An association between moenomycin resistance and vancomycin intermediate resistance inStaphylococcus aureus was demonstrated previously. Thus, to elucidate the mechanism of vancomycin intermediate resistance, we searched for factors contributing to moenomycin resistance. Random Tn551 insertional mutagenesis of methicillin-resistant S. aureus strain COL yielded three mutants with decreased susceptibilities to moenomycin. Correspondingly, these mutants also exhibited slightly decreased susceptibilities to vancomycin. Genetic analysis revealed that two of the mutants had Tn551 insertions in the fmtC (mprF) gene, which is associated with the synthesis of lysyl-phosphatidylglycerol. The third Tn551 insertion was located in the lysC gene, which is involved in the biosynthesis of lysine from aspartic acid. Consequently, mutations in both of these loci reduced the lysyl-phosphatidylglycerol content in the cell membrane, giving it a more negative net charge. The positively charged antibiotic gentamicin and cationic antimicrobial peptides such as ␤-defensins and CAP18 were more effective against the mutants. The levels of moenomycin and vancomycin binding to intact cells was also greater in the mutants than in the wild type, while the binding affinity was not altered when cells boiled in sodium dodecyl sulfate were used, indicating that both agents had higher affinities for the negatively charged membranes of the mutants. Therefore, the membrane charge of S. aureus appears to influence the efficacies of moenomycin, vancomycin, and other cationic antimicrobial agents.

SummaryGlucosamine-6-P occupies a central position between cell wall synthesis and glycolysis. In the initial steps leading to peptidoglycan precursor formation glucosamine-6-P is processed sequentially to UDP-Nacetylglucosamine, while to enter the glycolysis pathway, glucosamine-6-P is isomerized by NagB to fructose-6-P. Although we could not demonstrate NagB activity, nagB inactivation significantly reduced growth. Mutational analysis showed that NagA was involved in glucosamine-6-P formation from Nacetylglucosamine-6-P, and GlmS in that from fructose-6-P. Inactivation of glmS prevented growth on glucose as sole carbon source, which resumed after complementation with N -acetylglucosamine. Transcription of glmS as well as the amount of GlmS was reduced in the presence of N -acetylglucosamine. This and the preferential incorporation of Nacetylglucosamine over glucose into cell wall material showed that N -acetylglucosamine was used exclusively for cell wall synthesis, while glucose served both cell wall synthesis and glycolysis. These observations suggest furthermore GlmS to be the key and only enzyme leading from glucose to cell wall synthesis in Staphylococcus aureus, and show that there exists a tight regulation and hierarchy in sugar utilization. Inactivation of nagA , nagB or glmS affected the susceptibility of S. aureus to cell wall synthesis inhibitors, suggesting an interdependence between efficiency of cell wall precursor formation and resistance levels.

Abstract-Several mutations of cardiac ␤-myosin heavy chain (␤-MHC) gene were reported in patients with hypertrophic cardiomyopathy (HCM). Involvement of proto-oncogenes has been shown in the mechanism of experimental cardiac hypertrophy. This study sought to examine the effects of c-H-ras and c-myc expression in the steady-state myocardium on hypertrophic changes and to evaluate the possible interaction between ␤-MHC mutation and proto-oncogene expression in HCM. Endomyocardial biopsy was performed in 17 HCM patients (5 ␤-MHC mutations and 1 troponin T mutation) and 7 control subjects (no mutation). Reverse transcription-polymerase chain reaction analysis revealed c-H-ras expression in all members of both groups. Cardiomyocyte size was correlated with the expression level of c-H-ras (PϽ0.001), and c-H-ras expression was upregulated in HCM patients (PϽ0.01). HCM patients with a ␤-MHC mutation had the higher c-H-ras expression than did control subjects or patients without a mutation (PϽ0.01). c-myc mRNA was expressed in 7 of 17 HCM patients but not in control subjects. Myocyte size was greater in c-myc-positive HCM patients than in control subjects and c-myc-negative HCM patients (PϽ0.001 and PϽ0.05, respectively). The proto-oncogene expression did not affect clinical findings, myocardial fibrosis, or disarray. In conclusion, c-H-ras and c-myc expression in the steady-state myocardium may play a role in the hypertrophic mechanism in HCM. It is possible that ␤-MHC gene mutation has some effect on the regulation of proto-oncogene expression in HCM. (Circ Res. 1998;83:594-601.)

Clinical strains of MRSA, but not MSSA, that demonstrated an increased net charge also showed elevated resistance to LL-37, but not to hBD3.

Th1 and Th2 cytokines such as interferon-gamma (IFN-gamma ) , tumor necrosis factor- alpha (TNF-alpha ), and IL-4 are expressed in T-cell-mediated inflammation in the oral cavity. We tested the hypothesis that those cytokines may act on CXCR3-agonistic chemokines, T-cell recruiting factors, and on neighboring cells, including oral keratinocytes and fibroblasts. Human immortalized oral keratinocytes (RT7) and fibroblasts (GT1) after 24-hour stimulation with IFN-gamma showed increased mRNA levels of CXCL9 (600- and 700-fold), CXCL10 (10,000- and 150-fold), and CXCL11 (5000- and 300-fold), respectively. In contrast, TNF-alpha caused an increase in CXCL9 (300-fold), CXCL10 (2000-fold), and CXCL11 (2000-fold) mRNA levels in GT1, but not RT7 cells, at 24 hrs. IL-4 reinforced the promotion of CXCL9, CXCL10, and CXCL11 expression by IFN-gamma in RT7 cells, whereas IL-4 inhibited the increased levels by IFN-gamma and TNF-alpha in GT1 cells. Thus, IFN-gamma , TNF-alpha , and IL-4 appear cooperatively to regulate CXCR3-agonistic chemokines in oral keratinocytes and fibroblasts in T-cell-mediated oral inflammation sites.