Conformational Regulation of alpha2beta1 Integrin in Ligand Binding

Integrins are heterodimeric cell adhesion receptors involved in cell-cell and cell-extracellular matrix (ECM) interactions. They transmit bidirectional signals across the cell membrane. This results in a wide range of biological events from cell differentiation to apoptosis. alpha2beta1 integrin is an abundant collagen receptor expressed on the surface of several cell types. In addition to ECM ligands, alpha2beta1 integrins are bound by echovirus 1 (EV1) which uses alpha2beta1 as a receptor to initiate its life cycle in the infected cell. The aim of this thesis project was to provide further insight into the mechanisms of alpha2beta1 integrin ligand recognition and receptor activation. Collagen fibrils are the principal tensile elements of the ECM. Yet, the interaction of alpha2beta1 integrin with the fibrillar form of collagen I has received relatively little attention. This research focused on the ability of alpha2beta1 integrin to act as a receptor for type I collagen fibrils. Also the molecular requirements of the EV1 interaction with alpha2beta1 were studied. Conventionally, ligand binding has been suggested to require integrin activation and the binding may further trigger integrin signalling. Another main objective of this study was to elucidate both the inside-out and outside-in signalling mechanisms of alpha2beta1 integrin in adherent cells. The results indicated that alpha2beta1 integrin is the principal integrin-type collagen receptor for type I collagen fibrils, and alpha2beta1 may participate in the regulation of pericellular collagen fibrillogenesis. Furthermore, alpha2beta1 integrin inside-out activation appeared to be synergistically regulated by integrin clustering and conformational activation. The triggering of alpha2beta1 integrin outside-in signalling, however, was shown to require both conformational changes and clustering. In contrast to ECM ligands, EV1 appeared to take advantage of the bent, inactive form of alpha2beta1 integrin in initiating its life cycle in the cell. This research together with other recent studies, has shed light on the molecular mechanisms of integrin activation. It is becoming evident that large ligands are able to bind to the bent form of integrin, which has been previously considered to be physiologically inactive. Consequently, our understanding of the conformational modulation of integrins upon activation is changing.

Effect of epithelial debridement on human cornea proteoglycans

Corneal transparency is attributed to the regular spacing and diameter of collagen fibrils, and proteoglycans may play a role in fibrillogenesis and matrix assembly. Corneal scar tissue is opaque and this opacity is explained by decreased ultrastructural order that may be related to proteoglycan composition. Thus, the objectives of the present study were to characterize the proteoglycans synthesized by human corneal explants and to investigate the effect of mechanical epithelial debridement. Human corneas unsuitable for transplants were immersed in F-12 culture medium and maintained under tissue culture conditions. The proteoglycans synthesized in 24 h were labeled metabolically by the addition of 35S-sulfate to the medium. These compounds were extracted by 4 M GuHCl and identified by a combination of agarose gel electrophoresis, enzymatic degradation with protease and mucopolysaccharidases, and immunoblotting. Decorin was identified as the main dermatan sulfate proteoglycan and keratan sulfate proteoglycans were also prominent components. When the glycosaminoglycan side chains were analyzed, only keratan sulfate and dermatan sulfate were detected (~50% each). Nevertheless, when these compounds were 35S-labeled metabolically, the label in dermatan sulfate was greater than in keratan sulfate, suggesting a lower synthesis rate for keratan sulfate. 35S-Heparan sulfate also appeared. The removal of the epithelial layer caused a decrease in heparan sulfate labeling and induced the synthesis of dermatan sulfate by the stroma. The increased deposit of dermatan sulfate proteoglycans in the stroma suggests a functional relationship between epithelium and stroma that could be related to the corneal opacity that may appear after epithelial cell debridement.

