Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange.

Streptococcus mutans produces several enzymes which metabolize sucrose. Three glucosyltransferase genes (gtfB, gtfC, and gtfD) and a single fructosyltransferase gene (ftf) encode enzymes which are important in formation of exopolysaccharides. Mutants of S. mutans V403 carrying single and multiple mutations of the gtfB, gtfC, gtfD, and ftf genes recently have been constructed by allelic exchange in our laboratory. Using selected strains from this panel of mutants, we examined the importance of water-insoluble glucan, water-soluble glucan, and fructan production in cariogenicity while controlling for the effects of strain and species variability. Genetic and biochemical characterization of mutants and assays of glucosyltransferase and fructosyltransferase activities were performed to ensure that the phenotypes of strains coincided with deficiencies predicted by genotype. The young gnotobiotic rat model of cariogenicity was used to assess virulence of the wild-type strain and isogenic mutants. Mutant strains were less virulent than the wild type in almost every location examined for caries on tooth surfaces and level of involvement of lesions (depth and severity). Inactivation of either gtfB and gtfC or ftf dramatically reduced virulence; the subsequent inactivation of gtfD did not enhance the effect of reduced virulence.     

Evidence for a disseminated plasmid in Streptococcus mutans

Based on a survey of 86 isolates, approximately 5% of all naturally occurring strains of Streptococcus mutans contains a 3.6 X 10(6)-dalton (3.6-megadalton) multicopy plasmid of unknown function. The amount of plasmid deoxyribonucleic acid per chromosome varies from 2 to 6% depending on the host strain. About 13% of the total covalently closed circular deoxyribonucleic acid in each of the four plasmid-containing strains consists of dimeric molecules, with interlocked circular forms predominating. Site-specific restriction endonucleases have been identified that cleave this 3.6-megadalton plasmid at single and at multiple sites. Each of the four plasmids is cleaved once by the HindIII and BamHI restriction enzymes. The HpaI, TaqI, and HhaI enzymes generate two, five, and six components, respectively, and the digestion products of each of the four plasmids are identical. Because the four plasmid-containing S. mutans strains are physiologically unique with respect to one another, we conclude this plasmid to be a disseminated extrachromosomal element in S. mutans.     

Repeated DNA sequence involved in mutations affecting transport of sucrose into Streptococcus mutans V403 via the phosphoenolpyruvate phosphotransferase system.

Mutants of Streptococcus mutans V403 defective in the intracellular sucrose-6-phosphate hydrolase (product of the scrB gene) are sensitive to sucrose because of the intracellular accumulation of the phosphorylated sugar. Using a scrB mutant prepared by allelic exchange, we have isolated and characterized a number of sucrose-resistant revertants. One such mutant was found to lack the ability to transport sucrose into the cell via the phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS). Genetic analysis of this strain revealed this lesion to be linked to the scrB gene. This was corroborated by the physical demonstration of an insertion mutation very near scrB. Taken together with DNA sequence information (Y. Sato, F. Poy, G. R. Jacobson, and H. K. Kuramitsu, J. Bacteriol. 171:263-271, 1989), our results indicated that all of the mutations characterized were located in the adjoining scrA gene which encodes the membrane-associated, sugar-specific enzyme II (EIIsucrose) component of the sucrose PTS in S. mutans. Biochemically, such a genetic lesion disables the sucrose PTS and prevents sucrose from entering the cell by this system. In this paper, we detail the nature of two independent insertion mutations and conclude them to be the result of duplicative transposition events into the scrA gene. This region of the chromosome was amplified and purified in large quantities by using the polymerase chain reaction. Examination of the amplified DNA revealed that the two independent insertion mutations were composed of sequences that were indistinguishable by size and by restriction site endonuclease maps. Their insertion points in the scrA gene were approximately 200 bp apart. The amplified DNA fragment was also used as a probe to demonstrate the presence of five copies of this element on the S. mutans V403 chromosome. A second strain, S. mutans V310, also was found to carry similarly arranged, multiple copies of this sequence on its chromosome, suggesting a clonal origin of V403 and V310. The small size of this sequence, its presence in multiple copies on the V403 chromosome, and its ability to duplicate itself semiconservatively into remote sites argue compellingly that it is an insertion sequence element. One such insertion mutant, with a defective sucrose PTS, was tested for virulence in rats and was found to cause caries at levels similar to those of the wild-type strain.     

