Effects of DNA methylation on expression of virulence genes in Streptococcus mutans

Bacteria produce a variety of enzymes capable of methylating DNA. In many species, the majority of adenine methylation is accomplished by the DNA adenine methylase Dam. In Escherichia coli the Dam methylase plays roles in the initiation of replication, mismatch repair, and gene regulation. In a number of other bacterial species, mutation or overexpression of Dam leads to attenuation of virulence. Homologues of the dam gene exist in some members of the Firmicutes, including Streptococcus mutans, a dental pathogen. An S. mutans strain inactivated in the dam gene (SMU.504; here designated damA) was engineered, and phenotypes linked to cariogenicity were examined. A prominent observation was that the damA mutant produced greater amounts of glucan than the parental strain. Real-time PCR confirmed upregulation of gtfB. To determine whether other loci were affected by the damA mutation, a microarray analysis was carried out. Seventy genes were upregulated at least 2-fold in the damA mutant, and 33 genes were downregulated at least 2-fold. In addition to gtfB (upregulated 2.6-fold; 1.7-fold when measured by real-time PCR), other upregulated virulence factors included gbpC (upregulated 2.1-fold) and loci predicted to encode bacteriocins (upregulated 2- to 7-fold). Various sugar transport operons were also upregulated, the most extreme being the cellobiose operon (upregulated nearly 40-fold). Expression of sacB, encoding fructosyltransferase, was downregulated 2.4-fold. The sequence 5'-GATC-3' appeared to constitute the recognition sequence for methylation. These results provide evidence that DNA methylation in S. mutans has a global effect on gene expression, including that of genes associated with cariogenic potential.     

Reliable Identification of Deep Sulcal Pits: The Effects of Scan Session,Scanner, and Surface Extraction Tool

Sulcal pit analysis has been providing novel insights into brain function and development. The purpose of this study was to evaluate the reliability of sulcal pit extraction with respect to the effects of scan session, scanner, and surface extraction tool. Five subjects were scanned 4 times at 3 MRI centers and other 5 subjects were scanned 3 times at 2 MRI centers, including 1 test-retest session. Sulcal pits were extracted on the white matter surfaces reconstructed with both Montreal Neurological Institute and Freesurfer pipelines. We estimated similarity of the presence of sulcal pits having a maximum value of 1 and their spatial difference within the same subject. The tests showed high similarity of the sulcal pit presence and low spatial difference. The similarity was more than 0.90 and the spatial difference was less than 1.7 mm in most cases according to different scan sessions or scanners, and more than 0.85 and about 2.0 mm across surface extraction tools. The reliability of sulcal pit extraction was more affected by the image processing-related factors than the scan session or scanner factors. Moreover, the similarity of sulcal pit distribution appeared to be largely influenced by the presence or absence of the sulcal pits on the shallow and small folds. We suggest that our sulcal pit extraction from MRI is highly reliable and could be useful for clinical applications as an imaging biomarker.     

α-Amino acid containing degradable polymers as functional biomaterials: rational design, synthetic pathway, and biomedical applications

Currently, biomedical engineering is rapidly expanding, especially in the areas of drug delivery, gene transfer, tissue engineering, and regenerative medicine. A prerequisite for further development is the design and synthesis of novel multifunctional biomaterials that are biocompatible and biologically active, are biodegradable with a controlled degradation rate, and have tunable mechanical properties. In the past decades, different types of α-amino acid-containing degradable polymers have been actively developed with the aim to obtain biomimicking functional biomaterials. The use of α-amino acids as building units for degradable polymers may offer several advantages: (i) imparting chemical functionality, such as hydroxyl, amine, carboxyl, and thiol groups, which not only results in improved hydrophilicity and possible interactions with proteins and genes, but also facilitates further modification with bioactive molecules (e.g., drugs or biological cues); (ii) possibly improving materials biological properties, including cell-materials interactions (e.g., cell adhesion, migration) and degradability; (iii) enhancing thermal and mechanical properties; and (iv) providing metabolizable building units/blocks. In this paper, recent developments in the field of α-amino acid-containing degradable polymers are reviewed. First, synthetic approaches to prepare α-amino acid-containing degradable polymers will be discussed. Subsequently, the biomedical applications of these polymers in areas such as drug delivery, gene delivery and tissue engineering will be reviewed. Finally, the future perspectives of α-amino acid-containing degradable polymers will be evaluated.     

