Prevalence of the C282Y, H63D, and S65C mutations of the HFE gene in 1,146 newborns from a region of Northern Spain

In Spain, 85% of patients with genetic hemochromatosis (GH) are homozygous for the C282Y mutation of the HFE gene. H63D and S65C mutations of HFE may also play some role in the disease. The aim of this study was to establish the prevalence of C282Y, H63D, and S65C mutations of the HFE gene in newborns in Catalonia, Spain. One thousand one hundred forty-six newborn screening cards were selected randomly. DNA from these cards was extracted and HFE mutations were analyzed with the LightCycler equipment (Roche Diagnostics Gmbh, Mannheim, Germany). Sufficient DNA sample was obtained to screen for the three mutations in 1,043 cases (91%). The allelic frequencies of C282Y, H63D, and S65C mutations were 0.03 (IC 95% 0.022-0.037), 0.2 (IC 95% 0.19-0.22), and 0.01 (95% confidence interval [CI] 0.006-0.015), respectively. The frequency of C282Y homozygous newborns was 0.001 (95% CI 0.0005-0.0014). The frequencies of newborns doubly heterozygous for C282Y/H63D and C282Y/S65C were 0.01 (95% CI 0.005-0.02) and 0.002 (95% CI 0.0002-0.01), respectively. The allelic frequency of C282Y mutation is similar to that observed in Southern France, in the Czech Republic and in some areas of Italy. The allelic frequency of H63D mutation in Catalonia is the highest reported to date. Nevertheless, S65C is infrequent. These data should be kept in mind when designing hemochromatosis genotypic screening programs in Catalonia.     

Changes in lipid class and fatty acid composition during development in white seabream (Diplodus sargus) eggs and larvae

To establish the changes which occur during embryogenesis and early larvae development, eggs, yolk-sac larvae (one day old larvae) and absorbed yolk-sac larvae (three day old larvae) of white sea bream were examined for lipid class and fatty acid composition. The development was characterized by a decrease in all lipid classes with the exception of phosphatidylserine (PS) and fatty free acids (FFA) which increased, and sphingomyelin (SM) which remained unchanged. The changes observed in lipid class content and the decrease in fatty acids in total lipid (TL) reflect the utilization and mobilization of lipids during both embryogenesis and early larvae development. Fluctuations in the relative composition of fatty acids in phosphatidylcholine (PC) during development suggest a selective bulk uptake and catabolism of fatty acids in this lipid class. Unlike PC, catabolism of triacylglycerol (TG) fatty acid appears to be non-selective. During development, the decrease in levels of polyunsaturated fatty acids (PUFA) eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA) in total lipid denotes their utilization as energy substrate by Diplodus sargus larvae.     

Lipid and fatty acid composition of muscle and liver from wild and captive mature female broodstocks of white seabream, Diplodus sargus

Total lipids (TL), lipid classes, and their associated fatty acids from muscle and liver of captive and wild mature female broodstocks were investigated in order to estimate the fatty acid requirements of white seabream (Diplodus sargus). The results showed that the percentage of triacylglycerol was higher in liver and muscle of captive fish than in wild fish. The distribution of phospholipid classes in liver and muscle of both fish groups was similar, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol being the predominant lipid classes. The general pattern of fatty acid distribution in total lipid of liver and muscle from captive and wild fish was similar. However, the relative percentage of specific fatty acids differed in captive and wild fish. The most noteworthy difference was the lower proportion of arachidonic acid (20:4n-6, AA) and the higher proportion of eicosapentaenoic acid (20:5n-3, EPA) in liver and muscle of captive fish with respect to those of wild fish. The proportion of docosahexaenoic acid (22:6n-3, DHA) did not differ between the two fish groups. The differences in EPA and AA proportions between captive and wild fish implied that captive fish presented a higher EPA/AA ratio and a lower DHA/EPA ratio than wild fish. In general terms, in both liver and muscle, the differences in fatty acid composition observed for TL were extended to all lipid classes. The results suggest that the different AA, EPA and DHA proportions in liver and muscle between captive and wild broodstocks are attributed to different levels of these fatty acids in broodstock diets.     

