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.

Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule

Lessa LM, Carraro-Lacroix LR, Crajoinas RO, Bezerra CN, Dariolli R, Girardi AC, Fonteles MC, Malnic G. Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule. Am J Physiol Renal Physiol 303: F1399-F1408, 2012. First published September 5, 2012; doi: 10.1152/ajprenal.00385.2011.-We previously demonstrated that uroguanylin (UGN) significantly inhibits Na+/H+ exchanger (NHE)3-mediated bicarbonate reabsorption. In the present study, we aimed to elucidate the molecular mechanisms underlying the action of UGN on NHE3 in rat renal proximal tubules and in a proximal tubule cell line (LLC-PK1). The in vivo studies were performed by the stationary microperfusion technique, in which we measured H+ secretion in rat renal proximal segments, through a H+-sensitive microelectrode. UGN (1 mu M) significantly inhibited the net of proximal bicarbonate reabsorption. The inhibitory effect of UGN was completely abolished by either the protein kinase G (PKG) inhibitor KT5823 or by the protein kinase A (PKA) inhibitor H-89. The effects of UGN in vitro were found to be similar to those obtained by microperfusion. Indeed, we observed that incubation of LLC-PK1 cells with UGN induced an increase in the intracellular levels of cAMP and cGMP, as well as activation of both PKA and PKG. Furthermore, we found that UGN can increase the levels of NHE3 phosphorylation at the PKA consensus sites 552 and 605 in LLC-PK1 cells. Finally, treatment of LLC-PK1 cells with UGN reduced the amount of NHE3 at the cell surface. Overall, our data suggest that the inhibitory effect of UGN on NHE3 transport activity in proximal tubule is mediated by activation of both cGMP/PKG and cAMP/PKA signaling pathways which in turn leads to NHE3 phosphorylation and reduced NHE3 surface expression. Moreover, this study sheds light on mechanisms by which guanylin peptides

Recombinant ADAMTS13 normalizes von Willebrand factor-cleaving activity in plasma of acquired TTP patients by overriding inhibitory antibodies

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 as observed in acquired thrombotic thrombocytopenic purpura (TTP) is caused by inhibitory and non-inhibitory autoantibodies directed against the protease. Current treatment with plasma exchange is considered to remove circulating antibodies and to concurrently replenish the deficient enzyme.

Inhibitory regulation of dendritic activity in vivo

The spatiotemporal control of neuronal excitability is fundamental to the inhibitory process. We now have a wealth of information about the active dendritic properties of cortical neurons including axonally generated sodium action potentials as well as local sodium spikelets generated in the dendrites, calcium plateau spikes, and NMDA spikes. All of these events have been shown to be highly modified by the spatiotemporal pattern of nearby inhibitory input which can drastically change the output firing mode of the neuron. This means that particular populations of interneurons embedded in the neocortical microcircuitry can more precisely control pyramidal cell output than has previously been thought. Furthermore, the output of any given neuron tends to feed back onto inhibitory circuits making the resultant network activity further dependent on inhibition. Network activity is therefore ultimately governed by the subcellular microcircuitry of the cortex and it is impossible to ignore the subcompartmentalization of inhibitory influence at the neuronal level in order to understand its effects at the network level. In this article, we summarize the inhibitory circuits that have been shown so far to act on specific dendritic compartments in vivo.

EEG marker of inhibitory brain activity correlates with resting-state cerebral blood flow in the reward system in major depression

Frontal alpha band asymmetry (FAA) is a marker of altered reward processing in major depressive disorder (MDD), associated with reduced approach behavior and withdrawal. However, its association with brain metabolism remains unclear. The aim of this study is to investigate FAA and its correlation with resting – state cerebral blood flow (rCBF). We hypothesized an association of FAA with regional rCBF in brain regions relevant for reward processing and motivated behavior, such as the striatum. We enrolled 20 patients and 19 healthy subjects. FAA scores and rCBF were quantified with the use of EEG and arterial spin labeling. Correlations of the two were evaluated, as well as the association with FAA and psychometric assessments of motivated behavior and anhedonia. Patients showed a left – lateralized pattern of frontal alpha activity and a correlation of FAA lateralization with subscores of Hamilton Depression Rating Scale linked to motivated behavior. An association of rCBF and FAA scores was found in clusters in the dorsolateral prefrontal cortex bilaterally (patients) and in the left medial frontal gyrus, in the right caudate head and in the right inferior parietal lobule (whole group). No correlations were found in healthy controls. Higher inhibitory right – lateralized alpha power was associated with lower rCBF values in prefrontal and striatal regions, predominantly in the right hemisphere, which are involved in the processing of motivated behavior and reward. Inhibitory brain activity in the reward system may contribute to some of the motivational problems observed in MDD.

