Effects of Probiotic Bacteria Bacillus on Growth Performance,Digestive Enzyme Activity,and Hematological Parameters of Asian Sea Bass,Lates calcarifer (Bloch)

Probiotics and Antimicrobial Proteins - This study was conducted to evaluate different doses of two species of Bacillus (Bacillus licheniformis and Bacillus subtilis), on growth parameters,...     

Expression and Purification of Membrane Proteins in Different Hosts

International Journal of Peptide Research and Therapeutics - Membrane proteins play important functions, such as cellular communication and transferring materials in the cell. Many membrane...     

Cryopreservation of tench, Tinca tinca, sperm: Sperm motility and hatching success of embryos

The aim of the present study was to elaborate cryopreservation methods for ex situ conservation of tench. Success of cryopreservation was tested during two series of experiments. The first set of experiments studied the effects of two types of cryoprotectants (DMSO and a combination of DMSO with propanediol at ratio 1:1) at concentrations of 8 and 10% and three different equilibration times in two different immobilization solutions (IS) (Kurokura 180 and Kurokura) before freezing (0.0, 2.0 and 4.0h after T(0)). The K4 cooling programme was used to freeze 1ml of cryoextended sperm using 1.8ml cryotubes. Main monitored parameter was hatching rate after using of cryopreserved sperm. The second set of experiments studied the volume effect of 0.5, 1 and 5ml straws and compared these with 1.8ml cryotubes as well as the effect of the cooling programme (K4 and L1). Following the results of the first study, a combination of DMSO and propanediol (ratio 1:1) at concentration of 10% was added to extended sperm in Kurokura 180 IS. Main monitored parameter was hatching rate after using cryopreserved sperm, supplementary parameters were sperm velocity and motility percentage assessed at 10s post-activation. Sperm was collected directly into IS and stored at 4 degrees C for 2.5h. Thereafter were sperm samples pooled, equlibred in IS (first set of experiments) or directly mixed with cryoprotectants (DMSO or a mixture of DMSO with propanediol at ratio 1:1) and transferred to 1.8ml cryotubes or straws (0.5, 1 and 5ml). Then the cryotubes/straws were directly transferred to pre-programmed PLANER Kryo 10 series III and cooled using two different cooling programmes including a slow cooling programme (a) named K4 (from +4 to -9 degrees C at a rate of 4 degrees Cmin(-1) and then from -9 to -80 degrees C at a rate of 11 degrees Cmin(-1)) and a rapid cooling programme (b) named L1 (directly from +4 to -80 degrees C at a rate of 20 degrees Cmin(-1)). Both slow (K4) and rapid (L1) cooled samples were held 6min at -80 degrees C. Finally, samples were transferred into liquid N(2). The frozen spermatozoa were thawed in a water bath (40 degrees C) according to the frozen volume and checked for fertilization and hatching rates. Percentage of sperm motility and sperm velocity were measured using video recorded frames. ANOVA showed a significant influence of frozen and fresh sperm in all treatments. The hatching rates of 33.8% were obtained when sperm was equilibrated for 0h before freezing in IS of Kurokura 180 and frozen with a 10% of mixture 1:1 of DMSO and propanediol into straws of 5ml and cooled using program L1. The velocity of frozen-thawed spermatozoa ranged from 31 to 46microms(-1) and in post-thawed sperm was not significantly different according to frozen sperm volume, but a higher velocity was obtained when sperm was fast frozen using programme L1. A large volume of frozen sperm could reveal the best procedure for freezing, but also for simulating methods of artificial propagation for future practical use of frozen tench sperm at a large scale.     

Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III

Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair.     

Apelin-13 Inhibits Apoptosis of Cortical Neurons Following Brain Ischemic Reperfusion Injury in a Transient Model of Focal Cerebral Ischemia

Stroke is the third leading cause of death world-wide, affecting 15 million people annually. Diminished blood supply to the brain cells is the main cause of damage following stroke. When focal ischemia occurs, the core of brain tissue influenced by reduced blood supply undergoes necrotic cell death. The adipocytokine Apelin is a peptide that was isolated from a bovine stomach for the first time. This peptide and its receptor are abundantly expressed in the nervous and cardiovascular systems. According to previous studies, Apelin-13 protects cardiomyocytes from ischemic injury and apoptosis. In addition, this peptide has neuroprotective effect on hippocampal and cultured mouse cortical neurons against NMDA receptor-mediated excitotoxicity as well as cortical neurons from ischemic injury. The present study was conducted to determine whether Apelin-13 inhibits apoptosis in the ischemic penumbra in transient focal cerebral ischemia. Focal cerebral ischemia was induced in male Wistar rats by 60 min middle cerebral artery occlusion (MCAO) using a filament method, followed by 23-h reperfusion. Saline as a vehicle and Apelin-13 at doses of 50 and 100 μg were injected intracerebro-ventriculary (ICV) at the beginning of ischemia. Apoptosis and neurological dysfunction were assessed 24-h after MCAO. Our results indicated that administration of Apelin-13 at doses of 50 and 100 μg ICV markedly reduced apoptosis by decreasing positive TUNEL cells (P < 0.001). In addition, Apelin-13 at doses of 100 μg significantly change neurological dysfunction (P < 0.05). Our findings demonstrate that treatment by Apelin-13 exerts its protective effects in ischemic models via blocking programmed cell-death. We suggest that Apelin-13 might be a promising therapeutic target for stroke, although more researches are necessary to take into account the potential therapeutic effects of Apelin-13 in stroke patients.     

