Influence of cortisol on osmoregulation and energy metabolism in gilthead seabream Sparus aurata

Gilthead seabream Sparus aurata were injected intraperitoneally with slow-release implants of coconut oil alone or containing cortisol (50 and 100 microg x g(-1) body weight), and sampled after two, five, and seven days to assess the simultaneous effects of cortisol on both osmoregulation and energy metabolism. Plasma cortisol levels increased in treated fish to 50-70 ng x ml(-1). An enhanced hypoosmoregulatory capacity of cortisol-implanted fish is suggested by the increase observed in gill Na+, K+-ATPase activity, and the decrease observed in plasma ion concentration (Na+ and Cl-) and osmolality. Cortisol also elicited metabolic changes in liver (increased gluconeogenic potential suggested by elevated FBPase activity, and decreased potential of glycolysis and pentose-phosphate shunt, suggested by the decreased activities of both PK and G6PDH) supporting changes in levels of plasma metabolites suitable for use in other tissues. Thus in this study, we demonstrate for the first time in fish that cortisol treatments elicit changes in the use of exogenous glucose in gills (decreased HK activity) and an increased glycolytic and glycogenic potential in brain (increased GPase, PK and PFK activities).     

Myostatin is an inhibitor of myogenic differentiation

Myostatin (MSTN), atransforming growth factor (TGF)- superfamily member, has been shownto negatively regulate muscle growth by inhibiting muscle precursorcell proliferation. Here, we stably transfectedC₂C₁₂ cells with mouse MSTN cDNA to investigateits possible role in myoblast differentiation. We found that MSTN cDNAoverexpression reversibly inhibits the myogenic process by downregulating mRNA levels of the muscle regulatory factors myoD andmyogenin, as well as the activity of their downstream target creatinekinase. Taking into consideration that MSTN expression duringdevelopment is restricted to muscle, our results suggest that MSTNprobably regulates myogenic differentiation by an autocrine mechanism.

     

Phylogeography of chamois (Rupicapra spp.) inferred from microsatellites

Evolutionary relationships among populations of chamois (Rupicapra spp.) across their current range from the Caucasus to the Cantabrian Mountains were investigated. The allelic variation in 23 microsatellite loci was assessed in eight geographical populations, recognised as subspecies of the two closely related species R. pyrenaica and R. rupicapra. Analysis of variance in allele frequencies (Fst, statistics) and in repeat numbers (Rst, statistics) showed these data to be highly structured. Two genetic distances between pairs of populations, Ds and (deltamu)(2), were computed and phylogenetic trees were constructed. Similar patterns were produced by the different statistics. All trees indicate a deep divergence between the two recognised species, which is compatible with archaeological data that place their split in the Riss-Würm interglacial period. Genetic distances between pairs of populations are highly correlated with geographical distance. This suggests that the history of the genus during Pleistocene glacial-interglacial periods was dominated by expansions and contractions within limited geographic regions, leading to alternate contact and isolation of contiguous populations. In addition, the alpine barrier has played a substantial role in West-East differentiation.     

Identification and cloning of a type III polyketide synthase required for diffusible pigment biosynthesis in Saccharopolyspora erythraea

The soluble, diffusible red-brown pigment produced by a Saccharopolyspora erythraea "red variant" has been shown to contain glycosylated and polymerized derivatives of 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). Flaviolin is a spontaneous oxidation product of 1,3,6,8-tetrahydroxynaphthalene (THN), which is biosynthesized in bacteria by a chalcone synthase-like (CS-like) type III polyketide synthase (PKS). A fragment of the gene responsible for THN biosynthesis in S. erythraea E_8-7 was amplified by polymerase chain reaction (PCR) using degenerate primers based on conserved regions of known plant CS and bacterial CS-like genes. From the isolated fragment, a suicide vector was prepared, which was subsequently used to disrupt the red-brown pigment-producing (rpp) locus in S. erythraea, generating a mutant that displayed an albino phenotype. Chromosomal DNA from the albino mutant was subsequently used in a vector-recapture protocol to isolate a plasmid that contained an insert spanning the entire rpp locus. Sequencing of the insert revealed that the disrupted open reading frame (ORF) encodes a CS-like protein displaying 69% sequence identity to the rppA gene of Streptomyces griseus. The S. griseus rppA gene encodes RppA, the first characterized bacterial CS-like protein, which is sufficient in vitro for the synthesis of THN from malonyl-CoA. The rppA disruption mutant and rppA sequence provided a means by which to address the mechanism of diffusible pigment biosynthesis, as well as to investigate any link between this and the modulation of erythromycin A titre, which has been observed for S. erythraea variants.     

