[Structural Analysis of Salivary Bacterial and Fungal Microbiome in Oral Lichen Planus Patients].

Objective: To explore the differences of oral mycobiome and bacteriome between the healthy controls (H) and oral lichen planus (OLP) patients, and the co-occurrence patterns of the salivary mycobiome and bacteriome and the association with host immunity. Methods: Saliva samples were collected from clinical OLP patients (n=35) and healthy volunteers (n=18). Microbiome DNA was extracted for bacterial 16S rRNA genes sequencing and fungal internal transcribed spacer 2 (ITS2) sequencing. Bioinformatics analysis was performed on the data.The levels of IL-17 and IL-23, two pro-inflammatory cytokines, in the saliva were examined, and their correlation with the bacteria was analyzed. Results: There was no significant difference in the overall community structure of the mycobiome and the bacteriome between OLP patients and healthy controls. The abundance of Prevotellaand Solobacterium in the saliva bacteriome was significantly increased in the OLP group (P<0.05), and the relative abundance of Candidaand Aspergillusin the saliva mycobiome was also significantly increased (P<0.05). The co-occurrence pattern of the salivary mycobiome and bacteriome showed that the aforementioned difference was not related. However, the correlation between Aspergillusand bacteria was altered in the H group and the OLP group, and co-occurrence was reduced in the latter group. The level of IL-17 in the saliva was significantly increased in the OLP group. IL-17 and clinical scores were significantly correlated with the abundance of Porphyromonas. Conclusion: The increased abundance of Prevotella, Solobacterium, Candida, and Aspergillus was associated with the pathogenesis of OLP, and the changes of the microbiome co-occurrence relationship and host immunity may be involved in the pathogenesis of OLP.

TCAB1: a potential target for diagnosis and therapy of head and neck carcinomas.

BACKGROUND: WRAP53, including α, ß and γ isoforms, plays an important role not only in the stability of p53 mRNA, but also in the assembly and trafficking of the telomerase holoenzyme. It has been considered an oncogene and is thought to promote the survival of cancer cells. The aim of this study was to detect the role of TCAB1 (except WRAP53α) in the occurrence and development of head and neck carcinomas. METHODS: Immunohistochemistry was used to detect the TCAB1 expression in clinical specimen sections and performed western blotting to check the TCAB1 expression levels in cell lines. TCAB1 was depleted using shRNA lentivirus and the knockdown efficiency was assessed using q-PCR and Western blotting. We performed CCK-8 assays and flow cytometry to check the cell proliferation potential and used the trans-well assay to test the invasion ability in vitro. Xenografts were used to detect the tumor formation potential in vivo. Moreover, we performed cDNA microarray to investigate the candidate factors involved in this process. RESULTS: We observed a notable overexpression of TCAB1 in head and neck carcinoma clinical specimens as well as in carcinoma cell lines. Knockdown of TCAB1 decreased the cellular proliferation potential and invasion ability in vitro. cDNA microarray analysis suggested the possible involvement of several pathways and factors associated with tumorigenesis and carcinoma development in the TCAB1-mediated regulation of cancers. Furthermore, the xenograft assay confirmed that the depletion of TCAB1 would inhibit tumor formation in nude mice. The immunohistochemistry results of the mice tumor tissue sections revealed that the cells in shTCAB1 xenografts showed decreased proliferation potential and increased apoptotic trend, meanwhile, the angiogenesis was inhibited in the smaller tumors form shTCAB1 cells. CONCLUSIONS: Our study demonstrated that depletion of TCAB1 decreased cellular proliferation and invasion potential both in vitro and in vivo. The data indicated that TCAB1 might facilitate the occurrence and development of head and neck carcinomas. In future, TCAB1 might be useful as a prognostic biomarker or a potential target for the diagnosis and therapy of head and neck carcinomas.

Elevated levels of triglyceride and triglyceride-rich lipoprotein triglyceride induced by a high-carbohydrate diet is associated with polymorphisms of APOA5-1131T>C and APOC3-482C>T in Chinese healthy young adults.

