. Among them. the allelic fragments (26kb and 3.8kb) in PvuⅡ/2b-3 pattern and the allelic fragments (10.0kb and 8.4kb) in Taq Ⅰ/5b-7 patterns had never been reported previously. Compared with the data from Caucasians and Japanese. it indicated that here was a significant difference (P <0.01) of the allelic fragment frequency in Taq Ⅰ/2b-3 and Xba Ⅰ/10 patterns between Chinese and Caucasians. The frequencies of allelic fragments A2 (5.6kb) in Taq Ⅰ/8 and A2 (10.7kb) in EcoR V/9 were high in Caucasians, yet had not been detected in Chinese, the differences were also highly significant. But in Chinese and Caucasians. the BIB2 allelic frequencies in Taq Ⅰ/5b-7 are the same. As to the frequency of the allelic fragments A1A2 and B1B2 in Pvu Ⅱ/1a. there was no significant difference between Chinese and Japenese.

RESTRICTION FRAGMENT LENGTH POLYMORPHISM（RFLP） ANALYSIS OF GENOMIC DNA OF 5 STRAINS OF TRICHINELLA SPIRALIS IN CHINA

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Differentiation of Helicobacter pylori isolates by polymerase chain reaction-restriction fragment length polymorphism

Objective: To investigate the association between the diversity of urease gene and urease activity of clinical isolates of Helicobacter pylori (H. pylori). Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of urease gene and rapid urease activity test were used to study the urease activity of different clinical isolates of H. pylori. Results: H. pylori clinical isolates were divided into 4 types according to their PCR-RFLP results of urease gene and urease activity. Type Ⅰ , possessing strong urease activity (0. 11) and presented 1 fragment of 1. 7 kb by PCR-RFLP, had close relations with gastric ulcer; type Ⅱ , with the weakest urease activity (0. 07) and 2 fragments (1. 3 and 0. 4 kb respectively) , was associated with duodenal bulb ulcer; type Ⅲ , with the strongest urease activity (0. 12) and 2 fragments (0. 4 and 0. 17 kb) with or without 1 fragment (0. 23 or 0. 37 kb) , was responsible for gastritis; type Ⅳ , with weak urease activity (0. 09) and 2

Association of TNF-α-238G/A and 308 G/A Gene Polymorphisms with Pulmonary Tuberculosis among Patients with Coal Worker’s Pneumoconiosis

Objectives Tumor necrosis factor-α （TNF-α） may play an important role in host＇s immune response to mycobacterium tuberculosis （M. tuberculosis） infection. This study was to investigate the association of TNF-α gene polymorphism with pulmonary tuberculosis （TB） among patients with coal worker＇s pneumoconiosis （CWP）. Methods A case-control study was conducted in 113 patients with confirmed CWP complicated with pulmonary TB and 113 non-TB controls with CWP. They were matched in gender, age, job, and stage of pneumoconiosis. All participants were interviewed with questionnaires and their blood specimens were collected for genetic determination with informed consent. The TNF-α gene polymorphism was determined with polymerase chain reaction of restriction fragment length polymorphism （PCR-RFLP）. Frequency of genotypes was assessed for Hardy-Weinberg equilibrium by chi-square test or Fisher＇s exact probability. Factors influencing the association of individual susceptibility with pulmonary TB were evaluated with logistic regression analysis. Gene-environment interaction was evaluated by a multiplieative model with combined OR. All data were analyzed using SAS version 8.2 software. Results No significant difference in frequency of the TNF-α-308 genotype was found between CWP complicated with pulmonary TB and non-TB controls （2,2=5.44, P=-0.07）. But difference in frequency of the TNF-α-308 A allele was identified between them （2,2-5.14, P=0.02）. No significant difference in frequencies of the TNF-α-238 genotype and allele （P=0.23 and P=0.09, respectively） was found between cases and controls either, with combined （GG and AA） OR of 3.96 （95% confidence interval of 1.30-12.09） at the -308 locus of the TNF-α gene, as compared to combination of the TNF-α-238 GG and TNF-α-308 GG genotypes. Multivariate-adjusted odds ratio of the TNF-α-238 GG and TNF-α-308 GA genotypes was 1.98 （95% CI of 1.06-3.71） for risk for pulmonary TB in patients with CWP. There was a synergic interaction between the TNF-a-308 GG genotype and body mass index （OR=4.92）, as well as an interaction between the TNF-α-308 GG genotype and history of BCG immunization or history of TB exposure. And, the interaction of the TNF-α-238 GG genotype and history of BCG immunization or TB exposure with risk for pulmonary TB in them was also indicated. Conclusions TNF-α-308 A allele is associated with an elevated risk for pulmonary TB, whereas TNF-α-238 A allele was otherwise.

Association of G+1688A Polymorphism of Platelet Endothelial Cell Adhesion Molecule-1 Gene with Myocardial Infarction in the Chinese Han Population

In order to investigate the association of G＋1688A （Ser563Asn） polymorphism of platelet endothelial cell adhesion molecule-1 （PECAM-1） gene with myocardial infarction （MI） in the Chinese Han population, the G＋1688A polymorphism in PECAM-1 gene was detected by polymerase chain reaction-restriction fragment-length polymorphism （PCR-RFLP） method among 502 subjects, including 218 patients with MI and 284 controls. The results showed that there was significant difference in AA frequencies of genotype G＋1688A polymorphism between case and control groups （39% vs 24%, P〈0.001）. A similar trend was observed on the allele frequencies （A/G： 62% vs 49%, P〈0.001）. Among the subjects with high serum total cholesterol level or high systolic blood pressure level, the variant AA genotype was associated with high risk of MI （adjusted OR, 2.13; 95% CI, 1.08 -4.41 and adjusted OR, 2.53; 95%CI, 1.63-3.63）. The single nucleotide polymorphism （SNP） at position ＋1688 in the exon 8 of PECAM-1 gene was associated with MI and the allele A might be a risk factor for MI in the Chinese Han population.

