Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones.

Disulfide bonds are normally formed after a polypeptide has been exported from the reducing environment of the cytoplasm into a more oxidizing compartment, such as the bacterial periplasm. Recently, we showed that in Escherichia coli trxB gor mutants, in which the reduction of thioredoxin and glutathione is impaired, the redox potential of the cytoplasm becomes comparable to that of the mammalian endoplasmic reticulum, thus allowing the formation of disulfide bonds in certain complex proteins (P. H. Bessette et al., 1999, Proc. Natl. Acad. Sci. USA 96, 13703-13708]. Here, we investigate the expression of a Fab antibody fragment in the bacterial cytoplasm. The effect of coexpressing cytoplasmic chaperones (GroEL/ES, trigger factor, DnaK/J), as well as signal sequenceless versions of periplasmic chaperones (DsbC and Skp), was examined. Skp coexpression was shown to have the most significant effect (five- to sixfold increase) on the yield of correctly folded Fab. A maximum yield of 0.8 mg Fab/L/OD(600) Fab was obtained, indicating that cytoplasmic expression may be a viable alternative for the preparative production of antibody fragments.     

Post-resistance training detraining: time-of-day effects on training and testing outcomes

The aim of this study was to examine the effects of 3 and 5 weeks of detraining after 14 weeks of resistance training at a specific time of day on performances during the squat jump (SJ) and the maximal voluntary contraction (MVC). Thirty-one healthy male physical education students (age: 23.1 ± 1.0 years; height: 176.1 ± 6.3 cm; weight: 74.9 ± 10.9 kg) were randomly assigned to either a morning training group (MTG, training between 07:00 and 08:00 h, n = 10), an evening training group (ETG, training between 17:00 and 18:00 h, n = 11) or a control group (CG, no training, n = 10). Participants then performed eight test sessions (twice per day, at 07:00 and 17:00 h) over the course of four phases: during pre-training, immediately post-training, and after 3 and 5 weeks of detraining. Before each test session, oral temperature was recorded. During the first 12 weeks of resistance training, participants performed 3 sets of 10 repetitions to failure (10-RM) for 4 exercises (squat, leg press, leg extension and leg curl, with 2 min of recovery between each exercise); during the last two weeks, training intensity increased to 8-RM with 3 min of recovery between each exercise. Oral temperature was significantly higher at 17:00 than 07:00 h during all test periods (p < 0.05). Likewise, SJ and MVC performances were significantly higher at 17:00 h than 07:00 h during all four test days in ETG and CG, and before training and 3 and 5 weeks after training in MTG (p < 0.05). For both training groups, most SJ and MVC performances (except MTG at 07:00 h and ETG at 17:00 h) returned to baseline values after 5, but not after 3, weeks of detraining. This study showed that 14 weeks of training at a specific time of day blunted the diurnal variation of MVC and SJ in the MTG. The improvement in performance brought about by resistance training was partially retained after 3 weeks of detraining (unless training had taken place at a non-habitual time of day) but was lost after 5 weeks of detraining. There was no effect of the time of training on core temperature.     

Pathological obstacles of newly-introduced fish species in Mediterranean mariculture: a review

Species diversification has been extensively employed in Mediterranean mariculture industry as a tool to relief the crisis resulting from market saturation and overproduction of the two main representatives, sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata). Numerous new promising candidates belonging to several fish families such as Sparidae, Polyprionidae, Serranidae, Sciaenidae, Soleidae, Carangidae and Scombridae have been introduced to provide alternative culture choices in Mediterranean. The farming potential of these species in production scale has received some or little success partly due to the fact that their entrance has not been accompanied without the presence of considerable pathological problems. Bacterial outbreaks causing serious problems have been mainly induced by Photobacterium damsella subsp. piscicida, Vibrio alginolyticus, V. harveyi, Tenacibaculum maritimum and Flavobacterium sp. Detrimental parasitic pathogens included the myxosporean Enteromyxum leei, the ciliates Cryptocaryon irritans and Uronema spp., the flagellate Amyloodinium occelatum, the monogenean Zeuxapta seriolae and the digeneans Paradeontacylix-like spp. Viral encephalopathy and retinopathy infections and the chlamydia-like bacteria causing Epitheliocystis disease are also of significance importance. Numerous other agents of lesser pathogenic significance as well as other occasionally recorded pathogens are also mentioned in this review. Additional research effort has to be devoted to the pathogenesis of important pathogens and the interactions with their hosts to assist the viability of new fish species farming in Mediterranean region.     

Thymus transplantation and disease prevention in the diabetes-prone Bio-Breeding rat

Bio-Breeding rat T lymphocytes proliferate poorly in response to alloantigen. Transplantation of Bio-Breeding rats with fetal thymus tissue from diabetes resistant rats leads to an improvement in the T cell proliferative response, but only if the thymus contains bone marrow-derived, radiation-resistant thymic antigen presenting cells of the diabetes-resistant phenotype. The current study provides evidence that thymus transplantation leading to the restoration of Bio-Breeding T cell proliferative function can also significantly reduce the incidence of insulitis and prevent the development of diabetes. It appears that a defect in the bone marrow-derived thymic APC population contributes to an abnormal maturation of Bio-Breeding T lymphocytes which in turn predisposes animals to insulitis and diabetic disease.     

Characterization of purified leaf cytosolic pyruvate kinase from the C4 plant Cynodon dactylon

Cytosolic pyruvate kinase (EC 2.7.1.40) from leaves of the C₄ plant Cynodon dactylon (L.) Pers. was purified 56-fold to apparent homogeneity by polyethylene glycol fractionation and column chromatography including Q-Sepharose anion exchanger, ADP-Agarose and gel filtration. Nondenaturing PAGE of the final preparation resulted in a single protein band that co-migrated with the pyruvate kinase activity. Gel filtration and SDS-PAGE (± DTT) showed that this enzyme has a molecular mass of 200 kDa and is a homotetramer with a subunit molecular mass of 50 kDa. The subunits are not associated to each other with S-S bonds. The enzyme has a pH optimum of 6.2 and is heat stable. Typical Michaelis-Menten kinetics was obtained for both substrates, PEP and ADP, with K_m values of 64 and 235 μ M , respectively. Initial velocity studies indicated a sequential binding of the substrates to the enzyme.