Design, Synthesis And Biological Evaluation Of Hybrid Bioisoster Derivatives Of N-acylhydrazone And Furoxan Groups With Potential And Selective Anti-trypanosoma Cruzi Activity.

Hybrid bioisoster derivatives from N-acylhydrazones and furoxan groups were designed with the objective of obtaining at least a dual mechanism of action: cruzain inhibition and nitric oxide (NO) releasing activity. Fifteen designed compounds were synthesized varying the substitution in N-acylhydrazone and in furoxan group as well. They had its anti-Trypanosoma cruzi activity in amastigotes forms, NO releasing potential and inhibitory cruzain activity evaluated. The two most active compounds (6, 14) both in the parasite amastigotes and in the enzyme contain the nitro group in para position of the aromatic ring. The permeability screening in Caco-2 cell and cytotoxicity assay in human cells were performed for those most active compounds and both showed to be less cytotoxic than the reference drug, benznidazole. Compound 6 was the most promising, since besides activity it showed good permeability and selectivity index, higher than the reference drug. Thereby the compound 6 was considered as a possible candidate for additional studies.

Antimycobacterial and cytotoxicity activity of synthetic and natural compounds

Antimycobacterial and cytotoxicity activity of synthetic and natural compounds. Secondary metabolites from Curvularia eragrostidis and Drechslera dematioidea, Clusia sp. floral resin, alkaloids from Pilocarpus alatus, salicylideneanilines, piperidine amides, the amine 1-cinnamylpiperazine and chiral pyridinium salts were assayed on Mycobacterium tuberculosis H37Rv. N-(salicylidene)-2-hydroxyaniline was the most effective compound with a minimal inhibitory concentration (MIC) of 8 µmol/L. Dihydrocurvularin was moderately effective with a MIC of 40 µmol/L. Clusia sp. floral resin and a gallocatechin-epigallocatechin mixture showed MIC of 0.02 g/L and 38 µmol/L, respectively. The cytotoxicity was evaluated for N-(salicylidene)-2-hydroxyaniline, curvularin, dihydrocurvularin and Clusia sp. floral resin, and the selectivity indexes were > 125, 0.47, 0.75 and 5, respectively.

Maximum inhibitory dilution of mouthwashes containing chlorhexidine and polyhexamethylene biguanide against salivary staphylococcus aureus

OBJECTIVE: The aim of the present study was to determine the in vitro maximum inhibitory dilution (MID) of two chlorhexidinebased oral mouthwashes (CHX): Noplak®, Periogard®, and one polyhexamethylene biguanide-based mouthwash (PHMB): Sanifill Premium® against 28 field Staphylococcus aureus strains using the agar dilution method. MATERIALS AND METHODS: For each product, decimal dilutions ranging from 1/10 to 1/655,360 were prepared in distilled water and added to Mueller Hinton Agar culture medium. After homogenization, the culture medium was poured onto Petri dishes. Strains were inoculated using a Steers multipoint inoculator and dishes were incubated at 37ºC for 24hours. For reading, MID was considered as the maximum dilution of the mouthwash still capable of inhibiting microbial growth. RESULTS: Sanifill Premium® inhibited the growth of all strains at 1/40 dilution and of 1 strain at 1/80 dilution. Noplak® inhibited the growth of 23 strains at 1/640 dilution and of all 28 strains at 1/320 dilution. Periogard® showed inhibited growth of 7 strains at 1/640 dilution and of all 28 strains at 1/320 dilution. Data were submitted to Kruskal-Wallis statistical test, showing significant differences between the mouthwashes evaluated (p<0.05). No significant difference was found between Noplak® and Periogard® (p>0.05). Sanifill Premium® was the least effective (p<0.05). CONCLUSION: It was concluded that CHX-based mouthwashes present better antimicrobial activity against S. Aureus than the PHMB-based mouthwash.

Determination of the maximum inhibitory dilution of cetylpyridinium chloride-based mouthwashes against staphylococcus aureus: an in vitro study

