Long-term inhibition of 5-alpha reductase and aromatase changes the cellular and extracellular compartments in gerbil ventral prostate at different postnatal ages

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

Effects of myenteric denervation on extracellular matrix fibers and mast cell distribution in normal stomach and gastric lesions

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

Biosynthesis of antimalarial lignans from Holostylis reniformis

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

Turbinatine, a potential key intermediate in the biosynthesis of corynanthean-type indole alkaloids

Extraction of the leaves of Chimarrhis turbinata has led to the isolation of turbinatine (1), a new corynanthean-type indole alkaloid, besides four known indole alkaloids, strictosidine, 5alpha-carboxystrictosidine, vallesiachotamine, and isovallesiachotamine. The structural determination of 1 was based on 1D and 2D spectroscopic data. An evaluation of the DNA-damaging activities of the isolates was performed by means of a bioassay using mutant strains of Saccharomyces cerevisiae, which indicated these compounds were weakly active.

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

Production of extracellular alkaline proteases by Aspergillus clavatus

The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37degreesC. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25degreesC for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH4NO3 showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40degreesC and the half-lives at 40, 45 and 50degreesC were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.

GNN is a self-glucosylating protein involved in the initiation step of glycogen biosynthesis in Neurospora crassa

The initiation of glycogen synthesis requires the protein glycogenin, which incorporates glucose residues through a self-glucosylation reaction, and then acts as substrate for chain elongation by glycogen synthase and branching enzyme. Numerous sequences of glycogenin-like proteins are available in the databases but the enzymes from mammalian skeletal muscle and from Saccharomyces cerevisiae are the best characterized. We report the isolation of a cDNA from the fungus Neurospora crassa, which encodes a protein, GNN, which has properties characteristic of glycogenin. The protein is one of the largest glycogenins but shares several conserved domains common to other family members. Recombinant GNN produced in Escherichia coli was able to incorporate glucose in a self-glucosylation reaction, to trans-glucosylate exogenous substrates, and to act as substrate for chain elongation by glycogen synthase. Recombinant protein was sensitive to C-terminal proteolysis, leading to stable species of around 31 kDa, which maintained all functional properties. The role of GNN as an initiator of glycogen metabolism was confirmed by its ability to complement the glycogen deficiency of a S. cerevisiae strain (glg1 glg2) lacking glycogenin and unable to accumulate glycogen. Disruption of the gnn gene of N. crassa by repeat induced point mutation (RIP) resulted in a strain that was unable to synthesize glycogen, even though the glycogen synthase activity was unchanged. Northern blot analysis showed that the gnn gene was induced during vegetative growth and was repressed upon carbon starvation. (C) 2004 Elsevier B.V. All rights reserved.

CHEMICAL SYMPATHECTOMY BLOCKS ANDROGEN BIOSYNTHESIS DURING PREPUBERTY

Twenty-one-day old male Wistar rats were injected subcutaneously with guanethidine (GUA) at doses of 5 and 10 mg kg(-1) day(-1) for 20 days. Animals were sacrificed by decapitation during the prepubertal (41 days of age) and early-pubertal (51 days of age) periods of sexual development. The testes were collected, frozen in liquid N-2 and stored at -70 degrees C until determination of testicular progesterone (P): androstenedione (A) and testosterone (T). Higher levels of P (2.18 +/- 0.24 ng/g. control = 1.24 +/- 0.16 ng/g) associated with decreased levels of androgens (A = 0.26 +/- 0.06 ng/g and T = 2.05 +/- 0.19 ng/g; control = 1.86 +/- 0.76 ng/g and 8.48 +/- 1.16 ng/g, respectively) were observed in 10 mg GUA-treated rats of prepubertal age, while only P levels (3.12 +/- 0.51 ng/g control = 1.73 +/- 0.27 ng/g) were increased in rats of early pubertal age. It is important to note that in 41-day old male rats both 5 and 10 mg were effective in decreasing testicular concentration of testosterone. These results suggest that the sympathetic innervation of the testis is involved in the modulation of androgen biosynthesis, acting through a selective step in the steroid biochemical pathway during the pubertal process and that under the conditions employed the blockage in androgen biosynthesis in the prepubertal stage of sexual maturation is dependent on the dose of GUA.