BACKGROUND/AIMS: Changes in lipid profiles have been shown to be associated with diet and apolipoprotein (APO) polymorphisms. Therefore, 2 polymorphisms, i.e. APOA5-1131T>C and APOC3-482C>T, and serum lipids were examined in a Chinese healthy young population with high-carbohydrate/low-fat (HC/LF) diet intervention. METHODS: After a wash-out diet for 7 days, 56 young adults (22.89 ± 1.80 years) received the HC/LF diet for 6 days. Body mass index (BMI) and fasting serum lipid profiles at baseline, after the wash-out diet, and after the HC/LF diet were measured. RESULTS: APOA5-1131C carriers had higher triglyceride (TG) and TG-rich lipoprotein TG (TRL-TG) levels at baseline and after the HC/LF diet, though this mainly corresponded to the female cohort. APOC3-482T carriers had higher TRL-TG levels following the wash-out and HC/LF diets, but these were not directly attributable to a single gender. CONCLUSIONS: Both polymorphisms may play an important role in the elevated TG and TRL-TG levels induced by the HC/LF diet, especially in females, thus indicating a potential dietary prevention of coronary heart disease in this Chinese cohort.

Analysis of Soluble Sugar Content in Minute Quantities of Rice Tissues by GC-MS.

Soluble sugars play key roles in plant growth, development, and adaption to the environment. Characterizing sugar content profiling of plant tissues promotes our understanding of the mechanisms underlying these plant processes. Several technologies have been developed to quantitate soluble sugar content in plant tissues; however, it is difficult with only minute quantities of plant tissues available. Here, we provide a detailed protocol for gas chromatography mass spectrometry (GC-MS)-based soluble sugar profiling of rice tissues that offers a good balance of sensitivity and reliability, and is considerably more sensitive and accurate than other reported methods. We summarize all the steps from sample collection and soluble sugar extraction to derivatization procedures of the soluble extracted sugars, instrumentation settings, and data analysis.

Plasmodesmata play pivotal role in sucrose supply to Meloidogyne graminicola-caused giant cells in rice.

On infection, plant-parasitic nematodes establish feeding sites in roots from which they take up carbohydrates among other nutrients. Knowledge on how carbohydrates are supplied to the nematodes' feeding sites is limited. Here, gene expression analyses showed that RNA levels of OsSWEET11 to OsSWEET15 were extremely low in both Meloidogyne graminicola (Mg)-caused galls and noninoculated roots. All the rice sucrose transporter genes, OsSUT1 to OsSUT5, were either down-regulated in Mg-caused galls compared with noninoculated rice roots or had very low transcript abundance. OsSUT1 was the only gene up-regulated in galls, at 14 days postinoculation (dpi), after being highly down-regulated at 3 and 7 dpi. OsSUT4 was down-regulated at 3 dpi. No noticeable OsSUTs promoter activities were detected in Mg-caused galls of pOsSUT1 to -5::GUS rice lines. Loading experiments with carboxyfluorescein diacetate (CFDA) demonstrated that symplastic connections exist between phloem and Mg-caused giant cells (GCs). According to data from OsGNS5- and OsGSL2-overexpressing rice plants that had decreased and increased callose deposition, respectively, callose negatively affected Mg parasitism and sucrose supply to Mg-caused GCs. Our results suggest that plasmodesmata-mediated sucrose transport plays a pivotal role in sucrose supply from rice root phloem to Mg-caused GCs, and OsSWEET11 to -15 and OsSUTs are not major players in it, although further functional analysis is needed for OsSUT1 and OsSUT4.

Identification of a novel gene, MSAG, regulated by high levels of glucose and insulin.

