Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

Purpose: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. Methods: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c. 3277C>T, a nonsense mutation, and c. 3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. Results: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. Conclusions: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

Leishmania amazonensis: Xylitol as inhibitor of macrophage infection and stimulator of macrophage nitric oxide production

Xylitol is a sugar alcohol being explored for clinical uses. The aim was to evaluate the effects of xylitol on Leishmania amazonensis-infected J774A.1 macrophages. Macrophages were infected with L. amazonensis for 3 It, washed and incubated with 2.5 or 5.0% xylitol for 24, 48, and 72 h at 37 degrees C. Infection indexes for macrophages incubated only in medium were compared to those treated with xylitol. Cell viability and nitric oxide production were determined each time. Xylitol did not affect L. amazonensis or J774A.1 cell viabilities. Xylitol at 5.0% stimulated nitric oxide production by macrophages at 72 h (p < 0.01). At 2.5 and 5.0%, xylitol inhibited nitric oxide production by L. amazonensis at 48 h. (p < 0.05) when compared to control. Infection indexes were significantly lower at 72 h (P < 0.05), (16.9% and 9.6%) in cells cultivated with 2.5 and 5.0% xylitol, respectively, compared to control (38.4%). Results suggest a potential leishmanicidal action of the xylitol on infected macropliages. (C) 2008 Elsevier Inc. All rights reserved.

Immobilization and stabilization of microbial lipases by multipoint covalent attachment on aldehyde-resin affinity: Application of the biocatalysts in biodiesel synthesis

Microbial lipase preparations from Thermomyces lanuginosus (TLL) and Pseudomonas fluorescens (PFL) were immobilized by multipoint covalent attachment on Toyopearl AF-amino-650M resin and the most active and thermal stable derivatives used to catalyze the transesterificanon reaction of babassu and palm oils with ethanol in solvent-free media For this different activating agents mainly glutaraldehyde glycidol and epichlorohydrin were used and immobilization parameters were estimated based on the hydrolysis of olive oil emulsion and butyl butyrate synthesis ILL immobilized on glyoxyl-resin allowed obtaining derivatives with the highest hydrolytic activity (HA(der)) and thermal stability between 27 and 31 times more stable than the soluble lipase Although PFL derivatives were found to be less active and thermally stables similar formation of butyl butyrate concentrations were found for both ILL and PFL derivatives The highest conversion into biodiesel was found in the transesterification of palm oil catalyzed by both ILL and PFL glyoxyl-derivatives (c) 2010 Elsevier B V All rights reserved

Effect of Antioxidants and Corrosion Inhibitor Additives on the Quenching Performance of Soybean Oil

Cooling curve analysis was used to evaluate the effect of corrosion inhibitor additives and antioxidants on the quenching properties of soybean oil. The results showed that addition of corrosion inhibitors provided significant changes in the cooling curve behavior and of the yellow metal corrosion inhibitors evaluated tolyltriazole exhibits the greatest rate acceleration of heat transfer. However, the presence of antioxidants did not exhibit a significant effect on quenching properties of soybean oil. (C)2010 Journal of Mechanical Engineering. All rights reserved.

Dodigen 213-N as corrosion inhibitor for ASTM 1010 mild steel in 10% HCL

This article describes a study of the behavior of a mixture of amines and amides, commercially known as Dodigen 213-N (D-213 N), as a corrosion inhibitor for ASTM 1010 mild steel in 10% w/w HCl solution. The concentration range used was 1 x 10(-5) M to 8 x 10(-4) M. The weight loss and electrochemical techniques used were corrosion potential measurement, anodic and cathodic polarization curves, and electrochemical impedance spectroscopy (EIS). The solution temperature was 50 +/- A 1 A degrees C and it was naturally aerated. The corrosion potential values shifted to slightly more positive values, thus indicating mixed inhibitor behavior. The anodic and cathodic polarization curves showed that D-213 N is an effective corrosion inhibitor, since both the anodic and the cathodic reactions were polarized in comparison with those obtained without inhibitor. For all concentrations the cathodic polarization curves were more polarized than the anodic ones. The inhibition efficiency was in the range 75-98%, calculated from values of weight loss and corrosion current density, i (corr), obtained by extrapolation of Tafel cathodic linear region.

