Effects of roscovitine on the nuclear and cytoskeletal components of calf oocytes and their subsequent development

Roscovitine, a potent inhibitor of M-phase promoting factor kinase activity, was used to maintain calf oocytes at the germinal vesicle stage for a 24h culture period. Cumulus-oocyte complexes were first prematured for 24h in the presence of different levels of roscovitine (12.5, 25, 50 and 100 microM). Roscovitine was shown to block germinal vesicle breakdown in calf oocytes in a concentration dependent manner. Significantly greater inhibitory effect was observed at 50 and 100 microM with 64.6% and 63.2% oocytes being blocked in the germinal vesicle stage when compared to the control (0.0%) and the 12.5 microM (2.9%) and 25 microM (18.8%) groups. However, this inhibitory effect of roscovitine was fully reversible since a substantial number of the oocytes resumed meiosis and reached the metaphase II stage after a further 24h of culture in a permissive medium. Cleavage rates and blastocyst yields were not significantly different for oocytes cultured under 50 microM roscovitine inhibition compared to oocytes not subjected to prematuration culture (rates of 76.7% cleavage and 8.7% blastocysts for control oocytes compared to 69.8% and 6.3%, respectively, for oocytes pretreated with 50 microM roscovitine). The morphology of the meiotic spindle was typical of metaphase II in 75.8% and 82.1% of the oocytes reaching the metaphase II stage after pretreatment with 50 microM roscovitine compared to control, respectively. A normal distribution of actin filaments was observed in 97.0% and 98.2% of oocytes exposed to 50 microM roscovitine compared to control, respectively. These results demonstrate the feasibility of maintaining calf oocytes in artificial meiotic arrest without compromising their subsequent developmental competence.     

Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes

Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs' role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.     

Diacylglycerol kinase alpha regulates the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes

Fas ligand (FasL) mediates both apoptotic and inflammatory responses in the immune system. FasL function critically depends on the different forms of FasL; soluble Fas ligand lacking the transmembrane and cytoplasmic domains is a poor mediator of apoptosis, whereas full-length, membrane-associated FasL (mFasL) is pro-apoptotic. mFasL can be released from T lymphocytes, via the secretion of mFasL-bearing exosomes. mFasL in exosomes retains its activity in triggering Fas-dependent apoptosis, providing an alternative mechanism of cell death that does not necessarily imply cell-to-cell contact. Diacylglycerol kinase alpha (DGKalpha), a diacylglycerol (DAG)-consuming enzyme, is involved in the attenuation of DAG-derived responses initiated at the plasma membrane that lead to T lymphocyte activation. Here we studied the role of DGKalpha on activation-induced cell death on a T cell line and primary T lymphoblasts. The inhibition of DGKalpha increases the secretion of lethal exosomes bearing mFas ligand and subsequent apoptosis. On the contrary, the overactivation of the DGKalpha pathway inhibits exosome secretion and subsequent apoptosis. DGKalpha was found associated with the trans-Golgi network and late endosomal compartments. Our results support the hypothesis that the DGKalpha effect on apoptosis occurs via the regulation of the release of lethal exosomes by the exocytic pathway, and point out that the spatial orchestration of the different pools of DAG (plasma membrane and Golgi membranes) by DGKalpha is crucial for the control of cell activation and also for the regulation of the secretion of lethal exosomes, which in turn controls cell death.     

The incorporation of glucosamine into enterobacterial core lipopolysaccharide: two enzymatic steps are required

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to l-glycero-d-manno-heptopyranose II (ld-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of d-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to ld-HeppII.     

Differential effects of strength training leading to failure versus not to failure on hormonal responses, strength, and muscle power gains.

The purpose of this study was to examine the efficacy of 11 wk of resistance training to failure vs. nonfailure, followed by an identical 5-wk peaking period of maximal strength and power training for both groups as well as to examine the underlying physiological changes in basal circulating anabolic and catabolic hormones. Forty-two physically active men were matched and then randomly assigned to either a training to failure (RF; n = 14), nonfailure (NRF; n = 15), or control groups (C; n = 13). Muscular and power testing and blood draws to determine basal hormonal concentrations were conducted before the initiation of training (T0), after 6 wk of training (T1), after 11 wk of training (T2), and after 16 wk of training (T3). Both RF and NRF resulted in similar gains in 1-repetition maximum bench press (23 and 23%) and parallel squat (22 and 23%), muscle power output of the arm (27 and 28%) and leg extensor muscles (26 and 29%), and maximal number of repetitions performed during parallel squat (66 and 69%). RF group experienced larger gains in the maximal number of repetitions performed during the bench press. The peaking phase (T2 to T3) after NRF resulted in larger gains in muscle power output of the lower extremities, whereas after RF it resulted in larger gains in the maximal number of repetitions performed during the bench press. Strength training leading to RF resulted in reductions in resting concentrations of IGF-1 and elevations in IGFBP-3, whereas NRF resulted in reduced resting cortisol concentrations and an elevation in resting serum total testosterone concentration. This investigation demonstrated a potential beneficial stimulus of NRF for improving strength and power, especially during the subsequent peaking training period, whereas performing sets to failure resulted in greater gains in local muscular endurance. Elevation in IGFBP-3 after resistance training may have been compensatory to accommodate the reduction in IGF-1 to preserve IGF availability.     

