Effect of deletion of the rgpA gene on selected virulence of Porphyromonas gingivalis.

BACKGROUND/PURPOSE: The most potent virulence factors of the periodontal pathogen Porphyromonas gingivalis are gingipains, three cysteine proteases (RgpA, RgpB, and Kgp) that bind and cleave a wide range of host proteins. Considerable proof indicates that RgpA contributes to the entire virulence of the organism and increases the risk of periodontal disease by disrupting the host immune defense and destroying the host tissue. However, the functional significance of this proteinase is incompletely understood. It is important to analyze the effect of arginine-specific gingipain A gene (rgpA) on selected virulence and physiological properties of P. gingivalis. MATERIALS AND METHODS: Electroporation and homologous recombination were used to construct an rgpA mutant of P. gingivalis ATCC33277. The mutant was verified by polymerase chain reaction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cell structures of the mutant were examined by transmission electron microscopy and homotypic biofilm formation was examined by confocal laser scanning microscopy. RESULTS: Gene analysis revealed that the rgpA gene was deleted and replaced by a drug resistance gene marker. The defect of the gene resulted in a complete loss of RgpA proteinase, a reduction of out membrane vesicles and hemagglutination, and an increase in homotypic biofilm formation. CONCLUSION: Our data indicate that an rgpA gene deficient strain of P. gingivalis is successfully isolated. RgpA may have a variety of physiological and pathological roles in P. gingivalis.

[Construction of recombinant plasmid with Porphyromonas gingivalis FimA deficiency].

OBJECTIVE: To construct the recombinant plasmid pPHU281_A_Spec_B, which knock out Porphyrmonas gingivalis (Pg) FimA gene. METHODS: Genomic DNA was extracted from PgATCC33277 which was cultured in anaerobic condition. The upstream and downstream gene of FimA was cloned from Pg genenomic DNA with specific restriction sites by polymerase chain reaction. Suicide vector pPHU281 was inserted by three fragments: upstream, downstream of FimA gene and spectinomycin resistance gene. The recombinant plasmid was confirmed by electrophoresis and sequenced after amplification in compentent cells DH-5α. RESULTS: The gene sequence was identified by DNA sequencing analysis. The recombinant plasmid pPHU281_A_Spec_B was successfully constructed. CONCLUSIONS: The recombinant plasmid pPHU281_A_Spec_B was constructed, which may be used for the constructon of FimA deficient Pg.

Amorphous calcium phosphate and its application in dentistry.

Amorphous Calcium Phosphate (ACP) is an essential mineral phase formed in mineralized tissues and the first commercial product as artificial hydroxyapatite. ACP is unique among all forms of calcium phosphates in that it lacks long-range, periodic atomic scale order of crystalline calcium phosphates. The X-ray diffraction pattern is broad and diffuse with a maximum at 25 degree 2 theta, and no other different features compared with well-crystallized hydroxyapatite. Under electron microscopy, its morphological form is shown as small spheroidal particles in the scale of tenths nanometer. In aqueous media, ACP is easily transformed into crystalline phases such as octacalcium phosphate and apatite due to the growing of microcrystalline. It has been demonstrated that ACP has better osteoconductivity and biodegradability than tricalcium phosphate and hydroxyapatite in vivo. Moreover, it can increase alkaline phosphatase activities of mesoblasts, enhance cell proliferation and promote cell adhesion. The unique role of ACP during the formation of mineralized tissues makes it a promising candidate material for tissue repair and regeneration. ACP may also be a potential remineralizing agent in dental applications. Recently developed ACP-filled bioactive composites are believed to be effective anti-demineralizing/remineralizing agents for the preservation and repair of tooth structures. This review provides an overview of the development, structure, chemical composition, morphological characterization, phase transformation and biomedical application of ACP in dentistry.

[Effect of longterm lower Porphyromonas gingivalis infection on the progression of atherosclerosis in apolipoprotein E-knocked out mice].