Adenanthera pavonina TRYPSIN INHIBITOR RETARD GROWTH OF Anagasta kuehniella (LEPIDOPTERA: PYRALIDAE)

Anagasta kuehniella is a polyphagous pest that feeds on a wide variety of stored products. The possible roles suggested for seed proteinase inhibitors include the function as a part of the plant defensive system against pest via inhibition of their proteolytic enzymes. In this study, a trypsin inhibitor (ApTI) was purified from Adenanthera pavonina seed and was tested for insect growth regulatory effect. The chronic ingestion of ApTI did result in a significant reduction in larval survival and weight. Larval and pupal developmental time of larvae fed on ApTI diet at 1% was significantly longer; the larval period was extended by 5 days and pupal period was 10 days longer, therefore delaying by up to 20 days and resulting in a prolonged period of development from larva to adult. As a result, the ApTI diet emergence rate was only 28% while the emergence rate of control larvae was 80%. The percentage of surviving adults (%S) decreased to 62%. The fourth instar larvae reared on a diet containing 1% ApTI showed a decrease in tryptic activity of gut and that no novel proteolytic form resistant to ApTI was induced. In addition, the tryptic activity in ApTI -fed larvae was sensitive to ApTI. These results suggest that ApTI have a potential antimetabolic effect when ingested by A. kuehniella. (C) 2010 Wiley Periodicals, Inc.

Regulatory effects of an inhibitor from Plathymenia foliolosa seeds on the larval development of Anagasta kuehniella (Lepidoptera)

The Mediterranean flour moth, Anagasta kuehniella, is one of the most important insect pests of grains, reported worldwide, feeding on stored grains and products of rice, rye, corn and wheat. Plants synthesize a variety of molecules, including trypsin inhibitors, to defend themselves against attack by insects. In this study, a trypsin inhibitor (PFTI) was purified from Plathymenia foliolosa (Benth.) seeds and was tested for insect growth regulatory effect. The survival and mass of A. kuehniella larvae feeding on control seeds were about 82.7% and 5 ring, respectively, whereas survival on seeds containing 0.7% PFTI was about 56%, while a 66.1% reduction in the average mass of the larvae was observed. The results from dietary utilization experiments with A. kuehniella larvae showed a reduction in efficiency of conversion of ingested food and digested food, and an increase in approximate digestibility and metabolic cost. The level of trypsin was significantly decreased in larval midgut and increased in the feces of larvae reared on a diet containing 0.7% PFTI. Results indicate that PFTI possesses a toxic effect against A. kuehniella larvae. (C) 2008 Elsevier Inc. All rights reserved.

Purification and Characterization of a Trypsin Inhibitor from Plathymenia foliolosa Seeds

A novel trypsin inhibitor (PFTI) was isolated from Plathymenia foliolosa (Benth.) seeds by gel filtration chromatography on a Sephadex G-100, DEAE-Sepharose, and trypsin-Sepharose columns. By SDS-PAGE, PFTI yielded a single band with a M(r) of 19 kDa. PFTI inhibited bovine trypsin and bovine chymotrypsin with equilibrium dissociation constants (K(i)) of 4 x 10(-8) and 1.4 x 10(-6) M, respectively. PFTI retained more than 50% of activity at up to 50 degrees C for 30 min, but there were 80 and 100% losses of activity at 60 and 70 degrees C, respectively. DTT affected the activity or stability of PFTI. The N-terminal amino acid sequence of PFTI showed a high degree of homology with various members of the Kunitz family of inhibitors. Anagasta kuehniella is found worldwide; this insect attacks stored grains and products of rice, oat, rye, corn, and wheat. The velvet bean caterpillar (Anticarsia gemmatalis) is considered the main defoliator pest of soybean in Brazil. Diatraea saccharalis, the sugar cane borer, is the major pest of sugar cane crops, and its caterpillar-feeding behavior, inside the stems, hampers control. PFTI showed significant inhibitory activity against trypsin-like proteases present in the larval midguts on A. kuehniella and D. saccharalis and could suppress the growth of larvae.

