Comparative quantitative LC-MS/MS analysis of 13 amylase/trypsin inhibitors in ancient and modern Triticum species.

Amylase/trypsin inhibitors (ATIs) are major wheat allergens and they are also implicated in causing non-celiac gluten sensitivity and worsening other inflammatory conditions. With only few studies on ATI contents in different Triticum species available so far, we developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method based on stable isotope dilution assays to quantitate the 13 most important ATIs in a well-defined sample set of eight cultivars of common wheat and durum wheat (modern species), as well as spelt, emmer and einkorn (ancient species) grown at three locations in Germany, respectively. Only few ATIs with low contents were detected in einkorn. In contrast, spelt had the highest total ATI contents. Emmer and common wheat had similar total ATI contents, with durum wheat having lower contents than common wheat. Due to the lack of correlation, it was not possible to estimate ATI contents based on crude protein contents. The wheat species had a higher influence on ATI contents than the growing location and the heritability of this trait was high. Despite comparatively low intra-species variability, some cultivars were identified that may be promising candidates for breeding for naturally low ATI contents.

Analysis on evolutionary relationship of amylases from archaea, bacteria and eukaryota.

Amylase is one of the earliest characterized enzymes and has many applications in clinical and industrial settings. In biotechnological industries, the amylase activity is enhanced through modifying amylase structure and through cloning and expressing targeted amylases in different species. It is important to understand how engineered amylases can survive from generation to generation. This study used phylogenetic and statistical approaches to explore general patterns of amylases evolution, including 3118 α-amylases and 280 ß-amylases from archaea, eukaryota and bacteria with fully documented taxonomic lineage. First, the phylogenetic tree was created to analyze the evolution of amylases with focus on individual amylases used in biofuel industry. Second, the average pairwise p-distance was computed for each kingdom, phylum, class, order, family and genus, and its diversity implies multi-time and multi-clan evolution. Finally, the variance was further partitioned into inter-clan variance and intra-clan variance for each taxonomic group, and they represent horizontal and vertical gene transfer. Theoretically, the results show a full picture on the evolution of amylases in manners of vertical and horizontal gene transfer, and multi-time and multi-clan evolution as well. Practically, this study provides the information on the surviving chance of desired amylase in a given taxonomic group, which may potentially enhance the successful rate of cloning and expression of amylase gene in different species.

Hydrolytic enzymes expressivity in different parts of the Rapana digestive system.

The relevance of comprehensive studies of the Rapana vital functions is determined by its considerab­le negative impact on the ecosystem of the Black Sea. The aim of the work was to find out the polymorphism and activity of the main hydrolases in the different parts of the digestive system of Rapana. Hydrolases (proteases, amylases, esterases, lipases and phosphatases) in glandular structures of the Rapana digestive system were studied by electrophoresis. It was found that different sets of hydrolytic enzymes are functioning in certain parts of the Rapana digestive tract. The gland of Leiblein and hepatopancreas played the most important role in the digestion of food components. The salivary glands had the significant influence on proteolysis.

Diástase dos músculos retos abdominais em puérperas na fase hospitalar / Diastasis of the rectus abdominis muscle in puerperium during hospitalization period

Introdução: Uma diástase do músculo reto abdominal (DMRA) de 3 cm ou mais em puérperas é considerada acima dos padrões de normalidade e poderão trazer maiores complicações como dores lombares, limitações funcionais e herniações das vísceras abdominais. Objetivo: Mensurar a DMRA em puérperas, na Maternidade do Hospital Satélite, e correlacionar com paridade, idade da puérpera, peso do recém nascido (RN) e sedentarismo. Material e métodos: Estudo transversal, observacional, com uma amostra de 30 puérperas, examinadas no mês de novembro de 2008 em relação à paridade, ao sedentarismo e ao peso do RN. Resultados: A multiparidade foi o que apresentou uma maior relação ao aumento de DMRA, quando comparada ao grupo primigestas/secundigestas (p = 0,003). A média da DMRA em sedentárias foi de 3,7 cm e para as não sedentárias foi de 2,8 cm (p < 0,05). O peso do RN tem influência de 11,47 por cento sobre a diástase (p < 0,067). A variável que mais evidenciou uma correlação positiva com o aumento da DMRA foi a faixa etária da gestante com influência de 42,99 por cento sobre DMRA (p < 0,008).Conclusão: A paridade, a prática de atividade física, o peso do RN e, principalmente, a idade da gestante influenciam diretamente na variação do tamanho da DMRA durante o período de gestação.