OBJECTIVE: To assess the effect of longterm and lower oral inoculation with Porphyromonas gingivalis (Pg) on the progression of atherosclerosis in apolipoprotein E-knocked out (ApoE(-/-)) mice. METHODS: Six-week-old male ApoE(-/-) mice were inoculated orally with 0.1 ml live Pg(10(13)/L) or bouillon culture-medium quintic per week for 15 consecutive weeks, altogether 75 times of inoculations. The lesion area of atherosclerosis in the aortic tree was measured by en face quantification by red oil O staining method. The atherosclerotic lesion was examined by histopathology. The levels of total cholesterol and triglycerides were compared. RESULTS: At 22 weeks after inoculation, the mean atherosclerotic lesion area in inoculated mice was (98 363.68 ± 12 043.00) µm(2), which was significantly greater than that in noninoculated mice, which was (62 985.06 ± 7419.64) µm(2) (P = 0.035). CONCLUSIONS: Longterm lower oral inoculation of Pg can accelerate the progression of atherosclerosis in apolipoprotein E-knocked out mice.

[Surveillance and forecast of schistosomiasis transmission in Chaohu Lake area in Anhui Province, 2008-2010].

OBJECTIVE: To understand the dynamic changes of the potential prevalent factors of schistosomiasis in Chaohu Lake area so as to provide forecast information on the outbreak of schistosomiasis in the area. METHODS: From 2008 to 2010, fixed and mobile surveillance sites in potential endemic areas of Juchao District in Chaohu City, which was located in the southeast side of Chaohu Lake, were selected, and the schistosomiasis infection situation of local people, mobile population and livestock were investigated by immunological assays and/or stool examinations. The distribution of Oncomelania snails was surveyed in risk areas and suspicious areas, the spreading patterns of Oncomelania snails were observed in rivers that directly connected with the Yangtze River, and the Oncomelania snails were raised in the cages on the beaches of Chaohu Lake and a control area, and their survival and reproduction capacity was observed. RESULTS: In 2008, a total of 301 local people were screened by IHA, and there were no positives. From 2008 to 2010, a total of 321, 362 and 306 mobile population were examined by IHA, respectively, and the positive rate of antibody were 3.74%, 4.97% and 2.94%, respectively. The antibody positives were tested by stool examinations, and the positive rates were 66.67%, 50% and 55.56%, respectively. A total of 91 local livestock and 92 livestock from endemic areas were examined respectively by the miracidium hatching method, and there were no positives. A total of 97.8 hm2 risk areas and 193.62 hm2 suspicious areas in the potential endemic area were surveyed respectively, but no Oncomelania snails were found. The investigation results on snail spreading patterns indicated that snails could spread into Chaohu Lake by adsorbing on floating debris. The field study revealed that Oncomelania snails could survive and reproduce in the Lake. CONCLUSIONS: The imported infectious sources of schistosomiasis have been found in Chaohu Lake area, and the higher possibility of imported Oncomelania snails spreading into the Lake and surviving and reproducing in the lake is predicted. Therefore, effective measures should be taken to decrease the risks of schistosomiasis transmission in the potential endemic area.

[Effect of Porphyromonas gingivalis on nitric oxide in cultured human umbilical vein endothelial cells].

OBJECTIVE: To observe the effect of Porphyromonas gingivalis (Pg) on the production of nitric oxide (NO) in cultured human umbilical vein endothelial cells (HUVEC), and to investigate the pathway of damaging endothelial function by Pg. METHODS: Pg ATCC33277 was cultured in anaerobic jar, and HUVEC was treated with various concentrations of Pg ATCC33277 at multiplicity of infection (MOI) of 1:10, 1:100 and 1:1000 for 4, 8, 12, 24 h respectively. The cells supernatants were collected and stored at -70 degrees C and NO concentration in the cells supernatants was measured by nitrate reductase assay. RESULTS: Within 24 h, Pg at MOI of 1:10 and 1:100 stimulated the release of nitric oxide in cultured HUVEC. Within 12 h, Pg at an MOI of 1:1000 group increased NO production, and NO decreased at 24 h. CONCLUSIONS: Pg has an effect on the production of NO. Low concentrations of Pg stimulated release of nitric oxide in endothelial cells but high concentrations can decrease the release of NO.

[Preliminary study on herpes simplex virus type 1 infection of human oral epithelial cell in vitro].

