New vectors for co-expression of proteins: structure of Bacillus subtilis ScoAB obtained by high-throughput protocols

The Bacillus subtilis genes scoA and scoB encode subunits of the heteromeric enzyme ScoAB, a putative succinyl-CoA:acetoacetate coenzyme A transferase. High-throughput, ligation-independent cloning (LIC) vectors used extensively for production and purification of single proteins were modified to allow simultaneous expression of interacting proteins and selective purification of functional complexes. Transfer of the LIC region of vector pMCSG7 (L. Stols, M. Gu, L. Dieckman, R. Raffen, F.R. Collart, M.I. Donnelly. A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr. Purif. (2002) 25, 8–15) into commercial vectors with alternative, compatible origins of replication allowed introduction of standard LIC PCR products into the vectors by uniform protocols. Replacement of the His-tag encoding region of pMCSG7 with a sequence encoding the S-tag enabled selective purification of interacting proteins based on the His-tag associated with one member of the complex. When expressed separately and mixed, the ScoAB subunits failed to interact productively; no transferase activity was detected, and S-tagged ScoB failed to co-purify with His-tagged ScoA. Co-expression, in contrast, generated active transferase that catalyzed the predicted reaction. The ScoAB complex was purified by standard high-throughput metal-ion affinity chromatography procedures, crystallized robotically, and its structure was determined by molecular replacement.     

Regulation of protein tyrosine phosphatase 1B by sumoylation

Protein-tyrosine phosphatase 1B (PTP1B) is an ubiquitously expressed enzyme that negatively regulates growth-factor signalling and cell proliferation by binding to and dephosphorylating key receptor tyrosine kinases, such as the insulin receptor. It is unclear how the activity of PTP1B is regulated. Using a yeast two-hybrid assay, a protein inhibitor of activated STAT1 (PIAS1) was isolated as a PTP1B-interacting protein. Here, we show that PIAS1, which functions as a small ubiquitin-like modifier (SUMO) E3 ligase, associates with PTP1B in mammalian fibroblasts and catalyses sumoylation of PTP1B. Sumoylation of PTP1B reduces its catalytic activity and inhibits the negative effect of PTP1B on insulin receptor signalling and on transformation by the oncogene v-crk. Insulin-stimulated sumoylation of endogenous PTP1B results in a transient downregulation of the enzyme; this event does not occur when the endogenous enzyme is replaced with a sumoylation-resistant mutant of PTP1B. These results suggest that sumoylation, which has been implicated primarily in processes in the nucleus and nuclear pore, also modulates a key enzyme-substrate signalling complex that regulates metabolism and cell proliferation.     

Chronic renal failure-induced multiple-organ injury in rats is alleviated by the selective CysLT1 receptor antagonist montelukast

Chronic renal failure (CRF) is associated with oxidative stress that promotes production of reactive oxygen species and cytokine release. We aimed to investigate the possible protective effect of montelukast, a CysLT1 receptor antagonist, against oxidative damage in a rat model of CRF, induced by 5/6 reduction of renal mass. Male Wistar albino rats were randomly assigned to either the CRF group or the sham-operated control group, which received saline or montelukast (10mg/kg, i.p.) for 4 weeks. At the end of the 4 weeks, rats were decapitated and trunk blood was collected. Creatinine, blood urea nitrogen and lactate dehydrogenase (LDH) activity were measured in the serum samples, while leukotriene B(4), TNF-alpha, IL-1 beta, IL-6, total antioxidant capacity (AOC) and leukocyte apoptosis were assayed in plasma samples. Kidney, lung, heart and brain tissue samples were taken for the determination of tissue malondialdehyde (MDA), glutathione (GSH) levels, and myeloperoxidase (MPO) activity. Oxidant-induced tissue fibrosis was determined by tissue collagen contents, and the extent of tissue injuries was analyzed microscopically. CRF caused significant decreases in tissue GSH and plasma AOC, which were accompanied with significant increases in MDA levels, MPO activities, and collagen contents of all the studied tissues, while the circulating levels of the pro-inflammatory mediators, LDH activity, creatinine and BUN were elevated. Montelukast treatment reversed all these biochemical indices, as well as histopathological alterations induced by CRF. Similarly, flow cytometric measurements revealed that leukocyte apoptosis was increased in CRF group, while montelukast reversed this effect. In conclusion, CRF-induced oxidative tissue injury occurs via the activation of pro-inflammatory mediators and by neutrophil infiltration into tissues, and that protective effects of montelukast on CRF-induced injury can be attributed to its ability to inhibit neutrophil infiltration and apoptosis, to balance oxidant-antioxidant status and to regulate the generation of pro-inflammatory mediators.     

A conformational switch‐based aptasensor for the chemiluminescence detection of microRNA

A simple microRNA (miRNA) aptasensor has been developed combining the conformational switch of a streptavidin aptamer and isothermal strand displacement amplification. In the presence of its target miRNA, the allosteric molecular beacon (aMB) probe immobilized on the plate can be ‘switched on' and release the streptavidin aptamer. At the same time, Klenow fragment (3′→5′ exo‐) is utilized to initiate DNA‐strand displacement, which starts the target recycling process. Based on the aptamer' high binding affinity and subsequent catalytic chemiluminescence (CL) detection, this CL strategy is highly specific in distinguishing mature miRNAs in same family. It exhibits a dynamic range of four orders of magnitude with a detection limit of 50 fM, and shows great potential for miRNA‐related clinical practices and biochemical research.     

