The Role Of Lipids In The Structure And Function Of Yeast Membranes

Considerable attention has focused on the relationships that may exist between membrane lipids and membrane-associated enzyme activities. To explore these associations and to provide a better understanding of the role that lipids play in membrane function, a study was undertaken using Saccharomyces cerevisiae as a model system to study the lipid-protein interactions of membrane-bound enzymes. Using nutritional supplementation, the membrane level of ergosterol was increased which resulted in increased membrane tolerance against hyperthermia. Yeast mutants cho1 and opi3-3, defective in specific enzymes in the phospholipid biosynthetic pathway, were employed to study the effect of altered membrane phospholipid levels on the activities of cytochrome c oxidase, oligomycin-sensitive ATPase and plasma membrane-ATPase. The electron transfer capability of cytochrome c oxidase was found to be influenced by the altered ratio of phosphatidylcholine and phosphatidylethanolamine, indicating that a critical level of these phospholipids is required for the enzyme activity. To probe the structural specificity of phospholipids, analogues of choline and ethanolamine were used to enrich mitochondrial membranes physiologically. Kinetic analyses of the oxidase under these conditions revealed that the charge and size of phospholipid polar head groups markedly impaired electron flow from cytochrome c to the enzyme. Although DPH fluorescent polarization experiments revealed no major changes in membrane \u27fluidity\u27, possible membrane restructuring was observed when these membranes were probed with ANS. To assess the effect of fatty acids on cytochrome c oxidase, a double fatty acid auxotroph, FAI-4C, was used to manipulate the level of saturated and unsaturated fatty acids in mitochondrial membranes. Similar to phospholipid enrichment, the elevation of both saturated and unsaturated fatty acids in mitochondria also changed the low affinity kinetic phase of the oxidase. It may, therefore, follow that this enzyme site may be embedded in, or juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane. These results indicate that changes in membrane lipid composition can effect membrane-bound enzyme activities by changing enzyme conformation or possibly by altering subunit assembly. (Abstract shortened with permission of author.

Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Material

Aim To develop a reagent kit that enables multiplex polymerase chain reaction (PCR) amplification of 18 short tandem repeats (STR) and the canine sex-determining Zinc Finger marker. Methods Validation studies to determine the robustness and reliability in forensic DNA typing of this multiplex assay included sensitivity testing, reproducibility studies, intra- and inter-locus color balance studies, annealing temperature and cycle number studies, peak height ratio determination, characterization of artifacts such as stutter percentages and dye blobs, mixture analyses, species- specificity, case type samples analyses and population studies. Results The kit robustly amplified domesticated dog samples and consistently generated full 19-locus profiles from as little as 125 pg of dog DNA. In addition, wolf DNA samples could be analyzed with the kit. Conclusion The kit, which produces robust, reliable, and reproducible results, will be made available for the forensic research community after modifications based on this study’s evaluation to comply with the quality standards expected for forensic casework

Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Materials

Aim To develop a reagent kit that enables multiplex polymerase chain reaction (PCR) amplification of 18 short tandem repeats (STR) and the canine sex-determining Zinc Finger marker. Methods Validation studies to determine the robustness and reliability in forensic DNA typing of this multiplex assay included sensitivity testing, reproducibility studies, intra- and inter-locus color balance studies, annealing temperature and cycle number studies, peak height ratio determination, characterization of artifacts such as stutter percentages and dye blobs, mixture analyses, species- specificity, case type samples analyses and population studies. Results The kit robustly amplified domesticated dog samples and consistently generated full 19-locus profiles from as little as 125 pg of dog DNA. In addition, wolf DNA samples could be analyzed with the kit. Conclusion The kit, which produces robust, reliable, and reproducible results, will be made available for the forensic research community after modifications based on this study’s evaluation to comply with the quality standards expected for forensic casework

Pharmacodynamics of Fluoroquinolones against Streptococcus pneumoniae in Patients with Community-Acquired Respiratory Tract Infections

Fluoroquinolone antibiotic agents have demonstrated efficacy in the treatment of respiratory tract infections. This analysis was designed to examine the relationship between drug exposure, as measured by the free-drug area under the concentration-time curve at 24 h (AUC(24))/MIC ratio, and clinical and microbiological responses in patients with community-acquired respiratory tract infections involving Streptococcus pneumoniae. The study population included 58 adult patients (34 males, 24 females) who were enrolled in either of two phase III, randomized, multicenter, double-blind studies of levofloxacin versus gatifloxacin for the treatment of community-acquired pneumonia or acute exacerbation of chronic bronchitis. Clearance equations from previously published population pharmacokinetic models were used in conjunction with dose and adjusted for protein binding to estimate individual patient free-drug AUC(24)s. In vitro susceptibility was determined in a central laboratory by broth microdilution in accordance with NCCLS guidelines. Pharmacodynamic analyses were performed on data from all evaluable patients with documented S. pneumoniae infection using univariate and multivariable logistic regression; pharmacodynamic breakpoints were estimated using Classification and Regression Tree analysis. A statistically significant (P = 0.013) relationship between microbiological response and the free-drug AUC(24)/MIC ratio was detected. At a free-drug AUC(24)/MIC ratio of <33.7, the probability of a microbiological response was 64%, and at a free-drug AUC(24)/MIC ratio of >33.7, it was 100% (P < 0.01). These findings may provide a minimum target free-drug AUC(24)/MIC ratio for the treatment of infections involving S. pneumoniae with fluoroquinolone antibiotics and provide a paradigm for the selection of fluoroquinolones to be brought forward from drug discovery into clinical development and dose selection for clinical trials. Further, when target free-drug AUC(24)/MIC ratios are used in conjunction with stochastic modeling techniques, these findings may be used to support susceptibility breakpoints for fluoroquinolone antibiotics and S. pneumoniae

The clinicopathological and gene expression patterns associated with ulceration of primary melanoma

Ulceration of primary melanomas is associated with poor prognosis yet is reported to predict benefit from adjuvant interferon. To better understand the biological processes involved, clinicopathological factors associated with ulceration were determined in 1804 patients. From this cohort, 348 primary tumor blocks were sampled to generate gene expression data using a 502-gene cancer panel and 195 blocks were used for immunohistochemistry to detect macrophage infiltration and vessel density. Gene expression results were validated using a whole genome array in two independent sample sets. Ulceration of primary melanomas was associated with more proliferative tumors, tumor vessel invasion, and increased microvessel density. Infiltration of tumors with greater number of macrophages and gene expression pathways associated with wound healing and up-regulation of pro-inflammatory cytokines suggests that ulceration is associated with tumor-related inflammation. The relative benefit from interferon reported in patients with ulcerated tumors may reflect modification of signaling pathways involved in inflammation