Introduction: A diastasis recti of the rectus abdominis muscle (RAMD) of 3 cm width or more is considered out of the acceptable patterns and can cause other commitments such as back pain, functional limitations and abdominal visceral herniation. Objective: To measure the RAM in post childbirth woman at the Maternity of Satélite Hospital in Teresina, Piauí, Brazil, and to correlate it to parity, mother’s age at delivery, newborn weight and sedentarism. Method: This was a transversal and observational study with 30 women examined at immediate post-partum on November 2008 in terms of parity, sedentarism and newborn weight. Results: High parity factor showed a higher relation to the diastase increase when was compared first pregnancy and second pregnancy group (p = 0.003). The average of RAMD in sedentary women was 3.7 cm comparing to 2.8 cm in non-sedentaries (p < 0.05). The newborn weight has 11.47 percent of influence over diastase (p < 0,067). The variable that most influenced with a positive correlation with RAMD was the mother age at delivery (42.99 percent over RAMD, p < 0.008). Conclusion: Parity, physical activity, newborn weight and, mainly, age of pregnant woman are factors that influence RAMD size range during pregnancy.

Electrophoretically unique amylases in rat livers: phylogenic and ontogenic study on the mammalian liver.

Liver amylase activity in rodents was assayed with Blue Starch as substrate, and found to be higher than in humans or pigs. Based on the result of concanavalin A affinity chromatography, we found that the sugar moieties of amylase molecules increased in parallel with amylase activity in the tested mammals. However, the amounts of amylase proteins determined by Western bloting with anti-human salivary-type antibody as the probe, were similar to the levels in mammalian livers. Moreover, a similar expression of amylase mRNA was also detected in the mammalian livers by a reverse transcriptional-polymerase chain reaction using primers specific for the human salivary and/or pancreatic amylase complementary DNA (cDNA) sequences. The amylase was detected at the catalytic activity, protein molecule and mRNA levels in rat liver at all ages from fetus to adult. Salivary-type liver amylase activity increased up to one week after birth, and was maintained at the adult level thereafter. However, based on the results of the electrophoretic mobility test, livers with accelerated amylase activity, e.g., at 2-4 weeks after birth or during liver regeneration after partial hepatectomy, were also found to express an amylase electrophoretical identical to pancreatic-type amylase in addition to salivary-type activity. These results suggest that the liver may express an etopic amylase in a certain condition.

Suitability and limitations of methods for characterisation of activity of malto-oligosaccharide-forming amylases.

The suitability and limitations of essential methods and reference substrates used for characterisation of activity of amylolytic enzymes is investigated. Saccharogenic, chromogenic and chromatographic methods are included. The results are discussed in relation to the measurement of reaction rates, determination of action mode and product specificity and the impact on identification and nomenclature of malto-oligosaccharide-forming amylases. An accurate determination of reaction rates using the saccharogenic methods strongly depends on the degree of polymerisation (DP) of the standards used and the hydrolysis products formed by the amylase. Particularly the use of glucose as standard can lead to overestimates due to the differences in the reducing potential of glucose and malto-oligosaccharides. The reliability of the chromogenic methods for determination of action mode depends on the DP of the substrate and the specificity of the amylase. For a characterisation of the starch hydrolysis products and the variation in the DP during hydrolysis, high performance anion-exchange chromatography with pulsed amperometric detection provided a fast and reliable method. A literature survey revealed varying and inconsistent use of nomenclature of malto-oligosaccharide forming amylases. Therefore a systematic approach identifying three main classes of activity is suggested using not only the mode of action and the DP of the major product but also the stage of hydrolysis at which this product is formed.