OBJECTIVE: To investigate the way of herpes simplex virus type 1 (HSV-1) infecting human oral epithelial cell in vitro. METHODS: Abundance of HSV-1 strains were used to infect human oral epithelial cells. The culture supernatant was collected to infect Vero cells again. Morphology of HSV-1 was identified by inverted microscope and transmission electron microscope. Nucleic acid of the virus was detected by PCR. RESULTS: The infected human oral epithelial cells didn't display obvious cytopathic effect (CPE) while Vero cells infected by the culture supernatant showed typical CPE. The virus particles were observed in Vero cells. The nucleic acid of HSV-1 could be detected in infected human oral epithelial cells by PCR. CONCLUSIONS: HSV-1 could successfully infect human oral epithelial cells.

[The expression and activity of alkaline phosphatase in human periodontal ligament cells with nanometer hydroxyapatite].

OBJECTIVE: To investigate the effect of nanometer hydroxyapatite on the proliferation and the osteogenetic differentiation of periodontal ligament cells (PDLC). METHODS: Nano-hydroxyapatite powders were fabricated with sol-gel method. The fourth passage periodontal ligament cells were cultured with nanometer hydroxyapatite powder (nano-HA), dense hydroxyapatite powder (dense-HA) and only medium as control respectively. On the 5th, 8th day of culture, the osteogenetic differentiation of human periodontal ligament cells was evaluated though alkaline phosphatase (ALP) activity, ALP immunohistochemical stain and ALP positive flow cytometry. RESULTS: There were significant differences among nano-HA group, dense-HA group and control group on the 5th and 8th day of culture. A majority of nano-HA group and dense-HA group cells sample showed positive ALP stain. But the ALP positive stain of nano-HA group cells sample was denser than that of dense-HA group. In FCM, the distribution of ALP positive cells cultured with nanoparticles were significantly more than that of other groups. CONCLUSIONS: The nano-HA, as a calcium phosphate biomaterial, has ability to promote the activity of osteogenetic differentiation for periodontal ligament cells compared with dense-HA.

[The biological changes of NIH3T3 cells co-cultured with human bone morphogenetic protein-2 gene transfecting cells].

OBJECTIVE: To investigate the ultrastructure and the alkaline phosphatase (ALP) activity changes of NIH3T3 cells incubated with secretive human bone morphogenetic protein-2 (hBMP-2) that is induced by gene transfection through transwell system. METHODS: Eukaryotic expression vector (pcDNA3.1-B2) was transduced into NIH3T3 cells by Sofast, a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expression of BMP-2 in the NIH3T3 cells were determined by immunohistochemical and enzyme-linked immunosorbent assay (ELISA). NIH3T3 cells were co-cultured with hBMP-2 gene transfecting cells through transwell system, and the ultrastructure and ALP activity (the markers of osteogenetic differentiation) changes were observed. RESULTS: There were cytoplasmic and extracellular expression of BMP-2 in transfecting NIH3T3 cells. The ultrastructure changes and the high expression of ALP suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells. CONCLUSIONS: Secretive BMP-2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells.

[A new method of molecular marker--RMAPD].

Random microsatellite amplify polymorphic DNA (RMAPD) is a new method of molecular marker. It can reveal genome DNA polymorphisms of organism only when random primer and microsatellite forward or reverse primer are combined together as a pair of primers by the PCR reaction system with Taq DNA polymerase, MgCl2, dNTPs and contemplate DNA. The core question of RMAPD is validity of the primers used. A lot of experiments of RMAPD in 69 Xinong Saanen dairy goats showed that RMAPD primers were effective. Comparison of RMAPD, microsatellite and RAPD markers demonstrated that RMAPD was different from each other in primers, amplification protocol and repetition. RMAPD is not equal to RAPD, but a extendable RAPD. Therefore, RMAPD is regarded as a new method of molecular marker. As it has many characteristics of DNA marker, RMAPD has a widest potential application in animal breeding and genetics, such as analysis of genetic structure and relationship and MAS.

[Research on the genetic polymorphism in crested ibis by RMAPD].