Identification and characterization of novel genes involved in derangement of metabolisms of glucose and triglycerides are important in understanding the development of metabolic syndrome (MS) and atherosclerosis. Model rats with certain phenotypes of MS were fed a high-carbohydrate diet. The rat hepatic subtracted cDNA libraries were constructed and screened. A novel cDNA of full length was identified by screening of a human hepatic cDNA library with a mixture of probes of the differentially expressed fragments from the rat hepatic subtracted cDNA libraries. The corresponding gene of the cDNA was temporarily named metabolic syndrome-associated gene (MSAG). The predicted protein encoded by MSAG contains 110 amino acids and has a theoretical molecular weight of 11667.04 and an isoelectric point of 4.91. Compared with the housekeeping gene of beta-actin, MSAG had low transcription activity. However, the mRNA level of MSAG in HepG2 cells, a human hepatoma cell line, was significantly increased by glucose and decreased by insulin concentrations higher than physiological levels. These results suggest that MSAG may be involved in the metabolism and/or its regulation of glucose, the functioning of insulin under non-physiological conditions, and further in the development of metabolic syndrome.

[Therapeutic effects of hTR-siRNA adenovirus on human cervical cancer xenografts in nude mice].

OBJECTIVE: To study the anti-tumor effects and its mechanism of hTR-siRNA adenovirus on human cervical cancer in vivo. METHODS: The in vivo model of human cervical cancer was established by the subcutaneous inoculation of HeLa cells at the right armpit of BALB/c nu/nu mice. After successful implantation, the mice were randomized into four groups, in which the mice were intratumorally injected with 0.1 mL of Ad-hTR-siRNA (10(13) pfu/L), or Ad-NT-siRNA (10(13) pfu/L), or Cisplatin (1.20 g/L), or serum-free DMEM alone respectively. These treatments were given once every 3 days for 6 times. After the last injection, the mice were observed for 7 days continuously and sacrificed at the end. The tumors were harvasted, weighed, and sectioned. The TUNEL assay was used to assess the apoptosis of these tumor cells. RESULTS: Tumors-implanted were established successfully by 100% with HeLa cells. As compared with Ad-NT-siRNA, Ad-hTR-siRNA could slow down tumor growth, decrease tumor volume (45.48%) and tumor weight (34.68%), as well as promote the apoptosis and necrosis of tumor cells. The TUNEL positive cells were about 11.8%. But the anti-tumor activity of Ad-hTR-siRNA didn't catch on Cisplatin's. CONCLUSION: This study indicated that the hTR-siRNA adenovirus could suppress cervical cancer-xenografted growth in vivo and induce tumor cell apoptosis or necrosis.

Association of Val66Met polymorphism at brain derived neurotrophic factor gene with depression among Chinese adolescents after Wenchuan earthquake: An 18months longitudinal study.

To longitudinally investigate the association of Val66Met polymorphism at brain derived neurotrophic factor (BDNF) gene (BDNF) with depression in Chinese Han adolescents after the 2008 Wenchuan earthquake, BDNF Val66Met was identified by polymerase chain reaction-restriction fragment length polymorphism analyses and verified by DNA sequencing. Depression was assessed by Beck Depression Inventory (BDI) among high school students at 6, 12 and 18months after the earthquake. The results showed that the females constantly had higher depression prevalence than the males during the follow-up in the Met allele carriers, but not in the Val/Val homozygotes. When compared to that at 6months, the prevalence was lowered at 12months in the male Met allele carriers, and at 18months in all the females and the male Met allele carriers. Moreover, the Met allele carriers had higher BDI scores than the Val/Val homozygotes only in the females at 18months. The females had higher BDI scores than the males constantly during the follow-up in the Met allele carriers and at 12months only in the Val/Val homozygotes. When compared to those at 12months, the scores decreased at 18months in all the females and the male Met allele carriers. In addition, the potential factors of prevalence or predictors of severity of depression were different between the Val/Val homozygotes and the Met allele carriers at different times after the earthquake. The results suggest that interactions may occur after stresses among BDNF Val66Met, gender and time course to influence depression. This may be one of the explanations for the inconsistent relationships reported before between depression and BDNF Val66Met and need to take into account for precision medical and more effective interference of depression in adolescents after disasters.