New approaches to establish optimum moisture content for compostable materials

This paper reports on the persistence of total and immobilised enzyme activities (urease and phosphatase) in a soil amended with organic wastes containing high levels of total-urease and phosphatase activity. Fresh organic materials showed the highest values for both total-enzymatic activities. The addition of organic waste to soil increased both total-enzymatic activities in the soil, which, after 360 days, showed values above those of the control. Immobilised enzymes were also higher in the fresh wastes than in the soil with compost, while the specific enzymatic activity levels (enzymatic activity per unit of carbon) were similar. The immobilised urease activity was greater in the amended soil than in the control. At the beginning of the incubation period, the immobilised urease activity was significantly higher in the soil amended with fresh organic wastes than with compost. However, this activity decreased with incubation, whilst the compost-immobilised urease activity increased with time. The effect of organic amendment on immobilised phosphatase activity was similar to that shown by immobilised urease but less pronounced. The persistence of both enzymes was significantly higher in the soil amended with compost than in that amended with fresh materials.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Studies of cadmium binding to hexokinase: structural and functional implications

The interaction between cadmium and yeast hexokinase was studied. Cadmium produces changes in the aggregation state of the protein and large structures with a large molecular mass were formed. This phenomenon occurs without large modifications to the secondary structure. During this change the enzyme maintains a high level of activity in the monomer as well as in aggregate form. This implies that the enzyme function is not greatly affected by the change and it maintains its active sites without significant modifications. According to kinetic measurements with both glucose and ATP as a variable substrate, cadmium causes a mixed-type inhibition with a main uncompetitive component. Binding experiments show that the protein presents negative cooperative binding with cadmium at various temperatures (298, 303 and 313 K) and a progressive loss in metal union with concentration depending on the temperature. The total union percentage decreases as the metal concentration increases. This is probably due to the aggregation process, which affects the binding sites for the metal and also for the substrate. Labile interactions are more persistent than specific interactions in accordance with the solvation parameter.     

Determination of the topography and biometry of chlorosomes by atomic force microscopy

Isolated chlorosomes of several species of filamentous anoxygenic phototrophic bacteria (FAPB) and green sulfur bacteria (GSB) were examined by atomic force microscopy (AFM) to characterize their topography and biometry. Chlorosomes of Chloroflexus aurantiacus, Chloronema sp., and Chlorobium (Chl.) tepidum exhibited a smooth surface, whereas those of Chl. phaeobacteroides and Chl. vibrioforme showed a rough one. The potential artifactual nature of the two types of surfaces, which may have arisen because of sample manipulation or AFM processing, was ruled out when AFM images and transmission electron micrographs were compared. The difference in surface texture might be associated with the specific lipid and polypeptide composition of the chlorosomal envelope. The study of three-dimensional AFM images also provides information about the size and shape of individual chlorosomes. Chlorosomal volumes ranged from ca. 35000 nm³ to 247000 nm³ for Chl. vibrioforme and Chl. phaeobacteroides, respectively. The mean height was about 25 nm for all the species studied, except Chl. vibrioforme, which showed a height of only 14 nm, suggesting that GSB have 1–2 layers of bacteriochlorophyll (BChl) rods and GFB have 4. Moreover, the average number of BChl molecules per chlorosome was estimated according to models of BChl rod organisation. These calculations yielded upper limits ranging from 34000 BChl molecules in Chl. vibrioforme to 240000 in Chl. phaeobacteroides, values that greatly surpass those conventionally accepted.This revised version was published online in October 2005 with corrections to the Cover Date.