Evaluation of mannitol salt agar for detection of oxacillin resistance in Staphylococcus aureus by disk diffusion and agar screening

Mannitol salt agar was evaluated for detection of oxacillin resistance in 136 Staphylococcus aureus isolates. All mecA-positive isolates (n = 54) were correctly categorized as oxacillin resistant by the disk diffusion test (1-microgram disk; zone diameter, < 16 mm); the specificity was 97.6%. Agar screening (2 micrograms of oxacillin per ml) revealed a sensitivity of 98.1% and a specificity of 95.1%.     

pi-Stacking interactions. Alive and well in proteins

A representative set of high resolution x-ray crystal structures of nonhomologous proteins have been examined to determine the preferred positions and orientations of noncovalent interactions between the aromatic side chains of the amino acids phenylalanine, tyrosine, histidine, and tryptophan. To study the primary interactions between aromatic amino acids, care has been taken to examine only isolated pairs (dimers) of amino acids because trimers and higher order clusters of aromatic amino acids behave differently than their dimer counterparts. We find that pairs (dimers) of aromatic side chain amino acids preferentially align their respective aromatic rings in an off-centered parallel orientation. Further, we find that this parallel-displaced structure is 0.5-0.75 kcal/mol more stable than a T-shaped structure for phenylalanine interactions and 1 kcal/mol more stable than a T-shaped structure for the full set of aromatic side chain amino acids. This experimentally determined structure and energy difference is consistent with ab initio and molecular mechanics calculations of benzene dimer, however, the results are not in agreement with previously published analyses of aromatic amino acids in proteins. The preferred orientation is referred to as parallel displaced pi-stacking.     

Quantitative determination of endogenous retinoids in mouse embryos by high-performance liquid chromatography with on-line solid-phase extraction, column switching and electrochemical detection

An isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid and all-trans-retinol in mouse embryos using on-line solid-phase extraction and column switching in combination with electrochemical detection has been developed. The method was validated using retinoids in albumin solutions and 13-cis-acitretin was used as internal standard. About 370 microliters of albumin solution was injected on a 10 x 2.1-mm I.D. pre-column packed with Bondapak C18, 37-53-micron particles. The proteins were washed to waste within 5 min using as mobile phase, a 1:3 dilution of mobile phase 2, which consisted of acetonitrile-methanol-2% ammonium acetate-glacial acetic acid (79:2:16:3, v/v). Components retained on the pre-column were back-flushed to and separated on the 250 x 4.6-mm I.D. Suplex pKb-100 analytical column using mobile phase 2. The retinoids were detected electrochemically at +750 mV using a coulometric electrochemical detector. The total analysis time was about 20 min. Recoveries were in the range of 86-103%. The mass limits of detection were about 10 pg and 25 pg for the retinoic acids and all-trans-retinol, respectively. The intra-assay precision, reported as relative standard deviation, was in general better than 4% (n = 6) for the four retinoids. Inter-assay precision was in the range 3-4% (n = 10). The method was applied for determination of endogenous retinoids in 9.5 day-old mouse embryos. A 340-microliter solution containing 100 microliters of embryo homogenate (1.64 embryos) was analyzed. The concentrations of all-trans-retinol and all-trans-retinoic acid were found to be 279 pg per embryo and 75.8 pg per embryo, respectively. The amount of 13-cis-retinoic acid and 9-cis-retinoic acid was below the detection limit.     

Enrichment of the more hydrophilic bile acid ursodeoxycholic acid in the fecal water-soluble fraction after feeding to rats with colon polyps

We recently showed that feeding the cytoprotective bile acid ursodeoxycholic acid (UDCA) to rats resulted in significant reduction in polyps and especially cancers, both in number and size (D. L. Earnest et al., Cancer Res., 54: 5071-5074, 1994). Because fecal secondary bile acids [particularly deoxycholic acid (DCA)] are considered to promote formation of colon adenomas and cancer, we have now attempted to find a relationship between polyp reduction and fecal secondary bile acids after feeding UDCA to these rats. We examined the fecal bile acids in rats with polyps and compared them with fecal bile acids in control rats and also determined the bile acid composition in fecal aqueous phase, which is in direct contact with the colon epithelium and may be physiologically more active. Treatment with azoxymethane did not significantly alter fecal bile acid composition in the rats. Cholic acid feeding resulted in greatly increased proportions of DCA (82% of total bile acids versus 18% in control rats). On the other hand, UDCA feeding significantly reduced the proportion of fecal DCA (2% in control rats fed UDCA and 3% in rats also treated with azoxymethane). In control rats, 96% of the bile acids were present in the water-insoluble fraction and 4% in the water-soluble fraction. The major insoluble bile acids included DCA and hyodeoxycholic acid (73% of total bile acids). In contrast, the muricholic acids were concentrated in the soluble fraction (37%). When 0.4% UDCA was added to the diet, lithocholic acid increased in the insoluble fraction (40 versus 1%), but the hydrophilic UDCA and muricholic acids were enriched in the water-soluble fraction (37 and 43%, respectively). Thus, the hydrophobic bile acids were distributed predominantly in the water-insoluble fraction, whereas the hydrophilic bile acids were distributed preferentially in the water-soluble fraction. These data suggest that UDCA may prevent colon tumors and polyps by countering the toxic effect of DCA and enhancing the possible cytoprotective effects of UDCA and muricholic acids in the water-soluble fraction in the feces of rat.     

