A cleavable affinity biotinylating agent reveals a retinoid binding role for RPE65

Retinal pigment epithelial (RPE) membranes contain the full biochemical apparatus capable of processing all-trans-retinol (vitamin A) into 11-cis-retinal, the visual chromophore. As many of these proteins are integral membrane proteins and resistant to traditional methods of identification, alternate methods of identifying these proteins are sought. The approach described here involves affinity biotinylation with alkali cleavable linkers. A vitamin A containing affinity-labeling haloacetate is described which facilitates the identification of retinoid binding proteins (RBPs). Treatment of crude bovine RPE membranes with (3R)-3-[boc-lys(biotinyl)-O]-all-trans-retinol chloroacetate 1 in the low micromolar range led to the specific labeling of RPE65 and lecithin retinol acyltransferase (LRAT). Only RPE65 is labeled at 5 microM 1 at 4 degrees C. Labeled RPE65 was readily isolated by binding the labeled protein to avidin-containing beads, followed by cleavage of the protein from the beads at pH 11. Trypsin digestion of RPE65 modified by 1, followed by mass spectrometry, demonstrates that C231 and C448 are alkylated by 1. These studies validate the approach that was used, and furthermore demonstrate that RPE65, a major membrane-associated protein of the RPE, is a RBP.     

Biosynthesis of protease by Pseudomonas aeruginosa MN7 grown on fish substrate

Fish powders and fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were prepared and tested as growth media for alkaline protease production by Pseudomonas aeruginosa MN7. Cultivated in fish substrate as carbon source, the strain exhibited a slightly greater protease production (about 7800 U ml^–1) than that obtained with commercial peptones (about 7222 U ml^–1). Furthermore, P. aeruginosa MN7 produced the same amount of protease when cultivated in medium containing only fish substrate or that containing all ingredients, indicating that the strain can obtain its carbon and nitrogen requirements directly from whole fish proteins. Moreover, it was found that extensive hydrolysis of fish proteins did not increase protease formation. Protease production in media containing only FPH prepared by Alcalase was about 70% of those obtained with MN7 protease digest of fish protein or with meat-fish powder. These results indicate that sardinella substrates are an excellent carbon and nitrogen source for the growth of P. aeruginosa MN7 and the production of protease.     

Fatty acid composition of two Tunisian pine seed oils

Oils were extracted from fully ripen Pinus pinea L. and Pinus halepensis Mill seeds and fatty acid composition has been established by capillary gas chromatography. Seeds are rich in lipids, 34.63-48.12% on a dry weight basis. Qualitatively, fatty acid composition of both species is identical. For P. halepensis linoleic acid is the major fatty acid (56.06% of total fatty acids) followed by oleic (24.03%) and palmitic (5.23%) acids. For P. pinea, the same fatty acids are found with the proportions 47.28%, 36.56%, and 6.67%, respectively. Extracted fatty acids from both species are mainly unsaturated, respectively, 89.87% and 88.01%. Pinus halepensis cis-5 olefinic acids are more abundant (7.84% compared to 2.24%). Results will be important as a good indication of the potential nutraceutical value of Pinus seeds as new sources of fruit oils rich in polyunsaturated fatty acids and cis-5 olefinic acids.     

Faunistic data and biogeography of terrestrial isopods from Tunisian wetlands

In Tunisia, while wetlands are considered as remarkable habitats for their faunal and floral diversity, few studies on the biogeography and the diversity of terrestrial isopods were performed. To fill this gap, we carried out a field study in the supralittoral zone of 146 Tunisian wetlands belonging to eight types (lagoon, river, lake, sebkha, dam, hill reservoir, beach and chott) and to five bioclimatic zones (humid, subhumid, semi‐arid, arid and Saharan). Field work was carried out in spring 2010. Terrestrial isopods were collected in the morning by hand search each time with the same sampling effort. During the study, 22 species of terrestrial isopods belonging to nine families were collected. Porcellio variabilis, Porcellio laevis, Chaetophiloscia elongata and Armadillidium pelagicum are the most common species identified. From the northern to the southern regions, a gradual decrease in species richness has been shown. The identified species belonged to different biogeographical categories: Mediterranean, Mediterranean‐Atlantic, North Africa with circum‐Sicilian islands, semi‐arid and Saharan species. Some of these species are endemic to Tunisia (Porcellio dominici) or to North Africa (Armadillidium sulcatum, Armadillidium tunisiense, Porcellio marginenotatus and P. variabilis). Terrestrial isopod species differ according to wetland type and bioclimatic zone.     

Appropriateness of empiric antimicrobial therapy with imipenem/colistin in severe septic patients: observational cohort study

Background

Empiric antimicrobial therapy (EAMT) using imipenem/colistin is commonly prescribed as a first line therapy in critically ill patients with severe sepsis. We aimed to assess the appropriateness of prescribing imipenem/colistin as EAMT in ICU patients.

Methods

A 3-year observational prospective study included ICU patients that required imipenem/colistin as EAMT. The EAMT was assessed according to microbiological and clinical outcomes. The outcomes were: delay in apyrexia, delay in the decrease of the biological inflammatory parameters (BIP), the requirement for vasoactive agents, bacteriological eradication, length of stay, ventilator days and 30-day mortality.

