葡聚糖结合蛋白A抗体对变形链球菌黏附影响的研究

目的：了解葡聚糖结合蛋白(CbP)A抗体对变形链球菌黏附的影响情况。方法：利用核素闪烁计数法，测定不同浓度的葡聚糖结合蛋白抗体影响两种变形链球菌对羧基磷灰石(HA)的黏附率。结果：葡聚糖结合蛋白抗体可明显抑制两种变形链球菌对羧基磷灰石的黏附率，并且随着葡聚糖结合蛋白抗体浓度的提高，抑制作用增强。结论：葡聚糖结合蛋白抗体可抑制变形链球菌对羧基磷灰石的黏附，在免疫防龋方面具有一定的意义。     

A IIIman protein is involved in the transport of glucose,mannose and fructose by oral streptococci

We show in this article that the transport of glucose, mannose and fructose by the phosphoenolpyruvate: mannose phosphotransferase system of oral streptococci requires the participation of a protein component that we have called III_man. This protein was purified from Streptococcus salivarius by chromatography on DEAE-cellulose, DEAE-TSK, hydroxyapatite, and Dyematrex Green A. The purified protein migrated as a 38,900 molecular weight protein on a sodium dodecyl sulfate polyacrylamide gel. However, electrophoretic analysis of phosphoproteins and Western blot experiments indicated the presence in membrane-free cellular extracts of S. salivarius of 2 different forms of III_man having molecular weights of 38,900 and 35,200. The presence of the high-molecular-weight form of III_man was observed by immunodiffusion. Western blot and phosphorylation by [³²]PEP in S. salivarius, Streptococcus mutans, Streptococcus sobrinus, and Streptococcus lactis but not in Streptococcus faecium, Staphylococcus aureus, Bacillus subtilis and Lactobacillus casei. Antibodies directed against the III_man of S. salivarius did not react with the III_man of Escherichia coli.     

变形链球菌表面蛋白多肽片段在转基因番茄中的表达

目的在获得含编码变形链球菌表面蛋白唾液粘附区片段基因的转基因番茄原代种子的基础上，应用分子生物学的方法检测外源基因在转基因植株中的表达。方法用CTAB法提取子代番茄总DNA，PCR筛选含变形链球菌PAc基因唾液粘附区片段的转基因番茄子代植株；用Trizol提取番茄总RNA，RT- PCR检测外源基因的转录情况；提取番茄果实总蛋白，用Bradford法测定番茄果实总蛋白含量；通过Western blot检测外源蛋白的表达情况，并用ELISA法对外源蛋白含量进行定量测定。结果获得植物细胞染色体上整合有外源基因并发生表达的转基因番茄子代植株；Western blot和ELISA分析表明含编码变形链球菌表面蛋白唾液粘附区片段基因的转基因番茄子代植株能有效表达外源蛋白，该蛋白含量占番茄可溶性总蛋白的1.2%。结论含编码变形链球菌表面蛋白唾液粘附区片段基因的转基因番茄子代植株能有效表达外源蛋白。     

Antimicrobial activity of garlic,tea tree oil,and chlorhexidine against oral microorganisms

OBJECTIVE: To compare the antimicrobial activity of tea tree oil, garlic, and chlorhexidine solutions against oral microorganisms. METHOD: The five-week study consisted of thirty subjects. The first week was considered baseline. All subjects used a control solution (second week), and were randomly divided into the three groups (third week): G1-0.12% chlorhexidine; G2 - 2.5% garlic (Allium sativum, L.); and G3 - 0.2% tea tree oil (Melaleuca alternifolia). Dishes containing blood agar and Mitis Salivarius Bacitracin agar (MSB) were inoculated with the subjects' saliva (collected twice a week). Total microorganisms and mutans streptococci were counted in blood agar and MSB, respectively. RESULTS: Chlorhexidine and garlic groups showed antimicrobial activity against mutans streptococci, but not against other oral microorganisms. The tea tree oil group showed antimicrobial activity against mutans streptococci and other oral microorganisms. Maintenance of reduced levels of microorganisms was observed only for garlic and tea tree oil during the two consecutive weeks (fourth and fifth). Unpleasant taste (chlorhexidine 40%, tea tree oil 30%, garlic 100%), burning sensation (chlorhexidine 40%, tea tree oil 60%, garlic 100%), bad breath (chlorhexidine 40%, tea tree oil 20%, garlic 90%), and nausea (chlorhexidine 0%, tea tree oil 10%, garlic 30%) were reported. CONCLUSION: Garlic and tea tree oil might be an alternative to chlorhexidine.     

Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction

Igarashi T, Yamamoto AA, Goto N. Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction.
Streptococcus mutans is an etiological agent in human dental caries. A method for the detection of S. mutans directly from human dental plaque by polymerase chain reaction has been developed. Oligonucleotide primers specific for a portion of the dextranase gene (dexA) of S. mutans Ingbritt (serotype c) were designed to amplify a 1272-bp DNA fragment by polymerase chain reaction. The present method specifically detected S. mutans (serotypes c, e and f), but none of the other mutans streptococci: S. cricetus (serotype a), S. rattus (serotype b), S. sobrinus (serotypes d and g), and S. downei (serotype h), other gram-positive bacteria (16 strains of 12 species of cocci and 18 strains of 12 species of bacilli) nor gram-negative bacteria (1 strain of 1 species of cocci and 20 strains of 18 species of bacilli). The method was capable of detecting 1 pg of the chromosomal DNA purified from S. mutans Ingbritt and as few as 12 colony-forming units of S. mutans cells. The S. mutans cells in human dental plaque were also directly detected. Seventy clinical isolates of S. mutans isolated from the dental plaque of 8 patients were all positive by the polymerase chain reaction. These results suggest that the dexA polymerase chain reaction is suitable for the specific detection and identification of S. mutans.     

聚乙二醇抑制变形链球菌生长的实验研究

目的 :探讨聚乙二醇 (polyethyleneglycol,PEG)与变形链球菌作用后 ,其抑制细菌生长的效果。方法 :采用 2种低分子量的PEG(Mr分别为 2 0 0和 4 0 0 ,简称PEG2 0 0和PEG4 0 0 ) ,各选 4种不同浓度与细菌作用 ,分别经过 2 4和 4 8h培养 ,用分光光度计测试细菌生长的A540 值。结果 :经双因素无重复实验方差分析结果显示 :PEG的分子量大小和浓度高低及培养时间对实验结果均有较大的影响 ,两实验组之间以及实验组与对照组之间结果相差非常显著 (P <0 .0 0 1) ,即认为在培养时间为 2 4h时 ,浓度越大 ,分子量越小 (Mr为 2 0 0 ) ,抑制细菌生长的效果越好。结论 :低分子量的PEG作为非离子型表面活性剂 ,在一定时间内对抑制变链球菌的生长是有效的 ,值得我们对其防龋作用作进一步的研究探讨     

变形链球菌表面蛋白和葡糖基转移酶基因疫苗免疫防龋实验研究:唾液和血清抗体水平测定

目的　将基因疫苗pcDNA3-pac和pcDNA3-gtfB经腺周注射免疫定菌大鼠,观察唾液和血清抗体的变化,证实基因疫苗的免疫原性。方法　36只Wistar大鼠分为6个免疫组:pcDNA3-pac,pcDNA3-gtfB,pcDNA3-pac联合 pcDNA3-gtfB,阳性对照灭活变形链球菌,阴性对照pcDNA3及PBS液组。建立龋攻击定菌鼠模型3个月,各组大鼠均以100μg剂量免疫定菌鼠3次,ELISA法检测唾液SIgA和血清IgG水平。结果　疫苗组在首次免疫后第33天 (第5周)都出现较高水平的唾液SIgA,在第75天(第11周)后达最高水平,并持续至实验结束。成组单因素方差分析唾液SIgA水平在总体上各实验组间存在差异(P<0·01或P<0·05),q检验分析在联合免疫组和全菌细胞组最高,单基因疫苗免疫组次之,pcDNA3与PBS组最低;血清IgG水平在4个免疫组之间没有差异(P>0·05),但都高于空白及阴性对照组(P<0·05)。结论　pcDNA3-pac和pcDNA3-gtfB腺周途径免疫定菌Wistar大鼠,能有效诱导唾液 SIgA和血清IgG抗体产生,两种疫苗联合免疫优于单基因疫苗。     