Muc-1 promotes migration and invasion of oral squamous cell carcinoma cells via PI3K-Akt signaling.

Muc-1 is a member of the carbohydrate-binding protein family that contributes to neoplastic transformation, tumor survival, angiogenesis, and metastasis. The aim of this study is to investigate the role of muc-1 in human oral squamous cell carcinoma progression. In this study, we tested our hypothesis that muc-1 regulate oral squamous cell carcinoma cells (SCC-9) malignant biological behaviors, and silencing muc-1 reduced SCC-9 cellular colony forming ability, migration and invasion. Moreover, silenced cells present defects in phosphatidylinositol 3-kinase (PI3K)-serine/threonine kinase (Akt) signaling, and reduced expression/activity of matrix metallopeptidase (MMP)-2/9. Furthermore, in muc-1 siRNA-transfected cells, we detected a decrease in signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation. In vivo, muc-1 siRNA cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and PI3K-Akt signaling inhibition. These results indicate that muc-1 is a key factor in SCC-9 tumor migration, invasion, and suggesting that muc-1 can be a novel therapeutic target in oral squamous cell carcinoma.

Associations of the SREBP-1c gene polymorphism with gender-specific changes in serum lipids induced by a high-carbohydrate diet in healthy Chinese youth.

We investigated the possible association between the sterol regulatory element-binding protein-1c gene (SREBP-1c) rs2297508 polymorphism and the changes in lipid profiles in a high-carbohydrate and low-fat (high-CHO/LF) diet in a Chinese population well characterized by a lower incidence of coronary heart disease and a diet featuring higher carbohydrate and lower fat. Fifty-six healthy youth (aged 22.89 ± 1.80 years) were given wash-out diets of 31% fat and 54% carbohydrate for 7 days, followed by the high-CHO/LF diet of 15% fat and 70% carbohydrate for 6 days, without total energy restriction. Fasting blood samples were collected. Serum variables of lipid and glucose metabolism after the wash-out and high-CHO/LF diets, as well as the rs2297508 polymorphism, were analyzed. Compared with the male subjects on the wash-out diet, significantly elevated levels of high-density lipoprotein cholesterol (HDL-C) and decreased levels of apolipoprotein B-100 were observed in the male carriers of the C allele after the high-CHO/LF diet. In the female subjects, significantly increased triacylglycerol levels, insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) were found in the GG genotype after the high-CHO/LF diet. These results suggest that the C allele of the rs2297508 polymorphism is associated with a retardation of the increases in serum triacylglycerol, serum insulin, and HOMA-IR in females and with the elevated serum HDL-C in males after the high-CHO/LF diet.

A high-carbohydrate diet enhances the adverse effect of the S2 allele of APOC3 SstI polymorphism on the TG/HDL-C ratio only in young Chinese females.