Protective role of ETA endothelin receptors during the acute phase of Trypanosoma cruzi infection in rats

Chagas' disease, caused by Trypanosoma cruzi, has an acute phase characterized by blood-circulating trypomastigotes and amastigote proliferation in several cell types, especially muscle cells. In the chronic phase, around 70% of infected people are asymptomatic (latent form). The remainder develop chagasic cardiomyopathy and/or digestive syndromes. There is evidence for aggravation of the chronic cardiac pathology by endothelin-mediated vasoconstriction. Holtzman rats have proven to be a good model for Chagas' disease acute phase and latent chronic phase. Now, we investigate the effects of prolonged treatment with an endothelin ET(A) receptor antagonist, BSF 461314, during the acute phase on parasitemia, coronary flow, tissue parasitism and the inflammatory process. Using isolated heart in Langendorff's preparation, endothelial dysfunction was observed only in non-treated infected animals. Histoquantitative analyses carried out in heart and diaphragm showed higher tissue parasitism and/or inflammatory process in BSF 461314-treated animals. Our data indicate that endothelin ET(A) receptors contribute to the initial mechanisms of parasite control. Impairment of the endothelium-dependent vasodilatation favors hazardous effects. However, blocking endothelin ET(A) receptors can prevent the latter.     

Glucose ingestion attenuates the exercise-induced increase in circulating heat shock protein 72 and heat shock protein 60 in humans

Heat shock protein (Hsp) 72 is a cytosolic stress protein that is highly inducible by several factors including exercise. Hsp60 is primarily mitochondrial in cellular location, plays a key role in the intracellular protein translocation and cytoprotection, is increased in skeletal muscle by exercise, and is found in the peripheral circulation of healthy humans. Glucose deprivation increases Hsp72 in cultured cells, whereas reduced glycogen availability elevates Hsp72 in contracting human skeletal muscle. To determine whether maintained blood glucose during exercise attenuates the exercise-induced increase in intramuscular and circulating Hsp72 and Hsp60, 6 males performed 120 minutes of semirecumbent cycling at approximately 65% maximal oxygen uptake on 2 occasions while ingesting either a 6.4% glucose (GLU) or sweet placebo (CON) beverage throughout exercise. Muscle biopsies, obtained before and immediately after exercise, were analyzed for Hsp72 and Hsp60 protein expression. Blood samples were simultaneously obtained from a brachial artery, a femoral vein, and the hepatic vein before and during exercise for the analysis of serum Hsp72 and Hsp60. Leg and hepatosplanchnic blood flow were measured to determine Hsp72-Hsp60 flux across these tissue beds. Neither exercise nor glucose ingestion affected the Hsp72 or Hsp60 protein expression in, or their release from, contracting skeletal muscle. Arterial serum Hsp72 increased (P < 0.05) throughout exercise in both trials but was attenuated (P < 0.05) in GLU. This may have been in part because of the increased (P < 0.05) hepatosplanchnic Hsp72 release in CON, being totally abolished (P < 0.05) in GLU. Serum Hsp60 increased (P < 0.05) after 60 minutes of exercise in CON before returning to resting levels at 120 minutes. In contrast, no exercise-induced increase in serum Hsp60 was observed in GLU. We detected neither hepatosplanchnic nor contracting limb Hsp60 release in either trial. In conclusion, maintaining glucose availability during exercise attenuates the circulating Hsp response in healthy humans.     