Collagen type VI is a component of the extracellular matrix microfibril network of the prostatic stroma

Stroma-epithelium relationships are of great relevance in prostatic morphogenesis and physiology, However, little knowledge exists about either stromal cells or extracellular matrix composition and arrangement in this system, Ultrastructural analysis revealed the existence of a microfibrillar system which occupies large areas of the rat prostatic stroma, In this work, we have applied immunocytochemistry and an ATP treatment for the ultrastructural identification of collagen type VI microfibrils, aiming at examining its participation in the prostatic microfibrillar network. Immunocytochemistry was also extended to a human case of prostatic nodular hyperplasia, Both methods succeeded in identifying collagen type VI in the rat ventral prostate, Collagen type VI is evenly distributed throughout the stroma but mainly associated with the basal lamina, collagen fibrils, and around the stromal cells, the use of ATP treatment allowed for the discrimination between collagen type VI and elastin-associated microfibrils, and demonstrated that these two classes of microfibrils establish an extended, mixed, and open network. The same aspects of association with the basal lamina, with stromal cells (particularly with smooth muscle cells), and with fibrillar components of the stroma were observed in the human tissue, We suggest that the collagen type VI and elastin-associated microfibril system may be involved in the control of some aspects of cellular behavior and may also play a structural role, maintaining the organ integrity after the deformations occurring under smooth muscle contraction.

Biosynthesis of friedelane and quinonemethide triterpenoids is compartmentalized in Maytenus aquifolium and Salacia campestris

Maytenus aquifolium (Celastraceae) and Salacia campestris (Hippocrateaceae) species accumulate friedelane and quinonemethide triterpenoids in their leaves and root bark, respectively. Enzymatic extracts obtained from leaves displayed cyclase activity with conversion of the substrate oxidosqualene to the triterpenes, 3 beta -friedelanol and friedelin. In addition, administration of (+/-)5-H-3 mevalonolactone in leaves of M. aquifolium seedlings produced radio labelled friedelin in the leaves, twigs and stems, while the root bark accumulated labelled maytenin and pristimerin. These experiments indicated that the triterpenes once biosynthesized in the leaves are translocated to the root bark and further transformed to the antitumoral quinonemethide triterpenoids. (C) 2000 Elsevier B.V. Ltd. All rights reserved.

Extracellular matrix of porcine pericardium: Biochemistry and collagen architecture

Pericardial tissue has been used to construct bioprostheses employed in the repair of different kinds of injuries, mostly cardiac. However, calcification and mechanical failure have been the main causes of the limited durability of cardiac bioprostheses constructed with bovine pericardium. In the course of this work, a study was conducted on porcine fibrous pericardium, its microscopic structure and biochemical nature. The general morphology and architecture of collagen were studied under conventional light and polarized light microscopy. The biochemical study of the pericardial matrix was conducted according to the following procedures: swelling test, hydroxyproline and collagen dosage, quantification of amino acids in soluble collagen, component extraction of the extracellular matrix of the right and left ventral regions of pericardium with different molarities of guanidine chloride, protein and glycosaminoglycan (GAG) dosage, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and total GAG analysis. Microscopic analysis showed collagen fibers arranged in multidirectionally oriented layers forming a closely knit web, with a larger number of fibers obliquely oriented, initiating at the lower central region toward the upper left lateral relative to the heart. No qualitative differences were found between proteins extracted from the right and left regions. Likewise, no differences were found between fresh and frozen material. Protein dosages from left frontal and right frontal pericardium regions showed no significant differences. The quantities of extracted GAGs were too small for detection by the method used. Enzymatic digestion and electrophoretic analysis showed that the GAG found is possibly dermatan sulfate. The proteoglycan showed a running standard very similar to the small proteoglycan decorin.

Protease activity in extracellular products secreted in vitro by trophozoites of Giardia duodenalis

There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.

Characterization of a thermostable extracellular tannase produced under submerged fermentation by Aspergillus ochraceus

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