Effects of detergents and choline-containing phospholipids on human spleen glucocerebrosidase.

1. Glucocerebrosidase, extracted from human spleen lysosomal membrane by sodium cholate and recovered in a high speed centrifugation supernatant, aggregated following removal of the detergent. 2. Re-solubilization of the enzymatic activity from the aggregate was achieved by treatment with the non-ionic detergents Triton X-100 and Tween 20. The anionic detergents sodium cholate and sodium taurocholate and the cationic detergents cetyltrimethylammonium bromide and cetylpyridinium chloride were also effective. The solubilizing capacity of the anionic detergents was smaller than that of the nonionic detergents. Quantitative evaluation of the solubilizing capacity of the cationic detergents was not feasible because of their being potent inhibitors of glucocerebrosidase activity. 3. Treatment of the enzyme aggregate with acetone rendered it buffer-soluble. 4. In addition to the above cationic detergents some choline-containing and highly hydrophobic phospholipids were found to inhibit the glucocerebrosidase activity.     

The results after transrectal prostate biopsy with 12 biopsy cores taken

This report describes the clinical value of transrectal prostate biopsy during which 12 biopsy cores are taken in comparison to the classical sextant method. There were 106 patients included in the study, who had transrectal prostate biopsy (TRB) due to abnormal finding after digitorectal examination (DRE) and/or values of PSA > 4 ng/ml in the period from 4 October 2001 till 14 August 2002. There were 117 biopsies with 12 biopsy cores taken, 6 cores from each lobe. Prostate cancer was confirmed in 49 patients (46%). Out of total number of confirmed cancer cases, initial biopsy detected 94%. There were three patients who had suspicious DRE finding, with PSA value of < 4 ng/ml, but cancer was not detected in any of them. In the patient group with PSA value between 4-10 ng/ml, cancer was detected in 26% of them and in the group with PSA value > 10 ng/ml cancer was detected in 58%. The most common Gleason score in the case of cancer was 7 (43%). During the biopsy procedure, 3 patients experienced strong vasovagal reactions, meaning that out of 117 biopsies incidence of complications was 2.6%. Few days after the biopsy, two patients developed urogenital tract infections (1.7%) and right after the procedure, there was one case of strong hematuria (0.8%) and strong rectal bleeding (0,8%) that needed hospitalization. Our results regarding the incidence of complications do not differ much from the results in the literature. According to data in the literature regarding sextant biopsy, 15-34% of cancer cases remain undiagnosed at initial biopsy. The method of 12 biopsy cores fails to diagnose only 6% of all cancers, but it is important to note that in the mentioned period, re-biopsy was indicated only in 11 from 60 patients with negative biopsies.     

The chemical composition of the meat of fat dormice (<Emphasis Type="Italic">Glis glis</Emphasis> L.)

The fat dormouse (Glis glis) is a traditional game species in the Republic of Croatia. Although today the fat dormouse is not frequently caught as game, it is still a source of animal protein in human nutrition in certain rural areas of Croatia. In this paper the chemical analysis of fat dormouse meat is presented. The average values for the quantity of water, fat, protein and ash in dormouse meat are comparable with the chemical composition of the meat of rabbits and brown hares, except for the important fact that rabbit and hare meat contains a greater quantity of fat on average. According to our results, the meat of fat dormice can be categorised as dietary food, characterised by a small percentage of fat (mean 2.83%) and a high amount of protein (mean 21.01%).     

Comparative morphology of incisor enamel and dentin in humans and fat dormice (Glis glis)

The structure of teeth in all living beings is genetically predetermined, although it can change under external physiological and pathological factors. The author's hypothesis was to indicate evolutional shifts resulting from genetic, functional and other differences. A comparative study about certain characteristics of incisors in humans and myomorpha, the fat dormouse (Glis glis) being their representative as well, comprised measurements of enamel and dentin thickness in individual incisor segments, evaluation of external enamel index, and also assessment of histological structure of enamel and dentin. The study results involving dormice showed the enamel to be thicker in lower than in the upper teeth, quite contrary to enamel thickness in humans. In the upper incisors in dormice the enamel is the thickest in the medial layer of the crown, and in the cervical portion of the crown in the lower incisors. The thickness of dentin in dormice is greater in the oral than in the vestibular side. These findings significantly differ from those reported in reference literature, but they are based on the function of teeth in dormice. Histological characteristics of hard dental tissues in dormice are similar to those in humans, with exception of uniserial structure of enamel and appearance of dentinoenamel junction.     

Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis

The ability to identify and quantitate integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and facilitate derivatization of these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified, of which 40 were integral membrane proteins containing from one to nine mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g., ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g., pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.     

Polynitroxyl albumin plus tempol attenuates liver injury and inflammation after hepatic ischemia and reperfusion

PNA+Tempol, albumin containing conjugated (polynitroxyl albumin; PNA) and free (4-hydroxyl-2,2,6,6-tetramethyl-piperidinyl-1-oxyl; Tempol) nitroxide may protect against injury caused by reactive oxygen species. Therefore, the actions of PNA+Tempol on liver injury and inflammation induced by hepatic ischemia and reperfusion (I/R) were examined. Rats were subjected to 1 h ischemia followed by 24 h reperfusion in the absence (I/R) or presence of PNA+Tempol (25%; 15 mL/kg, i.v.) (I/R+PNA+Tempol) or human serum albumin (23%; 13.5 mL/kg, i.v.) (I/R+HSA). Test solutions were administered prior to and for 2 h during reperfusion. Sham-operated rats underwent surgery with neither ischemia nor infusion. I/R+PNA+Tempol rats had significantly less liver injury and inflammation than I/R rats. I/R+PNA+Tempol livers exhibited focal lesions whereas I/R livers exhibited global necrosis. Likewise, plasma ALT activity was significantly lower in I/R+PNA+Tempol rats. PNA+Tempol reduced I/R-induced neutrophil accumulation and intercellular adhesion molecule-1 (ICAM-1) expression. HSA did not alter I/R-induced liver injury or inflammation. Sham-operated rats exhibited normal liver morphology and no inflammation. Attenuation of I/R liver injury by PNA+Tempol may be mediated by its effect on inflammation, the major contributor to I/R injury. Reduction of inflammation by PNA+Tempol is most likely due to the antioxidative nature of the nitroxides.     

