Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Disruption of the peritrophic matrix by exogenous chitinase feeding reduces fecundity in Lutzomyia longipalpis females

Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.

Combined effect of three insect growth regulators on the digestive enzymatic profiles of callosobruchus maculatus [coleoptera: bruchidae]

Insect growth regulators [IGRs] are insecticides that mimic insect produced hormones by regulating developmental process. They have little or no mammalian toxicity, and are considered reduced-risk insecticides that are often exempt from tolerance requirements of regulatory agencies. IGRs, especially, chlorfluazuron, hydroprene and hexaflumuron [benzoylphenylurea] are currently studied because of possibility of using in stored products protection. Many of IGRs compounds used in insect pests control are known to affect digestive enzymes. Chlorfluazuron, hydroprene and hexaflumuron were tested topically at doses of 0.25%, 0.5% and1% for chlorfluazuron and hydroprene and 0.5, 1 and 2microg/ml of hexaflumuron to investigate its effects on the activities of the digestive enzymes protease, amylase and lipase in Callosobruchusmaculatus larvae, which were affected by IGRs individually and in combination. When combined, the effect was more severe at low concentration. There were statistically significant differences [P

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (ì = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (ì = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

comparative study on the inhibitory trend of triphenyltin chloride on the activity of digestive enzymes of two insects

Organotin compounds are widely used mostly as antifeedant. However, in the present investigation the inhibitory effects of one of the organotin compound [Triphenyltin Chloride] on the digestive enzymes of Hieroglyplus nigrorepletus and Leptocorisa varicornis have been studied. It was found [that .05% to 0.10% concentrations inhibited invertase and amylase enzymes of both the insects in progressive manner. The inhibition in the activity of both the enzymes was more in H. nigrorepletus than L. varicornis i.e. 55% and 61% and 44 and 46%, which suggest that this chemical may be useful for the control of pests of agricultural crops

Purification of elastase-like chymotrypsin from cardamom shoot and Capsule borer [corrected]

An elastase-like chymotrypsin was purified by aprotinin-agarose affinity chromatography from the midgut extract of cardamom shoot and capsule borer, Conogethes punctiferalis. The purified enzyme had a Vmax of 687.6 +/- 22.1 nmole pNA released/min/mg protein, Km of 0.168 +/- 0.012 mM with SAAPLpNA as substrate and gave a single band on SDS-PAGE with a molecular mass of 72.1 kDa. Casein zymogram revealed one clear zone of proteolytic activity, which corresponded to the band obtained with SDS-PAGE indicating that this could be a single-polypeptide enzyme.

Cloning and characterization of a V-ATPase subunit C from the American visceral leishmaniasis vector Lutzomyia longipalpis modulated during development and blood ingestion

Visceral leishmaniasis (VL) is a serious tropical disease that affects approximately 500 thousand people worldwide every year. In the Americas, VL is caused by the parasite Leishmania (Leishmania) infantum chagasi mainly transmitted by the bite of the sand fly vector Lutzomyia longipalpis. Despite recent advances in the study of interaction between Leishmania and sand flies, very little is known about sand fly protein expression profiles. Understanding how the expression of proteins may be affected by blood feeding and/or presence of parasite in the vector's midgut might allow us to devise new strategies for controlling the spread of leishmaniasis. In this work, we report the characterization of a vacuolar ATPase subunit C from L. longipalpis by screening of a midgut cDNA library with a 220 bp fragment identified by means of differential display reverse transcriptase-polymerase chain reaction analysis. The expression of the gene varies along insect development and is upregulated in males and bloodfed L. longipalpis, compared to unfed flies.

Development and mechanisms of resistance to Bacillus thuringiensis endotoxin Cry1Ac in the American bollworm, Helicoverpa armigera (Hübner).

The American bollworm, H. armigera, evolved 31-fold resistance to selection pressure of B. thuringiensis endotoxin Cry1Ac within six generations. The Cry1Ac selected larvae of H. armigera showed cross-resistance to Cry1Aa and Cry1Ab both in terms of mortality and growth reduction. Studies on mechanisms of resistance to Cry1Ac showed that proteases of resistant insects degraded Cry1Ac faster than those of susceptible insects, which led to the relative unavailability of toxin of about 58 kDa for binding and perforation of midgut epithelial membrane of the target insect. Besides, resistant and susceptible populations of H. armigera differed in the binding of their receptors with Cry1Ac toxin. These results suggest the possibility of both mechanisms existing in imparting resistance. These findings mandate the necessity of B. thuringiensis resistance management for usage of B. thuringiensis either as a conventional insecticide or through transgenic crops.

Effect of a non-hormonal feed additive, Cholymbi on growth, body composition and digestive enzyme activity of common carp, Cyprinus carpio.

Supplementation of pelleted feeds (30% crude protein) consisting of practical ingredients with 250, 375 and 500 mg Cholymbi/kg diet resulted significantly improved the growth of common carp, Cyprinus carpio. Highest final average weight was recorded with diet containing 500 mg Cholymbi/kg. Feeding Cholymbi to fish showed increased carcass protein and fat. Gut protease and hepatopancreas amylase activity were the highest with 500 mg Cholymbi/kg incorporated diet (T3). The results of the present study suggest that Cholymbi, a non-hormonal growth promoter could be effectively used in the diet of common carp. Further investigations are required to work out the optimal dosage required in the diet of common carp.