Analysis of Rb gene Xba Ⅰ polymorphism in Shaanxi aged atherosclerosis population

Objective:To investigate variable number tandem repeat (VNTR) polymorphism of the 17th intron of Rb gene in Shaanxi aged population and the relationship between the polymorphism of Rb gene and atherosclerosis(AS) genetic suscepti- bility. Methods: VNTR polymorphism of the 17th intron of Rb gene were examined in 100 Shaanxi aged AS patients and 100 Shaanxi aged control individuals by PCR-Rb-Xba Ⅰ-RFLP. Results::Two alleles were found both in AS group and control group, which were separately 945 bp(S1) and 630bp + 315bp(S2). S1S2 genotype was the most frequent one in the two populations. Significant difference in allele frequency was not found between AS group and control group, and allele frequency was no significant difference between Chinese and Caucasian. Conclusion: Xba Ⅰ enzyme site of Rb gene could have been certainly stable in AS population, and it was inferred that the polymorphism locus was not liable to cause mutation, which might not implicated in the formation of AS.

POLYMORPHISMS OF THE HUMAN LIPOPROTEIN LIPASE GENE：POSSIBLE ASSOCIATION WITH LIPID LEVELS IN PATIENTS WITH CORONARY HEART DIS

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Association between estrogen receptor β gene Rsa1 polymorphism and depressive disorder in peri-menopausal and menopausal women

Objective: To investigate estrogen receptor β (ERβ) gene Rsa1 polymorphism and concentration of estrogen, FSH and LH in serum in peri-menopausal and menopausal women with depressive disorder. Methods: Seventy-four peri-menopausal and menopausal women with depressive disorder met ICD-10 and CCMD-3 assessment criteria for depressive disorder were recruited. ERβ gene Rsa1 polymorphism was analyzed with PCR-RFLP. Serum levels of estrogen, FSH and LH were measured by magnetism-ELISA. Results: The respective frequency of ERβ gene Rsa1 polymorphism was no significant difference between women with depressive disorder and the healthy women (χ 2=1.106,P>0.05). The serum level of estrogen was lower in women with depressive disorder than in the healthy women (P<0.05). No difference was found for FSH and LH between two groups. Conclusion: ERβ gene Rsa1 polymorphism may be not associated with depressive disorder in the peri-menopausal and menopausal women. The serum level of estrogen is associated with depressive disorder in the peri-menopausal and menopausal women.

Cytotoxic T lymphocyte associated antigen-4 gene exon 1A49→G polymorphisms confer susceptibility to idiopathic dilated cardiomyopathy

Autoimmune mechanisms, including cellular and humoral immune, are likely to participate in the pathogenesis of at least a subgroup of idiopathic dilated cardiomyopathy (IDC), in which cellular immune-mediation plays a more important role. Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) is the major negative regulatory factor of cellular immunity. This study was conducted to investigate the association of CTLA-4 gene exon 1 A49→G polymorphism with susceptibility to IDC in Han Chinese and its influences on serum soluble CTLA-4 (sCTLA-4) and Th1/Th2 cytokine bias. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques were used to analyze the dimorphism of CTLA-4 exon 1 in the unrelated Han ethnic population in Heilongjiang Province (including 48 IDC patients and 50 normal controls). Serum sCTLA-4, IFN-γ and IL-4 were evaluated by ELISA, with the ratio of IFN-γ/IL-4 as indicator for Th1/Th2 bias. Compared with controls, the frequencies of GG genotype (0.6042 and 0.3600, P=0.012) and the G allele (0.7396 and 0.5600, P=0.008) were significantly increased in IDC patients. Increased serum sCTLA-4 was found in the IDC group compared with the controls [(1.87±1.06) μg/L vs. (0.54±0.19) μg/L, P<0.05]. IFN-γ was much lower in IDC patients than that of the controls [(16.38±6.25) ng/L vs. (29.81±10.66) ng/L (P<0.05)], whereas no statistical difference of IL-4 was found between the two groups [(12.85±1.86) ng/L vs. (12.11±2.76) ng/L], so the ratio of IFN-γ/IL-4 declined significantly (1.63±0.50 vs. 3.01±0.89, P<0.05). Linear regression analysis manifested a significant interrelationship between the GG genotype, G allele frequencies and serum sCTLA-4, IFN-γ/IL-4 in the IDC group (r=0.57, P=0.021 and r=0.32, P=0.036). CTLA-4 gene A49→G substitution was associated with an increased IDC risk, which implicated that the CTLA-4 gene exon 1 may have a considerable role in autoimmune cardiac damage, possibly via a Thr→Ala change in signal peptide, which influences the protein synthesis and modification processes, with a result of functional alteration of sCTLA-4. The bias of Th1/Th2 paradigm was associated with the increased sCTLA-4 under certain background of immunogenetics.