A Genetic Analysis of Lysosomal Enzyme Activities in Brahman Cattle

Analyses of variance and co variance were carried out on the activities of three lysosomal enzymes in mononuclear blood cells from Brahman cattle. These were hexosaminidase (HEX), beta-D-galacto-sidase (GAL) and acid alpha-glucosidase (GLU) which had been measured in blood mononuclear cells from 1752 cattle from 6 herds in a Pompe's disease control programme. Herd of origin and date of bleeding significantly affected the level of activity of all enzymes. In addition, HEX and GAL were affected by age and HEX by the sex of the animal bled. Estimates of heritability from sire variances were 0.29:t 0.09 for HEX, 0.31 :t 0.09 for GAL and 0.44:t 0.09 for GLU. Genetic correlations between all enzymes were positive. The data indicate the existence of a major gene causing Pompe's disease and responsible for 16% of the genetic variation in GLU. One standard deviation of selection differential for high GLU should almost eliminate Pompe's disease from the population. The effi-ciency of selection would be aided by estimating the breeding value for GLU using measurements of HEX and GLU and taking account of an animal's sex, age, date of bleeding and herd of origin.

The preparation and bioactivity research of agaro-oligosaccharides

Agaro-oligosaccharides were hydrolytically obtained from agar using hydrochloric acid, citric acid, and cationic exchange resin (solid acid). The FT-IR and NMR data showed that the hydrolysate has the structure of agaro-oligomers. Orthogonal matrix method was applied to optimize the preparation conditions based on alpha-naphthylamine end-labeled HPLC analysis method. The optimal way for oligosaccharides with different degree of polymerization (DP) was achieved by using solid acid degradation, which could give high yield and avoid solution neutralization process. Agaro-oligosaccharides with high purity were consequently obtained by activated carbon column isolation. Furthermore, the antioxidant and alpha-glucosidase inhibitory activity of three fractions were also investigated. The result indicated that 8% ethanol-eluted fraction showed highest activity against alpha-glucosidase with IC50 of 8.84 mg/mL, while 25% ethanol-eluted fraction possessed excellent antioxidant ability.

Investigation on key molecular targets responsible for antidiabetic properties of Symplocos cochinchinensis (Lour.) S. Moore

Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycemia with disturbances in carbohydrate, protein and lipid metabolism resulting from defects in insulin secretion, insulin action or both. Currently there are 387 million people with diabetes worldwide and is expected to affect 592 million people by 2035. Insulin resistance in peripheral tissues and pancreatic beta cell dysfunction are the major challenges in the pathophysiology of diabetes. Diabetic secondary complications (like liver cirrhosis, retinopathy, microvascular and macrovascular complications) arise from persistent hyperglycemia and dyslipidemia can be disabling or even life threatening. Current medications are effective for control and management of hyperglycemia but undesirable effects, inefficiency against secondary complications and high cost are still serious issues in the present prognosis of this disorder. Hence the search for more effective and safer therapeutic agents of natural origin has been found to be highly demanding and attract attention in the present drug discovery research. The data available from Ayurveda on various medicinal plants for treatment of diabetes can efficiently yield potential new lead as antidiabetic agents. For wider acceptability and popularity of herbal remedies available in Ayurveda scientific validation by the elucidation of mechanism of action is very much essential. Modern biological techniques are available now to elucidate the biochemical basis of the effectiveness of these medicinal plants. Keeping this idea the research programme under this thesis has been planned to evaluate the molecular mechanism responsible for the antidiabetic property of Symplocos cochinchinensis, the main ingredient of Nishakathakadi Kashayam, a wellknown Ayurvedic antidiabetic preparation. A general introduction of diabetes, its pathophysiology, secondary complications and current treatment options, innovative solutions based on phytomedicine etc has been described in Chapter 1. The effect of Symplocos cochinchinensis (SC), on various in vitro biochemical targets relevant to diabetes is depicted in Chapter 2 including the preparation of plant extract. Since diabetes is a multifactorial disease, ethanolic extract of the bark of SC (SCE) and its fractions (hexane, dichloromethane, ethyl acetate and 90 % ethanol) were evaluated by in vitro methods against multiple targets such as control of postprandial hyperglycemia, insulin resistance, oxidative stress, pancreatic beta cell proliferation, inhibition of protein glycation, protein tyrosine phosphatase-1B (PTP-1B) and dipeptidyl peptidase-IV (DPPxxi IV). Among the extracts, SCE exhibited comparatively better activity like alpha glucosidase inhibition, insulin dependent glucose uptake (3 fold increase) in L6 myotubes, pancreatic beta cell regeneration in RIN-m5F and reduced triglyceride accumulation in 3T3-L1 cells, protection from hyperglycemia induced generation of reactive oxygen species in HepG2 cells with moderate antiglycation and PTP-1B inhibition. Chemical characterization by HPLC revealed the superiority of SCE over other extracts due to presence of bioactives (beta-sitosterol, phloretin 2’glucoside, oleanolic acid) in addition to minerals like magnesium, calcium, potassium, sodium, zinc and manganese. So SCE has been subjected to oral sucrose tolerance test (OGTT) to evaluate its antihyperglycemic property in mild diabetic and diabetic animal models. SCE showed significant antihyperglycemic activity in in vivo diabetic models. Chapter 3 highlights the beneficial effects of hydroethanol extract of Symplocos cochinchinensis (SCE) against hyperglycemia associated secondary complications in streptozotocin (60 mg/kg body weight) induced diabetic rat model. Proper sanction had been obtained for all the animal experiments from CSIR-CDRI institutional animal ethics committee. The experimental groups consist of normal control (NC), N + SCE 500 mg/kg bwd, diabetic control (DC), D + metformin 100 mg/kg bwd, D + SCE 250 and D + SCE 500. SCEs and metformin were administered daily for 21 days and sacrificed on day 22. Oral glucose tolerance test, plasma insulin, % HbA1c, urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total protein etc. were analysed. Aldose reductase (AR) activity in the eye lens was also checked. On day 21, DC rats showed significantly abnormal glucose response, HOMA-IR, % HbA1c, decreased activity of antioxidant enzymes and GSH, elevated AR activity, hepatic and renal oxidative stress markers compared to NC. DC rats also exhibited increased level of plasma urea and creatinine. Treatment with SCE protected from the deleterious alterations of biochemical parameters in a dose dependent manner including histopathological alterations in pancreas. SCE 500 exhibited significant glucose lowering effect and decreased HOMA-IR, % HbA1c, lens AR activity, and hepatic, renal oxidative stress and function markers compared to DC group. Considerable amount of liver and muscle glycogen was replenished by SCE treatment in diabetic animals. Although metformin showed better effect, the activity of SCE was very much comparable with this drug. xxii The possible molecular mechanism behind the protective property of S. cochinchinensis against the insulin resistance in peripheral tissue as well as dyslipidemia in in vivo high fructose saturated fat diet model is described in Chapter 4. Initially animal were fed a high fructose saturated fat (HFS) diet for a period of 8 weeks to develop insulin resistance and dyslipidemia. The normal diet control (ND), ND + SCE 500 mg/kg bwd, high fructose saturated fat diet control (HFS), HFS + metformin 100 mg/kg bwd, HFS + SCE 250 and HFS + SCE 500 were the experimental groups. SCEs and metformin were administered daily for the next 3 weeks and sacrificed at the end of 11th week. At the end of week 11, HFS rats showed significantly abnormal glucose and insulin tolerance, HOMA-IR, % HbA1c, adiponectin, lipid profile, liver glycolytic and gluconeogenic enzyme activities, liver and muscle triglyceride accumulation compared to ND. HFS rats also exhibited increased level of plasma inflammatory cytokines, upregulated mRNA level of gluconeogenic and lipogenic genes in liver. HFS exhibited the increased expression of GLUT-2 in liver and decreased expression of GLUT-4 in muscle and adipose. SCE treatment also preserved the architecture of pancreas, liver, and kidney tissues. Treatment with SCE reversed the alterations of biochemical parameters, improved insulin sensitivity by modifying gene expression in liver, muscle and adipose tissues. Overall results suggest that SC mediates the antidiabetic activity mainly via alpha glucosidase inhibition, improved insulin sensitivity, with antiglycation and antioxidant activities.

Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays

Short-chain fructooligosaccharides (scFOS) and other prebiotics are used to selectively stimulate the growth and activity of lactobacilli and bifidobacteria in the colon. However, there is little information on the mechanisms whereby prebiotics exert their specific effects upon such microorganisms. To study the genomic basis of scFOS metabolism in Lactobacillus plantarum WCFS1, two-color microarrays were used to screen for differentially expressed genes when grown on scFOS compared to glucose (control). A significant up-regulation (8- to 60-fold) was observed with a set of only five genes located in a single locus and predicted to encode a sucrose phosphoenolpyruvate transport system (PTS), a beta-fructofuranosidase, a fructokinase, an alpha-glucosidase, and a sucrose operon repressor. Several other genes were slightly overexpressed, including pyruvate dehydrogenase. For the latter, no detectable activity in L. plantarum under various growth conditions has been previously reported. A mannose-PTS likely to encode glucose uptake was 50-fold down-regulated as well as, to a lower extent, other PTSs. Chemical analysis of the different moieties of scFOS that were depleted in the growth medium revealed that the trisaccharide 1-kestose present in scFOS was preferentially utilized, in comparison with the tetrasaccharide nystose and the pentasaccharide fructofuranosylnystose. The main end products of scFOS fermentation were lactate and acetate. This is the first example in lactobacilli of the association of a sucrose PTS and a beta-fructofuranosidase that could be used for scFOS degradation.

Estudo da funcionalidade de espécies comestíveis do semiárido nordestino e estratégias para sua utilização como ingredientes para fins alimentícios

The Brazilian caatinga is characterized by low annual rainfall and arid soils. Several cactaceae, either native or adapted species, grow in this semi-arid region, including the prickly pear (Opuntia fícus indica) and facheiro ((Philosocereus pachycladus Ritter) which produce underexploited edible fruits. In addition to these species, the algaroba is a leguminous with little studied technological applications and bioactive potential so far. Therefore, this research aims to investigate the physicochemical, bioactive and functional attributes of the prickly pear and facheiro fruit pulps and the algaroba flour. Specifically, this study approaches the physicochemical characterization, total phenolic compounds (TPC) and the betalain identification and quantification by HPLC-DAD-ESI-MS. It is also investigated the DPPH antioxidant capacity and the antienzymatic activities against alpha-amylase and alphaglucosidase of water and ethanolic extracts of these food material. In order to address their potential to be used as food ingredients, juice blends prepared with mixtures of cajá and prickly pear, biofilms with facheiro and cereal bars with algaroba flour were elaborated and analyzed. The prickly pear fruits presented low acidity and high sugar content when compared to facheiro. The Philosocereus pachycladus Ritter fruits had higher protein and ash content, but the algaroba flour was the species with higher protein and sugar content among all. The algaroba flour also presented outstanding food fiber content, which reveals its potentiality to be used as a natural intestinal regulator. The TPC of water and ethanol extracts ranged from 3.87 to 16.21 mg GAE/100g for algaroba flour, 79.24 to 110.20 GAE/ 100g for prickly pear and 412.23 to 539.14 mg GAE/100g for facheiro. The 70% (w/v) ethanol extract reached the highest DPPH antioxidant activity, which was linearly correlated to its high TPC content. In regard to the enzymatic inhibitory activities, the best performance was observed for the prickly pear extracts which presented a moderate inhibition for both investigated enzymes, but interestingly, no alpha-glucosidase inhibition was observed for facheiro extracts. This work shows, for the first time in the literature, the functional attributes of facheiro fruits, as well as the presence of betacianins and isobetanin in the pulp of this exotic fruit. When it comes to the food products developed here, the sensory attributes that better described the juice blend cajá-prickly pear were sweetness, acidity, color yellow-orange, body, turbidity and cajá flavor. The discriminative test applied for cereal bars produced with and without algaroba revealed that the texture was the only sensory attribute that differed (p<0.05) between these two samples. It was also observed that the addition of facheiro extracts did not influence the visual characteristics of the biofilms. Overall, this work unveils the physicochemical and bioactive attributes of these commercial and technologically underexploited species widely found in the Brazilian caatinga and presents alternatives for their rational use

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Profiling the proteome complement of the secretion from hypopharyngeal gland of Africanized nurse-honeybees (Apis mellifera L.)