Collagens and proteoglycans of the corneal extracellular matrix

The cornea is a curved and transparent structure that provides the initial focusing of a light image into the eye. It consists of a central stroma that constitutes 90% of the corneal depth, covered anteriorly with epithelium and posteriorly with endothelium. Its transparency is the result of the regular spacing of collagen fibers with remarkably uniform diameter and interfibrillar space. Corneal collagen is composed of heterotypic fibrils consisting of type I and type V collagen molecules. The cornea also contains unusually high amounts of type VI collagen, which form microfibrillar structures, FACIT collagens (XII and XIV), and other nonfibrillar collagens (XIII and XVIII). FACIT collagens and other molecules, such as leucine-rich repeat proteoglycans, play important roles in modifying the structure and function of collagen fibrils.Proteoglycans are macromolecules composed of a protein core with covalently linked glycosaminoglycan side chains. Four leucine-rich repeat proteoglycans are present in the extracellular matrix of corneal stroma: decorin, lumican, mimecan and keratocan. The first is a dermatan sulfate proteoglycan, and the other three are keratan sulfate proteoglycans. Experimental evidence indicates that the keratan sulfate proteoglycans are involved in the regulation of collagen fibril diameter, and dermatan sulfate proteoglycan participates in the control of interfibrillar spacing and in the lamellar adhesion properties of corneal collagens. Heparan sulfate proteoglycans are minor components of the cornea, and are synthesized mainly by epithelial cells. The effect of injuries on proteoglycan synthesis is discussed.

Myxobolus cordeiroi n. sp., a parasite of Zungaro jahu (Siluriformes: Pimelodiade) from Brazilian Pantanal: Morphology, phylogeny and histopathology

This work is part of an ongoing investigation into the characteristics of Myxozoan parasites of freshwater fish in Brazil and was carried out using morphology, histopathology and molecular analysis. A new Myxosporea species (Myxobolus cordeiroi) is described infecting the jau catfish (Zungaro jahu). Fifty jau specimens were examined and 78% exhibited plasmodia of the parasite. The plasmodia were white and round, measuring 0.3-2.0 mm in diameter and the development occurred in the gill arch, skin, serosa of the body cavity, urinary bladder and eye. The spores had an oval body and the spore wall was smooth. Partial sequencing of the 18S rDNA gene resulted in a total of 505 bp and the alignment of the sequences obtained from samples in different organs revealed 100% identity. In the phylogenetic analysis, the Myxobolus species clustered into two clades-one primarily parasites of freshwater fish and the other primarily parasites of marine fish. M. cordeiroi n. sp. was clustered in a basal position in the freshwater fish species clade. The histological analysis revealed the parasite in the connective tissue of the different infected sites, thereby exhibiting affinity to this tissue. The plasmodium was surrounded by an outer collagen capsule of fibers with distinct orientation from the adjacent connective tissue and an inner layer composed of delicate collagen fibrils-more precisely reticular fibers. The development of the parasite in the cornea and urinary bladder caused considerable stretching of the epithelium. (C) 2009 Elsevier B.V. All rights reserved.

Effects of exercise on the morphology of the myenteric neurons of the duodenum of Wistar rats during the ageing process

This study aimed to evaluate the effects of regular physical activity on the morphology of the myenteric plexus of the duodenum in rats during the ageing process. To this end, 45 Wistar rats were divided into three groups: C (sedentary - 6 months old), S (sedentary - 12 months old) and T (trained - 12 months old). The animals of group S were given with a physical activity programme consisting of a 10-min-treadmill workout once a week. The animals of group T were submitted to the physical activity programme five times a week. Their duodenums were collected and submitted to the techniques of nicotinamide adenine dinucleotide (NADH)-diaphorase enzyme histochemistry for whole-mount preparations and transmission electron microscopy. No differences in the constitution of the myenteric plexuses were found when the sedentary and trained groups were compared with the control group. The ultrastructural features were similar for the three groups. However, it was verified that the physical activity of the trained animals resulted in a similar myenteric neuron morphology to that of the adult animals (6 months old), thereby confirming its beneficial effect, as the sedentary animals had larger alterations in the collagen fibrils and the basal membrane that occur through ageing. The quantitative analysis showed that the NADH-diaphorase positive neurons decreased with ageing and increased with physical activity (P > 0.05). No significant alteration (P > 0.05) in the neuronal profile area of the NADH-diaphorase positive neurons has been observed with ageing.