Apparent digestibility coefficients of selected feed ingredients for dourado Salminus brasiliensis

The excellence of its flesh and fast growth makes the dourado, Salminus brasiliensis, a carnivorous fish native to the Prata basin, a potential candidate for intensive fish farming. This study evaluated the apparent digestibility coefficients (ADC) of energy and nutrients of animal and plant protein sources for the carnivorous Characin dourado S. brasiliensis. Fish (19.5 +/- 5.0 g) were stocked in plastic cages (80-L) and fed pelleted test diets containing 30% of the test ingredient [fish meal (FM), poultry by-product meal (PBM), soybean meal (SBM), and corn gluten meal (CGM) plus 70% of a reference diet (481.4 g kg(-1) of crude protein and 18.63 kJ of gross energy per gram]. After the last daily meal, cages were transferred to cylindrical, conical-bottomed aquarium (200-L) where faeces were collected by sedimentation in a refrigerated container. Except for ADC of protein and energy, all other ADC of nutrients showed significant differences (P < 0.01). ADC values were: 94.3%, 91.3%, 93.1%, and 93.5% for crude protein; 91.0%, 90.3%, 87.8%, and 88.8% for gross energy; 92.1%, 84.5%, 80.6%, and 79.3% for ash; 83.9%, 80.3%, 84.3%, and 84.6% for dry matter; 97.4%, 96.7%, 93.3%, and 91.5% for lipid for FM, PBM, SBM, and CGM, respectively. The average amino acid ADC was: 93.6%, 90.0%, 92.1%, and 92.5% of FM, PBM, SBM, and CGM, respectively. All test ingredients were efficiently used and can partially replace FM in diets for carnivorous dourado.

Apparent digestibility coefficient of protein and amino acids of acid, biological and enzymatic silage for Nile tilapia (Oreochromis niloticus)

Using the fish silage to partially replace proteic feedstuff in aquafeeds is an alternative to mitigate sanitary and environmental problems caused by the lack of adequate destination for fisheries residues. It would also lower feed costs, consequently improving fish culture profitability. However, using fish silages in aquafeeds depends on determination of its apparent digestibility coefficients (ADC). This work aimed to determining the ADC of crude protein and amino acids of acid silage (AS), biological silage (BS) and enzymatic silage (ES) for juvenile Nile tilapia (94.5 +/- 12.7 g). The ADC(CP) was: 92.0%, 89.1% and 93.7% for AS, BS and SE respectively. The average ADC of amino acids was: 91.8%, 90.8% and 94.6% for AS, BS and ES respectively. Results encourage the use of AS, BS and ES to partially replace protein sources in balanced diets for neotropical fish.

An Apparent Paradox: Attenuation of Phenylephrine-mediated Calcium Mobilization and Hyperreactivity to Phenylephrine in Contralateral Carotid After Balloon Injury

Balloon catheter injury promotes hyperreactivity to phenylephrine (Phe) in the contralateral carotid. Phe-induced contraction involves calcium mobilization, a process that may be sensitive to reactive oxygen species. In this study, we investigated whether increased reactivity to Phe in the contralateral carotid is due to alterations in calcium mobilization by Phe and reactive oxygen species signaling. Concentration-response curves to Phe were obtained in control and contralateral arteries 4 days after balloon injury. Tiron did not modify E(max) to Phe in control arteries but reduced this parameter in the contralateral carotid to control levels. Moreover, immunofluorescence to dihydroethydine showed increased basal oxidative stress in the contralateral artery compared with control artery. Intracellular calcium mobilization by Phe in the contralateral artery was not different from control, but Phe-induced extracellular calcium mobilization was reduced in the contralateral artery compared with that in the control. These data were confirmed by confocal microscopy using Fluo 3-AM. Tiron and SC-236 increased Phe-induced calcium influx in the contralateral artery, which was similar to controls in the same conditions. However, catalase did not modify this response. Taken together, our results suggest that superoxide anions and prostanoids from cyclooxygenase-2 alter pathways downstream of alpha(1)-adrenoceptor activation in the contralateral carotid in response to injury. This results in reduced Phe-induced calcium influx, despite hyperreactivity to Phe.