Both genetic background and diet have profound effects on plasma lipid profiles. We hypothesized that a high-carbohydrate (high-CHO) diet may affect the ratios of serum lipids and apolipoproteins (apo) differently in subjects with different genotypes of the SstI polymorphism in the apoCIII gene (APOC3). Fifty-six healthy university students (27 males and 29 females, 22.89 ± 1.80 years) were given a washout diet of 54% carbohydrate for 7 days, followed by a high-CHO diet of 70% carbohydrate for 6 days without total energy restriction. Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apoB100, apoAI, and the APOC3 SstI polymorphism were analyzed. The ratios of serum lipids and apoB100/apoAI were calculated. At baseline, the TG/HDL-C ratio was significantly higher in females, but not in males, with the S2 allele. The differences in the TG/HDL-C ratio between genotypes remained the same after the washout and the high-CHO diet in females. When compared with those before the high-CHO diet, the TC/HDL-C (male S2 carriers: 3.13 ± 1.00 vs 2.36 ± 0.65, P = 0.000; male subjects with the S1S1 genotype: 2.97 ± 0.74 vs 2.09 ± 0.55, P = 0.000; female S2 carriers: 2.68 ± 0.36 vs 2.24 ± 0.37, P = 0.004; female subjects with the S1S1 genotype: 2.69 ± 0.41 vs 2.09 ± 0.31, P = 0.000) and LDL-C/HDL-C (male S2 carriers: 1.44 ± 0.71 vs 1.06 ± 0.26, P = 0.012; male subjects with the S1S1 genotype: 1.35 ± 0.61 vs 1.01 ± 0.29, P = 0.005; female S2 carriers: 1.18 ± 0.33 vs 1.00 ± 0.18, P = 0.049; female subjects with the S1S1 genotype: 1.18 ± 0.35 vs 1.04 ± 0.19, P = 0.026) ratios were significantly decreased after the high-CHO diet regardless of gender and of genotype of the APOC3 SstI polymorphism. However, in female S2 carriers, the TG/HDL-C (1.38 ± 0.46 vs 1.63 ± 0.70, P = 0.039) ratio was significantly increased after the high-CHO diet. In conclusion, the high-CHO diet has favorable effects on the TC/HDL-C and LDL-C/HDL-C ratios regardless of gender and of genotype of the APOC3 SstI polymorphism. Somehow, it enhanced the adverse effect of the S2 allele on the TG/HDL-C ratio only in females.

[The latest progress in studies of human oral microbiome].

With the successful implementation of Human Genome Project, more and more scientists started to pay attention on the second genome of human body: Microbiome. This paper will briefly introduce the latest developments of the Human Microbiome Project, the human oral microbiome research, and new technologies of microbial gene research.

TaqIB polymorphism in the CETP gene modulates the impact of HC/LF diet on the HDL profile in healthy Chinese young adults.

The aim of this study was to investigate the interactions of genetic variants in the genes of cholesterol ester transfer protein (CETP) and low-density lipoprotein receptor (LDLR) with high carbohydrate and low fat (HC/LF) diet on lipid profiles in a young and healthy Chinese Han population. Fifty-six healthy subjects (22.89±1.80 years) were given washout diets of 31% fat and 54% carbohydrate for 7 days, followed by HC/LF diets of 15% fat and 70% carbohydrate for 6 days, with no total energy restriction. Serum lipid profiles at baseline, after washout and following HC/LF diets, as well as CETP and LDLR polymorphisms were analyzed. Carriers of B2 allele of CETP TaqIB polymorphism had significantly higher levels of high density lipoprotein cholesterol (HDL-C) and apo A-I in the whole study population after the diet intervention. Notably, males with CETP TaqIB B1B1 experienced significantly increased HDL-C and apo A-I after HC/LF diet. Regarding the LDLR Pvu II polymorphism, both P1P1 subjects and P2 carriers experienced decreased total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels after HC/LF diet with no statistically significant differences between the genotypes. Our results demonstrate that the elevated HDL-C levels after HC/LF diet in healthy Chinese Han youth are associated with CETP TaqI B2 allele while males with B1B1 genotype are more susceptible to the influence of HC/LF diet on their HDL-C levels. The decreased TC and LDL-C levels after HC/LF diet are not associated with LDLR Pvu II polymorphism.

[Discrimination of common oral streptococcus with metabonomics method].

OBJECTIVE: To evaluate the feasibility of identifying oral streptococcus by comparing their metabolic profiling, and to find a convenient and rapid way to discriminate oral microorganisms. METHODS: The pure cultivation of Streptococcus sanguis ATCC 10556 and Streptococcus sobrinus 6715 (reference strain) from solid culture were respectively inoculated in TPY liquid medium. Then the growth quantity was measured periodically by turbidimetry and the growth curves of the inoculated bacteria were completed. The culture solutions in the stationary phase of the two bacteria were centrifuged, and then tested with the 1H-nuclear magnetic resonance (1H-NMR) spectrometer respectively. The gained free induction decay (FID) data were all inputted into MestReC Soft and finally transformed into metabolic profiling. The metabolic profiles were integrated segmentingly and the results were inputted into SIMCA-P Soft for principal components analysis (PCA). RESULTS: The PCA results showed the obvious clustering phenomena and the points of two group data differentially centralized in two clusters. Therefore, the NMR-based metabonomics profiles can discriminate the two different kinds of bacteria. CONCLUSION: The metabonomics can be expected to be a kind of promising useful method in quick discrimination of oral streptococcus.