Functional characterization of Ape1 variants identified in the human population

Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions. Ape1 is the major human repair enzyme for abasic sites and incises the phosphodiester backbone 5′ to the lesion to initiate a cascade of events aimed at removing the AP moiety and maintaining genetic integrity. Through resequencing of genomic DNA from 128 unrelated individuals, and searching published reports and sequence databases, seven amino acid substitution variants were identified in the repair domain of human Ape1. Functional characterization revealed that three of the variants, L104R, E126D and R237A, exhibited ~40–60% reductions in specific incision activity. A fourth variant, D283G, is similar to the previously characterized mutant D283A found to exhibit ~10% repair capacity. The most common substitution (D148E; observed at an allele frequency of 0.38) had no impact on endonuclease and DNA binding activities, nor did a G306A substitution. A G241R variant showed slightly enhanced endonuclease activity relative to wild-type. In total, four of seven substitutions in the repair domain of Ape1 imparted reduced function. These reduced function variants may represent low penetrance human polymorphisms that associate with increased disease susceptibility.     

Recombinant Production and Antimicrobial Assessment of <Emphasis Type="Italic">Beta</Emphasis> Casein- IbAMP<Subscript>4</Subscript> as a Novel Antimicrobial Polymeric Protein and its Synergistic Effects with Thymol

There is a growing research interest on products with antimicrobial activity. Antimicrobial polymers are one of the most surefire procedures to combat microbes. In the present study, the ability of Βeta-casein- one of the milk major self assembly proteins with high polymeric film production capability—as a fusion partner of Ib-AMP₄ antimicrobial peptide was investigated. Also, the antimicrobial activities of Βeta-casein- IbAMP₄ fusion protein antimicrobial against common food pathogens were assessed. The pET21a-BCN-Ib-AMP ₄ construct was transformed to Escherichia coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg mL^?1 fusion protein by ultrafiltration. 5 μg mL^?1 H₂O₂ was applied for accelerating the formation of two necessary disulfide bonds. Antimicrobial assays were performed against E. coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus and Candida albicans. Results of antimicrobial tests confirmed the efficiency of BCN-IbAMP₄ against all tested microorganisms. Overall, the combination of thymol plus BCN-IbAMP₄ increased their antimicrobial activities. MIC, MBC, MFC, FICI and FBCI values showed strong synergistic activity between the two examined compounds. Time kill and growth kinetic studies indicated significant reduction of cell viability during first period of exposure to BCN-IbAMP₄ and thymol combination.     

Combined evidence annotation of transposable elements in genome sequences

Transposable elements (TEs) are mobile, repetitive sequences that make up significant fractions of metazoan genomes. Despite their near ubiquity and importance in genome and chromosome biology, most efforts to annotate TEs in genome sequences rely on the results of a single computational program, RepeatMasker. In contrast, recent advances in gene annotation indicate that high-quality gene models can be produced from combining multiple independent sources of computational evidence. To elevate the quality of TE annotations to a level comparable to that of gene models, we have developed a combined evidence-model TE annotation pipeline, analogous to systems used for gene annotation, by integrating results from multiple homology-based and de novo TE identification methods. As proof of principle, we have annotated "TE models" in Drosophila melanogaster Release 4 genomic sequences using the combined computational evidence derived from RepeatMasker, BLASTER, TBLASTX, all-by-all BLASTN, RECON, TE-HMM and the previous Release 3.1 annotation. Our system is designed for use with the Apollo genome annotation tool, allowing automatic results to be curated manually to produce reliable annotations. The euchromatic TE fraction of D. melanogaster is now estimated at 5.3% (cf. 3.86% in Release 3.1), and we found a substantially higher number of TEs (n = 6,013) than previously identified (n = 1,572). Most of the new TEs derive from small fragments of a few hundred nucleotides long and highly abundant families not previously annotated (e.g., INE-1). We also estimated that 518 TE copies (8.6%) are inserted into at least one other TE, forming a nest of elements. The pipeline allows rapid and thorough annotation of even the most complex TE models, including highly deleted and/or nested elements such as those often found in heterochromatic sequences. Our pipeline can be easily adapted to other genome sequences, such as those of the D. melanogaster heterochromatin or other species in the genus Drosophila.     

Abnormal hemoglobins among Kurdish population of Western Iran: hematological and molecular features

The type and frequency of structural hemoglobin variants and their hematological and molecular characteristics were identified using PCR-RFLP and sequencing techniques in 66 individuals from 33 unrelated families who referred to the two clinics of Kermanshah University of Medical Sciences from 2005 to 2006. We detected 28 subjects carrier for Hb D-Punjab (42.4%), 21 individuals carrier of Hb Q-Iran (31.8%), 12 subjects heterozygous for Hb Setif (18.2%), four cases with sickle cell disease (6.1%), and one case with Hb C (1.5%). All β^S genes (4 genes) were linked to the Benin haplotype with negative Taq I site 5′ to γ^A gene. All β^D-Punjab genes (29 genes) were in linkage disequilibrium with haplotype I. The only β^C chromosome was linked to haplotype II. Both β⁰-thalassemia chromosomes with CD15 (G → A) mutation had haplotype background I. Three β⁺-thalassemia chromosomes with IVSI.110 (G → A) mutation were associated with haplotype I [+ − − − − + +]. In turn, the three β-thalassemia chromosomes with IVS II.1 G → A mutation were associated with atypical haplotype [− + + + + + −]. Hematological indices of carriers of Hb D-Punjab, Hb Q-Iran and Hb Setif were lower than those reported for normal individuals. For the first time, we have reported the haplotype background of β^S gene among Kurdish population of Iran. Our results revealed that Hb D-Punjab is the most prevalent β-globin chain structural variant in this area and that is followed in frequency by an α-chain variant, Hb Q-Iran. The result of present study is useful for clinical management and the establishment of screening programmes in Western Iran.