Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production

Background  

The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.3 antibody fragment in E. coli BL21 and evaluated the overall physiological and global gene expression changes upon Skp or FkpA co-expression.     

ITS2 sequences as barcodes for identifying and analyzing spider mites (Acari: Tetranychidae)

The use of DNA barcodes, short DNA sequences from a standardized region of the genome, has recently been proposed as a tool to facilitate species identification and discovery. Here we show that second internal transcribed spacer of nuclear ribosomal DNA (rDNA-ITS2) barcodes effectively discriminate among 16 species of spider mites (Acari: Tetranychidae) from Israel. The barcode sequences of each species were unambiguously distinguishable from all other species and formed distinct, nonoverlapping monophyletic groups in the maximum-parsimony tree. Sequence divergences were generally much greater between species than within them. Using a 0.02 (2%) threshold for species diagnosis in our data set, 14 out of 16 species recognized by morphological criteria would be accurately identified. The only exceptions involved the low divergence, 0.011–0.015 (1.1–1.5%), between Tetranychus urticae and Tetranychus turkestani, where speciation may have occurred only recently. Still, these species had fixed alternative rDNA-ITS2 variants, with five diagnostic nucleotide substitutions. As a result, we tentatively conclude that rDNA-ITS2 sequence barcodes may serve as an effective tool for the identification of spider mite species and can be applicable as a diagnostic tool for quarantine and other pest management activities and decision-making. We predict that our work, together with similar efforts, will provide in the future the platform for a uniform, accurate, practical and easy-to-use method of spider mite species identification.     

Biofilms of As(III)-oxidising bacteria: formation and activity studies for bioremediation process development

The formation and activity of an As(III)-oxidising biofilm in a bioreactor, using pozzolana as bacterial growth support, was studied for the purpose of optimising fixed-bed bioreactors for bioremediation. After 60 days of continuous functioning with an As(III)-contaminated effluent, the active biofilm was found to be located mainly near the inflow rather than homogeneously distributed. Biofilm development by the CAsO1 bacterial consortium and by Thiomonas arsenivorans was then studied both on polystyrene microplates and on pozzolana. Extra-cellular polymeric substances (EPS) and yeast extract were found to enhance bacteria attachment, and yeast extract also appears to increase the kinetics of biofilm formation. Analysis of proteins, sugars, lipids and uronic acids indicate that sugars were the main EPS components. The specific As(III)-oxidase activity of T. arsenivorans was higher (by ninefold) for planktonic cells than for sessile ones and was induced by As(III). All the results suggest that the biofilm structure is a physical barrier decreasing As(III) access to sessile cells and thus to As(III)-oxidase activity induction. The efficiency of fixed-bed reactors for the bioremediation of arsenic-contaminated waters can be thus optimised by controlling different factors such as temperature and EPS addition and/or synthesis to increase biofilm density and activity.     

Effects of altered stride frequency and contact time on leg-spring behavior in human running

Many studies have demonstrated that contact time is a key factor affecting both the energetics and mechanics of running. The purpose of the present study was to further explore the relationships between contact time (t(c)), step frequency (f) and leg stiffness (k(leg)) in human running. Since f is a compound parameter, depending on both contact and aerial time, the specific goal of this study was to independently vary f and t(c) and to investigate their respective effects on spring-mass characteristics during running, seeking to determine if the changes in k(leg) observed when running at different f are mainly due to inherent changes in t(c). We compared three types of constant 3.33 m s(-1) running conditions in 10 male subjects: normal running at the subject's freely chosen f, running with decreased and increased f, and decreased and increased t(c) at the imposed freely chosen f. The data from the varied f trials showed that the variation of t(c) was strongly correlated to that of k(leg) (r(2)=0.90), and the variation of f was also significantly correlated to that of k(leg) (r(2)=0.47). Further, changes in t(c) obtained in various t(c) conditions were significantly correlated to changes in k(leg) (r(2)=0.96). These results confirm that leg stiffness was significantly influenced by step frequency variations during constant speed running, as earlier demonstrated, but our more novel finding is that compared to step frequency, the effect of contact time variations appears to be a stronger and more direct determinant of k(leg). Indeed, 90-96% of the variance in k(leg) can be explained by contact time, whether this latter parameter is directly controlled, or indirectly controlled through its close relationship with step frequency. In conclusion, from the comparison of two experimental procedures, i.e. imposing various step frequency conditions vs. asking subjects to intentionally vary contact time at their freely chosen step frequency, it appears that changes in leg stiffness are mainly related to changes in contact time, rather than to those in step frequency. Step frequency appears to be an indirect factor influencing leg stiffness, through its effect on contact time, which could be considered a major determinant of this spring-mass characteristic of human running.     