A hint to search for metalloproteins in gene banks

MOTIVATION: With the advent of genome sequencing, a huge database of protein primary sequences has been accumulating. In parallel, a number of tools to investigate and expand upon this information, e.g. reconstructing and building relationships between protein families and superfamilies, have been developed. Metalloproteins are proteins capable of binding one or more metal ions, which are required for their biological function or for regulation of their activities or for structural purposes. Sometimes, metal binding can be observed in vitro but not be physiologically relevant. At present, there is a lack of specific tools to address the matter of the identification of metalloproteins in databases of gene sequences. RESULTS: In the present work, an approach exploiting metal-binding patterns (MBPs) of metalloproteins present in the Protein Data Bank to search gene banks for new metalloproteins is presented and applied to copper proteins. Nearly 100 different MBPs have been identified and then used for subsequent applications. The ensemble of sequences of the whole PDB is used to assess the potentiality and limits of the method and to identify levels of confidence for the predictions output by the search. It appears that copper-binding capabilities are identified with a confidence >90% when the percentage of identical amino acids aligned around the MBP by PHI-BLAST is at least 20% with respect to the entire protein domain length. If this percentage is between 10% and 20%, the level of confidence is approximately 50%. Application of the methodology to the entire genome sequences of Pyrococcus furiosus, Escherichia coli, Drosophila melanogaster and Homo sapiens suggests some differentiation between prokaryotes and eukaryotes. SUPPLEMENTARY INFORMATION: A table reporting statistics on the MBP identified; a list of all hits retrieved for the four organisms considered; a figure showing the number of hits for the four organisms as a function of I(d)(Global).     

Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies

Genetic transformation has been achieved for several citrus genotypes. However, regeneration of escapes at high frequency is a major problem, making the available procedures rather inefficient. Attempts to improve selection by increasing the concentration of kanamycin, used as the selective agent, or substituting it by geneticin have been unsuccessful. Here, we have critically assessed the actual frequency and origin of escapes in citrus by using visual screening with -glucuronidase (gusA) and green fluorescent protein (gfp) markers, by studying the persistence of engineered Agrobacterium in the explants, and by characterising through Southern blot analysis all the regenerants obtained under kanamycin selection. Our results show that inefficient selection could be attributed to the protection of the non-transformed cells from the selective agent by the surrounding transformed cells, and to the persistence of kanamycin-resistance Agrobacterium in explant tissues over long periods of time after co-cultivation. This also explained the high frequency (12%) of chimeric shoots that were commonly recovered. High frequency regeneration of chimeras that resulted from the fusion of different transformation events is reported for the first time. On the other hand, molecular analysis of all the regenerants reveals that transformation frequency is underestimated when based on the expression of a screenable marker gene, and that low expressors and silenced lines could account for at least 25% of those plants considered escapes based on selectable and screenable marker analysis. Consequences of these results at the practical level are also discussed.     

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum (L.) Thell

A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the high-density Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.     

Modulation of adenyl cyclase activity in the gills of Tapes philippinarum

Adenyl cyclase (AC) plays a pivotal role in cell signaling. The AC system of bivalves has received little attention so far, and our study has been addressed to the characterization of AC properties in the gills of T. philippinarum. The enzyme showed a Km value of 0.77 mM for ATP in the presence of 5 mM Mg2+; in the absence of agonists, it was poorly affected by GTP, while it was stimulated by GTPgammaS and GppNHp up to 14-fold and 4-fold, respectively. Similarly to other invertebrates, the enzyme activity was scarcely stimulated by forskolin. The receptor agonist serotonin (5-HT) significantly stimulated the AC activity, and the pharmacological profile of the 5-HT receptor/s was as follows: (+)butaclamol > dihydroergocryptine > methysergide > prazosin > yohimbine. The AC activity was assessed in vitro in the presence of tributyltin chloride and HgCl2, which reduced the AC activity only at the highest dose tested (10-100 microM). Our data indicate the presence of a membrane-bound AC in gill membranes of T. philippinarum, coupled to Gs proteins and to a specific class of 5-HT receptors. Such receptors show a pharmacological profile slightly different from that reported for 5-HT invertebrate receptors cloned so far.