Colostrogenesis: Role and Mechanism of the Bovine Fc Receptor of the Neonate (FcRn)

Journal of Mammary Gland Biology and Neoplasia - Colostrogenesis is a separate and unique phase of mammary epithelial cell activity occurring in the weeks before parturition and rather abruptly...     

Regulation of MCF-7 breast cancer cell growth by beta-estradiol sulfation

Estrogen stimulation is an important factor in human breast cancer cell growth and development. Metabolism of -estradiol (E₂), the major endogenous human estrogen, is important in regulating both the level and activity of the hormone in breast tissues. Conjugation of E₂ with a sulfonate moiety is an inactivation process since the sulfate ester formed by this reaction can not bind and activate the estrogen receptor. In human tissues including the breast, estrogen sulfotransferase (EST, SULT1E1) is responsible for high affinity E₂ sulfation activity. EST is expressed in human mammary epithelial (HME) cells but not in most cultured breast cancer cell lines, including estrogen responsive MCF-7 cells. Stable expression of EST in MCF-7 cells at levels similar to those detected in HME cells significantly inhibits cell growth at physiologically relevant E₂ concentrations. The mechanism of cell growth inhibition involves the abrogation of responses observed in growth factor expression in MCF-7 cells following E₂ stimulation. MCF-7 cells expressing EST activity did not show a decrease in estrogen receptor- levels, nor a characteristic increase in progesterone receptor or decrease in transforming growth factor- expression upon exposure to 100 pM or 1 nM E₂. The lack of response in these MCF-7 cells is apparently due to the rapid sulfation and inactivation of free E₂ by EST. These results suggest that loss of EST expression in the transformation of normal breast tissues to breast cancer may be an important factor in increasing the growth responsiveness of preneoplastic or tumor cells to estrogen stimulation.     

Left ventricular thrombosis after blunt chest trauma

A 22-year-old man was admitted to our observation with left ventricular thrombus arising after blunt chest trauma occurring during a ski accident one year before. None was obtained from a review of instrumental and laboratory data at trauma time. Transesophageal echocardiography showed an intraventricular thrombus and severe hypokinesia at the apex. Standard cardiac surgery procedure was performed and postoperative period was uneventful. Echocardiography controls at 6/12 months showed a normal apex kinesia. This case shows the importance of hospitalization, hemodynamics monitorization and late serial echocardiographic controls for timely diagnosis and management of myocardial contusion and consecutive ventricular thrombus formation to prevent life-threatening complications.     

Genetic characterization of a Streptococcus mutans LraI family operon and role in virulence

Proteins belonging to the LraI (for "lipoprotein receptor antigen") family function as adhesins in several streptococci, as a virulence factor for endocarditis in at least one of these species, and potentially as metal transporters in many bacteria. We have identified and characterized the chromosomal locus containing the LraI family gene (designated sloC) from Streptococcus mutans, an agent of dental caries and endocarditis in humans. Northern blot analysis indicated that sloC is cotranscribed with three other genes. As with other LraI operons, the sloA and sloB genes apparently encode components of an ATP-binding cassette transport system. The product of the fourth gene, sloR, has homology to the metal-dependent regulator from Corynebacterium diphtheriae, DtxR. A potential binding site for SloR was identified upstream from the sloABCR operon and was conserved upstream from LraI operons in several other streptococci. Potential SloR homologs were identified in the unfinished genomic sequences from two of these, S. pneumoniae and S. pyogenes. Mutagenesis of sloC in S. mutans resulted in apparent loss of expression of the entire operon as assessed by Northern blot analysis. The sloC mutant was indistinguishable from its wild-type parent in a gnotobiotic rat model of caries but was significantly less virulent in a rat model of endocarditis. Virulence for endocarditis was restored by correction of the sloC mutation but not by provision of the sloC gene in trans, suggesting that virulence requires the expression of other genes in the sloC operon.     