The genetic polymorphism of Yangxian artificially reared 43 crested ibises (Nipponia nippon) was firstly investigated by RMAPD (random microsatellite amplify polymorphic DNA) marker. The results showed that RMAPD was more stable and polymorphic than RAPD. 2147 bands were amplified by 12 pairs of primers,of which 93 bands had polymorphisms. The band sharing frequency and the genetic diversity index were 0.718 and 3.664, respectively, indicating that the genetic structure of crested ibis population was simple and poor genetic variations existed in crested ibis population in Yangxian. It is imperious to protect the genetic diversity of crested ibis population.

[Effects of secretive bone morphogenetic protein 2 induced by gene transfection on the biological changes of NIH3T3 cells].

BACKGROUND: Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta superfamily, are powerful regulators of cartilage and bone formation. This study investigated the biological changes of NIH3T3 cells incubated with secretive BMP2 that was induced by gene transfection through transwell. METHODS: Eukaryonic expression vector (pcDNA3.1-B2) was transfered into NIH3T3 cells with Sofast, a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expressions of BMP2 were determined by immunohistochemical stain and enzyme-linked immunosorbent assay. NIH3T3 cells were co-cultured with hBMP2 gene transfecting cells through transwell, and the ultrastructure, alkaline phosphatase activity and the expression of osteocalcin (the marker of osteogenetic differentiation) changes were observed. RESULTS: There were cytoplasmic and extracellular expressions of BMP2 in transfecting NIH3T3 cells. The ultrastructural changes, the high activity of alkaline phosphatase and the positive stain of osteocalcin suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells. CONCLUSION: Secretive BMP2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells.

[The effect of CSN1 S2, CSN3 and beta-lg genes on milk performance in Xinong Saanen dairy goat].

PCR-RFLP technique was applied to analyze correlation between the polymorphisms of CSN1 S2 (alpha(s2) casein), CSN3 (kappa casein) and beta-lg (beta-lactoglobulin) genes and milk performance in 69 individuals of Xinong Saanen dairy goat. The results showed that there was significant correlation between different genotypes of CSN1 S2 locus and milk yield:average milk yield of individuals with genotype FF was less than that of genotype NN (P < 0.05); the second lactation milk yield of individuals with genotype NN was over 90 kg higher than that with genotype FF (P < 0.01). This indicates that allele F of CSN1 S2 locus probably has significant negative effect on milk yield. The results of CSN3 gene digested with endonuclease Hind III cleavage showed that no significant difference of milk yield between genotype DE and genotype EE was detected in first, second, third and fourth lactation milk yield and average milk yield (P > 0.05). The results of CSN3 gene with endonuclease Taq I cleavage showed that no significant difference of milk yield among individuals with genotype TT, TC and CC was detected (P > 0.05). No polymorphism was detected in PCR products of CSN3 gene digested with endonuclease Hae III. The analysis of beta-lg gene's 5' flanking region (710 bp) by PCR-RFLP in Xinong Saanen dairy goat showed that milk yield of individuals with genotype AA was higher than that with genotype AB in second, third lactation milk yield and average milk yield (P < 0.05). The results implied that allele A of beta-lg gene's 5' flanking region is probably related to high milk yield.

[Study on mitochondrial DNA D-loop polymorphism in Chinese donkeys].