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N-acetyl-beta-D-glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa. which is purified by affinity, with immobilized N-acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels or TNF alpha. and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-1,4-homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin-affinity purification or paracoccin. This procedure provided higher yields than those achieved by means of the technique based oil the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS-PAGE and Western blot analysis revealed similarities between the N-acetylglucosamine- and chitin-bound fractions, confirmed by MALDI-TOF-MS of trypsinic peptides. Western blot of two-dimensional gel electrophoresis of the yeast extract showed a major spot with M(r) 70000 and pl approximately 5.63. Moreover, an N-acetyl-beta-D-glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright (C) 2009 John Wiley & Sons. Ltd.

Computer-aided Drug Design of Novel PLA(2) Inhibitor Candidates for Treatment of Snakebite

Phospholipases A(2) (PLA(2)) are enzymes commonly found in snake venoms from Viperidae and Elaphidae families, which are major components thereof. Many plants are used in traditional medicine its active agents against various effects induced by snakebite. This article presents the PLA(2) BthTX-I structure prediction based on homology modeling. In addition, we have performed virtual screening in a large database yielding a set of potential bioactive inhibitors. A flexible docking program was used to investigate the interactions between the receptor and the new ligands. We have performed molecular interaction fields (MIFs) calculations with the phospholipase model. Results confirm the important role of Lys49 for binding ligands and suggest three additional residues as well. We have proposed a theoretically nontoxic, drug-like, and potential novel BthTX-I inhibitor. These calculations have been used to guide the design of novel phospholipase inhibitors as potential lead compounds that may be optimized for future treatment of snakebite victims as well as other human diseases in which PLA(2) enzymes are involved.

Real-time monitoring and kinetic parameter estimation of the affinity interaction of jArtinM and rArtinM with peroxidase glycoprotein by the electrogravimetric technique

ArtinM is a D-mannose binding lectin that has been arousing increasing interest because of its biomedical properties, especially those involving the stimulation of Th1 immune response, which confers protection against intracellular pathogens The potential pharmaceutical applications of ArtinM have motivated the production of its recombinant form (rArtinM) so that it is important to compare the sugar-binding properties of jArtinM and rArtinM in order to take better advantage of the potential applications of the recombinant lectin. In this work, a biosensor framework based on a Quartz Crystal Microbalance was established with the purpose of making a comparative study of the activity of native and recombinant ArtinM protein The QCM transducer was strategically functionalized to use a simple model of protein binding kinetics. This approach allowed for the determination of the binding/dissociation kinetics rate and affinity equilibrium constant of both forms of ArtinM with horseradish peroxidase glycoprotein (HRP), a N-glycosylated protein that contains the trimannoside Man alpha 1-3[Man alpha 1-6]Man, which is a known ligand for jArtinM (Jeyaprakash et al, 2004). Monitoring of the real-time binding of rArtinM shows that it was able to bind HRP, leading to an analytical curve similar to that of jArtinM, with statistically equivalent kinetic rates and affinity equilibrium constants for both forms of ArtinM The lower reactivity of rArtinM with HRP than jArtinM was considered to be due to a difference in the number of Carbohydrate Recognition Domains (CRDs) per molecule of each lectin form rather than to a difference in the energy of binding per CRD of each lectin form. (C) 2010 Elsevier B V. All rights reserved

The crustacean gill (Na(+),K(+))-ATPase: Allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na(+), K(+))-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na(+) and K(+) of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na(+),K(+))-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na(+) and K(+). However, in contrast to Na(+), a threshold K(+) concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations. Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na(+),K(+))-ATPase compared to the vertebrate enzyme. (C) 2008 Elsevier Inc. All rights reserved.