[Initial study on the discrimination of oral common Actinomycetes with metabonomics method].

OBJECTIVE: The method of metabonomics based on 1H-nuclear magnetic resonance (1H-NMR) was preliminarily applied to discriminate the oral common Actinomycetes, Actinomyces naeslundii ATCC12104 and Actinomyces israelii ATCC12102. METHODS: Solutions of Actinomyces naeslundii and Actinomyces israelii with same density were made and cultured respectively at BHI liquid culture medium. The concentration of bacteria was determined periodically, and then the growth curves were drawn. The culture solutions in stationary phase of the two bacteria were used to test with the 1H-NMR spectroscopy respectively. The data of 1H-NMR spectroscopy results were analyzed by principal components analysis (PCA). RESULTS: The PCA showed the obvious clustering phenomena and the points of two groups data stayed differentially together by two clusters. Therefore, the NMR-based metabolomics profiles can discriminate the two different kinds of bacteria. CONCLUSION: The analysis technology of metabonomics is expected to be applied to rapid identification of actinomycetes.

[Preliminary study on the discrimination of putative periodontal pathogens with a metabonomics method].

OBJECTIVE: To evaluate the feasibility of identifying oral pathogenic bacteria by comparing the metabolic profiling of putative periodontal pathogens and try to find a convenient and rapid way to discriminate oral microorganisms. METHODS: Suspensions of Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum with same density were prepared and cultured respectively at liquid BHI medium. Then the growth quantity was measured periodically through turbidimetry and the growth curves of the inoculated bacteria were completed. The culture solutions of stable growth phase were sampled and characterized by 1H-nuclear magnetic resonance 1H-NMR). The data of 1H-NMR spectroscope results were analyzed by principal components analysis (PCA). RESULTS: The PCA showed the obvious clustering phenomena and the points of three groups differentially centralized to three clusters. Therefore, the NMR-based metabonomics profiles could discriminate the three different kinds of bacteria. CONCLUSION: The metabonomics is a potential classable method to identify the oral pathogenic bacteria.

[Initial study on the discrimination of oral microorganisms with a metabonomics method].

OBJECTIVE: To establish the spectra of metabolites that coued be employed in identification of oral pathogenic bacteria, and try to find a convenient and rapid way to discriminate oral microorganisms. METHODS: Suspensions of Streptococcus mutans ATCC 25175, Streptococcus sanguis ATCC 10556 and Lactobacillus acidophilus ATCC 4356 with same density were preparecd and cultured respectively at improved TPY liquid culture medium. The growth quantity were measured periodically by a turbidimeter. And the growth curves of the inoculated bacteria were completed. The culture solutions in stationary phase of the three bacteria were tested with 1H-nuclear magnetic resonance (1H-NMR) spectroscopy respectively. The data of 1H-NMR spectroscopy results were analyzed by principal components analysis (PCA). RESULTS: The PCA showed the obvious clustering phenomena and the points of three group differentially centralized to three clusters. Therefore, the NMR-based metabonomics profiles could discriminate the three different kinds of bacteria. CONCLUSION: The metabonomics is a promising new technology for developing to a rapid discrimination method of oral pathogenic bacteria.

[Construction of adenovirus expressing siRNA against hTR and its anti-tumor activity in vitro].

AIM: To study the effect of the hTR-siRNA adenovirus on hTR mRNA gene silence, telomerase activity inhibition and anti-tumor in vitro. METHODS: RNAi adenovirus vector, Ad-hTR-siRNA, and negative control Ad-NT-siRNA were constructed by an improved ligation method. Different tumor cells and liver cell line, HL-7702, were infected with 100 MOI of the recombinant adenoviruses. TRAP-ELISA, Real-time PCR and FCM were used to analyze telomerase activity, hTR mRNA, apoptosis rate and hTERT protein expression. RESULTS: As compared with Ad-NT-siRNA, Ad-hTR-siRNA reduced both hTR mRNA levels (70.21%) and telomerase activity (58.87%) of HeLa cells significantly, increased apoptosis rate (29.7%). But the telomerase activity of HL-7702 and hTERT protein didn't show the tendency of decrease. CONCLUSION: It is supposed that the hTR-siRNA adenovirus could knockdown hTR gene specifically and suppress the tumor cell growth in vitro efficiently. Maybe this siRNA expressing recombinant adenovirus system could be a new method for cancer gene therapy.

[Cloning of ureI gene from Helicobacter pylori and its expression in E. coli].

OBJECTIVE: To clone the urea membrane channel gene (ureI) from Helicobacter pylori (Hp) for its expression in E. coli, and evaluate the expression conditions and immunological features of the fusion protein. METHODS: ureI gene cloned by PCR from Hp was inserted into the plasmid pET32a (+) to construct the recombinant plasmid pET32a/ureI, followed by identification by BglII and HindIII digestion and sequencing. E. coli BL-21+(DE3) was transformed with pET32a/ureI to obtain the engineered bacterium BL21+/UreI, which was cultured at different temperatures and induced with 1.0 mmol/L IPTG for expression of the recombinant protein. The expressed proteins were identified by SDS-PAGE and analyzed by Pro-gel analyzer 4.0. Western blotting was performed to evaluate the immunogenicity of the expressed protein. RESULTS: The cloned gene fragment was about 650 bp in length, and BglII and HindIII digestion of pET32a/ureI yielded a 650-bp band. Sequence analysis revealed that the cloned ureI gene contained 646 bp without reading frame alterations. Comparison against GenBank indicated a homology of 100% of the cloned gene with ureI gene of the corresponding Hp strains, and also one no less than 98.5% with ureI gene from other strains. The engineered E. coli BL21+/UreI could express recombinant UreI (rUreI) with His tag, and the target protein accounted for 20.2% of the total bacterial proteins after 1.0 mmol/L IPTG induction of the bacterium at 37 degrees C for 14 h. SDS-PAGE and Western blotting showed that the recombinant UreI protein was produced mainly in the inclusion bodies and fused with his-tag (rUreI/his), which could react with human anti-Hp and mAb to his tag but not with mAb to Hp UreB. CONCLUSIONS: We have successfully cloned ureI gene and constructed the prokaryotic expression plasmid for efficient rUreI expression, and the fusion protein rUreI/his expressed in the inclusion bodies can react specifically with both Hp antibody and his-tag antibody.

[Initial study on discrimination of oral microorganisms with the metabonomics technique].

OBJECTIVE: To evaluate the feasibility of employing metabonomics method in identification of oral pathogenic bacteria. METHODS: The Streptococcus mutans ATCC25175 and Actinomyces viscosus ATCC15987 were respectively inoculated in same certain culture medium. The growth curves of the inoculated bacteria were drown by turbidimetry. The culture solutions in four different growth phases of the both bacteria were used to test with the 1H-Nuclear magnetic resonance (1H-NMR) spectroscopy respectively. The data of 1H-NMR spectroscopy results were analyzed by principal components analysis (PCA). RESULTS: The PCA showed the obvious clustering phenomena and the points of two group data stayed differentially together by two clusters. Therefore, the NMR-based metabonomics profiles can discriminate the two different kind of bacteria. CONCLUSION: The metabonomics can be expected to be a kind of promising useful method in quick discrimination of oral pathogenic bacteria.