The mitochondrial DNA (mtDNA) D-loop sequences with 399 bp in 26 individuals from 5 donkey breeds in China were analyzed. Aligned by Clustal W software,the results showed that 23 polymorphic nucleotide sites and only transition with the percentage of 5.76% in 399 bp were observed. In reference to mtDNA D-loop sequences of European domestic donkey as a control, the average percentage of mtDNA D-loop nucleotide variation in 5 Chinese donkey breeds was 1.80%. The average percentages of D-loop nucleotide variation from Liangzhou donkey (LZ), Yunnan donkey (YN), Guanzhong donkey (GZ), Xinjiang donkey (XJ) and Jiami donkey (JM) were 0. 35%, 1.25%, 2.30%, 2.91% and 2.20% respectively. The average sequence divergence estimated from D-loop sequences varied from 0.25% - 5.01% within breeds and 4.51% - 5.51% among breeds, respectively, demonstrating that there existed rather abundant mitochondrial genetic diversity in Chinese donkeys. Comparisons of the 26 sequences revealed 11 mitochondrial haplotypes; the percentage of haplotype was 42.31%. This phenomenon demonstrated that the mitochondrial genetic diversity in Chinese donkey breeds is being reduced. It is urgent to protect the genetic resources of Chinese donkey. The molecular phylogenetic tree of mtDNA D-loop sequences in 5 Chinese donkey breeds,6 sequences of Asian wild ass (Equus asinus kiang, Equus asinus kulan, Equus asinus hemionus;) and 4 sequences of European domestic donkeys from GenBank was constructed by Neighbor-Joining method. It was the first time proved in molecular level that the origin of Chinese donkey breeds was from African wild ass (Equus africanus africanus and Equus africanus somaliensis), not from Asian wild ass as bescribed in the paper.

[Study on mitochondrial DNA genetic diversity of some cattle breeds in China].

The complete mitochondrial D-loop sequences, 910 bp in length, in 22 individuals from 8 cattle breeds in China were analyzed. The results showed that A% + T% was about 61.65%. Comparisons of these 22 sequences revealed 66 polymorphic sites, and 5 types of mutation, transition, transversion, insertion, deletion and coexistence site of transition and transversion were observed,with the percentage of 81.82%, 6.06%, 7.57%, 3.03%, 1.52%, respectively. In reference to complete mtDNA D-loop of the European cattle as a control, eight Chinese cattle breeds were classified into 3 groups according to the average percentage of D-loop nucleotide variations. The lowest average percentage of mtDNA D-loop nucleotide variation was in Xizhen cattle, Mongolian cattle, Holstein, Qinchuan cattle with 0.37%, 0.44%, 0.52%, 0.66%, the mediate in Nanyang cattle and Jiaxian cattle with 1.91%, 2.02% and the highest in Jinnan cattle and Yueyang cattle with 4.47%, 4.73%, respectively. The average sequence divergence estimated from D-loop region within breeds and among breeds in China varied from 0.55% - 5.39% and 1.21% - 6.59%, respectively. Comparisons of these 22 sequences revealed 19 mitochondrial haplotypes, the percentage of haplotype was 86.36%, showing that abundant mitochondrial genetic diversity exists in Chinese cattle. The molecular phylogenetic tree of mtDNA D-loop of 8 Chinese cattle breeds was constructed by Neighbor-Joining method. The NJ tree indicated that these mtDNA sequences fell into 3 distinct haplotype groups, it also suggested in molecular level that there were probably 3 maternal origins, of which the main origins of Chinese cattle were from Bos taurus and Bos indicus.

[Polymorphisms of insulin-like growth factor binding protein 3 gene and its associations with several carcass traits in Qinchuan cattle].

DNA samples from 60 Qinchuan cattle (Bos taurus) were analyzed with PCR-RFLPs and sequencing for insulin-like growth factor binding protein 3 (IGFBP3) gene. Fragments of 651 bp were amplified with two primers and the products of PCR were digested with restriction endonuclease HaeIII. The produced fragments showed three genotypes, namely AA,AB and BB after electrophoresis. Frequencies of the genotype AA,AB,BB and allele A,B were 0.7,0.28,0.02,and 0.84,0.16,respectively. Sequence analysis showed that a transversion of C-->A at 299 nt resulted in loss of the cleaved site of restriction endonuclease HaeIII and produced this polymorphism. This polymorphic locus of IGFBP3 gene was at Hardy-Weinberg equilibrium (P>0.05). The genotypes of AA,AB,BB slightly affected several slaughter and carcass traits of Qinchuan cattle. Dressing percentage,net meat percentage, striplion percentage, tenderloin percentage, ribeye percentage and tender shoulder percentage were decreased with the genotypes of AA,AB and BB in Qinchuan cattle, but it was not significant (P>0.05). Average ribeye area in individuals of AA genotype was significantly higher than that in individuals of BB genotype (P<0.05), and beef fat content in individuals of genotype AB and BB was significantly higher than that in individuals of AA genotype (P<0.01).