Lateral wedge with medial only cardiac shielding (LEMONADE) technique in left chest wall adjuvant radiotherapy

BackgroundThis dosimetric study compared lateral wedge with medial only cardiac shielding (LEMONADE) technique, for left chest wall (LCW) irradiation against three other commonly used techniques.Materials and methodsDosimetric parameters of 22 consecutive LBC patients treated using the P1 (LEMONADE technique) were compared with 3 other virtually reconstructed plans : no cardiac shielding with paired wedges; P2 (paired wedges and medial only Y-direction shielding) and P3 (paired wedges and bilateral Y-direction shielding).ResultsP1 showed better target volume (TV) coverage with the mean 90% isodose coverage of 85.59% ± 5.44 compared to 78.90% ± 8.59 and 74.22% ± 9.50 for P2 and P3, respectively. Compared to no cardiac shielding, for a 4.65% drop in TV coverage the V26Gy of heart dropped from 6.68% to a negligible 0.85% for P1. TV receiving < 30Gy is also significantly lesser for P1 compared to P2 and P3 (5.42% vs 10.64% and 15.8%), whilst there is a small difference of 2.75% between no cardiac shielding and P1.ConclusionWith the improvement in BC survival rate, cardiac toxicity associated with adjuvant irradiation for LBC is a major concern. P1 (LEMONADE) technique has a good compromise between cardiac sparing and target coverage and should suffice for most LCW irradiations. Furthermore, the LEMONADE technique is a simple, reproducible and involves fast planning for cardiac sparing, which is ideal for under-resourced departments with heavy workload.     

Photoluminescence and thermoluminescence dosimetry properties of Ag/Y co-doped ZnO nanophosphor for radiation measurements

This work explores the thermoluminescence (TL) and photoluminescence (PL) properties of Ag/Y co-doped zinc oxide (ZnO) nanophosphor. The proposed dosimeter was prepared by the coprecipitation method and sintered at temperatures from 400°C to 1000°C in an air atmosphere. Raman spectroscopy was studied to investigate the structural features of this composition. The new proposed dosimeter revealed two peaks at 150°C and 175°C with a small shoulder at high temperature (225°C). The PL spectrum showed strong green emissions between 500 to 550 nm. The Raman spectrum showed many bands related to the interaction between ZnO, silver (Ag), and yttrium oxide (Y₂O₃). The rising sintering temperature enhanced the TL glow curve intensity. The Ag/Y co-doped ZnO nanophosphor showed an excellent linearity index within a dose from 1 to 4 Gy. The minimum detectable dose (MDD) of the Ag/Y co-doped ZnO nanopowder (pellets) equaled 0.518 mGy. The main TL properties were achieved in this work as follows: thermal fading (37% after 45 days at 1 and 4 Gy), optical fading (53% after 1 h and 68% after 6 h by exposure to sunlight), effective atomic number (27.6), and energy response (flat behavior from 0.1 to 1.3 MeV). Finally, the proposed material shows promising results nominated to be used for radiation measurements.     

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.     

Plasmid profiling and curing of Lactobacillus strains isolated from the gastrointestinal tract of chicken

In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, 17%, 58%, and 25% were found to exhibit a high degree of resistance to 200 microg/ml of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least 50 microg/ml of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least 50 microg/ml of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.     

Phospholipid and triacylglycerol fatty acid content and pattern in the cardiac endothelium of rats depleted in long-chain polyunsaturated omega 3 fatty acids

Rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3-depleted rats) display several features of the metabolic syndrome including hypertension and cardiac hypertrophy. This coincides with alteration of the cardiac muscle phospholipid and triacylglycerol fatty acid content and/or pattern. In the present study, the latter variables were measured in the cardiac endothelium of normal and omega3-depleted rats. Samples derived from four rats each were obtained from 16 female normal fed rats and three groups of 36-40 female fed omega3-depleted rats each aged 8-9, 15-16 and 22-23 weeks. At comparable mean age, the ratio between the square root of the total fatty acid content of phospholipids and cubic root of the total fatty acid content of triacylglycerols was lower in omega3-depleted rats than in control animals. The total fatty acid content of triacylglycerols was inversely related to their relative content in C20:4omega6. Other differences between omega3-depleted rats and control animals consisted in a lower content of long-chain polyunsaturated omega3 fatty acids in both phospholipids and triacylglycerols, higher content of long-chain polyunsaturated omega6 fatty acids in phospholipids, higher activity of delta9-desaturase (C16:0/C16:1omega7 and C18:0/C18:1omega9 ratios) and elongase [(C16:0 + C16:1omega7)/(C18:0 + C18:1omega9) and C20:4omega6/C22:4omega6 ratios], but impaired generation of C22:6omega3 from C22:5omega3 in the former rats. These findings support the view that cardiovascular perturbations previously documented in the omega3-depleted rats may involve impaired heart endothelial function.