The Estrous Cycle Modulates Small Leucine-Rich Proteoglycans Expression in Mouse Uterine Tissues

In the pregnant mouse uterus, small leucine-rich proteoglycans (SLRPs) are drastically remodeled within a few hours after fertilization, suggesting that ovarian hormone levels modulate their synthesis and degradation. In this study, we followed by immunoperoxidase approach, the presence of four members of the SLRP family (decorin, lumican, biglycan, and fibromodulin) in the uterine tissues along the estrous cycle of the mouse. All molecules except fibromodulin, which predominates in the myometrium, showed a striking modulation in their distribution in the endometrial stroma, following the rise in the level of estrogen. Moreover, notable differences in the distribution of SLRPs were observed between superficial and deep stroma, as well as between the internal and external layers of the myometrium. Only biglycan and fibromodulin were expressed in the luminal and glandular epithelia. All four SLRPs were found in cytoplasmic granules of mononucleated cells. The pattern of distribution of the immunoreaction for these molecules in the uterine tissues was found to be estrous cycle-stage dependent, suggesting that these molecules undergo ovarian hormonal control and probably participate in the preparation of the uterus for decidualization and embryo implantation. In addition, this and previous results from our laboratory suggest the existence of two subpopulations of endometrial fibroblasts that may be related to the centrifugal development of the decidua. Anat Rec, 292:138-153, 2009. (c) 2008 Wiley-Liss, Inc.

Role of the oblique ligament in the integrity of the medial collateral ligament of the canine elbow joint

Foram estudados o arranjo e o diâmetro médio das fibrilas colágenas do ligamento colateral medial da articulação do cotovelo do cão, isolado ou associado ao ligamento oblíquo e tracionado até a ruptura. Dezoito articulações foram divididas em três grupos. O primeiro grupo teve o ligamento colateral medial coletado, mas não tracionado; o segundo grupo teve o ligamento colateral medial tracionado isoladamente; o terceiro grupo teve os ligamentos colateral medial e oblíquo tracionados associadamente. O ligamento colateral medial não submetido ao ensaio de tração apresentou um padrão ondulado das fibras colágenas, o qual não foi totalmente destruído quando foi tracionado, associado ao ligamento oblíquo, e perdeu totalmente o padrão reticular das fibras colágenas quando testado isoladamente. Quando o ligamento colateral medial foi submetido à tensão isoladamente, o diâmetro médio das fibrilas colágenas aumentou em relação ao grupo não submetido à tensão. Associado ao ligamento oblíquo, o diâmetro médio das fibrilas colágenas foi o maior na região de inserção e o menor na região média, em relação aos outros grupos. Concluiu-se que o ligamento oblíquo pode favorecer a integridade da região de inserção do ligamento colateral medial, aumentando a eficácia de sua reconstrução após a lesão.

Effect of amniotic membrane transplantation on corneal healing and proteoglycan expression in an experimental model of limbal deficiency in rabbits

PURPOSE. Amniotic membrane transplantation (AMT) has been used as a graft or as a dressing in ocular surface reconstruction, facilitating epithelization, maintaining normal epithelial phenotype, and reducing inflammation, vascularization, and scarring. The corneal transparency is due, at least in part, to the arrangement in orthogonal lamellae of collagen fibrils, surrounded by proteoglycans (PGs). These PGs regulate fibrilogenesis, the matrix assembly, and ultimately the corneal transparency. The purpose of the present study was to investigate the effects of AMT upon the corneal PGs after severe limbal injury.METHODS. Experiments were performed on the right corneas of 22 New Zealand female albino rabbits, and their left corneas were used as matched controls. These animals were divided into 3 groups: G1 (n = 10): total peritomy and keratolimbectomy, followed by application of 0.5 M NaOH; G2 (n = 10): submitted to the same trauma as G1, and treated by AMT; G3: no trauma, only AMT (n = 2). The right corneas of G2 and G3 were covered by DMSO 4 cryopreserved human amniotic membrane, fixed by interrupted 9-0 mononylon sutures, with its stromal face toward the ocular surface. After 7 or 30 days, the corneas were removed and PGs were extracted.RESULTS. Normal corneas contained approximately 9 mg of PGs per gram of dry tissue. AMT on intact cornea (G3) did not cause any changes in the concentration of PGs. In contrast, injured corneas contained much less PGs, both on the seventh and on the 30th day posttrauma. The PG concentration was even lower in injured corneas treated by AMT. This decrease was due almost exclusively to dermatan sulfate PGs, and the structure of dermatan sulfate was also modified, indicating changes in the biosynthesis patterns.CONCLUSIONS. Although beneficial effects have been observed on clinical observation and concentration of soluble proteins after AMT, the normal PG composition of cornea was not attained, even 30 days postinjury, indicating that the normal ocular surface reconstruction, if possible, is a long-term process. (Eur J Ophthalmol 2010; 20: 290-9)