Antioxidant defense in hibernation: cloning and expression of peroxiredoxins from hibernating ground squirrels, Spermophilus tridecemlineatus

Mammalian hibernation is characterized by prolonged torpor bouts interspersed by brief arousal periods. Adequate antioxidant defenses are needed both to sustain cell viability over weeks of deep torpor and to defend against high rates of oxyradical formation associated with massive oxygen-based thermogenesis during arousal. The present study shows that up-regulation of peroxiredoxins contributes to antioxidant defense during torpor in thirteen-lined ground squirrels, Spermophilus tridecemlineatus. Expression levels of three isozymes of the 2-Cys peroxiredoxin (Prdx) family were quantified by Western blotting, the results showing 4.0- and 12.9-fold increases in Prdx1 protein in brown adipose tissue (BAT) and heart, respectively, during hibernation compared with euthermia. Comparable increases in Prdx2 were 2.4- and 3.7-fold whereas Prdx3 rose by 3.1-fold in heart of torpid animals. Total 2-Cys peroxiredoxin enzymatic activity also rose during hibernation by 1.5-fold in heart and 3.5-fold in BAT. Furthermore, RT-PCR showed that prdx2 mRNA levels increased by 1.7- and 3.7-fold in BAT and heart, respectively, during hibernation. A partial nucleotide sequence of prdx2 from ground squirrels was obtained by PCR amplification, the deduced amino acid sequence showing 96-97% identity with Prdx2 from other mammals. Some unique amino acid substitutions were identified that might contribute to stabilizing Prdx2 conformation at the near 0 degrees C body temperatures during torpor.     

Relaxing effects of 5-oxo-ETE on human bronchi involve BK Ca channel activation

The present study investigated the ability of 5-oxo-EicosaTetraEnoic acid (5-oxo-ETE) for modulating airway smooth muscle (ASM) tone in human bronchi. 5-Oxo-ETE induced a concentration-dependent relaxing effect on human bronchi pre-contracted with methacholine (MCh) and arachidonic acid (AA). This relaxing response was highly sensitive to Iberiotoxin (IbTx), a large conducting Ca(2+)-activated K(+) channel (BK(Ca)) inhibitor. Furthermore, microelectrode measurements revealed that 5-oxo-ETE (0.1-10 microM) hyperpolarizes the membrane potential of human bronchial ASM cells. These hyperpolarizing effects were also inhibited in the presence of 10nM IbTx. Lastly, 5-oxo-ETE was shown to directly activate reconstituted BK(Ca) channels derived from human airway smooth muscles. In summary, the 5-oxo-ETE eicosanoid activates a specific K(+) conductance, involved in membrane hyperpolarization, which in turn reduces Ca(2+) entry and facilitates relaxation of smooth muscle cells.     

Nuclear cysteine-protease involved in male chromatin remodeling after fertilization is ubiquitously distributed during sea urchin development

Previously we have identified a cysteine-protease involved in male chromatin remodeling which segregates into the nuclei of the two blastomeres at the first cleavage division. Here we have investigated the fate of this protease during early embryogenesis by immunodetecting this protein with antibodies elicited against its N-terminal sequence. As shown in this report, the major 60 kDa active form of this protease was found to be present in the extracts of chromosomal proteins obtained from all developmental stages analyzed. In morula and gastrula the 70 kDa inactive precursor, which corresponds to the major form of the zymogen found in unfertilized eggs, was detected. In plutei larvas, the major 60 kDa form of this enzyme was found together with a higher molecular weight precursor (90 kDa) which is consistent with the less abundant zymogen primarily detected in unfertilized eggs. As reported here, either the active protease or its zymogens were visualized in most of the embryonic territories indicating that this enzyme lacks a specific pattern of spatial-temporal developmental segregation. Taken together our results indicate that this protease persists in the embryo and is ubiquitously distributed up to larval stages of development, either as an active enzyme and/or as an inactive precursor. These results suggest that this enzyme may display yet unknown functions during embryonic development that complement its role in male chromatin remodeling after fertilization.