Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.

Convergent synthesis and cruzain inhibitory activity of novel 2-(N `-benzylidenehydrazino)-4-trifluoromethyl-pyrimidines

To search for new cruzain inhibitors, the synthesis of a series of novel 2-(N`-benzylidenehydrazino)-4-trifluoromethyl-pyrimidines in a convergent manner is presented. The cruzain inhibitory activity of some of these compounds was evaluated and a binding model was proposed. All derivatives tested were active and the most significant inhibitory effect (80% at 100 mu M) and IC(50) value (85 mu M) were obtained from the 2-(N`-4-chloro-benzylidenehydrazino)-4-trifluoromethyl-pyrimidine. Although further structural optimization to improve solubility is necessary, the molecular docking studies suggest that these inhibitors occupy the S2 pocket without irreversible enzyme inactivation, through hydrophobic interactions, thus, indicating a desirable mode of interaction for the design of novel inhibitors. (C) 2008 Elsevier Ltd. All rights reserved.

SBTX, a new toxic protein distinct from soyatoxin and other toxic soybean [Glycine max] proteins, and its inhibitory effect on Cercospora sojina growth

SBTX, a novel toxin from soybean, was purified by ammonium sulfate fractionation followed by chromatographic steps DEAE-Cellulose, CM-Sepharose and Superdex 200 HR fast-protein liquid chromatography (FPLC). Lethality of SBTX to mice (LD50 5.6 mg/kg) was used as parameter in the purification steps. SBTX is a 44-kDa basic glycoprotein composed of two polypeptide chains (27 and 17 kDa) linked by a disulfide bond. The N-terminal sequences of the 44 and 27 kDa chains were identical (ADPTFGFTPLGLSEKANLQIMKAYD), differing from that of 17 kDa (PNPKVFFDMTIGGQSAGRIVMEEYA). SBTX contains high levels of Glx, Ala, Asx, Gly and Lys and showed maximum absorption at 280 nm, epsilon(1 cm) (1%) of 6.3, and fluorescence emission in the 290-450nm range upon excitation at 280nm. The secondary structure content was 35% alpha-helix, 13% beta-strand and beta-sheet, 27% beta-turn, 25% unordered, and 1% aromatic residues. Immunological assays showed that SBTX was related to other toxic proteins, such as soyatoxin and canatoxin, and cross-reacted weekly with soybean trypsin inhibitor and agglutinin, but it was devoid of protease-inhibitory and hemagglutinating activities. The inhibitory effect of SBTX on growth of Cercospora sojina, fungus causing frogeye leaf spot in soybeans, was observed at 50 mu g/ml, concentration 112 times lesser than that found to be lethal to mice. This effect on phytopathogenic fungus is a potential attribute for the development of transgenic plants with enhanced resistance to pathogens. (c) 2007 Elsevier Ltd. All rights reserved.

Synthesis of the Macrolactone of Migrastatin and Analogues with Potent Cell-Migration Inhibitory Activity

The synthesis of the macrolactone core of migrastatin 2, its potent anti-metastasis analogue 34, and ester derivatives 35 and 38 are reported. The approach involves the use of a dihydroxylation reaction to establish the desired C-8 stereocenter followed by a metathesis cyclization reaction. The effects of the compounds on the migration and invasion of human breast cancer cells were evaluated by using the wound-healing and the Boyden-chamber cell-migration and cell-invasion assays. The results revealed a high potency of the macrolactones 2 and 34 and the ester analogues 35 and 38, which suggests they have potential as antimetastatic agents.

Chemical modification of a nanocrystalline TiO(2) film for efficient electric connection of glucose oxidase

A novel biosensor for glucose was prepared by adsorption of 1,1`-bis(4-carboxybenzyl)-4,4`-bipyridinium di-bromide compound (H(2)BpybcBr(2)) onto the surface of a nanocrystalline TiO(2) film deposited onto FTO glasses, which was used as a platform to assemble the enzyme glucose oxidase to the electrode surface. The H(2)BpybcBr(2)/TiO(2)/FTO modified electrode was characterized by scanning electron microscopy, X-ray fluorescence image, cyclic voltammograms and spectroelectrochemical measurements. The immobilization of GOD on functionalized TiO(2) film led to stable amperometric biosensing for glucose with a linear range from 153 mu mol L(-1) to 1.30 mmol L(-1) and a detection limit of 51 mu mol L(-1). The apparent Michaelis-Menten constant (K(m)) was estimated to be 3.76 mmol L(-1), which suggested a high enzyme-substrate affinity. The maximum electrode sensitivity was 1.25 mu A mmol L(-1). The study proved that the combination of viologen mediators with TiO(2) film retains the electrocatalytic activity of the enzyme, and also enhances the electron transfer process, and hence regenerating the enzyme in the reaction with glucose. (C) 2010 Elsevier Inc. All rights reserved.

