Thymidylate synthase with a C-terminal deletion catalyzes partial reactions but is unable to catalyze thymidylate formation.

The V316Am mutant of Lactobacillus casei thymidylate synthase has a single amino acid deletion at the C-terminus which abolishes catalysis of dTMP formation. However, V316Am catalyzes two partial reactions which require covalent catalysis: a CH2H4folate-dependent exchange of the 5-hydrogen of dUMP for protons in water and a thiol-dependent dehalogenation of 5-bromo- and 5-iodo-dUMP. These reactions proceed with kcat and Km values similar to those of the wild-type TS-catalyzed reactions. dUMP, dTMP, and FdUMP are competitive inhibitors of the debromination reaction with Ki values similar to those obtained with wild-type enzyme. These results show that removal of the terminal valine does not alter the ability of the enzyme to bind to or form covalent bonds with nucleotide ligands. V316Am also forms a covalent ternary complex with FdUMP and CH2H4folate. However, the affinity of the TS-FdUMP complex for the cofactor is reduced, and the rate of covalent ternary complex formation and its stability are significantly lower than with wild-type TS. These results allow us to place the major defects of the mutation on steps that occur subsequent to initial CH2H4folate binding.     

Rapid radiation and cryptic speciation in mediterranean triplefin blennies (Pisces: Tripterygiidae) combining multiple genes

The genus Tripterygion is the unique genus of the family Tripterygiidae in the Mediterranean Sea and in the northeastern Atlantic coast. Three species and four subspecies had been described: Tripterygion tripteronotus and Tripterygion melanurus (T. m. melanurus and T. m. minor) are endemic of the Mediterranean, and T. delaisi (T. d. delaisi and T. d. xanthosoma) is found in both areas. We used five different genes (12S, 16S, tRNA-val, COI, and 18S) to elucidate their taxonomy status and their phylogenetic relationships. We employed different phylogenetic reconstructions that yielded different tree topologies. This discrepancy may be caused by the speciation process making difficult the reconstruction of a highly supported tree. All pair comparisons between these three species showed the same genetic divergence indicating that the speciation process could have been resolved by a rapid radiation event after the Messinian Salinity Crisis (5.2Mya) leading to a trichotomy. Our molecular data revealed two clearly supported clades within T. tripteronotus, whose divergence largely exceeded that found between other fish species, consequently these two groups should be considered two cryptic species diverging 2.75-3.32Mya along the Pliocene glaciations. On the contrary, none of the genes studied supported the existence of two subspecies of T. melanurus. Finally, the two subspecies of T. delaisi were validated and probably originated during the Quaternary climatic fluctuations (1.10-1.23Mya), however their distribution ranges should be redefined.     

Interleukin-15 stimulates adiponectin secretion by 3T3-L1 adipocytes: evidence for a skeletal muscle-to-fat signaling pathway

Interleukin-15 (IL-15) is a cytokine which is highly expressed in skeletal muscle tissue, and which has anabolic effects on skeletal muscle protein dynamics both in vivo and in vitro. Additionally, administration of IL-15 to rats and mice inhibits white adipose tissue deposition. To determine if the action of IL-15 on adipose tissue is direct, the capacity of cultured murine 3T3-L1 preadipocytes and adipocytes to respond to IL-15 was examined. IL-15 administration inhibited lipid accumulation in differentiating 3T3-L1 preadipocytes, and stimulated secretion of the adipocyte-specific hormone adiponectin by differentiated 3T3-L1 adipocytes. The latter observation constitutes the first report of a cytokine or growth factor which stimulates adiponectin production. IL-15 mRNA expression by cultured 3T3-L1 adipogenic cells and C2C12 murine skeletal myogenic cells was also examined. Quantitative real-time PCR indicated IL-15 mRNA was expressed by C2C12 skeletal myogenic cells, and was upregulated more than 10-fold in differentiated skeletal myotubes compared to undifferentiated myoblasts. In contrast, 3T3-L1 cells expressed little or no IL-15 mRNA at either the undifferentiated preadipocyte or differentiated adipocyte stages. These findings provide support for the hypothesis that IL-15 functions in a muscle-to-fat endocrine axis which modulates fat:lean body composition and insulin sensitivity.     

