Genetic Responses to Nanostructured Calcium-phosphate-coated Implants

Nanostructured calcium phosphate (CaP) has been histologically and biomechanically proven to enhance osseointegration of implants; however, conventional techniques were not sufficiently sensitive to capture its biological effects fully. Here, we compared the conventional removal torque (RTQ) evaluation and gene expression in tissues around nanostructured CaP-coated implants, using real-time RT-PCR, with those of uncoated implants, in a rabbit model. At 2 wks, RTQ values were significantly higher, alkaline phosphatase (ALP) expression was significantly higher, and runt-related transcription factor 2 and tumor necrosis factor-alpha expressions were significantly lower in the coated than in the uncoated implants. This indicates that inflammatory responses were suppressed and osteoprogenitor activity increased around the CaP-coated surface. At 4 wks, although RTQ values did not significantly differ between the 2 groups, ALP and osteocalcin (OCN) were significantly up-regulated in the coated group, indicating progressive mineralization of the bone around the implant. Moreover, an osteoclast marker, adenosine triphosphatase, which indicates acidification of the resorption lacunae, was significantly higher for the coated implants, suggesting gradual resorption of the CaP coating. This study reveals detailed genetic responses to nanostructured CaP-coated implants and provides evidence that the effect of nanotopography is significant during the osseointegration cascade.

Maternal protein restriction induce skeletal muscle changes without altering the MRFs MyoD and myogenin expression in offspring

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

In situ evaluation of anticancer drug methotrexate-DNA interaction using a DNA-electrochemical biosensor and AFM characterization

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Structures of human purine nucleoside phosphorylase complexed with inosine and ddI

Human purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effect on B-cell function. PNP is highly specific for 6-oxopurine nucleosides and exhibits negligible activity for 6-aminopurine nucleosides. The catalytic efficiency for inosine is 350,000-fold greater than for adenosine. Adenine nucleosides and nucleotides are deaminated by adenosine deaminase and AMP deaminase to their corresponding inosine derivatives which, in turn, may be further degraded. Here we report the crystal structures of human PNP in complex with inosine and 2',3'-dideoxymosine, refined to 2.8 Angstrom resolution using synchrotron radiation. The present structures provide explanation for ligand binding, refine the purine-binding site, and can be used for future inhibitor design. (C) 2003 Elsevier B.V. All rights reserved.

Seasonal changes in blood oxygen transport and acid-base status in the tegu lizard, Tupinambis merianae

Oxygen-binding properties, blood gases, and acid-base parameters were studied in tegu lizards, Tupinambis merianae, at different seasons and temperatures. Independent of temperature and pH, blood oxygen affinity was higher in dormant lizards than in those active during the summer. Haematocrit (Hct) and hemoglobin content ([Hb]) were greater in active lizards resulting in a higher oxygen-carrying capacity. Nucleoside triphosphate content ([NTP]) was reduced during dormancy, but the ratio between [NTP] and [Hb] remained unchanged. Dormancy was accompanied by an increase in plasma bicarbonate ([HCO(3)(-)]PI) and an elevation of arterial CO(2) partial pressure (P(aCO2)) and CO(2) content in the plasma (C(PlCO2)). These changes in acid-base parameters persist over a broad range of body temperatures. In vivo, arterial O(2) partial pressure (Pa(O2)) and O(2) content (Ca(O2)) were not affected by season and tended to increase with temperature. Arterial pH (pH(a)) of dormant animals is reduced compared to active lizards at body temperatures below 15 degreesC, while no significant difference was noticed at higher temperatures. (C) 2003 Elsevier B.V. All rights reserved.

Detection of DNA Nucleotides on Pretreated Boron Doped Diamond Electrodes

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

Alchornea glandulosa Ethyl Acetate Fraction Exhibits Antiangiogenic Activity: Preliminary Findings from In Vitro Assays Using Human Umbilical Vein Endothelial Cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Aerobic training, but not creatine supplementation, alters the gluteus medius muscle

The aim of the present study was to investigate the effect of oral supplementation of creatine on the muscular responses to aerobic training. Twelve purebred Arabian horses were submitted to aerobic training for 90 d, with and without creatine supplementation, and evaluated with respect to BW and BCS and to the area and frequency of the different types of muscle fibers in the gluteus medius. Supplementation consisted of the daily administration of 75 g of creatine monohydrate mixed into the ration for the 90 d of training. Physical conditioning was conducted on a high-performance treadmill, and training intensity was stipulated by calculating the velocity at which blood lactate reaches 4 mmol/L, determined monthly for each animal. The individual intensity of physical force at 80% of aerobic threshold was established. Morphometry of glutens medius muscle fibers was performed on frozen sections processed for histochemical analysis of myosin adenosine triphosphatase and immunohistochemistry of slow-contracting myosin. The results demonstrated that the animals maintained a moderate BCS without alteration of BW during the course of training, providing evidence of equilibrium between food intake and caloric expenditure during the study period. The present study demonstrated that aerobic training for 90 d caused hypertrophy of fiber types I (P = 0.04), IIA (P = 0.04), and IIX (P = 0.01), as well as an increase in the relative area occupied by type I fibers (P = 0.02) at the expense of type IIX fibers (P = 0.03), resulting in modifications of the contractile and metabolic characteristics of the gluteus medius muscle. It was not possible to show any beneficial effect from creatine on the skeletal muscle characteristics examined.

