A new GH13 subfamily represented by the α-amylase from the halophilic archaeon Haloarcula hispanica

Extremophiles - α-Amylase catalyzes the endohydrolysis of α-1,4-glucosidic linkages in starch and related α-glucans. In the CAZy database, most α-amylases have been classified...     

Altered expression of claudin family proteins in Alzheimer’s disease and vascular dementia brains

Claudins (Cls) are a multigene family of transmembrane proteins with different tissue distribution, which have an essential role in the formation and sealing capacity of tight junctions (TJs). At the level of the blood–brain barrier (BBB), TJs are the main molecular structures which separate the neuronal milieu from the circulatory space, by a restriction of the paracellular flow of water, ions and larger molecules into the brain. Different studies suggested recently significant BBB alterations in both vascular and degenerative dementia types. In a previous study we found in Alzheimer’s disease (AD) and vascular dementia (VaD) brains an altered expression of occludin, a molecular partner of Cls in the TJs structure. Therefore in this study, using an immunohistochemical approach, we investigated the expression of Cl family proteins (Cl‐2, Cl‐5 and Cl‐11) in frontal cortex of aged control, AD and VaD brains. To estimate the number of Cl‐expressing cells, we applied a random systematic sampling and the unbiased optical fractionator method. We found selected neurons, astrocytes, oligodendrocytes and endothelial cells expressing Cl‐2, Cl‐5 and Cl‐11 at detectable levels in all cases studied. We report a significant increase in ratio of neurons expressing Cl‐2, Cl‐5 and Cl‐11 in both AD and VaD as compared to aged controls. The ratio of astrocytes expressing Cl‐2 and Cl‐11 was significantly higher in AD and VaD as compared to aged controls. The ratio of oligodendrocytes expressing Cl‐11 was significantly higher in AD and the ratio of oligodendrocytes expressing Cl‐2 was significantly higher in VaD as compared to aged controls. Within the cerebral cortex, Cls were selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings suggest a new function of Cl family proteins which might be linked to response to cellular stress.     

Developmental changes in gut microbiota and enzyme activity predict obesity risk in rats arising from reduced nests

The aim of the study was to assess the impact of preweaning overnutrition upon the ontogeny of intestinal microbiota, alkaline phosphatase activity (AP) and parameters of growth and obesity in male Sprague-Dawley rats. We tested whether intestinal characteristics acquired in suckling pups could programme the development of enhanced fat deposition during normalized nutrition beyond weaning. Postnatal nutrition was manipulated by adjusting the number of pups in the nest to 4 (small litters--SL) and 10 (normal litters--NL). In the postweaning period both groups were fed with a standard diet. The jejunal and colonic Lactobacillus/Enterococcus (LAB) and the Bacteroides/Prevotella (BAC) were determined using the FISH technique, and the jejunal AP activity was assayed histochemically. At 15 and 20 days of age the SL pups became heavier, displayed increased adiposity accompanied by significantly higher LAB and lower numbers of BAC and with higher AP activity in comparison with rats nursed in NL nests. These differences persisted to day 40 and withdrawal of the previous causal dietary influence did not prevent the post-weaning fat accretion. These results reveal the significance of early nutritional imprint upon the gut microbial/functional development and allow better understanding of their involvement in the control of obesity.     

Association of novel domain in active site of archaic hyperthermophilic maltogenic amylase from Staphylothermus marinus

Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1-4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N' domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N' domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes.     

Sequence fingerprints of enzyme specificities from the glycoside hydrolase family GH57

The glycoside hydrolase family 57 (GH57) contains five well-established enzyme specificities: α-amylase, amylopullulanase, branching enzyme, 4-α-glucanotransferase and α-galactosidase. Around 700 GH57 members originate from Bacteria and Archaea, a substantial number being produced by thermophiles. An intriguing feature of family GH57 is that only slightly more than 2 % of its members (i.e., less than 20 enzymes) have already been biochemically characterized. The main goal of the present bioinformatics study was to retrieve from databases, and analyze in detail, sequences having clear features of the five GH57 enzyme specificities mentioned above. Of the 367 GH57 sequences, 56 were evaluated as α-amylases, 99 as amylopullulanases, 158 as branching enzymes, 46 as 4-α-glucanotransferases and 8 as α-galactosidases. Based on the analysis of collected sequences, sequence logos were created for each specificity and unique sequence features were identified within the logos. These features were proposed to define the so-called sequence fingerprints of GH57 enzyme specificities. Domain arrangements characteristic of the individual enzyme specificities as well as evolutionary relationships within the family GH57 are also discussed. The results of this study could find use in rational protein design of family GH57 amylolytic enzymes and also in the possibility of assigning a GH57 specificity to a hypothetical GH57 member prior to its biochemical characterization.     

Intestinal microflora and obesity in rats

The relationship was evaluated between early nutritional experiences, the intestinal microflora and the small intestinal functions in the mechanism of predisposition to obesity development. Male Sprague-Dawley rats were used in which the quantity of nutrition was manipulated from birth to weaning (day 30) by adjusting the number of pups in the nest to 4 small litters (SL) and 10 normal litters (NL) and fed a standard diet from days 30 to 40 of age. After 40 d, the postnatally overfed SL pups became heavier, displayed significantly enhanced adiposity, body mass gain and food intake as well as a significantly higher jejunal alkaline phosphatase and maltase activity than in rats nursed in NL nests. The effect of different early nutrition was also accompanied by the appearance of significantly decreased Bacteroides and significantly increased enterococci and lactobacilli of obese rats than in lean NL rats. The amounts of Bacteroides were negatively correlated with fat pad mass, body mass, body-mass gain and food intake whereas enterococci and lactobacilli were correlated positively with the same parameters. Our results demonstrate that postnatal nutritional experience may represent a predisposing factor influencing ontogeny of small intestine function and development of intestinal microbial communities. The acquired changes and associated alterations in food digestion could be a component of regulatory mechanisms contributing to the development of obesity and its maintenance in later life.     

Heavy-metal stress induced accumulation of chitinase isoforms in plants

Plant chitinases belong to so-called pathogenesis related proteins and have mostly been detected in plants exposed to phytopathogenic viruses, bacteria or fungi. A few studies revealed that they might also be involved in plant defence against heavy metals. This work was undertaken to monitor the accumulation of chitinases in a set of heavy-metal stressed plants and bring evidence on their involvement during this kind of stress. Roots of different plant species including Vicia faba cvs. Aštar and Piešťansky, Pisum sativum, Hordeum vulgare, Zea mays and Glycine max were exposed to different concentrations of lead (300 and 500 mg l⁻¹ Pb²⁺), cadmium (100 and 300 mg l⁻¹ Cd²⁺) and arsenic (50 and 100 mg l⁻¹ As³⁺). In each case, the toxicity effects were reflected in root growth retardation to 80–10% of control values. The most tolerant were beans, most sensitive was barley. Extracts from the most stressed roots were further assayed for chitinase activity upon separation on polyacrylamide gels. Our data showed that in each combination of genotype and metal ion there were 2–5 different chitinase isoforms significantly responsive to toxic environment when compared with water-treated controls. This confirms that chitinases are components of plant defence against higher concentrations of heavy metals. In addition, accumulation of some isoforms in response to one but not to other metal ions suggests that these enzymes might also be involved in a more (metal) specific mechanism in affected plants and their biological role is more complex than expected.