The use of biochemical parameters for a qualitative and quantitative assessment of ischemic damage to the small-intestinal mucosa

Lactate dehydrogenase has been measured in the small-intestinal mucosa in order to assess its value as a marker for the effects of ischemia and of reperfusion. The decrease in specific activity of the enzyme illustrates the deleterious effect of reperfusion on the quality of the remaining epithelial cells. However, this parameter fails to detect the loss of epithelial cells, which is the major event during ischemia as well as during reperfusion. In contrast, the expression of enzyme activity per g protein of the underlying intestinal muscle allowed us, in addition, to assess quantitatively the loss of epithelial cells, in good agreement with the histological data.     

Metabolism of l-threonine and fatty acids and tylosin biosynthesis in Streptomyces fradiae

Abstract In Streptomyces fradiae l -threonine is catabolized by threonine dehydratase or threonine aldolase to 2-ketobutyrate or acetaldehyde and glycine, respectively. Threonine dehydratase synthesis is repressed and its activity is inhibited by NH₄⁺ ions. Threonine aldolase is not repressed by NH₄⁺ ions and its activity is slightly stimulated by these ions. The addition of threonine to the medium increased pronouncedly the fraction of non-branched fatty acids with an even carbon number under conditions when threonine dehydratase was repressed and inhibited. The results indicate that threonine serves as a source of propionyl-CoA and 2-methylbutyryl-CoA and also of acetyl-CoA required for tylosin and fatty acid biosynthesis.     

Repeatability of nest predation in passerines depends on predator species and time scale

It has been proposed that some specific locations of bird's nests have higher intrinsic chances of being depredated than other locations. This predicts that fates of consecutive nesting attempts at the same site should be repeatable. We used 20 pairs of old thrush nests to simulate repeated nesting attempts at the same sites, both within and between breeding seasons (n=40  sites×2  trials×2  years=160). Each nest was monitored by a camera to record multiple predation events and to identify predators. Predation by all predator species was repeatable during a 15-day trial. Predation by principal predators (jay Garrulus glandarius , marten Martes martes / foina ) and total predation (all species combined) was not correlated within pairs of simultaneously exposed nests or within samples of nests from particular study plot, and not repeatable for individual nests between-trials or between-years. These findings suggest short-term effect of predator memory causing revisitation of previously depredated nests during a current nesting trial (all predators); do not support an effect of nest site features on multiple nest discoveries and/or an effect of nest location on repeated random encounters with the same nest (principal predators). Long-term repeatability and correlation within pairs of simultaneously exposed nests was detectable only in occasional predators (great spotted woodpecker Dendrocopos major , possibly also squirrel Sciurus vulgaris ), which suggests effect of nest location combined with site fidelity and individual foraging specialization of these predators. We conclude that repeatability of nest predation depends on the time scale considered and the local predator community. We caution against spurious findings of repeatable nest predation resulting simply from statistical properties of correlation in binary data (nest fates).     

Characterization of the non-specific lipid transfer protein EP2 from carrot (Daucus carota L.)

The extracellular protein EP2 was previously identified as non-specific lipid transfer protein based on its cDNA-derived amino acid sequence. Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and gel permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This protein band was recognized by the antiserum raised against a EP2--galactosidase fusion-protein. Employing a fluorescent phospholipid analog, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic acid as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from their site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.     

Secondary alcohol dehydrogenase catalyzes the reduction of exogenous acetone to 2-propanol in Trichomonas vaginalis

Secondary alcohols such as 2-propanol are readily produced by various anaerobic bacteria that possess secondary alcohol dehydrogenase (S-ADH), although production of 2-propanol is rare in eukaryotes. Specific bacterial-type S-ADH has been identified in a few unicellular eukaryotes, but its function is not known and the production of secondary alcohols has not been studied. We purified and characterized S-ADH from the human pathogen Trichomonas?vaginalis. The kinetic properties and thermostability of T.?vaginalis S-ADH were comparable with bacterial orthologues. The substantial activity of S-ADH in the parasite's cytosol was surprising, because only low amounts of ethanol and trace amounts of secondary alcohols were detected as metabolic end products. However, S-ADH provided the parasite with a high capacity to scavenge and reduce external acetone to 2-propanol. To maintain redox balance, the demand for reducing power to metabolize external acetone was compensated for by decreased cytosolic reduction of pyruvate to lactate and by hydrogenosomal metabolism of pyruvate. We speculate that hydrogen might be utilized to maintain cytosolic reducing power. The high activity of Tv-S-ADH together with the ability of T.?vaginalis to modulate the metabolic fluxes indicate efficacious metabolic responsiveness that could be advantageous for rapid adaptation of the parasite to changes in the host environment.     