Localization and irregular distribution of Na,K-ATPase in myelin sheath from rat sciatic nerve

Sodium, potassium adenosine triphosphatase (Na,K-ATPase) is a membrane-bound enzyme that maintains the Na+ and K+ gradients used in the nervous system for generation and transmission of bioelectricity. Recently, its activity has also been demonstrated during nerve regeneration. The present study was undertaken to investigate the ultrastructural localization and distribution of Na,K-ATPase in peripheral nerve fibers. Small blocks of the sciatic nerves of male Wistar rats weighing 250-300g were excised, divided into two groups, and incubated with and without substrate, the para-nitrophenyl phosphate (pNPP). The material was processed for transmission electron microscopy, and the ultra-thin sections were examined in a Philips CNI 100 (TM) electron microscope. The deposits of reaction product were localized mainly on the axolemma, on axoplasmic profiles, and irregularly dispersed on the myelin sheath, but not in the unmyelinated axons. In the axonal membrane, the precipitates were regularly distributed on the cytoplasmic side. These results together with published data warrant further studies for the diagnosis and treatment of neuropathies with compromised Na,K-ATPase activity. (c) 2007 Elsevier Ltd. All rights reserved.

Granulocyte colony-stimulating factor expression from transduced vascular smooth muscle cells provides sustained neutrophil increases in rats

Granulocyte colony-stimulating factor (G-CSF) regulates granulocyte precursor cell proliferation, neutrophil survival, and activation. Cyclic hematopoiesis, a disease that occurs both in humans and grey collie dogs is characterized by cyclical variations in blood neutrophils. Although the underlying molecular defect is not known, long-term daily administration of recombinant G-CSF eliminates the severe recurrent neutropenia, indicating that expression of G-CSF by gene therapy would be beneficial. As a prelude to preclinical studies in affected collie dogs, we monitored hematopoiesis in rats receiving vascular smooth muscle cells transduced to express G-CSF. Cells transduced with LrGSN, a retrovirus expressing rat G-CSF, were implanted in the carotid artery and control animals received cells transduced with LASN, a retrovirus expressing human adenosine deaminase (ADA). Test animals showed significant increases in neutrophil counts for at least 7 weeks, with mean values of 3,670 +/- 740 cells/mu l in comparison to 1,870 +/- 460 cells/mu l in controls (p < 0.001). Thus, in rats G-CSF gene transfer targeted at vascular smooth muscle cells initiated sustained production of 1,800 neutrophils/mu l, a cell number that would provide clinical benefit to patients. Lymphocytes, red cells and platelets were not different between control and test animals (p > 0.05). These studies indicate that retrovirally transduced vascular smooth muscle cells can provide sustained clinically useful levels of neutrophils in vivo.

Signaling path of the action of AVP on distal K+ secretion

Background. Previous studies from our laboratory have shown that luminal perfusion with arginine vasopressin (AVP) stimulates distal tubule secretory potassium flux (J(K)) via V1 receptors (Am J Physiol 278: F809- F816, 2000). In the present work, we investigate the cell signaling mechanism of this process.Methods. In vivo stationary microperfusion was performed in rat cortical distal tubules and luminal K was measured using double K+ resin/reference microelectrodes.Results. In control conditions, J(K) was 0.71 +/- 0.05 nmol. cm(-2).second(-1); this process was inhibited (14%) by 10(-5) mol/L 8-bromo-cyclic adenosine monophosphate (cAMP), and increased by 35% with 10(-8) mol/L phorbol ester [phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC)]. During luminal perfusion with 10(-11) mol/L AVP, J(K) increased to 0.88 +/- 0.08 nmol. cm(-2).seconds(-1). In the presence of 10(-11) mol/L AVP, J(K) was not affected by 10(-4) mol/L H89, a blocker of protein kinase A (PKA), but was inhibited (45%) by 10(-5) mol/L staurosporine, an inhibitor of PKC, and by 41% during perfusion with 5 x 10(-5) mol/L of the cell Ca2+ chelator bis (2-aminophenoxy) ethane-tetraacetic acid (BAPTA). In order to study the role of Ca2+-dependent K channels in the luminal hormonal action, the tubules were perfused with 5 mmol/L tetraethylammonium chloride (TEA) or 10(-7) mol/L iberiotoxin, in the presence of AVP, and JK was significantly reduced by both agents. Iberiotoxin reduced AVP-stimulated J(K) by 36.4%, and AVP-independent J(K) (after blocking V1 receptors) by only 16%.Conclusion. The results suggest that the luminal V1-receptor effect of AVP on J(K) was mediated by the phospholipase C (PLC)/ Ca2+/PKC signaling path and not by adenylate cyclase/cAMP/PKA, therefore probably acting on maxi-potassium channels.

Morphometric and histochemical study of the human vocal muscle

The present study was conducted on vocal muscles removed at autopsy Rom adult individuals (10 men and 8 women, aes ranging from 48 to 78 years) with no laryngeal disease. Histologic analysis was performed with hematoxylin and eosin staining, and histochemical analysis was performed by nicotinamide-adenine-dinucleotide tetrazolium reductase, succinate dehydrogenase, and acid and alkaline myofibrillar adenosine triphosphatase reactions. The histochemical reactions showed that the muscle consists of slow-twitch oxidative (SO), fast-twitch glycolytic (FG), and fast-twitch glycolytic oxidative (FOG) fibers distributed in mosaic form. The frequencies of SO, FOG, and FG fibers were 40.50%, 54.75%, and 4.75%, respectively. The higher frequency of SO and FOG oxidative fibers characterizes the muscle as having aerobic metabolism, resistance to fatigue, and fast contraction. The mean minimum diameters were 31.37 mu m for SO fibers and 36.46 mu m for FOG and FG fibers.

Agents that inhibit histamine release: A review

It is well known that histamine is found in high concentration in mast cell granules(1). The histamine content of these granules may be released to the extracellular space if an appropriate stimulus is provided(2). Besides histamine, other preformed active substances like enzymes, chemotatic factors and proteoglycans, as well as newly generated mediators like eicosanoids, platelet activating factor and adenosine are released during the secretion process of mast cells(3). The activation of mast cell degranulation has been associated with a number of pathologic disorders, most frequently, diseases derived from the atopic state(4). It is now evident that mast cells are the primary effector cells in the early reaction in both allergic and non-allergic asthma(5,6), although some authors doubt that the late reaction of asthma is a mast cell dependent event(6). Other studies point towards basophils as cellular elements involved in the secondary phase of inflammation in allergic diseases(7). Secretion would depend on a histamine releasing factor, and on the presence of IgE on the basophil's surface(8). There is also evidence suggesting involvement of mast cells in some non-allergic inflammatory processes like arthritis(9). The pharmacological management of these diseases basically consists in the use of methylxantines, beta 2-adrenergic agonists, glucocorticoids, sodium cromoglycate-like drugs, anticholinergic and antihistaminic H 1 antagonists(10). Their therapeutic effects include bronchodilatation, receptor and physiological antagonism, prevention of inflammatory responses induced by secondary cells, and finally, inhibition of mast cell activation(11). This review is concerned with compounds having inhibitory action on mast cell activation, and their possible importance on the pathophysiology of mast cell-related diseases.

The Effects of N Helix Deletion and Mutant F29W on the Ca2+ Binding and Functional Properties of Chicken Skeletal Muscle Troponin C

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L.B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (±Ca2+) of F29W and F29W/ NHdel and titration of the Ca2+-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca2+-activated enzymic activities. These effects are cumulative in the double mutant F29W/ NHdel. On the other hand, maximal isometric tension development in Ca2+-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.

Evaluation of platelet number and function and fibrinogen level in patients bitten by snakes of the Bothrops genus.

Platelet function and plasma fibrinogen levels were evaluated in 14 patients, 10 males and 4 females, aged 13-59 years bitten by Bothrops genus snakes. There was a statistical difference (p < 0.05) among plasma fibrinogen levels evaluated 24 and 48 hours after envenomation. There was a tendency towards normalization after 48 hours of treatment. The low platelet number was clear in 24-48 hour evaluations with a tendency towards normalization after 48 hours of treatment (p < 0.05). When platelet function was stimulated by collagen and epinephrine, it appeared to be within normal values. On the other hand, when it was stimulated by adenosine diphosphate (ADP), platelet function was hypoaggregated by a single micromol concentration until 48 hours after treatment. At a 3 micromol concentration, there were alterations only before specific treatment (p < 0.05). Fibrinogen levels and fibrin degradation product (FDP) levels appeared to be altered in 83.33% of patients evaluated. The authors suggest that platelet hypoaggregation is related to decreased fibrinogen and increased FDP levels.