Identification of a glucan-binding protein C gene homologue in Streptococcus macacae

BACKGROUND/AIMS: The past few decades have seen the isolation of certain glucosyltransferases and a number of proteins from mutans streptococci. Some of these proteins have been shown to possess glucan-binding capabilities which confer an important virulence property on mutans streptococci for the role of these bacteria play in dental caries. Among these proteins is glucan-binding protein C, which is encoded by the gbpC gene, and which we have identified as being involved in the dextran-dependent aggregation of Streptococcus mutans. However, gbpC homologues have yet to be identified in other mutans streptococci. METHODS: We carried out polymerase chain reaction amplification of Streptococcus macacae using primers that were designed based on conserved sequences of S. mutans gbpC and identified a gbpC gene homologue. The gene of that homologue was then characterized. RESULTS: Nucleotide sequencing of the S. macacae gbpC homologue revealed a 1854 bp open reading frame encoding a protein with an N-terminal signal peptide. The molecular mass of the processed protein was calculated to be 67 kDa. We also found an LPxTG motif, the consensus sequence for gram-positive cocci cell wall-anchored surface proteins, which was followed by a characteristic sequence at the carboxal terminal region of the putative protein. This suggests that the S. macacae GbpC homologue protein was tethered to the cell wall. CONCLUSION: Based on these results, together with the demonstrated glucan-binding ability of the S. macacae GbpC homologue protein, we suggest that S. macacae cells are capable of binding dextran via the GbpC homologue protein, which is similar to the S. mutans GbpC protein. In addition, Southern hybridization analysis using the S. macacae gbpC homologue as a probe showed a distribution of gbpC homologues throughout the mutans streptococci.     

A searchable database for proteomes of oral microorganisms

An online database of proteomes for two-dimensional electrophoresis (2DE) gel data was constructed and it is now freely accessible through a web-based interface. Proteins from three oral bacteria, Streptococcus mutans UA159, Actinobacillus actinomycetemcomitans HK1651, and Porphyromonas gingivalis W83, whose genome databases are freely available, were separated by 2DE, and protein spots were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and identified. About 1000 spots from the gels of P. gingivalis W83 were extracted and analyzed by MALDI-TOF, and 330 proteins were identified. In addition, 160 of 240 spots of A. actinomycetemcomitans and 158 of 356 spots of S. mutans were identified. Information such as spot coordinates on the gels, protein names (predicted functions), molecular weights, isoelectroric points, and links to online databases, including Oral Pathogen Sequence Databases of the Los Alamos National Laboratory Bioscience Division (ORALGEN) and National Center for Biotechnology Information (NCBI) or The Institute Genomic Research (TIGR), were stored in tables accessible through the relational database management system MySQL on an Apache web server. To test for functionality of this database system, responses of S. mutans to environmental changes were analyzed using the database and 21 spots on the gel were identified as proteins whose expression had been increased or decreased by environmental pH change without in-gel trypsin digestion, protein extraction, or MALDI-TOF/TOF-MS (mass spectrometer) analysis. The identified proteins are agreement with those reported in previous papers on acid tolerance of S. mutans, demonstrating the usefulness of the system. This database is available at http://www.myamagu.dent.kyushu-u.ac.jp/~bioinformatics/index.html or http://www.bipos.mascat.nihon-u.ac.jp/index.html.