Digestive morphophysiology of Gryllodes sigillatus (Orthoptera: Gryllidae)

The evolution of the digestive system in the Order Orthoptera is disclosed from the study of the morphophysiology of the digestive process in its major taxa. This paper deals with a cricket representing the less known suborder Ensifera Most amylase and trypsin activities occur in crop and caeca. respectively. Maltase and aminopeptidase are found in soluble and membrane-bound forms in caeca, with aminopeptidase also occurring in ventriculus. Amaranth was orally fed to Gryllodes sigillatus adults or injected into their haemolymph. The experiments were performed with starving and feeding insects with identical results. Following feeding of the dye the luminal side of the most anterior ventriculus (and in lesser amounts the midgut caeca) became heavily stained. In injected insects, the haemal side of the most posterior ventriculus was stained This suggested that the anterior ventriculus is the main site of water absorption (the caeca is a secondary one). whereas the posterior ventriculus secretes water into the gut. Thus, a putative counter-current flux of fluid from posterior to anterior ventriculus may propel digestive enzyme recycling. This was confirmed by the finding that digestive enzymes are excreted at a low rate. The fine structure of midgut caeca and ventriculus cells revealed that they have morphological features that may be related to their involvement in secretion (movement from cell to lumen) and absorption (movement from lumen to cell) of fluids. Furthermore, morphological data showed that both merocrine and apocrine secretory mechanisms occur in midgut cells. The results showed that cricket digestion differs from that in grasshopper in having (1) more membrane-bound digestive enzymes; (2) protein digestion slightly displaced toward the ventriculus; (3) midgut fluxes, and hence digestive enzyme recycling, in both starved and fed insects. (C) 2009 Elsevier Ltd. All rights reserved.

Metabolic compartmentalization in the human cortex and hippocampus: evidence for a cell- and region-specific localization of lactate dehydrogenase 5 and pyruvate dehydrogenase.

BACKGROUND: For a long time now, glucose has been thought to be the main, if not the sole substrate for brain energy metabolism. Recent data nevertheless suggest that other molecules, such as monocarboxylates (lactate and pyruvate mainly) could be suitable substrates. Although monocarboxylates poorly cross the blood brain barrier (BBB), such substrates could replace glucose if produced locally.The two key enzymatiques systems required for the production of these monocarboxylates are lactate dehydrogenase (LDH; EC1.1.1.27) that catalyses the interconversion of lactate and pyruvate and the pyruvate dehydrogenase complex that irreversibly funnels pyruvate towards the mitochondrial TCA and oxydative phosphorylation. RESULTS: In this article, we show, with monoclonal antibodies applied to post-mortem human brain tissues, that the typically glycolytic isoenzyme of lactate dehydrogenase (LDH-5; also called LDHA or LDHM) is selectively present in astrocytes, and not in neurons, whereas pyruvate dehydrogenase (PDH) is mainly detected in neurons and barely in astrocytes. At the regional level, the distribution of the LDH-5 immunoreactive astrocytes is laminar and corresponds to regions of maximal 2-deoxyglucose uptake in the occipital cortex and hippocampus. In hippocampus, we observed that the distribution of the oxidative enzyme PDH was enriched in the neurons of the stratum pyramidale and stratum granulosum of CA1 through CA4, whereas the glycolytic enzyme LDH-5 was enriched in astrocytes of the stratum moleculare, the alveus and the white matter, revealing not only cellular, but also regional, selective distributions. The fact that LDH-5 immunoreactivity was high in astrocytes and occurred in regions where the highest uptake of 2-deoxyglucose was observed suggests that glucose uptake followed by lactate production may principally occur in these regions. CONCLUSION: These observations reveal a metabolic segregation, not only at the cellular but also at the regional level, that support the notion of metabolic compartmentalization between astrocytes and neurons, whereby lactate produced by astrocytes could be oxidized by neurons.

Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg(-) L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg(-) mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (arg Delta SKL) restored growth but failed to restore infectivity. Further study showed that the ARG Delta SKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration.

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.

A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.

Reduced Insulin Clearance And Lower Insulin-degrading Enzyme Expression In The Liver Might Contribute To The Thrifty Phenotype Of Protein-restricted Mice.

Nutrient restriction during the early stages of life usually leads to alterations in glucose homeostasis, mainly insulin secretion and sensitivity, increasing the risk of metabolic disorders in adulthood. Despite growing evidence regarding the importance of insulin clearance during glucose homeostasis in health and disease, no information exists about this process in malnourished animals. Thus, in the present study, we aimed to determine the effect of a nutrient-restricted diet on insulin clearance using a model in which 30-d-old C57BL/6 mice were exposed to a protein-restricted diet for 14 weeks. After this period, we evaluated many metabolic variables and extracted pancreatic islet, liver, gastrocnemius muscle (GCK) and white adipose tissue samples from the control (normal-protein diet) and restricted (low-protein diet, LP) mice. Insulin concentrations were determined using RIA and protein expression and phosphorylation by Western blot analysis. The LP mice exhibited lower body weight, glycaemia, and insulinaemia, increased glucose tolerance and altered insulin dynamics after the glucose challenge. The improved glucose tolerance could partially be explained by an increase in insulin sensitivity through the phosphorylation of the insulin receptor/protein kinase B and AMP-activated protein kinase/acetyl-CoA carboxylase in the liver, whereas the changes in insulin dynamics could be attributed to reduced insulin secretion coupled with reduced insulin clearance and lower insulin-degrading enzyme (IDE) expression in the liver and GCK. In summary, protein-restricted mice not only produce and secrete less insulin, but also remove and degrade less insulin. This phenomenon has the double benefit of sparing insulin while prolonging and potentiating its effects, probably due to the lower expression of IDE in the liver, possibly with long-term consequences.

