The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint

In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. Here, we show that, in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/C(Cdh1), with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin, an activator of the DNA-damage checkpoint, and Wee1, an inhibitor of cell-cycle progression, and allows an efficient G2 checkpoint. As a by-product of APC/C(Cdh1) reactivation in DNA-damaged G2 cells, Claspin, which we show to be an APC/C(Cdh1) substrate in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA-damage-response checkpoint.     

Identification and partial characterization of alpha-1,4-glucosidase activity in equine epididymal fluid

The expression of alpha-1,4-glucosidase activity was fluorometrically and electrophoretically assessed in the epididymal fluid and seminal plasma of stallions. alpha-Glucosidase specific activity in the epididymis increased significantly from the proximal caput to the cauda. Stallion epididymal glucosidase maintained activity in a wide range of pH, with two distinct peaks (around pH 4.0 and 6.0, respectively). Enzyme activities at different pH, inhibition assays with sodium dodecyl sulfate (SDS) and maltotriose (MTT, selective inhibitors of alpha-glucosidases "acidic" and "neutral" isoforms, described in other tissues) and the electrophoretic analysis in native and native/SDS-PAGE conditions, indicated that stallion epididymal glucosidase was due to two catalytically active forms. These forms, analyzed by non-denaturing electrophoresis, exhibited different electrophoretic mobility and molecular weight. Samples from the proximal caput of the epididymis were rich in Form II or "neutral" form, whereas the "acid" or Form I seemed to be predominate in the cauda epididymal region. At physiological pH, Form II was predominant in the seminal plasma. The physiological role(s) of these forms is uncertain, but based on their ability to hydrolyze glucosidic linkage, they probably are involved in degradation/modifications of epididymal fluid and/or spermatozoa glycoconjugates, thereby participating in plasma membrane remodeling associated with sperm maturation.     

Red wine polyphenols influence carcinogenesis, intestinal microflora, oxidative damage and gene expression profiles of colonic mucosa in F344 rats

Polyphenols from tea and other beverages such as red wine have been regarded with interest as possible chemopreventive agents against cancer. Here we report that red wine polyphenols (50 mg/kg) administered with the diet to F344 rats for 16 weeks inhibited colon carcinogenesis induced by azoxymethane (AOM, 7.4 mg/kg, total dose 74 mg/kg) or dimethylhydrazine (DMH, 30 mg/kg, total dose, 300 mg/kg). Polyphenol-treated animals had a consistently lower tumour yield compared to controls. In polyphenol-treated rats, the main bacterial strains in the faeces at sacrifice were Bacteroides, Lactobacillus and Bifidobacterium spp., whereas microorganisms predominantly identified in control-fed rats were Bacteroides, Clostridium and Propionibacterium spp. Wine polyphenols (57 mg/kg for 10 days, by gavage), administered to rats not treated with carcinogens, produced a significant decrease in the basal level of DNA oxidative damage of the colon mucosa as measured with the comet assay (average pyrimidine oxidation was reduced by 62% and purine oxidation by 57%, p<0.05). To further explore the molecular effects of wine polyphenols we used the microarray technology to study gene expression profiles: rats were treated with 50 mg/kg wine polyphenols for 14 days, mixed in the diet. Global expression analysis of 5707 genes revealed an extensive down-regulation of genes involved in a wide range of physiological functions, such as metabolism, transport, signal transduction and intercellular signalling. By analysing metabolic pathways with the GenMAPP software program we observed that two major regulatory pathways were down-regulated in the colon mucosa of polyphenols-treated rats: inflammatory response and steroid metabolism. We also found a down-regulation of many genes regulating cell surface antigens, metabolic enzymes and cellular response to oxidative stress. In conclusion, reduction of oxidative damage, modulation of colonic flora and variation in gene expression may all concur in the modulation of intestinal function and carcinogenesis by wine polyphenols.     