The protein complement of the secretion from hypopharyngeal gland of nurse-bees (Apis mellifera L.) was partially identified by using a combination of 2D-PAGE, peptide sequencing by MALDI-PSD/MS and a protein engine identification tool applied to the honeybee genome. The proteins identified were compared to those proteins already identified in the proteome complement of the royal jelly of the honey bees. The 2D gel electrophoresis demonstrated this protein complement is constituted of 61 different polypepides, from which 34 were identified as follows: 27 proteins belonged to MRJPs family, 5 proteins were related to the metabolism of carbohydrates and to the oxido-reduction metabolism of energetic Substrates, I protein was related to the accumulation of iron in honeybee bodies and I protein may be a regulator of MRJP-1 oligomerization. The proteins directly involved with the carbohydrates and energetic metabolisms were: alpha glucosidase, glucose oxidase and alpha amylase, whose are members of the same family of enzymes, catalyzing the hydrolysis of the glucosidic linkages of starch; alcohol dehydrogenase and aldehyde dehydrogenase, whose are constituents of the energetic metabolism. The results of the present manuscript support the hypothesis that the most of these proteins are produced in the hypoharyngeal gland of nurse-bees and secreted into the RJ. (C) 2004 Elsevier Ltd. All rights reserved.

Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise funcional de um consórcio microbiano de solo e prospecção de genes envolvidos na desconstrução da biomassa

Pós-graduação em Microbiologia Agropecuária - FCAV

Bioactive compounds and phenolic-linked functionality of powdered tropical fruit residues

Tropical fruit residues consisting of seeds, peels and residual pulp generated as by-products of fruit processing industry were investigated for bioactive compounds, the in vitro antioxidant capacity as well as alpha-glucosidase and alpha-amylase inhibitory activities. Cyanidin, quercetin, ellagic acid (EA) and proanthocyanidins were found in acerola, jambolan, pitanga and caja-umbu residue powders. Acerola powder had the highest phenolic content (8839.33 mg catechin equivalents (CE)/100 g) and also high-ascorbic acid (AA) concentration (2748.03 mg/100 g), followed by jambolan and pitanga. The greatest 1,1-Diphenyl-2-picrylhydrazyl (DPPH) inhibition was observed for jambolan (436.76 mmol Trolox eq/g) followed by pitanga (206.68 mmol Trolox eq/g) and acerola (192.60 mmol Trolox eq/g), while acerola had the highest ferric reducing antioxidant power (FRAP) assay result (7.87 mmol Trolox eq/g). All fruit powders exhibited enzymatic inhibition against alpha-amylase (IC50 ranging from 3.40 to 49.5 mg CE/mL) and alpha-glucosidase (IC50 ranging from 1.15 to 2.37 mg CE/mL). Therefore, acerola, jambolan and pitanga dried residues are promising natural ingredients for food and nutraceutical manufacturers, due to their rich bioactive compound content.

Transcriptional response to GAA deficiency (Pompe disease) in infantile-onset patients

Pompe disease is a genetic disorder resulting from a deficiency of lysosomal acid alpha-glucosidase (GAA) that manifests as a clinical spectrum with regard to symptom severity and rate of progression. In this study, we used microarrays to examine gene expression from the muscle of two cohorts of infantile-onset Pompe patients to identify transcriptional differences that may contribute to the disease phenotype. We found strong similarities among the gene expression profiles generated from biceps and quadriceps, and identified a number of signaling pathways altered in both cohorts. We also found that infantile-onset Pompe patient muscle had a gene expression pattern characteristic of immature or regenerating muscle, and exhibited many transcriptional markers of inflammation, despite having few overt signs of inflammatory infiltrate. Further, we identified genes exhibiting correlation between expression at baseline and response to therapy. This combined dataset can serve as a foundation for biological discovery and biomarker development to improve the treatment of Pompe disease. (C) 2012 Elsevier Inc. All rights reserved.

Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae)

Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 mu M); CAL2 (17 kDa, pHo 5.5, km 11 mu M Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM L-Leucine-p-nitroanilide), alpha-glucosidase (90 kDa, pHo 5.0, km 5 mM with p-nitrophenyl alpha-D-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans. (C) 2012 Elsevier Ltd. All rights reserved.

Luminal substrate "brake" on mucosal maltase-glucoamylase activity regulates total rate of starch digestion to glucose

BACKGROUND: Starches are the major source of dietary glucose in weaned children and adults. However, small intestine alpha-glucogenesis by starch digestion is poorly understood due to substrate structural and chemical complexity, as well as the multiplicity of participating enzymes. Our objective was dissection of luminal and mucosal alpha-glucosidase activities participating in digestion of the soluble starch product maltodextrin (MDx). PATIENTS AND METHODS: Immunoprecipitated assays were performed on biopsy specimens and isolated enterocytes with MDx substrate. RESULTS: Mucosal sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM) contributed 85% of total in vitro alpha-glucogenesis. Recombinant human pancreatic alpha-amylase alone contributed <15% of in vitro alpha-glucogenesis; however, alpha-amylase strongly amplified the mucosal alpha-glucogenic activities by preprocessing of starch to short glucose oligomer substrates. At low glucose oligomer concentrations, MGAM was 10 times more active than SI, but at higher concentrations it experienced substrate inhibition whereas SI was not affected. The in vitro results indicated that MGAM activity is inhibited by alpha-amylase digested starch product "brake" and contributes only 20% of mucosal alpha-glucogenic activity. SI contributes most of the alpha-glucogenic activity at higher oligomer substrate concentrations. CONCLUSIONS: MGAM primes and SI activity sustains and constrains prandial alpha-glucogenesis from starch oligomers at approximately 5% of the uninhibited rate. This coupled mucosal mechanism may contribute to highly efficient glucogenesis from low-starch diets and play a role in meeting the high requirement for glucose during children's brain maturation. The brake could play a constraining role on rates of glucose production from higher-starch diets consumed by an older population at risk for degenerative metabolic disorders.

[Oral hypoglycaemic agents in 2009]

Various new oral hypoglycaemic agents have been developed recently and have changed the therapy of type 2 diabetes mellitus. Six different classes of agents are available: Biguanides, sulfonylureas, glinides, glitazones, alpha-glucosidase inhibitors and dipeptidyl peptidase-4 inhibitors. The increasing number of these drugs does not facilitate the choice of the best medication for an individual patient. In the article we describe the specific mechanisms of action, side effects, advantages and disadvantages of the different agents. Every drug therapy should be supported by lifestyle changes. Despite all the new drugs type 2 diabetes is still a chronic and slowly progressive disease without chance of cure. Therefore, it is important to prevent type 2 diabetes by normalizing body weight and increasing physical exercise.

Swiss national guideline for reimbursement of enzyme replacement therapy in late-onset Pompe disease.

Glycogen storage disease type II is a rare multi-systemic disorder characterised by an intracellular accumulation of glycogen due a mutation in the acid alpha glucosidase (GAA) gene. The level of residual enzyme activity, the genotype and other yet unknown factors account for the broad variation of the clinical phenotype. The classical infantile form is characterised by severe muscle hypotonia and cardiomyopathy leading to early death. The late-onset form presents as a limb girdle myopathy with or without pulmonary dysfunction. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) in infants is life saving. In contrast, therapeutic efficacy of rhGAA in the late-onset form is modest. High expenses of rhGAA, on-going infusions and poor pharmacokinetic efficacy raised a discussion of the cost effectiveness of ERT in late-onset Pompe disease in Switzerland. This discussion was triggered by a Swiss federal court ruling which confirmed the reluctance of a health care insurer not to reimburse treatment costs in a 67-year-old female suffering from Pompe disease. As a consequence of this judgement ERT was stopped by all insurance companies in late-onset Pompe patients in Switzerland regardless of their clinical condition. Subsequent negotiations lead to the release of a national guideline of the management of late-onset Pompe disease. Initiation and limitation of ERT is outlined in a national Pompe registry. Reimbursement criteria are defined and individual efficacy of ERT with rhGAA is continuously monitored.