Inactivation kinetics of polyphenol oxidase and peroxidase in green coconut water by microwave processing

The inactivation kinetics of enzymes polyphenol oxidase (PPO) and peroxidase (POD) was studied for the batch (discontinuous) microwave treatment of green coconut water. Inactivation of commercial PPO and POD added to sterile coconut water was also investigated. The complete time-temperature profiles of the experimental runs were used for determination of the kinetic parameters D-value and z-value: PPO (D(92.20 degrees C) = 52 s and z = 17.6 degrees C); POD (D(92.92 degrees C) = 16 s and z = 11.5 degrees C); PPO/sterile coconut water: (D(84.45 degrees C) = 43 s and z = 39.5 degrees C) and POD/sterile coconut water: (D(86.54 degrees C) = 20 s and z = 19.3 degrees C). All data were well fitted by a first order kinetic model. The enzymes naturally present in coconut water showed a higher resistance when compared to those added to the sterilized medium or other simulated solutions reported in the literature. The thermal inactivation of PPO and POD during microwave processing of green coconut water was significantly faster in comparison with conventional processes reported in the literature. (C) 2008 Elsevier Ltd. All rights reserved.

Triphenylmethane dyes, an alternative for mediated electronic transfer systems in glucose oxidase biofuel cells

The bioelectrochemical behavior of three triphenylmethane (TPM) dyes commonly used as pH indicators, and their application in mediated electron transfer systems for glucose oxidase bioanodes in biofuel cells was investigated. Bromophenol Blue, Bromothymol Blue, Bromocresol Green were compared bio-electrochemically against two widely used mediators, benzoquinone and ferrocene carboxy aldehyde. Biochemical studies were performed in terms of enzymatic oxidation, enzyme affinity, catalytic efficiency and co-factor regeneration. The different features of the TPM dyes as mediators are determined by the characteristics in the oxidation/reduction processes studied electrochemically. The reversibility of the oxidation/reduction processes was also established through the dependence of the voltammetric peaks with the sweep rates. All three dyes showed good performances compared to the FA and BQ when evaluated in a half enzymatic fuel cell. Potentiodynamic and power response experiments showed maxima power densities of 32.8 mu W cm(-2) for ferrocene carboxy aldehyde followed by similar values obtained for TPM dyes around 30 mu W cm(-2) using glucose and mediator concentrations of 10 mmol L(-1) and 1.0 mmol L(-1), respectively. Since no mediator consumption was observed during the bioelectrochemical process, and also good redox re-cycled processes were achieved, the use of triphenylmethane dyes is considered to be promising compared to other mediated systems used with glucose oxiclase bioanodes and/or biofuel cells. (C) 2011 Elsevier Inc. All rights reserved.

Metronidazole-containing gel for the treatment of periodontitis: an in vivo evaluation

The aim of this investigation was to monitor metronidazole concentrations in the gingival crevicular fluid (GCF) collected from periodontal pockets of dogs after treatment with an experimental 15% metronidazole gel. Five dogs had periodontitis induced by cotton ligatures placed subgingivally and maintained for a 30-day period. After the induction period, only pockets with 4 mm or deeper received the gel. Each pocket was filled up to the gingival margin by means of a syringe with a blunt-end needle. GCF was collected in paper strips and quantified in an electronic device before and after 15 minutes, 1 h, 6 h, 24 h and 48 h of gel administration. The GCF samples were assayed for metronidazole content by means of a high performance liquid chromatography method. Concentrations of metronidazole in the GCF of the 5 dogs (mean ± SD, in µg/mL) were 0 ± 0 before gel application and 47,185.75 ± 24,874.35 after 15 minutes, 26,457.34 ± 25,516.91 after 1 h, 24.18 ± 23.11 after 6 h, 3.78 ± 3.45 after 24 h and 3.34 ± 5.54 after 48 h. A single administration of the 15% metronidazole gel released the drug in the GCF of dogs in levels several-fold higher than the minimum inhibitory concentration for some periodontopathogens grown in subgingival biofilms for up to one hour, but metronidazole could be detected in the GCF at least 48 hours after the gel application.