Relation between oxidative stress markers and antioxidant endogenous defences during exhaustive exercise

Hydrogen peroxide (H₂O₂) could induce oxidative damage at long distance from its generation site and it is also an important signalling molecule that induces some genes related to oxidative stress. Our objective was to study the plasma and blood cells capability to detoxify H₂O₂ after intense exercise and its correlation with oxidative damage. Blood samples were taken from nine professional cycling, participating in a mountain stage, under basal conditions and 3 h after the competition. Catalase and glutathione peroxidase activities decreased (40 and 50% respectively) in neutrophils after the cycling stage, while glutathione peroxidase increased (87%) in lymphocytes. Catalase protein levels and catalase specific activity maintained basal values after the stage in plasma. Catalase protein levels decreased (48%) in neutrophils and its specific activity increased up to plasma values after exercise. Myeloperoxidase (MPO) increased (39%) in neutrophils after the cycling stage. Exercise-induced hemolysis and lymphopenia inversely correlated with cellular markers of oxidative stress. Plasma malondialdehyde (MDA) directly correlated with neutrophil MPO activity and erythrocytes MDA. Intense exercise induces oxidative damage in blood cells as erythrocytes and lymphocytes, but not in neutrophils.     

Abdominal zygomycosis in a bitch due to Absidia corymbifera

We report a case of abdominal zygomicosis in a Doberman bitch. Clinical signs consisted of urinary incontinence and hard abdominal masses detected by palpation. The masses were surgically removed by exploratory laparatomy and had a tumoral-like appearance. A granulomatous reaction containing coarse and non septate hyphae was the main histological finding. Direct microscopic examination revealed the presence of fungal structures. On Sabouraud honey agar the fungus developed fluffy, greyish white colonies that were identified as Absidia corymbifera on the basis of their macro and microscopic morphology.     

In vitro susceptibility of fungal and yeast clinical isolates to itraconazole and voriconazole

The interest on the in vitro susceptibility to itraconazole has recently increased due the availability of the intravenous formulation. In this study, comparative MICs of this antifungal with voriconazole were carried out in 62 clinical isolates of filamentous fungi and 100 yeasts isolates using the NCCLS microbroth methods described in M38-A and M27-A2 documents. A MIC90 of 0.125 micrograms per ml was observed for itraconazole and voriconazole against Aspergillus fumigatus. Higher susceptibility to itraconazole was found for the filamentous form of Sporotrhix schenckii (p = 0.001). Voriconazole was more effective against Scedosporium apiospermium while Scedosporium prolificans isolates were resistant to both azoles. Some isolates of Rhizopus stolonifer were susceptible to itraconazole and resistant to voriconazole, but without statistical significance. Susceptibility of nine species of Candida was similar for both triazoles used in this study. However, Candida glabrata was more susceptible to voriconazole. Some fluconazole-resistant Candida albicans isolates were susceptible to itraconazole and / or voriconazole. Cryptococcus neoformans was more susceptible to itraconazole than to voriconazole. Itraconazole and voriconazole showed very close in vitro activity against the tested fungal isolated, except against S. schenckii. In spite of this, there were some differences in susceptibility among isolates within the same fungal species.     

In vitro activity of the echinocandins. How should it be evaluated?

Echinocandins are a novel class of antifungal drugs. They have good activity against Candida spp and Aspergillus spp. Their low selective toxicity allows their administration at high doses with few secondary side effects. We have reviewed the available data on the endpoints for these drugs in their in vitro susceptibility testing on yeasts and moulds. The microdilution broth method is the most commonly used technique and MIC-1 (80% of growth inhibition) seems to be the most reliable endpoint when yeasts are tested. This endpoint also seems to be the most appropriate for the different drugs when they are combined with echinocandins using the checkerboard method for testing yeasts. By contrast, in the case of moulds, the minimum effective concentration (MEC) correlates better with the in vivo activity than the MIC when echinocandins are tested, and when these drugs are combined with other antifungals, MIC-2 (50% of growth inhibition) seems the most appropriate endpoint. Criteria based on drug pharmacodynamics is the most useful to define the echinocandin endpoints that best correlate with their in vivo efficacy.     

Isoprenoids: an evolutionary pool for photoprotection

Plants have evolved several mechanisms for getting rid of excess energy in photosynthetic membranes, some of which involve isoprenoid compounds. In all photosynthetic organisms, the carotenoids beta-carotene and zeaxanthin, and tocopherols serve an important photoprotective role, either by dissipating excess excitation energy as heat or by scavenging reactive oxygen species (ROS) and suppressing lipid peroxidation. Isoprene and some monoterpenes, diterpenes and other carotenoids also occur in some plant lineages. Compelling evidence indicates that these non-ubiquitous isoprenoids might be particularly relevant in adapting plants to adverse climatic conditions by serving as additional and/or alternative protection mechanisms.