The aim of this in vitro study was to determine the maximum inhibitory dilution (MID) of four cetylpyridinium chloride (CPC)-based mouthwashes: CPC+Propolis, CPC+Malva, CPC+Eucaliptol+Juá+Romã+Propolis (Natural Honey®) and CPC (Cepacol®), against 28 Staphylococcus aureus field strains, using the agar dilution method. Decimal dilutions ranging from 1/10 to 1/655,360 were prepared and added to Mueller Hinton Agar. Strains were inoculated using Steers multipoint inoculator. The inocula were seeded onto the surface of the culture medium in Petri dishes containing different dilutions of the mouthwashes. The dishes were incubated at 37ºC for 24 h. For readings, the MID was considered as the maximum dilution of mouthwash still capable of inhibiting microbial growth. The obtained data showed that CPC+Propolis had antimicrobial activity against 27 strains at 1/320 dilution and against all 28 strains at 1/160 dilution, CPC+Malva inhibited the growth of all 28 strains at 1/320 dilution, CPC+Eucaliptol+Juá+Romã+Propolis inhibited the growth of 2 strains at 1/640 dilution and all 28 strains at 1/320 dilution, and Cepacol® showed antimicrobial activity against 3 strains at 1/320 dilution and against all 28 strains at 1/160 dilution. Data were submitted to Kruskal-Wallis test, showing that the MID of Cepacol® was lower than that determined for the other products (p<0.05). In conclusion, CPC-mouthwashes showed antimicrobial activity against S. aureus and the addition of other substances to CPC improved its antimicrobial effect.

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.

Antiplatelet activity of Croton celditifolius

Croton celtidifolius Baill is a tree found in the Atlantic Forest South of Brazil, mainly in Santa Catarina. The bark and leaf infusions of this medicinal plant have been popularly used for the treatment of inflammatory diseases. The anti-aggregant activity of C. celtidifolius crude extract (CE) and the column chromatography (CC) isolated compounds flavonoids, catechin and gallocatechin were evaluated in human blood platelets. The platelet-rich plasma (PRP) was incubated with different concentrations of flavonóides (50 - 200 µg/mL) to be tested before platelet aggregation was induced by the agonists adenosine 5'diphosphate (ADP) and collagen. At 200 µg/mL the CE, catechin and gallocatechin markedly inhibited platelet aggregation with the aggregant agents. Using ATP production as an index of platelet secretory capacity, we observed a decreased production of ATP in platelets treated with flavonoids when stimulated by collagen. On the other hand, the flavonoids did not promote inhibitory effect on prothrombin time (PT), thromboplastin time (APTT) and thrombin time (TT). In conclusion, these observations suggest that C. celtidifolius is likely to exert an inhibitory action on platelets in vitro by suppressing secretion and platelet aggregation.

A new bianthron glycoside as inhibitor of Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase activity

A phytochemical investigation of the ethanolic extract of stalks of Senna martiana Benth. (Leguminoseae), native specie of northeast Brazil, resulted in the isolation and spectroscopic characterization of a new bianthrone glycoside, martianine 1 (10,10'-il-chrysophanol-10-oxi10,10'-bi-glucosyl). Its identification was established by HRMS, IR and 2D NMR experiments. The evaluation of martianine trypanocidal activity was carried out against gliceraldehyde 3-phosphate dehydrogenase enzyme from Trypanosoma cruzi. Its inhibitory constant (Ki) is in the low micromolar concentration and it was determined by isothermal titration calorimetry to be 27.3 ± 2.47 µmol L-1. The non-competitive mechanism is asserted to be putative of the mode of action martianine displays against T. cruzi GAPDH. Results show that martianine has a great potential to become new lead molecule by inhibiting this key enzyme and for the development of new drugs against Chagas disease.

Moxidectin interference on motor activity of rats

The present study investigated the effects of t moxidectin (MXD) in some parameters of rat motor function and neurochemical. The general activity in the open field and the motor coordination in the wooden beam were employed to evaluate the MXD effects. The results showed that, in the open field, even at high doses (2.0 and 20.0 mg/kg), the MXD did not alter the locomotion and the rearing frequencies. However, MXD was able to impair the motor coordination of the animals at wooden beam. Neurochemical studies of striatal GABA and dopamine neurotransmitters showed a reduced levels of dopamine and its metabolite, homovanillic acid, without interference on striatal GABA levels. Since GABAergic receptor stimulation had an inhibitory effect on dopaminergic striatal system, the decreased motor coordination could be attributed to an action of MXD on dopamine system via GABA activation.

Antimicrobial Activity of Diterpenes from Viguiera arenaria against Endodontic Bacteria

Six pimarane-type diterpenes isolated from Viguiera arenaria Baker and two semi-synthetic derivatives were evaluated in vitro against a panel of representative microorganisms responsible for dental root canal infections. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia, Prevotella buccae, Fusobacterium nucleatum, Bacteroides fragilis, Actinomyces naeslundii, Actinomyces viscosus, Peptostreptococcus micros, Enterococcus faecalis and Aggregatibacter actinomycetemcomitans. The compounds ent-pimara-8(14), 15-dien-19-oic acid, its sodium salt and ent-8(14), 15-pimaradien-3 beta-ol were the most active, displaying MIC values ranging from 1 to 10 mu g mL(-1). The results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new chemicals for use as a complement to instrumental endodontic procedures.