Influence of root-surface conditioning with acid and chelating agents on clot stabilization

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ultrastructural features of myenteric ganglia of adult Wistar rats (Rattus norvegicus)

The ultrastructural features of the ganglia of the myenteric plexus exhibit changes according to the animal species. These myenteric ganglia in the duodenum of adult rats of the Wistar strain were characterized ultrastructurally in this work. Those ganglia were depicted as compact structures, composed of neurones and glial cells, forming a dense neuropil surrounded by a continuous basal lamina and collagen fibrils. Glial cell bodies were smaller and apparently more frequent than neuronal cell bodies, being morphologically distinguished by nuclear features. In the neuronal extensions granular and agranular synaptic vesicles of different sizes predominate, in addition to mitochondria and myelinized profiles. Gliofilaments were not observed on the glial extensions of the rats.

Myocardial dysfunction induced by food restriction is related to morphological damage in normotensive middle-aged rats

Previous works from our laboratory have revealed that food restriction (FR) promotes discrete myocardial dysfunction in young rats. We examined the effects of FR on cardiac function, in vivo and in vitro, and ultrastructural changes in the heart of middle-aged rats. Twelve-month-old Wistar- Kyoto rats were fed a control (C) or restricted diet (daily intake reduced to 50% of the control group) for 90 days. Cardiac performance was studied by echocardiogram and in isolated left ventricular (LV) papillary muscle by isometric contraction in basal condition, after calcium chloride (5.2 mM) and beta- adrenergic stimulation with isoproterenol (10(-6) M). FR did not change left ventricular function, but increased time to peak tension, and decreased maximum rate of papillary muscle tension development. Inotropic maneuvers promoted similar effects in both groups. Ultrastructural alterations were seen in most FR rat muscle fibers and included, absence and/or disorganization of myofilaments and Z line, hyper-contracted myofibrils, polymorphic and swollen mitochondria with disorganized cristae, and a great quantity of collagen fibrils. In conclusion, cardiac muscle sensitivity to isoproterenol and elevation of extracellular calcium concentration is preserved in middle-aged FR rats. The intrinsic muscle performance depression might be related to morphological damage.

Long-term effects of developmental exposure to di-n-butyl-phthalate (DBP) on rat prostate: Proliferative and inflammatory disorders and a possible role of androgens

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

Fibrosis-related gene expression in the prostate is modulated by doxazosin treatment

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

Myxobolus cordeiroi n. sp., a parasite of Zungaro jahu (Siluriformes: Pimelodiade) from Brazilian Pantanal: Morphology, phylogeny and histopathology

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

Inhibition of 5-alpha-reductase activity induces stromal remodeling and smooth muscle de-differentiation in adult gerbil ventral prostate

Prostatic differentiation during embryogenesis and its further homeostatic state maintenance during adult life depend on androgens. Dihydrotestosterone, which is synthesized from testosterone by 5alpha-reductase (5alpha-r), is the active molecule triggering androgen action within the prostate. In the present work, we examined the effects of 5alpha-reductase inhibition by finasteride in the ventral prostate (VP) of the adult gerbil, employing histochemical and electron microscopy techniques to demonstrate the morphological and organizational changes of the organ. After 10 days of finasteride treatment at a dose of 100 mg/kg/day, the prostatic complex (VP and dorsolateral prostate) absolute weight was reduced to about 18%. The epithelial cells became short and cuboidal, with less secretory blebs and reduced acid phosphatase activity. The luminal sectional area diminished, suggestive of decreased secretory activity. The stromal/epithelial ratio increased, the stroma becoming thicker but less cellular. There was a striking accumulation of collagen fibrils, which was accompanied by an increase in deposits of amorphous granular material adjacent to the basal lamina and in the clefts between smooth muscle cells (SMC). Additionally, the periacinar smooth muscle became loosely packed. Some SMC were atrophic and showed a denser array of the cytoskeleton, whereas other SMC had a highly irregular outline with numerous spine-like projections. The present data indicate that 5alpha-r inhibition causes epithelial and stromal changes by affecting intra-prostatic hormone levels. These alterations are probably the result of an imbalance of the homeostatic interaction between the epithelium and the underlying stroma.