Prevalence and epidemiology of toenail onychomycosis in diabetic subjects: a multicentre survey

The number of individuals diagnosed with diabetes mellitus is increasing. The diabetic may present with complications involving all systems of the body. While onychomycosis is often observed in diabetics, there have been no large studies on the prevalence of the condition in this patient group. We examined the prevalence of onychomycosis in diabetics attending diabetes and dermatology clinics in London, Ontario, Canada and Boston, MA, U.S.A. Diabetic subjects seen in dermatology offices were for unrelated dermatoses; those referred specifically for the management of onychomycosis were excluded from the sample. A total of 550 diabetic subjects was evaluated (283 males and 267 females), age 56.1 +/- 0.7 years (mean +/- SEM). Patients with type I diabetes constituted 34% of the sample. The racial origin was: 531 Caucasians, 17 Asians, one African-American and one American-Indian. Abnormal-appearing nails and mycological evidence of onychomycosis (mostly due to dermatophytes) were present in 253 (46%) and 144 (26%), respectively, of 550 subjects. The development of onychomycosis was significantly correlated with age (P < 0.0001) and male gender (P < 0.0001). Males were 2.99 times more likely to have onychomycosis compared with females (95% confidence interval, CI 1.94-4 61). After controlling for age and sex, the risk odds ratio for diabetic subjects to have toenail onychomycosis was 2.77 times compared with normal individuals (95% CI 2.15-3.57). After controlling for age and sex, a stepwise logistic regression demonstrated that significant predictors for onychomycosis included a family history of onychomycosis (P = 0.0001), concurrent intake of immunosuppressive therapy (P = 0.035) and peripheral vascular disease (P = 0.023). Toenail onychomycosis was present in 26% of the sample and is projected to affect approximately one-third of subjects with diabetes. Predisposing factors include increasing age, male gender, family history of onychomycosis, concurrent intake of immunosuppressive agents and peripheral vascular disease.     

Characterization of recombinant human cathepsin B expressed at high levels in baculovirus

The lysosomal cysteine protease cathepsin B has been studied intensely for many years because of its unique characteristics and its potential involvement in disease states. A reproducible, high yield expression system for active recombinant protein is key to biochemical and biophysical studies as well as rational drug design. Although several microbial and mammalian expression systems for recombinant human cathepsin B have been described, these have been limited by low or variable yields. Further, in some of these systems hyper-glycosylation of the enzyme near the active site affects its activity. We describe a baculovirus expression system and purification scheme that solve all of these problems. Yields of active, protected enzyme were reproducibly in excess of 25 mg/L. Since this protein was not hyper-glycosylated, it had greater activity than cathepsin B produced in yeast systems as indicated by a threefold increase in Kcat. In addition, the biophysical properties of the baculovirus-expressed cathepsin B, as measured by dynamic light scattering, were more amenable to crystallographic study since the data indicated proteins of more uniform size. Therefore, this system for the production of recombinant human cathepsin B constitutes a major improvement in both quantity and quality over those previously reported. Further, we demonstrate that the manner of expression and purification of this enzyme has profound effects on its kinetic and physical parameters.     

Unfolding of diphtheria toxin. Identification of hydrophobic sites exposed on lowering of pH by photolabeling

We report here the use of a hydrophobic photoactivable reagent, 2-[3H]diazofluorene (DAF), to map the hydrophobic sites exposed when the pH is lowered in diphtheria toxin (DT). The reagent binds to DT, and on photolysis with light of wavelength >350 nm, it covalently attaches itself to DT. The labeling was observed to increase considerably when the pH was lowered from 7.4 to 5.2. Although both A- and B-chains were labeled to a similar degree at pH 7.4, at lower pH (5.2), B-chain was labeled to a much higher extent. Subsequent chemical and enzymatic fragmentation of DT followed by separation indicated that the putative transmembrane domain was labeled to its maximum extent at pH 5.2, with the bulk of labeling associated with residues 340-459. Protein sequencing analysis indicated that the two buried hydrophobic helices, identified in the crystal structure and suggested to insert and span the membrane bilayer, corresponding to residues 326-347 and 358-376, are strongly labeled. The Pro-345 residue was observed to be labeled maximally at lower pH values. Finally, the DAF labeling pattern indicated that the parent structural motifs are retained at low pH, suggesting that the low pH conformation of DT corresponds to an equilibrium molten globule state.     

Experiences with functional magnetic resonance imaging at 1 tesla

Functional magnetic resonance imaging (fMRI) has been performed on a standard 1 T system using a pulse sequence developed to utilize blood oxygen level dependent (BOLD) contrast and an off-line analysis routine using correlation techniques. The sequence and the data analysis routine have been validated by reproducing the conventional hand movement paradigm studies reported by numerous other workers. Our work has then been extended to investigate cerebral foci for a tonic pain stimulus and the cortical representation of oesophageal stimulation. Both these studies relate to paradigms where the expected BOLD signal is significantly less than that encountered for motor or visual cortex paradigms. The results show good agreement with other modalities (positron emission tomography, magnetoencephalography and cortical evoked potentials). Performing fMRI at 1 T is slightly controversial. However, our successful study of demanding paradigms, using a standard clinical 1 T imaging system, has important implications for many other users operating at this field strength.