Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and beta 1 integrin

We studied the induction of protease activity by the laminin alpha 1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha 1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M I cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta 1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta 1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta 1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta 1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta 1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells. (c) 2008 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-beta-1,3-glucanase from Thermotoga petrophila

1,3-beta-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal beta-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90 degrees C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Ric-8B interacts with G alpha olf and G gamma 13 and co-localizes with G alpha olf, G beta 1 and G gamma 13 in the cilia of olfactory sensory neurons

Olfactory sensory neurons are able to detect odorants with high sensitivity and specificity. We have demonstrated that Ric-8B, a guanine nucleotide exchange factor (GEF), interacts with G alpha olf and enhances odorant receptor signaling. Here we show that Ric-8B also interacts with G gamma 13, a divergent member of the G gamma subunit family which has been implicated in taste signal transduction, and is abundantly expressed in the cilia of olfactory sensory neurons. We show that G beta 1 is the predominant GP subunit expressed in the olfactory sensory neurons. Ric-8B and G beta 1, like G alpha olf and G gamma 13, are enriched in the cilia of olfactory sensory neurons. We also show that Ric-8B interacts with G alpha olf in a nucleotide dependent manner, consistent with the role as a GEF. Our results constitute the first example of a GEF protein that interacts with two different olfactory G protein subunits and further implicate Ric-8B as a regulator of odorant signal transduction. (C) 2008 Elsevier Inc. All rights reserved.

Characterization of a beta-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to beta-glucan-binding proteins

Spodoptera frugiperda beta-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against beta-1,3-glucan (laminarin), but cannot hydrolyze yeast beta-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive beta-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of beta-1,3-glucanases and beta-glucan-binding protein supports the assumption that the beta-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived beta-1,3-glucanases by the loss of an extended N-terminal region and the beta-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells. (C) 2010 Elsevier Ltd. All rights reserved.

Purification, characterization and sequencing of the major beta-1,3-glucanase from the midgut of Tenebrio molitor larvae

The major beta-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an encloglucanase that hydrolyzes beta-1,3-glucans as laminarin and yeast beta-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched beta-1,3-glucans or mixed beta-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections. (C) 2009 Elsevier Ltd. All rights reserved.

Enzymatic hydrolysis of botryosphaeran and laminarin by beta-1,3-glucanases produced by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeran, a (1 -> 3; 1 -> 6)-beta-D-glucan produced by Botryosphaeria rhodina, and laminarin were hydrolysed by two fungal beta-glucanases predominantly of the 1,3-type produced by B. rhodina and Trichoderma harzianum Rifai grown on botryosphaeran as sole carbon source. Both beta-glucanase preparations presented different modes of attack on botryosphaeran and laminarin. Laminarin was hydrolysed to the extent of similar to 50% in 1 hand 100% within 24 h, and its hydrolysis products were mainly glucose and gentiobiose, and lesser amounts of laminaribiose and oligosaccharides of DP 3-4 during the early stages of hydrolysis, while botryosphaeran 'yielded mainly glucose and gentiobiose with some trisaccharide, but no laminaribiose or tetrasaccharide when hydrolysed by the T. harzianum enzyme. By contrast, B. rhodina beta-1,3-glucanases produced predominantly glucose during all stages of botryosphaeran hydrolysis. Some physicochemical properties of the 1,3- and 1,6-beta-glucanases, and beta-glucosidases contained in the two fungal P-glucanase preparations are also described for the first time. (c) 2006 Elsevier Ltd. All rights reserved.

Botryosphaeran, a new substrate for the production of beta-1,3-glucanases by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeria rhodina and Trichoderma harzianum Rifai were grown on botryosphaeran (an exopolysaccharide (EPS) of the beta-1,3; 1,6-D-Glucan type produced by B. rhodina) as sole carbon source with the objective of producing beta-glucanases of the beta-type. Conditions for beta-1,3-glucanase production by T harzianum were examined by a statistical response surface method, and showed maximal enzyme production at 5 days growth in media containing 1.5 g/1 of EPS. Good agreement was obtained between the experimental values of beta-1, 3-glucanase activity and the corresponding values predicted by the mathernatical model. The crude beta-1,3-glucanase preparations were active towards a number of different beta-1,3-glucans and beta-glucosides. The mycelium of B. rhodina also proved to be a good substrate for beta-1,3-glucanase production by both fungal species. (c) 2005 Published by Elsevier Ltd.

Platelet aggregation and TGF-beta(1) plasma levels in pregnant women with preeclampsia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Infiltrating CD8(+) T lymphocytes, natural killer cells, and expression of IL-10 and TGF-beta 1 in chemically induced neoplasms in male Wistar rats

The present study aimed to estimate the number of CD8(+) T and natural killer (NK) infiltrating cells and the expression of interleukin-10 (IL-10) and transforming growth factor beta 1 (TGF-beta1) in chemically induced neoplasms in an initiation-promotion bioassay for carcinogenesis. Male Wistar rats were treated with N-nitrosodiethylamine, N-methyl-N-nitrosourea, N-butyl-N-(4-hydroxybutyl) nitrosamine, dihydroxy-di-N-propylnitrosamine, and 1,2-dimethylhydrazine for 4 weeks. Two groups were subsequently exposed through diet to phenobarbital (0.05%) or 2-acetylaminofluorene (0.01%) for 25 weeks. An untreated group was used as a control. Immune cells and cytokines were immunohistochemically evaluated in neoplasms and in surrounding normal tissues at the liver, kidneys, lung, and small and large intestines. When compared to the respective normal tissues, an increased number of NK cells was verified infiltrating the colon, lung, and kidney neoplasms, while the number of CD8+ T cells decreased in the intestine and lung neoplasms. Expression of IL-10 was found mainly in kidney tumors. TGF-beta1 was expressed mainly in the liver and kidneys tumors. The results indicate that the differential occurrence of immune cells between neoplastic and normal tissues could be dependent upon tumor microenvironment.