Stock Transfers in Spanish Brown Trout Populations: A Long-term Assessment

Synopsis Stocking of fish from other populations has been commonly employed for enhancement of wild brown trout, Salmo trutta, populations in north Spain. Young hatchery reared brown trout of central European origin were introduced into some Asturian rivers every year since 1984. Based on variation at the isozyme locus LDH-C1* and at the microsatellite locus BFRO 002, two genetic markers race-specific in Salmo trutta, we detected introgression of foreign genomes into native gene pools in some Spanish trout populations where only pure native individuals were present 10 years ago. We strongly suggest development of alternative management policies for conservation of Spanish natural brown trout populations without endangering the traditional recreational fisheries. Jorge I. Izquierdo, Ana G. F. Castillo: These two authors contributed equally to the article.     

Selective hypoxia-cytotoxins based on vanadyl complexes with 3-aminoquinoxaline-2-carbonitrile-N1,N4-dioxide derivatives

A new vanadyl complex with the formula VO(L1)2, where L1=3-amino-6(7)-chloroquinoxaline-2-carbonitrile N(1), N(4)-dioxide, has been synthesized and characterized by elemental analyses, conductometry, fast atom bombardment mass spectroscopy (FAB-MS) and electronic, Fourier transform infrared (FTIR), Raman, nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies. Results were compared with those previously reported for analogous vanadium complexes with other 3-aminoquinoxaline-2-carbonitrile N1,N4-dioxide derivatives as ligands. As an effort to develop novel metal-based selective hypoxia-cytotoxins and to improve bioavailability and pharmacological and toxicological properties of aminoquinoxaline carbonitrile N-dioxides bioreductive prodrugs, the new complex and VO(L)2 complexes, with L=3-amino-6(7)-bromoquinoxaline-2-carbonitrile N1,N4-dioxide (L2) and 3-amino-6(7)-methylquinoxaline-2-carbonitrile N1,N4-dioxide (L3), were subjected to cytotoxic evaluation in V79 cells in hypoxic and aerobic conditions. The complexes resulted in vitro more potent cytotoxins than the free ligands (i.e. potencies P(VO(L1)2)=3.0, P(L1)=9.0 microM) and Tirapazamine (P=30.0 microM) and showed excellent selective cytotoxicity in hypoxia, being no cytotoxic in oxia. In addition, the solubility in hydrophilic solvents resulted significantly higher for the vanadyl complexes than for the free ligands. These results could be indicative that complexation of the quinoxaline-2-carbonitrile N1,N4-dioxide derivatives with vanadium could improve their bioavailability. In addition, a new aspect of the series has been investigated. A detailed comparison of the electrochemical behavior of the free ligands and the complexes has been performed searching for a correlation between reduction potentials of the complexes and their activities and hypoxia selectivities.     

Moderate volume of high relative training intensity produces greater strength gains compared with low and high volumes in competitive weightlifters

The purpose of this study was to examine the effect of 3 volumes of heavy resistance, average relative training intensity (expressed as a percentage of 1 repetition maximum that represented the absolute kilograms lifted divided by the number of repetitions performed) programs on maximal strength (1RM) in Snatch (Sn), Clean & Jerk (C&J), and Squat (Sq). Twenty-nine experienced (>3 years), trained junior weightlifters were randomly assigned into 1 of 3 groups: low-intensity group (LIG; n = 12), moderate-intensity group (MIG; n = 9), and high-intensity group (HIG; n = 8). All subjects trained for 10 weeks, 4-5 days a week, in a periodized routine using the same exercises and training volume (expressed as total number of repetitions performed at intensities equal to or greater than 60% of 1RM), but different programmed total repetitions at intensities of >90-100% of 1RM for the entire 10-week period: LIG (46 repetitions), MIG (93 repetitions), and HIG (184 repetitions). During the training period, MIG and LIG showed a significant increase (p < 0.01-0.05) for C&J (10.5% and 3% for MIG and LIG, respectively) and Sq (9.5% and 5.3% for MIG and LIG, respectively), whereas in HIG the increase took place only in Sq (6.9%, p < 0.05). A calculation of effect sizes revealed greater strength gains in the MIG than in HIG or LIG. There were no significant differences between LIG and HIG training volume-induced strength gains. All the subjects in HIG were unable to fully accomplish the repetitions programmed at relative intensities greater than 90% of 1RM. The present results indicate that short-term resistance training using moderate volumes of high relative intensity tended to produce higher enhancements in weightlifting performance compared with low and high volumes of high relative training intensities of equal total volume in experienced, trained young weightlifters. Therefore, for the present population of weightlifters, it may be beneficial to use the MIG training protocol to improve the weightlifting program at least in a short-term (10 weeks) cycle of training.