Results

79 administrations of EAMT in 70 patients were studied. EAMT was appropriate in 52% of the studied cases. An ICU stay?>?6 days was related to inappropriateness, and chronic respiratory failure was associated with appropriateness. In the appropriate EAMT group, we showed: earlier apyrexia, shorter delay in the decrease of the BIP and a reduced significant vasopressors requirement. Furthermore, EAMT improved survival with a median gain of 4 days. Inappropriate EAMT increased the mortality risk by six. The acquisition of NI in ICU was also an independent factor of mortality.

Conclusions

EAMT using imipenem-colistin was appropriate in half of the cases and inappropriateness was associated with an increased ICU mortality risk.

     

Low-cost culture medium for the production of proteases by <Emphasis Type="Italic">Bacillus mojavensis</Emphasis> SA and their potential use for the preparation of antioxidant protein hydrolysate from meat sausage by-products

The present study aims to maximize proteases production by Bacillus mojavensis SA strain and their use to produce bioactive protein hydrolysates from a meat by-product. The production of SA bacteria proteases was maximized using a culture medium based on wheat bran, which offer an advantage in minimizing the production cost and enhancing the enzyme activity by using agro-industrial wastes. The composition of media and cultural conditions for optimal proteases production by B. mojavensis SA strain were investigated. A successful and significant improvement of the alkaline proteases production (four folds) by the SA strain was achieved using the medium composed of (g/l): wheat bran, 50.0; KH₂PO₄, 0.5; K₂HPO₄, 0.5; CaCl₂, 2.0; pH 6.0, where the growth conditions were monitored at 37 °C with an agitation speed of 200 rpm. Interestingly, the enzyme preparation of B. mojavensis was applied for the preparation of protein hydrolysates from a meat by-product. Hydrolysis was carried out for 180 min at pH 12.0. The resulting hydrolysate displayed an important antioxidant activity as evaluated by the radical scavenging capacity, the reducing power, and the β-carotene bleaching inhibition. The present study showed the high proteases’ producing level by B. mojavensis SA strain in a low-cost fermentation medium (wheat bran) and their potential use in the production of bioactive protein hydrolysate from meat by-products.     

Purification and biochemical characterization of a new alkali-stable laccase from Trametes sp. isolated in Tunisia: role of the enzyme in olive mill waste water treatment

A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes sp, was selected in a broad plate screening because of its ability to decolorize and dephenolize olive oil mill wastewater (OMW) efficiently. The major laccase was purified and characterized as a monomeric protein with apparent molecular mass of 61 kDa (SDS-PAGE). It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 4.0 and a temperature of 60 °C. The purified laccase is stable at alkaline pH values. The enzyme retained 50 % of its activity after 90 min of incubation at 55 °C. Using ABTS, this laccase presented K _m and V _max values of 0.05 mM and 212.73 μmoL min^?1 mg^?1, respectively. It has shown a degrading activity towards a variety of phenolic compounds. The purified laccase was partially inhibited by Fe²⁺, Zn²⁺, Cd²⁺ and Mn²⁺, while Cu²⁺ acted as inducer. EDTA (10 mM) and NaN₃ (10 mM) were found to completely inhibit its activity. 73 % OMW was dephenolized after 315 min incubation at 30 °C with 2 U mL^?1 of laccase and 2 mM HBT.     

Polymorphisms in glutathione S-transferase are risk factors for perioperative acute myocardial infarction after cardiac surgery: a preliminary study

In the present study we explored glutathione S-transferase (GST) polymorphisms in selected patients who experienced accelerated myocardial injury following open heart surgery and compared these to a control group of patients without postoperative complications. 758 Patients were enrolled from which 132 patients were selected to genotype analysis according to exclusion criteria. Patients were divided into the following groups: Group I: control patients (n = 78) without and Group II.: study patients (n = 54) with evidence of perioperative myocardial infarction. Genotyping for GSTP1 A (Ile105Ile/Ala113Ala), B (Ile105Val/Ala113Ala) and C (Ile105Val/Ala113Val) alleles was performed by using real-time-PCR. The heterozygous AC allele was nearly three times elevated (18.5 vs. 7.7 %) in the patients who suffered postoperative myocardial infarction compared to controls. Contrary, we found allele frequency of 14.1 % for homozygous BB allele in the control group whereas no such allele combination was present in the study group. These preliminary results may suggest the protective role for the B and C alleles during myocardial oxidative stress whereas the A allele may represent predisposing risk for cellular injury in patients undergoing cardiac surgery.     

Building of a composite virtual slide from contiguous tissue samples

Background

Currently available microscope slide scanners produce whole slide images at various resolutions from histological sections. Nevertheless, acquisition area and so visualization of large tissue samples are limited by the standardized size of glass slides, used daily in pathology departments. The proposed solution has been developed to build composite virtual slides from images of large tumor fragments.

Materials and methods

Images of HES or immunostained histological sections of carefully labeled fragments from a representative slice of breast carcinoma were acquired with a digital slide scanner at a magnification of 20×. The tiling program involves three steps: the straightening of tissue fragment images using polynomial interpolation method, and the building and assembling of strips of contiguous tissue sample whole slide images in × and y directions. The final image is saved in a pyramidal BigTiff file format. The program has been tested on several tumor slices. A correlation quality control has been done on five images artificially cut.

Results

Sixty tumor slices from twenty surgical specimens, cut into two to twenty six pieces, were reconstructed. A median of 98.71% is obtained by computing the correlation coefficients between native and reconstructed images for quality control.

Conclusions

The proposed method is efficient and able to adapt itself to daily work conditions of classical pathology laboratories.