Involvement of central alpha(1)-adrenoceptors on renal responses to central moxonidine and alpha-methylnoradrenaline

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Functional effects of castration on alpha(1)-adrenoceptors in rat vas deferens

The effects of castration on alpha(1)-adrenoceptors in rat vas deferens were investigated by determining the actions of selective antagonists against the contractions induced by noradrenaline. The results obtained in vas deferens from control rats suggest participation of alpha(1A)-adrenoceptors as judged by the pA(2) values for prazosin (9.6), benoxathian (9.5), 2(2,6-dimethoxyphenoxyethyl) amino-methyl-1,4-benzodioxone hydrochloride) (WB 4101) (9.6), phentolamine (8.4), 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dionedihydrochloride (BMY 7378) (6.7) and by the insensitivity to chloroethylclonidine (100 mu M, 45 min). In vas deferens from castrated rats, WE 4101 and spiperone showed slopes lower than 1.0 in the Schild plots, suggesting participation of multiple receptors. In these organs, noradrenaline contractions were partially inhibited by chloroethylclonidine (100 mu M, 45 min), indicating participation of alpha(1B)-adrenoceptors. After chloroethylclonidine treatment, WE 4101 showed a slope not different from 1.0 in the Schild plot, resulting in a pA(2) of 9.4, which indicates an interaction with alpha(1A)-adrenoceptors. It is suggested that castration modifies the functional alpha(1)-adrenoceptors subtypes in rat vas deferens. (C) 1998 Elsevier B.V. B.V. All rights reserved.

Functional characterisation of alpha(1)-adrenoceptors in denervated rat vas deferens

We investigated whether or not surgical denervation of the rat vas deferens changes the alpha(1)-adrenoceptor subtypes involved in the contractions to noradrenaline. Denervated vas deferens was approximate to22 times more sensitive to noradrenaline (pD(2)=7.35 +/- 0.04) than control vas (pD(2)= 6.01 +/- 0.03). This difference in noradrenaline potency was eliminated when cocaine (6 muM) was added to control vas (pD(2)=7.22 +/- 0.04). The noradrenaline-induced contractions of control and denervated vas deferens were insensitive to the alpha(1B)/alpha(1D)-adrenoceptor alkylating agent chloroethylclonidine (100 muM, 45 min). The concentration-response curves to noradrenaline in control and denervated vas deferens were competitively antagonised by prazosin (pA(2)approximate to9.6), WB-4101 (pA(2)approximate to9.5), 5-methyl urapidil (pA(2)approximate to8.4), phentolamine (pA(2)approximate to8.7), yohimbine (pA(2)approximate to6.9), BMY 7378 (pA(2)approximate to6.9) and indoramin (pA(2)approximate to8.7). After the treatment of control and denervated vas deferens with phenoxybenzamine, the partial agonist oxymetazoline antagonised competitively the concentration-response curves to noradrenaline showing pA(2) values approximate to7.4 in both groups. We conclude that noradrenaline-induced contractions in control and denervated rat vas deferens are mediated by alpha(1A)-adrenoceptors and that surgical denervation of the rat vas deferens is not able to change the alpha(1)-adrenoceptor subtypes involved in the contractions to noradrenaline.

Paralyzing and myotoxic effects of a recombinant bothropstoxin-1 (BthTX-I) on mouse neuromuscular preparations

As a first step to investigate the structure-function relationship of bothropstoxin-1 (BthTX-1), a myotoxin from Bothrops jararacussu snake venom, Our group previously cloned a recombinant toxin (rBthTX-1) in Escherichia coli. The aim or this work was to characterize the biological activities of this rBthTX-1 (1.0 mu M) in both phrenic-diaphragm and extensor digitorum longus preparations in vitro, by means of myographic and morphologic techniques. Native BthTX-1 (1.0 mu M) was used as a standard. The influence of heparin (27.5 mu g/ml) upon the biological activities of both toxins was also investigated. rBthTX-1 had similar effects to the native toxin inducing blockage of both directly and indirectly evoked contractions in phrenic-diaphragm preparations, and muscle damage characterized by edema, round fibers, and cell areas devoid of myofibrils. Interestingly the paralyzing activity of rBthTX-1 was slightly more potent than the native toxin. Heparin prevented paralyzing and myotoxic effects of both the native and recombinant toxins. This work shows that rBthTX-1 was expressed in a fully active form, and presents a biological profile similar to the native toxin. (c) 2005 Elsevier GmbH All rights reserved.

alpha(1)-Adrenoceptors in proximal segments of tail arteries from control and reserpinised rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)