The Amylase gene cluster on the evolving sex chromosomes of Drosophila miranda.

On the basis of chromosomal homology, the Amylase gene cluster in Drosophila miranda must be located on the secondary sex chromosome pair, neo-X (X2) and neo-Y, but is autosomally inherited in all other Drosophila species. Genetic evidence indicates no active amylase on the neo-Y chromosome and the X2-chromosomal locus already shows dosage compensation. Several lines of evidence strongly suggest that the Amy gene cluster has been lost already from the evolving neo-Y chromosome. This finding shows that a relatively new neo-Y chromosome can start to lose genes and hence gradually lose homology with the neo-X. The X2-chromosomal Amy1 is intact and Amy2 contains a complete coding sequence, but has a deletion in the 3'-flanking region. Amy3 is structurally eroded and hampered by missing regulatory motifs. Functional analysis of the X2-chromosomal Amy1 and Amy2 regions from D. miranda in transgenic D. melanogaster flies reveals ectopic AMY1 expression. AMY1 shows the same electrophoretic mobility as the single amylase band in D. miranda, while ectopic AMY2 expression is characterized by a different mobility. Therefore, only the Amy1 gene of the resident Amy cluster remains functional and hence Amy1 is the dosage compensated gene.

Clinical significance of chronic hyperamylasemia.

A longitudinal study of patients with persistent hyperamylasemia was carried out to evaluate the clinical significance of this condition. Twenty-five outpatients were studied by means of serum amylase, isoamylase (wheat germ-inhibition method), and lipase determination; macroamylase detection; and abdominal ultrasonography over a one-year period. Cellulose acetate electrophoresis was carried out to validate the wheat germ-inhibition tests; the results of the two assays were closely correlated, except in three patients with macroamylasemia. At the time of the study, none of the patients had evident signs or symptoms of pancreatic disease. At initial evaluation, wheat germ test demonstrated an elevation of only salivary isoamylase in 16 patients, both pancreatic and salivary isoenzyme in two, and only pancreatic isoamylase in six patients (three with macroamylasemia). Normal salivary and pancreatic isoenzymes were found in one with predominantly pancreatic isoamylase. At the 12-month follow-up, hyperamylasemia had disappeared in six cases and salivary isoamylase elevation in three; pancreatic isoamylase remained abnormally high in all eight patients in whom it was elevated at initial evaluation, and lipase was abnormally high in three patients with elevated pancreatic isoamylase. Of the five patients with true pancreatic hyperamylasemia, one had a juxtapapillary duodenal diverticulum, one showed a slight ultrasound alteration of the pancreas, and one had a past history of acute pancreatitis. In our study, most cases of chronic hyperamylasemia were of nonpancreatic origin. In the patients with elevated pancreatic isoamylase, there was no clinical evidence of pancreatic damage, although a subclinical pancreatic involvement could not be excluded in some.

Multiple forms of amylase in leaf extracts: electrophoretic transfer of the enzyme forms into amylose-containing polyacrylamide gels.

A method for the analysis of multiple forms of glucan-degrading enzymes is described. The procedure consists of the separation of the proteins by electrophoresis or isoelectric focusing in glucan-free polyacrylamide gels followed by the nondenaturing electrophoretic transfer into a second polyacrylamide layer which contains immobilized glucans. The method combines the resolving power of electrophoretic separations in glucan-free media with the sensitivity of amylase activity detection in amylose-containing polyacrylamide gels. The procedure is especially useful when samples containing low amylase activity, but a large number of multiple enzyme forms, are to be analyzed.