Rhizobial nod gene-inducing activity in pea nodulation mutants: dissociation of nodulation and flavonoid response

With the characterization of the total genomes of Arabidopsis thaliana and Oryza sativa , several putative plasma membrane components have been identified. However, a lack of knowledge at the protein level, especially for hydrophobic proteins, have hampered analyses of physiological changes. To address whether protein complexes may be present in the native membrane, we subjected plasma membranes isolated from Spinacia oleracea leaves to blue-native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is well established in the separation of functional membrane protein complexes from mitochondria and chloroplasts, but a resolved protein complex pattern from PM of eukaryotic cells has previously not been reported. Using this method, protein complexes from Spinacia oleracea PM could be efficiently solubilized and separated, including the highly hydrophobic aquaporin (apparent molecular mass 230 kDa), a putative tetramer of H⁺-ATPase, and several less abundant complexes with apparent masses around or above 750 kDa. After denaturation and separation of the complexes into their subunits in a second dimension (SDS-PAGE), several of the complexes were identified as hydrophobic membrane proteins. Large amounts of protein (up to 1 mg) can be resolved in each lane, which suggests that the method could be used to study also low-abundance protein complexes, e.g. under different physiological conditions.     

Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes

Stratum corneum ceramides play an essential role in the barrier properties of skin. However, their structure-activity relationships are poorly understood. We investigated the effects of acyl chain length in the non-hydroxy acyl sphingosine type (NS) ceramides on the skin permeability and their thermotropic phase behavior. Neither the long- to medium-chain ceramides (8-24 C) nor free sphingosine produced any changes of the skin barrier function. In contrast, the short-chain ceramides decreased skin electrical impedance and increased skin permeability for two marker drugs, theophylline and indomethacin, with maxima in the 4-6C acyl ceramides. The thermotropic phase behavior of pure ceramides and model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesterol sulfate was studied by differential scanning calorimetry and infrared spectroscopy. Differences in thermotropic phase behavior of these lipids were found: those ceramides that had the greatest impact on the skin barrier properties displayed the lowest phase transitions and formed the least dense model stratum corneum lipid membranes at 32°C. In conclusion, the long hydrophobic chains in the NS-type ceramides are essential for maintaining the skin barrier function. However, this ability is not shared by their short-chain counterparts despite their having the same polar head structure and hydrogen bonding ability.     

European colonization by the spined loach (Cobitis taenia) from Ponto-Caspian refugia based on mitochondrial DNA variation

In the last 20 years, new species, asexual reproduction, polyploidy and hybridization have all been reported within the genus Cobitis. An understanding of the current distribution and baseline phylogeographical history of 'true' nonhybrid Cobitis species is crucial in order to unravel these discoveries. In the present work, we investigated the phylogeography of the spined loach, Cobitis taenia, using 1126 bp of the mitochondrial cytochrome b gene from 174 individuals collected at 47 sites. In total, 51 haplotypes that differed at 49 positions (4.35%) were detected. We deduce that C. taenia survived European glaciations in at least three refugees in the Ponto-Caspian area. Two of these refugees each provided a major lineage that recolonized Europe in separate directions: one westward to England and the other spreading north into Russia before moving west. A third (minor) lineage that contributed little to the recolonization of Europe was also revealed--remaining near its Black Sea refuge. However, more recent history was difficult to resolve with colonization from a more western refugium during the last glacial maximum (LGM) a distinct possibility. Nested clade analysis indicates a pattern of restricted gene flow with isolation by distance at the first two levels and overall. Unlike many other European freshwater fish species, the Danube is not part of the current distribution of C. taenia, nor was it used as either a refuge or a source of colonization of Europe. Low genetic diversity within C. taenia suggests that its colonization of Europe is relatively recent. Demographic analyses revealed a history of recent expansion and isolation by distance.     

Inhibition of the Equilibrative Nucleoside Transporter 1 and Activation of A2A Adenosine Receptors by 8-(4-Chlorophenylthio)-modified cAMP Analogs and Their Hydrolytic Products

Cyclic AMP analogs containing hydrophobic modification of C⁸ at the adenine ring such as 8-(4-chlorophenylthio)-cAMP (8-pCPT-cAMP) and 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT-2′-O-methyl-cAMP) can penetrate membranes due to their high lipophilicity and directly activate intracellular cAMP effectors. Therefore, these cAMP analogs have been used in numerous studies, assuming that their effects reflect the consequences of direct activation of cAMP effectors. The present study provides evidence that 8-pCPT-modified cAMP analogs and their corresponding putative hydrolysis products (8-(4-chlorophenylthio)-adenosine (8-pCPT-ado) and 8-(4-chlorophenylthio)-2′-O-methyl-adenosine (8-pCPT-2′-O-methyl-ado)) inhibit the equilibrative nucleoside transporter 1 (ENT1). In PC12 cells, in which nucleoside transport strongly depended on ENT1, 8-pCPT-ado, 8-pCPT-2′-O-methyl-ado, and, to a smaller extent, 8-pCPT-2′-O-methyl-cAMP caused an increase of protein kinase A substrate motif phosphorylation and anti-apoptotic effect by an A_2A adenosine receptor (A_2AR)-dependent mechanism. In contrast, the effects of 8-pCPT-cAMP were mainly A_2AR-independent. In HEK 293 showing little endogenous ENT1-dependent nucleoside transport, transfection of ENT1 conferred A_2AR-dependent increase in protein kinase A substrate motif phosphorylation. Together, the data of the present study indicate that inhibition of ENT1 and activation of adenosine receptors have to be considered when interpreting the effects of 8-pCPT-substituted cAMP/adenosine analogs.