Polymorphism of kappa variable region (V kappa-1) genes in inbred mice: relationship to the Ig kappa-Ef2 serum light chain marker

We describe here the cloning and complete sequence analysis of the rearranged kappa variable region gene from the V kappa-1A-producing myeloma (C.AL20-TEPC-105). A 5'-flanking region probe from the V kappa-1A gene has been used to study the V kappa-1 germ-line gene family in strains of mice differing at the Ig kappa-Ef2 locus. All Ef2a strains examined possess an identical pair of BamHI restriction fragments strongly hybridizing to the 5' probe. Surprisingly, only two of the six strains of mice previously designated Ef2b (NZB and C58) possessed clearly altered restriction fragment sizes for V kappa-1 genes. The remaining four Ef2b strains, namely BDP/J, CE/J, I/LnJ, and P/J, appear to carry V kappa-1 genes similar to those of Ef2a strains. It is suggested that these strains may carry a third form of the V kappa-1A gene, differing in the protein coding region but indistinguishable at the DNA level with the use of BamHI or EcoRI. Alternatively, these strains may fail to express V kappa-1A light chains due to a regulatory defect involving this subgroup of kappa genes.     

Modulation of gamma delta T cells in mouse buccal epithelium following antigen priming

T cells using the gamma delta T cell receptor (TCR) are abundant in mucosal and epidermal tissues in mice. Most studies of mucosal gamma delta T cells, however, have examined cells from the intestinal mucosa, whereas little is known about the presence or function of gamma delta T cells in the oral cavity. To better understand the involvement of oral gamma delta T cells in immunity, we have characterized TCR variable gamma-gene usage in the buccal epithelium from normal mice, and from mice challenged locally with a non-replicating antigen (bovine serum albumin [BSA]) or by influenza-virus infection as a replicating antigen. Our findings demonstrate a restricted use of V gamma genes by buccal gamma delta T cells, consisting primarily of V gamma 1.2, V gamma 3, and V gamma 5, with minimal use of V gamma 2 and V gamma 4 genes. Of particular interest, 3-4 days post-antigen challenge with BSA, there was a precipitous drop in the level of expression of V gamma 1.2, V gamma 3, and V gamma 5 genes, and to a lesser extent for the V gamma 2 gene, whereas V gamma 4 gene expression increased between days 1 and 2 post-priming. In influenza-infected mice, a similar pattern was observed for the V gamma 2 and V gamma 5 genes, but not other V gamma genes. The immune-modulating effects of oral antigen exposure on buccal gamma delta T cells suggest that these cells are functionally involved in the local immune response to both replicating and non-replicating antigens in oral mucosal surfaces.     

Phylogenetic analysis of Amphioxus genes of the proprotein convertase family, including aPC6C, a marker of epithelial fusions during embryology

The proprotein convertases (PCs) comprise a family of subtilisin-like endoproteases that activate precursor proteins (including, prohormones, growth factors, and adhesion molecules) during their transit through secretory pathways or at the cell surface. To explore the evolution of the PC gene family in chordates, we made a phylogenetic analysis of PC genes found in databases, with special attention to three PC genes of the cephalochordate amphioxus, the closest living invertebrate relative to the vertebrates. Since some vertebrate PC genes are essential for early development, we investigated the expression pattern of the C isoform of the amphioxus PC6 gene (aPC6C). In amphioxus embryos and larvae, aPC6C is expressed at places where epithelia fuse. Several kinds of fusions occur: ectoderm-to-ectoderm during neurulation; mesoderm-to-ectoderm during formation of the preoral ciliated pit; and endoderm-to-ectoderm during formation of the mouth, pharyngeal slits, anus, and external opening of the club-shaped gland. Presumably, at all these sites, aPC6C is activating proteins favoring association between previously disjunct cell populations.     

Characterization of allelic V kappa-1 region genes in inbred strains of mice

Germ line genes encoding mouse Ig kappa-chains belonging to the V kappa-1 group have been isolated from BALB/c, NZB, and CE, three inbred strains of differing kappa haplotype. The V kappa-1A and V kappa-1C germ line genes isolated from BALB/c (Ig kappa c) were identical to those previously described. These are the two major V kappa-1 germ line genes in BALB/c and together account for 40 of the 53 expressed V kappa-1 sequences that have been reported to date. Allelic differences in a single germ line variable region gene (V kappa-1A) in different strains of mice explain the differences in L chain IEF patterns previously associated with the Ig kappa-Ef2 locus. The rearranged kappa-gene expressed in the BALB/c myeloma MOPC-460 has been isolated and found to represent a V kappa-1A somatic variant differing by three nucleotides from the germ line V kappa-1A gene. Germ line genes isolated from NZB (Ig kappa b) and CE (Ig kappa f) show greater than 95% identity with the BALB/c genes over the 1700 nucleotides compared. Comparison by region indicated the greatest conservation of sequence occurs in and around the leader exon followed by the V-region exon. The NZB gene encodes the amino acid sequence found in the myeloma PC-2205, previously designated V kappa-1B. The V kappa-1 gene isolated from CE is likely an allele of the BALB/c V kappa-1C gene as the two share greater than 96% identity over 1700 nucleotides. The CE gene has been designated V kappa-1Cf. Ancient remnants of LINE-1 repetitive elements were detected approximately 400 bp downstream of all of the V kappa-1 genes. These possess greater homology with repetitive elements found near other kappa genes than they do with the native L1Md sequence.     

Utilization of a mini-mu transposon to construct defined mutants in Streptococcus mutans

The gtfB gene coding for glucosyltransferase-I (GTF-I) activity previously isolated from Streptococcus mutans GS-5 was insertionally inactivated with the newly constructed transposon MudE in an Escherichia coli background. Insertion of MudE into various regions of the gtfB gene led to inactivation of GTF-I activity. The altered gene was introduced back into S. mutans GS-5 by transformation and produced mutants defective in insoluble glucan synthesis as well as the ability to colonize smooth surfaces in the presence of sucrose. Therefore, the MudE transposon can be utilized to produce specific mutants in oral streptococci as well as in other transformable Gram-positive bacteria expressing an erythromycin-resistance marker.     

Molecular basis for the spontaneous generation of colonization-defective mutants of Streptococcus mutans

Spontaneous mutants of Streptococcus mutans GS-5 defective in sucrose-dependent colonization of smooth surfaces are generated at frequencies above the spontaneous mutation rate. Southern blot analysis of such mutants suggested rearrangement of the genes coding for glucosyltransferase (GTF) activity. Two strain GS-5 homologous tandem genes, gtfB and gtfC, coding for GTF-I and GTF-S activities respectively, were demonstrated to undergo recombination when introduced into recombination-proficient Escherichia coli transformants. However, the two genes were quite stable when transformed on a single DNA fragment into a recA mutant of E. coli. The DNA fragment coding for GTF activity from one S. mutans colonization-defective mutant, SP2, was isolated and shown also to have undergone recombination between the gtfB and gtfC genes, resulting in reduced GTF activity. These results are discussed relative to the in vivo generation of colonization-defective mutants in cultures of S. mutans.     

Sequence analysis of the gtfC gene from Streptococcus mutans GS-5

The nucleotide sequence of the gtfC gene, which codes for glucosyltransferase synthesizing both water-soluble and water-insoluble glucans, and its flanking regions from Streptococcus mutans GS-5, was determined. Although the gtfC gene (4218 bp) is preceded by a Shine-Dalgarno (SD) sequence, a promoter-like sequence for this gene could not be identified. The gtfC gene product composed of 1375 amino acid residues (approx. 153 kDa) is generally hydrophilic with three small hydrophobic domains. Two direct repeating units were found near the C terminus of the peptide. The gtfC gene has extensive homology with the previously sequenced gtfB gene. The homologous regions correspond to the signal sequence, an internal region, and the direct repeating units of the peptide. An open reading frame preceded by an SD sequence and followed by an inverted repeat sequence was found immediately downstream from the gtfC gene. The combined sequences of the gtfB and gtfC genes as well as flanking regions suggest that the two gtf genes and the small downstream coding region could be coordinately expressed within an operon. The possible evolution of the gtfC gene in S. mutans GS-5 is also discussed.     

Microbial fructosyltransferases and the role of fructans

Microbial fructosyltransferases are polymerases that are involved in microbial fructan (levan, inulin and fructo-oligosaccharide) biosynthesis. Structurally, microbial fructosyltransferase proteins share the catalytic domain of glycoside hydrolases 68 family and are grouped in seven phylogenetically related clusters. Fructosyltransferase-encoding genes are organized in operons or in clusters associated with other genes related to carbohydrate metabolism or fructosyltransferase secretion. Fructosyltransferase gene expression is mainly regulated by two-component systems or phosphorelay mechanisms that respond to sucrose availability or other environmental signals. Microbial fructans are involved in conferring resistance to environmental stress such as water deprivation, nutrient assimilation, biofilm formation, and as virulence factors in colonization. As a result of the biological and industrial importance of fructans, fructosyltransferases have been the subject of extensive research, conducted to improve their enzymatic activity or to elucidate their biological role in nature.     

Design and biophysical characterization of bioresponsive degradable poly(dimethylaminoethyl methacrylate) based polymers for in vitro DNA transfection

Water-soluble, degradable polymers based on poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) with low cytotoxicity and good p-DNA transfection efficiency are highlighted in this article. To solve the nondegradability issue of PDMAEMA, new polymers based on DMAEMA and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) for gene transfection were synthesized. A poly(ethylene oxide) (PEO) azo-initiator was used as free-radical initiator. PEGylation was performed to improve water solubility and to reduce cytotoxicity of the polymers. The resulting polymers contain hydrolyzable ester linkages in the backbone and were soluble in water even with very high amounts of ester linkages. These degradable copolymers showed significantly less toxicity with a MTT assay using L929 cell lines and demonstrated promising DNA transfection efficiency when compared with the gold standard poly(ethyleneimine). Bioresponsive properties of the corresponding quaternized DMAEMA based degradable polymers were also studied. Although the quaternized DMAEMA copolymers showed enhanced water solubility, they were inferior in gene transfection and toxicity as compared to the unquaternized copolymers.     

The anti-biofouling effect of Lactobacillus fermentum-derived biosurfactant against Streptococcus mutans

Biofouling in the oral cavity often causes serious problems. The ability of Streptococcus mutans to synthesize extracellular glucans from sucrose using glucosyltransferases (gtfs) is vital for the initiation and progression of dental caries. Recently, it was demonstrated that some biological compounds, such as secondary metabolites of probiotic bacteria, have an anti-biofouling effect. In this study, S. mutans was investigated for the anti-biofouling effect of Lactobacillus fermentum (L.f.)-derived biosurfactant. It was hypothesized that two enzymes produced by S. mutans, glucosyltransferases B and C, would be inhibited by the L.f.-biosurfactant. When these two enzymes were inhibited, fewer biofilms (or none) were formed. RNA was extracted from a 48-h biofilm of S. mutans formed in the presence or absence of L.f. biosurfactant, and the gene expression level of gtfB/C was quantified using the real-time polymerase chain reaction (RT-PCR). L.f. biosurfactant showed substantial anti-biofouling activity because it reduced the process of attachment and biofilm production and also showed a reduction in gtfB/C gene expression (P value?相似文献   

Sucrose:sucrose fructosyltransferase and fructan:fructan fructosyltransferase from allium cepa

Sucrose: sucrose fructosyltransferase and fructan:fructan fructosyltransferase were isolated from the inner leaf bases of bulbing onion plants (Allium cepa) and separated by gel filtration on Bio-Gel P-150. Sucrose:sucrose fructosyltransferase produced only one trisaccharide, 1^F-fructosylsucrose, from sucrose. Fructan:fructan fructosyltransferase produced tetrasaccharide and higher polymers from trisaccharide. The trisaccharide found in the greatest concentration in onion, 6^G-fructosylsucrose, was produced from 1^F-fructosylsucrose by fructan:fructan fructosyltransferase and was not a product of sucrose:sucrose fructosyltransferase.     

Isolation of DNA encoding sucrase genes from Streptococcus salivarius and partial characterization of the enzymes expressed in Escherichia coli.

Restriction enzyme fragments containing two sucrase genes have been isolated from a cosmid library of Streptococcus salivarius DNA. The genes were expressed in Escherichia coli cells, and the properties of both enzymes were studied in partially purified protein extracts from E. coli. One gene encoding an invertase-type sucrase was subcloned on a 2.4-kilobase-pair fragment. The sucrase enzyme had a Km for sucrose of 48 mM and a pH optimum of 6.5. The S. salivarius sucrase clone showed no detectable hybridization to a yeast invertase clone. Two overlapping subclones which had 1 kilobase pair of DNA in common were used to localize a fructosyltransferase gene. The fructosyltransferase had a Km of 93 mM and a pH optimum of 7.0. The product of the fructosyltransferase was a levan. A fructosyltransferase clone from Bacillus subtilis did not hybridize to S. salivarius DNA. The properties of the enzymes were compared with those of previously characterized sucrases.     

颊粘膜癌与舌癌差异基因表达的对比分析

目的:研究颊粘膜癌(Buccal Mucosa Cancer,BMC)与舌癌(Tongue Cancer,TC)的基因差异表达,探讨BMC与TC发生发展的基因学基础。方法:应用cDNA芯片技术对5例BMC和5例TC组织mRNA检测,通过芯片杂交、生物信息学处理,找出两者间差异表达基因。结果:BioStarH-40芯片发现差异表达基因503条,差异表达基因占12.9%,其中表达增强274条(显著增强69条),表达降低229条(显著降低54条)。结论:MBC与TC基因表达比较,差异有统计学意义,这些差异可能在两类肿瘤不同的生物学行为中起重要作用。