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.     

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.     

In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of some Ebselen analogues

Four analogues of Ebselen were synthesized and their glutathione peroxidase activity and antioxidant property evaluated and compared to Ebselen. Among the studied compounds, only diselenide [3] exhibited both glutathione peroxidase activity and radical-scavenging capability. Compounds [3] and [4] showed a strong inhibitory effect (53% and 43%, respectively) on the lipid peroxidation of linoleic acid compared to Ebselen and selenide derivatives ([1] and [2]) which were less active (28%, 26% and 18% inhibition, respectively). A concentration-dependent inhibitory effect was also found in the model of the formation of ABTS*+ radical cation: 65% and 89% inhibition for compound [3] at 10(-4) M and 5 x 10(-5) M, respectively, and 68% and 90% for compound [4], compared to 14% and 52% inhibition for Ebselen and the diselenides [1] and [2] (29%, 46% and 45%, 68%, respectively). By EPR spin trapping technique, the following inhibitory profile of the Ebselen analogues was observed towards the formation of thiyl radicals: Ebselen = [3]>[1]>[2]>[4]. Studies with compound [3] are in progress on oxidative stress cell models.     

Active and inactive ecto-5'-nucleotidase variants in liver of control and dystrophic Lama2dy mice

The ecto-5'-nucleotidase (eNT) activity and the eNT protein content in liver of normal and merosin-deficient dystrophic Lama2dy mice were studied. After the solubilization procedure, the eNT activity in the final extract was 9.2+/-2.5U/mg (nmol of phosphate released from AMP per min and per mg protein) in normal liver, and it rose to 16.1+/-3.9U/mg (P=0.005) in dystrophic liver. The increase of activity was less pronounced in Lama2dy liver (1.7-fold) than the one reported in muscle (four-fold), which probably reflects the lower content of merosin in liver. Similarly to muscle, liver contained active and inactive eNT, as demonstrated by the higher level of immunoreactive protein in normal than in dystrophic liver in Western blots performed with samples containing the same units of eNT activity. PNGase F digestion decreased the size of liver and muscle eNT from 72 and 69kDa, to 63 and 60kDa. Oligoglycan cleavage did not alter eNT activity or the sedimentation coefficient, revealing that oligosaccharides are not required for catalysis or for maintaining the dimeric structure. The eNT protein content in samples of normal liver decreased by 55 or 80% after the trypsinolysis of native or deglycosylated enzyme, but the activity did not change. Such a high proportion of inactive eNT is unlikely to come from aged enzyme, which suggests the involvement of inactive enzyme in non-catalytic actions.     

Intraspecific diversity of the marine fish pathogen Tenacibaculum maritimum as determined by randomly amplified polymorphic DNA-PCR

AIM: The aim of the present study was to evaluate the intraspecific genetic variability within Tenacibaculum maritimum strains isolated from different species of marine fish. METHODS AND RESULTS: Twenty-nine strains isolated from five different fish species and three reference strains were characterized by randomly amplified polymorphic DNA (RAPD) method. Cluster analysis of RAPD-PCR profiles showed that the strains, regardless of the oligonucleotide primer employed (P2 and P6), were separated into two main groups that strongly correlated with the host species and/or O-serotypes described for this pathogen. One group composed all strains isolated from sole (Solea senegalensis and S. solea) and gilthead seabream (Sparus aurata), and the other compiled the T. maritimum isolates from yellowtail (Seriola quinqueradiata), Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus). An important exception was observed in the RAPD patterns of the reference strains, which were included in different genetic groups depending on the primer employed. CONCLUSIONS: The results obtained demonstrated genetic variability within the T. maritimum isolated from different marine fish. Such genetic variability proved to be strongly associated with the host and/or serogroups described for this pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD analysis constitutes a valuable molecular technique for epidemiological studies of T. maritimum. Interestingly, this is the first report of intraspecific differentiation and characterization of T. maritimum strains isolated from cultured fish.     

SQUARE--determining reliable regions in sequence alignments

The Server for Quick Alignment Reliability Evaluation (SQUARE) is a Web-based version of the method we developed to predict regions of reliably aligned residues in sequence alignments. Given an alignment between a query sequence and a sequence of known structure, SQUARE is able to predict which residues are reliably aligned. The server accesses a database of profiles of sequences of known three-dimensional structures in order to calculate the scores for each residue in the alignment. SQUARE produces a graphical output of the residue profile-derived alignment scores along with an indication of the reliability of the alignment. In addition, the scores can be compared against template secondary structure, conserved residues and important sites.     

Functional assessment of cryopreserved pig aortas for pharmaceutical research

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached.After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.