Prevalence of mutans streptococci isolated from complete dentures and their susceptibility to mouthrinses

The aims of this study were to evaluate the incidence of mutans streptococci (MS - sessile form) on complete maxillary dentures after use of a specific denture paste, and to determine the minimum inhibitory concentration (MIC) and maximum inhibitory dilution (MID) of 3 oral mouthrinses: Cepacol, Plax and Periogard. Seventy-seven complete denture wearers were randomly assigned into 2 groups, according to the product used for denture cleaning: Control group - conventional dentifrice (Kolynos-Super White); and Test group: experimental denture cleaning paste. Denture biofilm was collected at baseline and after 90 and 180 days after treatment by brushing the dentures with saline solution. After decimal serial dilution, samples were seeded onto agar sucrose bacitracin to count colonies with morphological characteristics of MS. MS identification was performed by the sugar fermentation tests. After this procedure, brain heart infusion broth (BHI) was added to oral mouthrinses (Plax, Cepacol e Periogard) and seeded on Petri dishes. The colonies were seeded using the Steers multiplier and, after the incubation, the MIC and MID of the mouthrinses were calculated. The results showed an incidence of 74.0% (n=57) of MS in the 77 complete dentures examined in the study, being 76.3% (n=29) of the Control group (conventional dentifrice) and 71.8% (28) of the Test group (experimental denture cleaning paste). In both groups, the number of positive cases for MS decreased from day 0 to day 180. In the Test group there was a slight decrease in the incidence of Streptococcus mutans 90 days after use of the experimental denture cleaning paste, which was not observed in the Control group. As regards to mouthrinses, for both groups, Periogard showed antimicrobial action with the highest dilution, followed by Cepacol and Plax. In conclusion, the incidence of MS in complete dentures was high and Periogard was the mouthrinse with the strongest antimicrobial action against MS. The experimental denture cleaning paste showed a slight action against S. mutans after 90 days of treatment.

Treatment with paracetamol, ketorolac or etoricoxib did not hinder alveolar bone healing: a histometric study in rats

Prostaglandins control osteoblastic and osteoclastic function under physiological or pathological conditions and are important modulators of the bone healing process. The non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity and consequently prostaglandins synthesis. Experimental and clinical evidence has indicated a risk for reparative bone formation related to the use of non-selective (COX-1 and COX-2) and COX-2 selective NSAIDs. Ketorolac is a non-selective NSAID which, at low doses, has a preferential COX-1 inhibitory effect and etoricoxib is a new selective COX-2 inhibitor. Although literature data have suggested that ketorolac can interfere negatively with long bone fracture healing, there seems to be no study associating etoricoxib with reparative bone formation. Paracetamol/acetaminophen, one of the first choices for pain control in clinical dentistry, has been considered a weak anti-inflammatory drug, although supposedly capable of inhibiting COX-2 activity in inflammatory sites. OBJECTIVE: The purpose of the present study was to investigate whether paracetamol, ketorolac and etoricoxib can hinder alveolar bone formation, taking the filling of rat extraction socket with newly formed bone as experimental model. MATERIAL AND METHODS: The degree of new bone formation inside the alveolar socket was estimated two weeks after tooth extraction by a differential point-counting method, using an optical microscopy with a digital camera for image capture and histometry software. Differences between groups were analyzed by ANOVA after confirming a normal distribution of sample data. RESULTS AND CONCLUSIONS: Histometric results confirmed that none of the tested drugs had a detrimental effect in the volume fraction of bone trabeculae formed inside the alveolar socket.

Antibacterial activity of four mouthrinses containing triclosan against salivary Staphylococcus aureus

The maximum inhibitory dilution (MID) of triclosan-based mouthwashes against 28 Staphylococcus aureus strains was evaluated. Dilutions ranging from 1/10 to 1/655,360 were prepared. Strains were inoculated using a Steers multipoint inoculator. The MID was considered as the maximum dilution capable of inhibiting microorganism growth. The mouthwashes presented different MIDs.

Benign focal seizures of adolescence and neuropsychological findings in patients from community