Meat quality of the longissimus lumborum muscle of Casertana and Large White pigs: metabolomics and proteomics intertwined

Longissimus lumborum muscles from high fat-deposing Casertana and lean meat Large White pigs were assayed for meat quality parameters, including early and ultimate post mortem pH, water holding capacity and Minolta L*a*b*values. These parameters were correlated to results from differential proteomic and targeted metabolomic analyses. Higher levels of glycolytic enzymes and lactate accumulation were related to slow pH drop in Casertana pigs, albeit not to rapid pH lowering in LW counterparts. On the other hand, the individuation of pyruvate kinaseM1 and tropomyosin levels in LW were related to water holding capacity and Minolta values at 24h after slaughter. Bioinformatic analyses strengthened the correlation between over-expression of structural proteins in LW and more accentuated growth aptitude in this breed. Conversely, enzymes taking part into branching glycolytic reactions, such as glycerol 3-phosphate and creatine kinase M, were related to accentuated lipogenesis and slower albeit prolonged glycolytic rate in Casertana, respectively. Breed-specific differences at the protein level were not only related to growth performances and fat accumulation tendency in vivo, but they also affected post mortem performances through a direct influence on the forcedly anaerobic behavior of pig muscles after slaughter.     

A microsatellite linkage map of the European sea bass Dicentrarchus labrax L

A genetic linkage map of the European sea bass (Dicentrarchus labrax) was constructed from 174 microsatellite markers, including 145 new markers reported in this study. The mapping panel was derived from farmed sea bass from the North Adriatic Sea and consisted of a single family including both parents and 50 full-sib progeny (biparental diploids). A total of 162 microsatellites were mapped in 25 linkage groups. Eleven loci represent type I (coding) markers; 2 loci are located within the peptide Y (linkage group 1) and cytochrome P450 aromatase (linkage group 6) genes. The sex-averaged map spans 814.5 cM of the sea bass genome. The female map covers 905.9 cM, whereas the male map covers only 567.4 cM. The constructed map represents the first linkage map of European sea bass, one of the most important aquaculture species in Europe.     

Association of tyrosine protein kinase activity with mitochondria in human fibroblasts

A tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from human fibroblasts. By enzymatic and sedimentation analysis this activity appeared to be localized in the mitochondrial outer membrane. Mitochondrial tyrosine phosphorylation was strictly dependent on the presence of Mn²⁺ ions. An inverse relationship between cell proliferation and mitochondrial protein phosphorylation on tyrosine residues has been found: a marked increase in the mitochondrial tyrosine kinase activity occurred when a significant reduction in the growth rate followed serum step-down. In mitochondria purified from resting cells, a protein band with apparent molecular weight of 50 kd appeared to be phosphorylated on tyrosine.     

Karyotype morphology in Hyperiella dilatata Stebbing 1888 (Amphipoda: Hyperiidae) from the Ross Sea (Antarctica)

Summary The chromosome complement and some karyological features were investigated in the pelagic amphipod Hyperiella dilatata Stebbing 1888 from the Ross Sea (Antarctica). The diploid karyotype consists of 48 metacentric and 10 submetacentric elements (2n = 58). The presence of secondary constrictions and supernumerary chromosomes is described. Available chromosome numbers of Hyperiidea exhibit a wide range of distribution, among which Hyperiella dilatata is the closest to the modal number of other amphipods.     

The hemocyanin of the shamefaced crab Calappa granulata: structural-functional characterization

Arthropod hemocyanins (Hcs) transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Calappa granulata Hc is found as a mixture of dodecamers (95%) and hexamers (5%). Removal of calcium ions and alkaline pH induce an incomplete partially reversible dissociation of dodecameric Hc. Two-dimensional electrophoretic pattern of dissociated Hc indicated a large heterogeneity in Hc subunit: most differences are likely to be explained by post-translational modifications. Dodecameric Hc showed a large Bohr effect (Deltalog P50/DeltapH = -0.95) and a normal cooperativity (h50 values = 2.7 +/- 0.2) in the presence of 10 mM CaCl2. The hexameric molecule displayed lower Bohr effect and cooperativity than the dodecamer. Lactate effect on the oxygen affinity (Deltalog P50 = 0.55) and the increase of lactate concentrations in animals kept in emersion were related to the increased oxygen requirements that occur during hypoxia in vivo. Calcium affects oxygen affinity only at high concentrations: this Hc appeared to lack the calcium high-affinity binding sites found in other species. The effect of temperature on both oxygen affinity and cooperativity was measured in the absence and presence of 10 mM lactate, allowing calculation of the exothermic contribution of lactate binding (DeltaH = -25 kJ mol(-1)).