Functional characterization of Staphylococcus epidermidis IcaB, a de-N-acetylase important for biofilm formation.

A polymer of partially de-N-acetylated ß-1,6-linked N-acetylglucosamine (dPNAG), also known as the polysaccharide intercellular adhesin (PIA), is an important component of many bacterial biofilm matrices. In Staphyloccocus epidermidis, the poly-N-acetylglucosamine polymer is partially de-N-acetylated by the extracellular protein IcaB. To understand the mechanism of action of IcaB, the enzyme was overexpressed and purified. IcaB demonstrates metal-dependent de-N-acetylase activity on ß-1,6-linked N-acetylglucosamine oligomers with a broad preference for divalent metals. Steady-state kinetic analysis reveals the low catalytic efficiency (pentasaccharide kcat/KM 0.03 M(-1) s(-1)) of the enzyme toward the oligomeric substrates. While IcaB displays similar rates of de-N-acetylation with tri- through hexasaccharide PNAG oligomers, position specific de-N-acetylation was only observed with penta- and hexasaccharides. The enzyme preferentially de-N-acetylates the second residue from the reducing terminus in the pentasaccharide and second and third residues from the reducing terminus in the hexasaccharide. The data described here represent an important step toward a detailed understanding of dPNAG biosynthesis in S. epidermidis, an important nosocomial pathogen, as well as in other Gram-positive bacteria. The low catalytic activity of IcaB is consistent with reports of other enzymes which act on biofilm-related polysaccharides, and this emerging trend may indicate a common feature among this group of polysaccharide processing enzymes.

Tat peptide-calmodulin binding studies and bioinformatics of HIV-1 protein-calmodulin interactions.

The human immunodeficiency virus type 1 (HIV-1) genome encodes 18 proteins and 2 peptides. Four of these proteins encode high-affinity calmodulin-binding sites for which direct interactions with calmodulin have already been described. In this study, the HIV-1 proteome is queried using an algorithm that predicts calmodulin-binding sites revealing seven new putative calmodulin-binding sites including residues 34-56 of the transactivator of transcription (Tat). Tat is a 101-residue intrinsically disordered RNA-binding protein that plays a central role in the regulation of HIV-1 replication. Interactions between a Tat peptide (residues 34-56), melittin, a well-characterized calmodulin-binding peptide, and calmodulin were examined by direct binding studies, mass spectrometry, and fluorescence. The Tat peptide binds to both calcium-saturated and apo-calmodulin with a low micromolar affinity. Conformational changes induced in the Tat peptide were determined by circular dichroism, and residues in calmodulin that interact with the peptide were identified by HSQC NMR spectroscopy. Multiple interactions between HIV-1 proteins and calmodulin, a highly promiscuous signal transduction hub protein, may be an important mechanism by which the virus controls cell physiology.

Attitudes and experience of women to common vaginal infections.

OBJECTIVE: To determine women's experience and knowledge of the 2 most common non-sexually transmitted vaginal infections, vulvovaginal candidiasis (VVC) and bacterial vaginosis (BV). MATERIALS AND METHODS: An online omnibus was conducted on 6,010 women aged 16 to 55 years to determine the incidence and awareness of VVC and BV in Europe (France, Germany, the Netherlands, Sweden, and the United Kingdom) and the United States, followed by an in-depth questionnaire on 1,945 women about experience and attitudes to VVC and BV. RESULTS: Almost all (97%) of the women who took part stated that they were aware of VVC and 44% reported having had VVC, whereas only 30% of women had heard of BV and only 9% thought they had experienced it. There was confusion between symptoms specifically related to each condition, and women thought they were caused by poor hygiene, ill health, or a sexually transmitted infection, with antibiotic use cited as a cause for VVC only. Diagnosis was generally by a health care professional, but there was also considerable self-diagnosis in countries where an over-the-counter treatment was available for VVC. Rates of reported examination and testing by the health care provider varied by country, with high rates in Germany and low rates in the United Kingdom. CONCLUSIONS: Women seem very aware and knowledgeable about VVC, but awareness of BV is low with self-reported incidence considerably less than prevalence rates, suggesting misdiagnosis. Increased education and better diagnosis of these 2 conditions is needed to remove the stigma and taboo, especially for BV, and to ensure correct diagnosis with appropriate treatment.

Safety assessment for hair-spray resins: risk assessment based on rodent inhalation studies.

The methods involved in the safety assessment of resins used in hair-spray products have received little peer review, or debate in the published literature, despite their widespread use, in both hairdressing salons and the home. The safety assessment for these resins currently involves determining the type of lung pathology that can be caused in animal inhalation exposure studies, and establishing the no-observable-effect level (NOEL) for these pathologies. The likely human consumer exposure is determined by techniques that model the simulated exposure under "in use" conditions. From these values it is then possible to derive the likely safety factors for human exposure. An important part of this process would be to recognize the intrinsic differences between rodents and humans in terms of the respiratory doses that each species experiences during inhalation exposures, for the purpose of the safety assessment. Interspecies scaling factors become necessary when comparing the exposure doses experienced by rats, compared to humans, because of basic differences between species in lung clearance rates and the alveolar area in the lungs. The rodent inhalation data and modeled human exposure to Resin 6965, a resin polymer that is based on vinyl acetate, has been used to calculate the safety factor for human consumer exposure to this resin, under a range of "in use" exposure conditions. The use of this safety assessment process clearly demonstrates that Resin 6965 is acceptable for human consumer exposure under the conditions considered in this risk assessment.