The Characterization Of The Endoglucanase Cel12a From Gloeophyllum Trabeum Reveals An Enzyme Highly Active On β-glucan.

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on β-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

Immobilization Of Papain On Chitin And Chitosan And Recycling Of Soluble Enzyme For Deflocculation Of Saccharomyces Cerevisiae From Bioethanol Distilleries.

Yeast flocculation (Saccharomyces cerevisiae) is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1-10% w·v(-1)), polyethyleneimine (0.5% v·v(-1)), and tripolyphosphate (1-10% w·v(-1)) inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L(-1). Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes.

Análise do perfil de humor e da enzima alfa-amilase salivar em indivíduos fisicamente ativos = : Analysis of profile of mood states and salivary alpha amylase enzyme in physically active individuals

Universidade Estadual de Campinas . Faculdade de Educação Física

Bleaching of melanin in the epidermis of South American fur seal and its application on enzyme immunohistochemistry

The South American fur seal (Arctocephalus australis) is an amphibious marine mammal distributed along the Atlantic and Pacific coasts of South America. The species is well adjusted to different habitats due to the morphology of its fin-like members and due to some adaptations in their integumentary system. Immunohistochemical studies are very important to evaluate the mechanisms of skin adaptation due the differential expression of the antigens present in the tissue depending of the region of the body surface. However, its strongly pigmented (melanin) epidermis prevents the visualization of the immuno-histochemical chromogens markers. In this study a melanin bleaching method was developed aimed to allow the visualization of the chromogens without interfering in the antigen-antibody affinity for immunohistochemistry. The analysis of PCNA (proliferating cell nuclear antigen) index in the epidermis of A. australis by immunohistochemistry with diaminobenzidine (DAB) as chromogen was used to test the method. The bleaching of the melanin allowed to obtain the cell proliferation index in epidermis and to avoid false positive results without affecting the immunohistochemical results.

Reduced cortical renal GLUT1 expression induced by angiotensin-converting enzyme inhibition in diabetic spontaneously hypertensive rats

Diabetes in spontaneously hypertensive rats is associated with cortical renal GLUT1 and GLUT2 overexpression. Our objective was to evaluate the effect of the angiotensin-converting enzyme blockade on cortical renal GLUT1 and GLUT2 expression, urinary albumin and urinary TGF-β1. Streptozotocin, 50 mg/kg, or citrate buffer (N = 16) was administered as a single injection into the tail vein in adult spontaneously hypertensive rats (~260 g). Thirty days later, these diabetic spontaneously hypertensive rats received ramipril by gavage: 0.01 mg·kg-1·day-1 (D0.01, N = 14), 1 mg·kg-1·day-1 (D1, N = 9) or water (D, N = 11) for 15 days. Albumin and TGF-β1 (24-h urine), direct arterial pressure, renal tissue angiotensin-converting enzyme activity (fluorometric assay), and GLUT1 and GLUT2 protein levels (Western blot, renal cortex) were determined. Glycemia and glycosuria were higher (P < 0.05) in the diabetic rats compared with controls, but similar between the diabetic groups. Diabetes in spontaneously hypertensive rats lowered renal tissue angiotensin-converting enzyme activity (40%), which was reduced further when higher ramipril doses were used. Diabetes associated with hypertension raised GLUT1 by 28% (P < 0.0001) and GLUT2 by 76% (P = 0.01), and both doses of ramipril equally reduced cortical GLUT1 (D vs D1 and vs D0.01, P ≤ 0.001). GLUT2 levels were reduced in D0.01 (P < 0.05 vs D). Diabetes increased urinary albumin and TGF-β1 urinary excretion, but the 15-day ramipril treatment (with either dose) did not reduce them. In conclusion, ramipril is effective in lowering renal tissue angiotensin-converting enzyme activity, as well as blocking cortical GLUT1 overexpression, which may be beneficial in arresting the development of diabetic nephropathy.

Enzyme kinetics, structural analysis and molecular modeling studies on a series of Schistosoma mansoni PNP inhibitors

The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive molecular target for the development of novel drugs against schistosomiasis, a neglected tropical disease that affects about 200 million people worldwide. In the present work, enzyme kinetic studies were carried out in order to determine the potency and mechanism of inhibition of a series of SmPNP inhibitors. In addition to the biochemical investigations, crystallographic and molecular modeling studies revealed important molecular features for binding affinity towards the target enzyme, leading to the development of structure-activity relationships (SAR).

Screening of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase enzyme inhibitors

The inhibitory activity of crude extracts of Meliaceae and Rutaceae plants on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) enzyme from Trypanosoma cruzi was evaluated at 100 μg/mL. Forty-six extracts were tested and fifteen of them showed significant inhibitory activity (IA % > 50). The majority of the assayed extracts of Meliaceae plants (Cedrela fissilis, Cipadessa fruticosa and Trichilia ramalhoi) showed high ability to inhibit the enzymatic activity. The fractionation of the hexane extract from branches of C. fruticosa led to the isolation of three flavonoids: flavone, 7-methoxyflavone and 3',4',5',5,7-pentamethoxyflavone. The two last compounds showed high ability to inhibit the gGAPDH activity. Therefore, the assayed Meliaceae species could be considered as a promising source of lead compounds against Chagas' disease.

Biotin/avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns.

Differentiation of Bovine coronavirus (BCoV) genotypes by a restriction enzyme assay

This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation.