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J A Andrades

Publications and source records attributed to J A Andrades.

17 recordsLinked to original sources
PubMed2003

A modified rhTGF-beta1 and rhBMP-2 are effective in initiating a chondro-osseous differentiation pathway in bone marrow cells cultured in vitro.

Rat bone marrow cells were cultured in vitro in a collagen-gel medium at 0.5% fetal bovine serum concentration for 10 days in the presence of recombinant human transforming growth factor-beta-1, genetically engineered to contain a collagen binding domain (rhTGF-beta1-F2), or a commercial rhTGF-beta1. To compare the effects of TGF-betas with other growth factors in which the osteogenic capacity has been widely documented, a recombinant human bone morphogenetic protein (rhBMP-2) was evaluated. Once serum conditions compatible with growth were re-established, the selected cells were cultured for 6 more days in the presence of the growth factor. In the last 2 days, dexamethasone (dex) and beta-glycerophosphate (beta-GP) were added to promote osteogenesis. After this 16-day period, cells were placed into diffusion chambers or demineralized bone matrix (DBM) implants, and implanted subdermally on the backs of rats for 28 days. Biochemical, histological, and immunohistochemistry analysis provided evidence of cartilage (commercial rhTGF-beta1-treated cells), osteoid (rhTGF-beta1-F2-treated cells), and bone tissues (rhBMP-2 treated cells), inside the diffusion chambers, whereas bone, cartilage, and osteoid were observed inside the DBM implants under any of the three growth factors effect. Our study advances the technology capable of selecting a cell population from bone marrow that, in the presence of rhTGF-beta1 or rhBMP-2 in vitro, achieves chondro-osteogenic potential in vitro and in vivo.

Animals↗
PubMed2001

Production of a recombinant human basic fibroblast growth factor with a collagen binding domain.

Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney, and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-F1) from Escherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of a purification tag, a protease-sensitive linker and collagen binding domain, and a cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains the collagen binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured by a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a nickel-nitrilotriacetic acid metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of human vein endothelial cells, monitored by [3H]thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. The high-affinity binding was demonstrated by the binding of [3H]collagen to the rhbFGF-F2 protein immobilized on a Ni-nitrilotriacetic acid column. The rhbFGF-F2 fusion protein bound to collagen-coated surfaces with high affinity. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for their high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type I collagen. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins for specific biomedical applications.

Collagen↗
PubMed2001

Immunocytochemical detection of Reissner's fiber-like glycoproteins in the subcommissural organ and the floor plate of wildtype and cyclops mutant zebrafish larvae.

The subcommissural organ (SCO) and the floor plate (FP) secrete high molecular weight glycoproteins that polymerize in the form of the Reissner's fiber (RF). To study to what extent the absence of the FP affects the expression of these glycoproteins, we have investigated the brain and spinal cord of 48-h and 72-h wildtype and cyclops (cyc) mutant zebrafish larvae by using a polyclonal antiserum against bovine RF. Wildtype larvae showed immunoreactivity in the SCO at the dorsal forebrain-midbrain boundary. In the ventricle, over the SCO surface, thin immunoreactive fibers aggregated into an RF that ran along the third and fourth ventricles and the central canal of the spinal cord until, at its caudal end, the fiber disintegrated and formed a strongly immunoreactive massa caudalis that left the neural tube and invaded the surrounding tissues of the tail fin. The rostral end of the FP, lining the pontine flexure, was also strongly immunoreactive, as was the caudal third of the FP. Cyc mutants showed an immunoreactive SCO and fibrous material in the ventricle, but an RF was missing. There was no label in the ventral midline of the neural tube except in some specimens in which the caudal FP persisted and was immunoreactive. It is concluded that the product of the cyc gene is not required for the expression of SCO glycoproteins but for their polymerization into an RF in the brain ventricles.

Animals↗
PubMed2001

Engineering, expression, and renaturation of a collagen-targeted human bFGF fusion protein.

Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-F1) from Escherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen-binding domain, and (iii) cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains a collagen-binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured using a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factors was demonstrated by their ability to stimulate proliferation of human vein endothelial cells (HVEC), monitored by [3H]-thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. Binding of the renatured hbFGF-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column. The high affinity binding was also demonstrated by the binding of [3H]-collagen to the rhbFGF-F2 protein immobilized on a Ni-NTA column. The rhbFGF-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen-binding domain effectively targets the recombinant growth factor to type I collagen. The clinical effect of rhbFGF-F2 on wound healing is also studied in streptozotocin-induced diabetic rats and evaluated by histological examination comparing with rhbFGF-F1 and commercial bFGF effects. The highly beneficial effects of rhbFGF-F2 on wound healing is suggested to be due to its extremely potent angiogenesis and granulation tissue formation activities, leading to a rapid reepithelialization of the wound. Topical application of rhbFGF-F2 mixed with type I collagen is a more effective method in accelerating closure of full-thickness excisional skin-wound in diabetic rats when compared with the fusion protein alone or commercial hbFGF at the same doses. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins as well as to develop new strategies for specific biomedical applications.

Amino Acid Sequence↗
PubMed2001

Selection and amplification of a bone marrow cell population and its induction to the chondro-osteogenic lineage by rhOP-1: an in vitro and in vivo study.

The differentiation and maturation of osteoprogenitor cells into osteoblasts are processes which are thought to be modulated by transforming growth factors-beta (TGF-beta) as well as by bone morphogenetic proteins (BMPs). Osteogenic protein-1 (OP-1, also known as BMP-7) is a member of the BMP family, and it is considered to have important regulatory roles in skeletal embryogenesis and bone healing. Rat bone marrow cells were cultured in vitro in a collagen-gel medium containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of 40 ng/ml recombinant human OP-1 (rhOP-1). Under these conditions, survival of the bone marrow cell population was dependent on the presence of rhOP-1. Subsequently, the selected cells were cultured-for 6 days in medium containing 40 ng rhOP-1 and 10% FBS. During the last 2 days, dexamethasone (10(-8) M) and beta-glycerophosphate (2 mM) were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels, colony number and size were determined. Chondro-osteogenic differentiation in vitro was evaluated in terms of the expression of alkaline phosphatase, the production of osteocalcin and the formation of mineralized matrix. After culturing in vitro, cells were placed inside diffusion chambers or inactivated demineralized bone matrix (DBM) cylinders and implanted subdermically into the backs of old rats for 28 days. Biochemical, histological and immunocytochemical analyses provided evidence of cartilage and osteoid tissue inside the diffusion chambers, whereas bone was also observed inside the DBM implants. In conclusion, this experimental procedure is capable of selecting a cell population from bone marrow which, in the presence of rhOP-1, achieves skeletogenic potential under in vitro as well as in vivo environments.

Alkaline Phosphatase↗
PubMed1999

A recombinant human TGF-beta1 fusion protein with collagen-binding domain promotes migration, growth, and differentiation of bone marrow mesenchymal cells.

A continuous source of osteoblasts for normal bone maintenance, as well as remodeling and regeneration during fracture repair, is ensured by the mesenchymal osteoprogenitor stem cells of the bone marrow (BM). The differentiation and maturation of osteoprogenitor cells into osteoblasts are thought to be modulated by transforming growth factors-beta (TGF-beta1 and TGF-beta2) and TGF-beta-related bone morphogenetic proteins (BMPs). To define the responses of mesenchymal osteoprogenitor stem cells to several growth factors (GFs), we cultured Fischer 344 rat BM cells in a collagen gel medium containing 0.5% fetal bovine serum for prolonged periods of time. Under these conditions, survival of BM mesenchymal stem cells was dependent on the addition of GFs. Recombinant hTGF-beta1-F2, a fusion protein engineered to contain an auxiliary collagen binding domain, demonstrated the ability to support survival colony formation and growth of the surviving cells, whereas commercial hTGF-beta1 did not. Initially, cells were selected from a whole BM cell population and captured inside a collagen network, on the basis of their survival response to added exogenous GFs. After the 10-day selection period, the surviving cells in the rhTGF-beta1-F2 test groups proliferated rapidly in response to serum factors (10% FBS), and maximal DNA synthesis levels were observed. Upon the addition of osteoinductive factors, osteogenic differentiation in vitro was evaluated by the induction of alkaline phosphatase (ALP) expression, the production of osteocalcin (OC), and the formation of mineralized matrix. Concomitant with a down-regulation of cell proliferation, osteoinduction is marked by increased ALP expression and the formation of colonies that are competent for mineralization. During the induction period, when cells organize into nodules and mineralize, the expression of OC was significantly elevated along with the onset of extracellular matrix mineralization. Differentiation of BM mesenchymal stem cells into putative bone cells as shown by increased ALP, OC synthesis, and in vitro mineralization required the presence of specific GFs, as well as dexamethasone (dex) and beta-glycerophosphate (beta-GP). Although rhTGF-beta1-F2-selected cells exhibited the capacity to mineralize, maximal ALP activity and OC synthesis were observed in the presence of rhBMPs. We further report that a novel rhTGF-beta1-F2 fusion protein, containing a von Willebrand's factor-derived collagen binding domain combined with a type I collage matrix, is able to capture, amplify, and stimulate the differentiation of a population of cells present in rat BM. When these cells are subsequently implanted in inactivated demineralized bone matrix (iDBM) and/or diffusion chambers into older rats they are able to produce bone and cartilage. The population of progenitor cells captured by rhTGF-beta1-F2 is distinct from the committed progenitor cells captured by rhBMPs, which exhibit a considerably more differentiated phenotype.

Alkaline Phosphatase↗
PubMed1998

Floor plate and the subcommissural organ are the source of secretory compounds of related nature: comparative immunocytochemical study.

The subcommissural organ of vertebrates secretes glycoproteins into the third ventricle that condense to form Reissner's fiber (RF). Antibodies raised against the bovine RF-glycoproteins reacted with the floor plate (FP) cells of two teleost (Oncorhynchus kisutch, Sparus aurata) and two amphibian (Xenopus laevis, Batrachyla taeniata) species. At the ultrastructural level, the immunoreactivity was confined to secretory granules, mainly concentrated at the apical cell pole. In the rostro-caudal axis, a clear zonation of the FP was distinguished, with the hindbrain FP being the most, or the only (Batrachyla taeniata), immunoreactive region of the FP. In all the species studied, the caudal FP lacked immunoreactivity. Both the chemical nature of the immunoreactive material and the rostro-caudal zonation of the FP appear to be conservative features. Evidence was obtained that the FP secretes into the cerebrospinal fluid a material chemically related to the RF-glycoproteins secreted by the subcommissural organ. Thus, in addition to being the source of contact-mediated and diffusible signals, the FP might also secrete compounds into the cerebrospinal fluid that may act on distant targets.

Animals↗
PubMed1997

Nitric oxide mediates hyperglycemia-induced defective migration in cultured endothelial cells.

PURPOSE: To examine the effects of elevated glucose on the migration and proliferation of vascular endothelial cells in an in vitro wound model and to investigate whether nitric oxide (NO) mediates the effects of elevated glucose. METHODS: Migration was investigated in monolayers of bovine aortic endothelial cells wounded by scraping and measuring the distance, the number of cells migrating, and the area covered by the migrating cells in the presence of various concentrations of glucose. The effects of NO were evaluated by adding to the cultures NG-monomethyl arginine (NMMA), an inhibitor of NO synthase, or S-nitrosylated penicillamine, which is a slow-release agent of NO. Proliferation was investigated in the presence of various concentrations of serum, glucose, or both. RESULTS: Elevated glucose levels (16.5 and 27.7 mmol/L) inhibited endothelial cell migration in a dose-dependent manner compared with cells cultured in the presence of 5.5 mmol/L glucose. Inhibition of migration was also observed when wounded mono-layers cultured in 5.5 mmol/L glucose were treated with S-nitrosylated penicillamine, which generates NO. Inhibition of NO synthase by NMMA prevented the inhibition of migration observed in media containing 27.7 mmol/L glucose. Elevated glucose levels did not affect cell proliferation except in the presence of 20% fetal bovine serum. CONCLUSIONS: An elevated glucose level inhibits endothelial cell migration in an in vitro wound model, and the inhibition appears to be mediated by increased levels of NO.

Animals↗
PubMed1996

Complement proteins are present in developing endochondral bone and may mediate cartilage cell death and vascularization.

Normal endochondral bone formation follows a temporal sequence: immature or resting chondrocytes move away from the resting zone, proliferate, flatten, become arranged into columns, and finally become hypertrophic, disintegrate, and are replaced by bone. The mechanisms that guide this process are incompletely understood, but they include programmed cell death, a stage important in development and some disease processes. Using immunofluorescence we have studied the distribution of various complement proteins to examine the hypothesis that this sequence of events, particularly cell disintegration and matrix dissolution, are complement mediated. The results of these studies show that complement proteins C3 and Factor B are distributed uniformly in the resting and proliferating zones. Properdin is localized in the resting and hypertrophic zone but not in the proliferating zone. Complement proteins C5 and C9 are localized exclusively in the hypertrophic zones. This anatomically segregated pattern of distribution suggests that complement proteins may be important in cartilage-bone transformation and that the alternate pathway is involved.

Animals↗
PubMed1996

Demineralized bone matrix mediates differentiation of bone marrow stromal cells in vitro: effect of age of cell donor.