Impact of ‘Stretch’ Targets for Cardiovascular Disease Management within a Local Pay-for-Performance Programme

Pay-for-performance programs are often aimed to improve the management of chronic diseases. We evaluate the impact of a local pay for performance programme (QOF+), which rewarded financially more ambitious quality targets (‘stretch targets’) than those used nationally in the Quality and Outcomes Framework (QOF). We focus on targets for intermediate outcomes in patients with cardiovascular disease and diabetes. A difference-in-difference approach is used to compare practice level achievements before and after the introduction of the local pay for performance program. In addition, we analysed patient-level data on exception reporting and intermediate outcomes utilizing an interrupted time series analysis. The local pay for performance program led to significantly higher target achievements (hypertension: p-value <0.001, coronary heart disease: p-values <0.001, diabetes: p-values <0.061, stroke: p-values <0.003). However, the increase was driven by higher rates of exception reporting (hypertension: p-value <0.001, coronary heart disease: p-values <0.03, diabetes: p-values <0.05) in patients with all conditions except for stroke. Exception reporting allows practitioners to exclude patients from target calculations if certain criteria are met, e.g. informed dissent of the patient for treatment. There were no statistically significant improvements in mean blood pressure, cholesterol or HbA1c levels. Thus, achievement of higher payment thresholds in the local pay for performance scheme was mainly attributed to increased exception reporting by practices with no discernable improvements in overall clinical quality. Hence, active monitoring of exception reporting should be considered when setting more ambitious quality targets. More generally, the study suggests a trade-off between additional incentive for better care and monitoring costs.     

Differential Effects of Tra2? Isoforms on HIV-1 RNA Processing and Expression

Balanced processing of HIV-1 RNA is critical to virus replication and is regulated by host factors. In this report, we demonstrate that overexpression of either Tra2α or Tra2β results in a marked reduction in HIV-1 Gag/ Env expression, an effect associated with changes in HIV-1 RNA accumulation, altered viral splice site usage, and a block to export of HIV-1 genomic RNA. A natural isoform of Tra2β (Tra2ß3), lacking the N-terminal RS domain, also suppressed HIV-1 expression but had different effects on viral RNA processing. The functional differences between the Tra2β isoforms were also observed in the context of another RNA substrate indicating that these factors have distinct functions within the cell. Finally, we demonstrate that Tra2ß depletion results in a selective reduction in HIV-1 Env expression as well as an increase in multiply spliced viral RNA. Together, the findings indicate that Tra2α/β can play important roles in regulating HIV-1 RNA metabolism and expression.     

Chemical composition and antimicrobial activity of volatiles from Degenia velebitica, a European stenoendemic plant of the Brassicaceae family

Free and glucosidic bound leaf volatiles of Degenia velebitica were isolated and fractionated simultaneously into H₂O‐soluble, H₂O‐insoluble, and highly volatile compounds by hydrodistillation–adsorption (HDA) and analyzed by GC/MS. Among the 24 constituents identified, the main compounds obtained by the HDA method were S‐ and/or N‐atom containing compounds, i.e., 6‐(methylsulfanyl)hexanenitrile ( 10 ; 26.78%), dimethyl trisulfide ( 6 ; 26.35%), 3,4,5‐trimethylpyrazole ( 17 ; 13.33%), hex‐5‐enenitrile ( 2 ; 10.11%), dimethyl tetrasulfide ( 8 ; 4.93%), and pent‐4‐enyl isothiocyanate ( 7 ; 4.45%). In addition, O‐glycosidically bound volatiles and free volatiles were isolated by solvent extraction. Sixteen volatile O‐aglycones and twelve free volatile components were identified. The main O‐aglycones were eugenol ( 19 ; 24.15%), 2‐methoxy‐4‐vinylphenol ( 11 ; 11.50%), and benzyl alcohol ( 20 ; 9.49%), and the main free volatiles were (9Z,12Z)‐octa‐9,12‐dienic acid (38.35%), hexadecanoic acid (22.64%), and phytol (5.80%). The H₂O‐soluble volatile fraction obtained by HDA, containing mostly glucosinolate degradation products and 3,4,5‐trimethylpyrazole ( 17 ), was evaluated for antimicrobial activity by determining inhibition zones with the diffusion method as well as minimal inhibitory concentrations (MIC) and minimal microbicidal concentrations (MMC) with the micro‐dilution method. The fraction expressed activity against the tested Gram‐positive and Gram‐negative bacteria as well as against yeast, with MIC values equal to or lower than 16.7 μg/ml.     

A phylostratigraphy approach to uncover the genomic history of major adaptations in metazoan lineages

Macroevolutionary trends traditionally are studied by fossil analysis, comparative morphology or evo-devo approaches. With the availability of genome sequences and associated data from an increasing diversity of taxa, it is now possible to add an additional level of analysis: genomic phylostratigraphy. As an example of this approach, we use a phylogenetic framework and embryo expression data from Drosophila to show that grouping genes by their phylogenetic origin can uncover footprints of important adaptive events in evolution.