Effect of formulated diet on digestive enzymes of Labeo rohita (Ham.).

Six sets of feeding experiments were carried out using formulated diets containing prawn head waste (PW), chicken intestine waste (CW), banana flower (BF), cauliflower waste (CAU) Dolicos lab lab (DLL) and groundnut leaf (GNL) in four levels of inclusion (15, 30, 45 and 60%) to assess the pattern of distribution and activities of digestive enzymes like cellulase, amylase, maltase, invertase, protease and lipase in the digestive tracts of Labeo rohita fingerlings. A control group of fish was fed with diets containing antibiotics to destroy the digestive tract microflora which may induce digestive functions. In general, the activity of digestive enzymes depended on the amount and type of the ingredients present in the diets ingested by the fish. Test animals showed both endogenous and bacterial cellulase activities which suggests the necessity for including cellulose (plant protein source) as dietary ingredient. Occurrence of higher amount of cellulase in the foregut and amylase in the fore and midgut influenced by DNL and GNL diets revealed the possibility of including less than 40% of the respective ingredients in the diet of rohu. Maltase and invertase were highly influenced by GNL, DLL and BF diets than PW and CW diets. More than 40% inclusion of PW and CW was found to increase protease and lipase secretion in the midgut and hindgut regions. The higher secretion of lipase in the midgut suggested the physiological versatility for lipid digestion in rohu fingerlings.

Induction of some digestive enzymes in the midgut of the sandfly phlebotomus langeroni after sugar and blood meals

Changes associated with blood and sugar meals digestion in the sandfly, Phlebotomus langeroni were characterized. Different types of sugars [sucrose, glucose, melibiose, cellobiose, lactose, starch, fig fruits, honey dew and a mixture of sources and sugar sources] were used for the sandfly feeding. Activities of glycosidases and proteases in the sandfly guts after blood and sugar meals were determined using the endpoint method. The results showed that glycosidases [alpha- glycosidase, beta-glycosidase, alpha-galactosidase and beta galactosidase] are present in the sandfly midguts. No activities of the glycosidases [alpha-mannosidase and alpha-amylase] were detected in the sandfly gut. Proteases [trypsin and aminopeptidase] showed activities in the sandfly midguts. It was concluded that the midgut glycosidase may play an important role in the vector-parasite interaction. Trypsin and aminopeptidase induction after a blood meal is controlled by a secretogogues mechanism which indirectly influences the outcome of the Leishmania parasite infection

Enzymes--a review.

Enzymes are essential to life and catalyze various metabolic activities. They find application in a number of diseased conditions, and their scope is being widened as knowledge develops.

Protective effects of antioxidant enzymes and GSH in vivo on iron mediated lipid peroxidation in gastrointestinal tract of rat.

The protective mechanisms operating in the gastrointestinal (GI) tract to counteract the potential oxidizing effects of excess free iron, was tested in rats fed with excess iron. The activities of some antioxidant enzymes, the levels of GSH and the extent of lipid peroxidation at the site of iron absorption were measured. Based on the amount of thiobarbituric acid reactive substances (TBARS) produced, it could be deduced that the duodenal segment of GI tract is resistant to iron mediated lipid peroxidation. The duodenal function as judged from the activities of marker enzymes, namely, alkaline phosphatase and Lys-Ala-dipeptidyl aminopeptidase was normal. There was depletion of GSH possibly due to the increased activities of Cu, Zn SOD and catalase. However, the activity of Gpx was decreased in the Fe fed group. It was also observed that the ratios of SOD/Gpx and Cat/Gpx had significantly increased in the treated group whereas SOD/Cat remained constant suggesting that antioxidative enzymes play a key role in rendering the intestinal mucosal cells resistant to iron induced oxidative damage in rats.

Má-digestäo e má-absorçäo de carboidratos na infância / Poor carbohidrate digestion and malabsorption in childhood

É apresentada uma extensa revisao abordando aspectos gerais, da fisiopatologia, do diagnóstico e do tratamento dos problemas clínicos decorrentes de alteraçoes na digestao e absorçao dos carboidratos.

Distribution of digestive enzymes in gastrointestinal tract of young rhesus monkeys.

The distribution of different mucosal digestive enzymes along the gastrointestinal tract of young rhesus monkeys were studied by biochemical analysis of homogenized specimen from 13 different segments (i.e. stomach-fundus, body, antrum; small intestine-D1, D2, proximal, middle, distal; caecum; colon-ascending, transverse, descending and rectum), obtained from 6 monkeys of approximate age 8-12 months. The activity of all the enzymes was lowest in gastric mucosa rising through duodenum to peak in small intestine and declining towards the large bowel. There was no significant difference in levels of any of the enzymes between different parts of stomach or large bowel. This study suggests that like humans in rhesus monkeys maximum activity of digestive enzymes is present in small intestine and much less in gastric and large bowel mucosa.