Procalcitonin is useful whereas C-reactive protein is not, to predict complications following coronary artery bypass surgery

BACKGROUND: The respective value of procalcitonin (PCT) and C-reactive protein (CRP) as markers of postoperative complications after coronary bypass surgery is unclear. Therefore, complications during one week after surgery were studied to evaluate the predictive role of PCT and CRP changes during the immediate postoperative period. METHODS: Thirty-two patients, in whom an uneventful immediate postoperative course was anticipated, were prospectively enrolled and followed-up to the 7th postoperative day. At the end of the follow-up, patients were divided into two groups. Group A were patients with an uncomplicated postoperative course and Group B were patients with a complicated postoperative course. RESULTS: Serum samples were drawn for PCT and CRP determination after induction of anesthesia (baseline), at the end of surgery and daily until postoperative day 2. Baseline serum PCT concentrations were 0.11 +/- 0.09 and 0.20 +/- 0.21 ng/mL in Groups A and B, respectively (NS). Serum PCT concentration increased compared with baseline in both groups during the first two days after surgery. The increase in serum PCT concentration was significantly greater in Group B than A patients (p < 0.0002). Considering a perioperative abnormal cut-off value of >0.5 ng/mL, there were none in Group A versus 57% in Group B (p < 0.0001). Baseline serum CRP concentrations were 1.44 +/- 1.30 and 1.58 +/- 1.35 ng/mL in Groups A and B, respectively (NS). After surgery, CRP increased significantly compared with baseline in both groups. When changes in time-varying variables were included in a logistic model, complications were predicted by changes (between baseline and end of surgery values) of PCT (coefficient = 9.410; t = 2.18) and heart rate (coefficient = 0.075; t = 1.57), whereas changes of CRP, white blood cells, mean blood and central venous pressures did not contribute statistically. The model constant was -4.827 (t = -2.43) and the ROC curve area was 0.8971. Thus, absolute PCT changes of 0.20, 0.40 and 0.60 ng/mL carry an approximate risk of 5, 26 and 69%, respectively, of postoperative complications in the time frame of this study. CONCLUSIONS: A postoperative serum PCT concentration of >0.5 ng/mL is highly suggestive of a postoperative complication. CRP changes do not contribute to predictive information.     

Bladder and sexual function among women with multiple sclerosis

OBJECTIVE: Genitourinary dysfunction is common in women with multiple sclerosis (MS), yet few studies have evaluated the association between bladder and sexual dysfunction in these women. The aim of this study was to determine factors, including demographic and bladder function, associated with sexual dysfunction in a sample of women with MS. METHODS: One hundred and thirty-three women with MS completed questionnaires related to overall heath status, bladder function and sexual function. Response frequencies and percentages were calculated for questionnaire responses. Multivariate logistic regression analyses were performed to determine predictors of sexual dysfunction. RESULTS: Sixty-one per cent of the sample indicated that they had a problem with bladder control. Forty-seven per cent of respondents indicated that their neurological problems interfered with their sex life. Over 70% of the sample reported that they enjoyed, felt aroused and experienced orgasm during sexual activity. Not having a sexual partner and the indication of bothersome neurological problems were the best predictors of sexual dysfunction. Interestingly, patients bothered by their urge incontinence had higher levels of orgasm compared to women not bothered by urge incontinence. CONCLUSIONS: Although over half of the women reported voiding symptoms, most still enjoyed, felt aroused and could experience orgasm. Neurological symptoms and lacking a sexual partner emerged as the best predictors of sexual dysfunction. Urge incontinence may not be a risk factor for an orgasm. Our findings elucidate the complex nature of sexual dysfunction in women with MS.     

Inhibition of Streptococcus mutans glucosyltransferase activity by antiserum to a subsequence peptide.

An antigenic 15-amino-acid peptide sequence (gtfB.1) from the glucosyltransferase B enzyme of the cariogenic bacterium Streptococcus mutans GS-5 was identified previously from the genetic fusion of this sequence to the B subunit of cholera toxin. The resulting chimeric protein was used to raise antiserum in rabbits. This antiserum was shown to recognize the native glucosyltransferase enzyme and to inhibit its activity. The antiserum inhibited the synthesis of water-soluble glucan by approximately 40% and the synthesis of water-insoluble glucan by greater than 90%. The antiserum was shown to partially inhibit fructosyltransferase activity as well. The ability of this antipeptide antiserum to inhibit several enzymes from S. mutans suggests that these enzymes share an epitope related to enzymatic activity.