Bone maintenance requires a continuous source of osteoblasts throughout life. Its remodeling and regeneration during fracture repair is ensured by osteoprogenitor stem cells which are part of the stroma of the bone marrow (BM). Many investigators have reported that in cultured BM stromal cells there is a cell population that will differentiate along an osteogenic lineage if stimulated by the addition of osteogenic inducers, such as dexamethasone (dex), beta-glycerophosphate (beta-GP), transforming growth factor beta-1 (TGF-beta 1) and bone morphogenetic protein-2 (BMP-2). Here we report the effects of demineralized bone matrix (DBM) on the osteogenic differentiation of BM stromal cells in vitro, using morphological criteria, alkaline phosphatase (AP) activity, and calcium accumulation. DBM and DBM-conditioned medium (DBMcm) enhanced bone formation in the presence of dex and beta-GP, whereas DBM particles caused changes in the cell phenotype. Temporal expression of total and skeletal AP by BM stromal cells from 4-week-old rats showed a biphasic pattern enhanced by DBM and suggesting the presence of two cell populations. In one population, AP synthesis reaches a maximum during the first week in culture, following which cells either die or loose their ability to synthesize AP. A second, less abundant population begins to proliferate and synthesize AP during the second and third weeks. The synthesis of AP, which often decreases by the third week, can be maintained at high levels only if DBM is added to the cultures. BM stromal cells isolated from 24- and 48-week-old rats showed a decrease or loss of this biphasic AP expression pattern compared with cells isolated from 4-week-old rats. The addition of DBM to cultures derived from 24- and 48-week-old rats stimulated mostly the second cell population to synthesize AP, suggesting that DBM contains a factor(s) that acts on a specific bone marrow cell population by increasing the proliferation of active cells or inducing the differentiation of dormant cells.

Aging↗
PubMed1994

Perinotochordal connective sheet of gilthead sea bream larvae (Sparus aurata, L.) affected by axial malformations: an histochemical and immunocytochemical study.

BACKGROUND: Spinal malformations in adult teleosts occur under natural conditions and, more frequently, in culture exploitations. Skeletal deformities are linked with dysfunctions in collagen metabolism. We studied axial deviations appearing in early larval stages of cultured sea bream (Sparus aurata, L.). METHODS: To evaluate connective tissue components of normal and lordotic fish we used histochemistry (alcian blue, picrosirius-polarization, clorhydric orcein, fuchsin resorcin), immunohistochemistry (anti-collagen I, II, III, and IV), and specific enzymatic digestions. The results were evaluated by semiquantitative methods. RESULTS: Lordosis appeared before a vertebral column was developed, thus affecting the only skeletal structure present in the animal body, the notochord. At this stage the animal depends on the vitelline sac and an inflated swim-bladder is missing. The region of the curvature showed strong alterations in the arrangement of the muscle bundles and irregularities in notochord and perinotochordal collagen sheet. Histochemical and immunocytochemical analysis of the perinotochordal sheet revealed the presence of type II collagen, non-sulfated glycosaminoglycans, and elastic fibers in normal and lordotic specimens. Low collagen-proteoglycan interactions occurred in lordotic animals. CONCLUSIONS: Lordosis in Sparus aurata originated during embryonic development and was characterized by disorganized connective tissue and muscle bundles. No major differences in connective tissue constituents were seen with respect to normal specimens.

Animals↗
PubMed1994

Combined use of immunocytochemistry and lectin histochemistry for the study of the hypothalamic neurosecretory system of the snake Natrix maura (L.).

Natrix maura snakes were processed for immunocytochemistry and lectin histochemistry at both light- and electron-microscopic levels. Antisera against bovine neurophysins, vasotocin and mesotocin were used as well as concanavalin A, wheat germ and Limax flavus agglutinin lectins. The hypothalamic supraoptic and paraventricular nuclei were studied. Vasotocin neurons should contain a glycopeptide and displayed large colloid droplets consisting of large cisternae filled with packed secretory material. Mesotocin was located in different neurons.

Animals↗
PubMed1994

Skeletal deformities of the gilthead sea bream (Sparus aurata, L.): study of the subcommissural organ (SCO) and Reissner's fiber (RF).

A high incidence of lordotic curvatures has been detected in commercial cultures of Sparus aurata. We have studied juvenile and adult lordotic specimens to elucidate whether the subcommissural organ and its secretion, the Reissner's fiber, play any role in the development of this syndrome. Animals were X-radiographed and then the brain and spinal cord dissected out and processed for light microscopy. Adult lordotic fishes had a well developed swim-bladder whereas juvenile did not. The central canal of the spinal cord showed dramatic alterations, and an altered Reissner's fiber was always present. Our histochemical results suggested a hyperactivity of the subcommissural organ in lordotic fishes.

Aging↗
PubMed1989

[Increase in the number of colloidal droplets in the hypothalamus of Natrix maura caused by dehydration].

The influence of osmotic stress on the number of colloid droplets in the magnocellular neurosecretory nuclei of the water snake Natrix maura, has been studied. Five experimental groups of five specimens each, were submitted to dehydration by immersion in sea water for several periods of time (3 to 60 h). The number of colloid droplets, identified by histochemical procedures, was counted in serial sections of the retrochiasmatic nuclei. The study of the mean of each group revealed that the amount of colloids increased with the time of permanence in the hyperosmotic environment (48 h elicited the greatest response). As a conclusion, dehydration seems to activate the hypothalamic magnocellular neurosecretory nuclei of N. maura and, consequently, increases the production of colloid droplets.

Animals↗