RATIONALE: Benign focal seizures of adolescence (BFSA) described by Loiseau et al in 1972, is considered a rare entity, but maybe underdiagnosed. Although mild neuropsychological deficits have been reported in patients with benign epilepsies of childhood, these evaluations have not so far been described in BFSA. The aim of this study is to evaluate neuropsychological functions in BFSA with new onset seizures (<12 months). METHODS: Eight patients with BFSA (according to Loiseau et al, 1972, focal or secondarily tonic clonic generalized seizures between the ages of 10-18 yrs., normal neurologic examination, normal EEG or with mild focal abnormalities) initiated in the last 12 months were studied between July 2008 to May 2009. They were referred from the Pediatric Emergency Section of the Hospital Universitário of the University of Sao Paulo, a secondary care regionalized facility located in a district of middle-low income in Sao Paulo city, Brazil. The study was approved by the Ethics Committee of the Institution. All patients performed neurological, EEG, brain CT and neuropsychological evaluation which consisted of Raven's Special Progressive Matrices - General and Special Scale (according to different ages), Wechsler Children Intelligence Scale-WISC III with ACID Profile, Trail Making Test A/B, Stroop Test, Bender Visuo-Motor Test, Rey Complex Figure, Rey Auditory Verbal Learning Test-RAVLT, Boston Naming Test, Fluency Verbal for phonological and also conceptual patterns - FAS/Animals and Hooper Visual Organization Test. For academic achievement, we used a Brazilian test for named "Teste do Desempenho Escolar", which evaluates abilities to read, write and calculate according to school grade. RESULTS: There were 2 boys and 6 girls, with ages ranging from 10 yrs. 9 m to 14 yrs. 3 m. Most (7/8) of the patients presented one to two seizures and only three of them received antiepileptic drugs (AEDs). Six had mild EEG focal abnormalities and all had normal brain CT. All were literate, attended regular public schools and scored in a median range for IQ, and seven showed discrete higher scores for the verbal subtests. There were low scores for attention in different modalities in six patients, mainly in alternated attention as well as inhibitory subtests (Stroop test and Trail Making Test part B). Four of the latter cases who showed impairment both in alternated and inhibitory attention were not taking AEDs. Visual memory was impaired in five patients (Rey Complex Figure). Executive functions analysis showed deficits in working memory in five, mostly observed in Digits Indirect Order and Arithmetic tests (WISC III). Reading and writing skills were below the expected average for school grade in six patients according to the achievement scholar performance test utilized. One patient of this series who had the best scores in all tests was taking phenobarbital. CONCLUSIONS: Neuropsychological imbalance between normal IQ and mild dysfunctions such as in attention domain and in some executive abilities like working memory and planning, as well as difficulties in visual memory and in reading and writing, were described in this group of patients with BFSA from community. This may reflect mild higher level neurological dysfunctions in adolescence idiopathic focal seizures probably caused by an underlying dysmaturative epileptogenic process. Although academic problems often have multiple causes, a specific educational approach may be necessary in these adolescents, in order to improve their scholastic achievements, helping in this way, to decrease the stigma associated to epileptic seizures in the community.

Morphine infusions into the rostrolateral periaqueductal gray affect maternal behaviors

It is well established that morphine inhibits maternal behaviors. Previous studies by our group have shown activation of the rostrolateral periaqueductal gray (rlPAG) upon inhibition-intended subcutaneous injections of morphine. In this context, we demonstrated that a single naloxone infusion into the rlPAG, following this opioid-induced inhibition, reactivated maternal behaviors. Since these data were obtained by using peripheral morphine injections, the present study was designed to test whether morphine injected directly into the rlPAG would affect maternal behaviors. Our hypothesis that morphine acting through the rlPAG would disrupt maternal behaviors was confirmed with a local infusion of morphine. The mothers showed shorter latency for locomotor behavior to explore the home cage (P = 0.049). Inhibition was especially evident regarding retrieving (P = 0.002), nest building (P = 0.05) and full maternal behavior (P = 0.023). These results support the view that opioidergic transmission plays a behaviorally meaningful inhibitory role in the rostrolateral PAG.

Expression of SMAD proteins, TGF-beta/activin signaling mediators, in human thyroid tissues

OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-β/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-β and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-β and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-β/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.

A fototerapia com diodo emissor de luz (LEDT) aplicada pré-exercício inibe a peroxidação lipídica em atletas após exercício de alta intensidade: um estudo preliminar

Estresse oxidativo é o termo geralmente utilizado para descrever os danos causados pelo desequilíbrio entre pró-oxidantes e antioxidantes no organismo. O aumento no consumo de O2 induzido pelo exercício físico está associado ao aumento das espécies reativas de oxigênio (EROs) sendo estas indutoras do estresse oxidativo. Embora as evidências indiquem um provável efeito inibitório da fototerapia com diodos emissores de luz (LEDT) sobre a produção das EROs, não existem estudos observando tal efeito em atletas. Este estudo preliminar destina-se a verificar os efeitos da aplicação de LEDT previamente ao exercício de alta intensidade sobre a peroxidação lipídica, mensurada através dos níveis sanguíneos de substâncias reativas ao ácido tiobarbitúrico (TBARS). Todos os seis atletas de voleibol do sexo masculino foram submetidos às duas situações: aplicação de LEDT efetiva e aplicação de LEDT placebo. O desempenho no protocolo de exercício adotado não revelou diferença (p > 0,05) entre as duas situações nas variáveis potência pico, potência média e índice de fadiga. Os resultados relacionados com a peroxidação lipídica foram: na situação LEDT efetiva, não foi possível observar diferença estatisticamente significante (p > 0,05) entre os níveis pré e pós-exercício (6,98 ± 0,81 e 7,02 ± 0,47nmol/mL); na situação LEDT (LBP) placebo, houve diferença estatisticamente significante (p = 0,05) entre os valores pré e pós-exercício (7,09 ± 1,28 e 8,43 ± 0,71nmol/mL). Tais resultados demonstram que a aplicação efetiva de LEDT parece ser eficaz no controle da peroxidação lipídica em atletas submetidos a exercício intenso