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Sample records for crecimiento transformante beta-3

  1. Immunological characterization of eristostatin and echistatin binding sites on alpha IIb beta 3 and alpha V beta 3 integrins.

    PubMed Central

    Marcinkiewicz, C; Rosenthal, L A; Mosser, D M; Kunicki, T J; Niewiarowski, S

    1996-01-01

    Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with alpha IIb beta 3 genes (A5 cells) and to CHO cells transfected with alpha v beta 3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the beta 3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (alpha IIb beta 3 complex dependent) and 7E3 (alpha IIb beta 3 and alpha v beta 3 complex dependent) to A5 cells, to resting and to activated platelets and to purified alpha IIb beta 3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on alpha IIb beta 3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on alpha v beta 3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified alpha v beta 3 suggesting that alpha v beta 3 and alpha IIb beta 3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on alpha v beta 3. PMID:8760368

  2. [Functional exploration of brown adipose tissue using beta3 agonists].

    PubMed

    Bertin, R; de Marco, F; Blancher, G; Portet, R

    1994-06-01

    In view to utilize beta 3 adrenoceptor agonists for the investigation of body lipid metabolism, a study of the effects of BRL 37344 on the functional activity of the brown adipose tissue was performed in the Rat. It is known that this tissue is the principal site of heat production for nonshivering thermogenesis mainly due to the oxidation of fatty acids under the control of norepinephrine (NA) released from the sympathetic nervous system. In order to stimulate the activity of the tissue, rats were reared at 16 degrees C. When they were one month old, they were divided in two groups; one group received a surgical sympathectomy of the interscapular brown adipose tissue (TABI) (S group); the other group was sham-operated (T group). The resting metabolism was estimated by the continuous measurement of O2 consumption and CO2 release, at an ambient temperature of 25 degrees C. The animal capacity for nonshivering thermogenesis was determined by increased O2 consumption following i.p. administration of NA or BRL 37344. In the S group a large decrease in TABI NA content and a decrease in resting metabolism were observed. In both groups VO2 was increased by the two drugs; the increase was linearly related to the dose of BRL (between 2.5 to 10 micrograms/kg); but it was 3 times as high in the T group as in the S group. Moreover, the effect of BRL was 40 fold greater than the effect of NA. These results seem to indicate that, in cold reared rats, a part of nonshivering thermogenesis may be mediated by the beta 3 receptors of the brown fat. It may be concluded that the rats born in cold conditions are good models to study the role of beta 3 receptors in the energetic activity of this tissue very profuse in infant but not in adult man. PMID:7994586

  3. Discovery of novel acetanilide derivatives as potent and selective beta3-adrenergic receptor agonists.

    PubMed

    Maruyama, Tatsuya; Onda, Kenichi; Hayakawa, Masahiko; Matsui, Tetsuo; Takasu, Toshiyuki; Ohta, Mitsuaki

    2009-06-01

    In the search for potent and selective human beta3-adrenergic receptor (AR) agonists as potential drugs for the treatment of obesity and noninsulin-dependent (type II) diabetes, a novel series of acetanilide-based analogues were prepared and their biological activities were evaluated at the human beta3-, beta2-, and beta1-ARs. Among these compounds, 2-pyridylacetanilide (2f), pyrimidin-2-ylacetanilide (2u), and pyrazin-2-ylacetanilide (2v) derivatives exhibited potent agonistic activity at the beta3-AR with functional selectivity over the beta1- and beta2-ARs. In particular, compound 2u was found to be the most potent and selective beta3-AR agonist with an EC(50) value of 0.11 microM and no agonistic activity for either the beta1- or beta2-AR. In addition, 2f, 2u, and 2v showed significant hypoglycemic activity in a rodent diabetic model. PMID:19232786

  4. Evidence for the presence of beta 3-adrenergic receptor mRNA in the human brain.

    PubMed

    Rodriguez, M; Carillon, C; Coquerel, A; Le Fur, G; Ferrara, P; Caput, D; Shire, D

    1995-04-01

    The beta 3-adrenergic receptor (AR) is widely distributed in peripheral tissues, but up to now it has not been detected in the central nervous system. By using the polymerase chain reaction (PCR) technique, we found the beta 3-AR mRNA to be present in all the regions of the human brain we investigated. The quantities found were very low compared to those of the beta 1-AR and beta 2-AR mRNAs, being hardly detectable in adult brain. In contrast, the brain of very young infants contained about 100 times more beta 3-AR mRNA than the adult brain, whereas the amounts of beta 1-AR and beta 2-AR transcripts were essentially the same. In addition, using PCR we have cloned a central beta 3-AR coding region from a human frontal cortex cDNA library and have found it to be identical to the corresponding peripheral sequence. PMID:7609625

  5. Targeted inhibition of {alpha}v{beta}3 integrin with an RNA aptamer impairs endothelial cell growth and survival

    SciTech Connect

    Mi Jing; Zhang Xiuwu; Giangrande, Paloma H.; McNamara, James O.; Nimjee, Shahid M.; Sarraf-Yazdi, Shiva; Sullenger, Bruce A.; Clary, Bryan M. . E-mail: mi001@duke.edu

    2005-12-16

    {alpha}v{beta}3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. {alpha}v{beta}3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of {alpha}v{beta}3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-{alpha}v{beta}3 that binds recombinant {alpha}v{beta}3 integrin, for its ability to bind endogenous {alpha}v{beta}3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-{alpha}v{beta}3 binds {alpha}v{beta}3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-{alpha}v{beta}3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-{alpha}v{beta}3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-{alpha}v{beta}3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.

  6. Localisation of Neuregulin 1-{beta}3 to different sub-nuclear structures alters gene expression

    SciTech Connect

    Wang, Ming; Trim, Carol M.; Gullick, William J.

    2011-02-15

    Neuregulins are growth factors that signal via the ErbB3 and ErbB4 receptors. Here we show using immunohistochemistry that they are often expressed in the nucleus of a range of tumour types including soft tissue and breast. The Neuregulin 1 type I-{beta}3 (NRG1-{beta}3) isoform localises to two sub-nuclear compartments in animal cells, nucleoli and spliceosomes. We used NRG1-{beta}3 tagged with photoactivatable GFP and demonstrated that this re-localised from nucleoli to spliceosomes over 90 min. Tyrosine kinase activity was not required for retaining the NRG1-{beta}3 within the nucleus. Mutation of the lysines 14 and 16 or 15 and 16 together prevented nucleolar uptake while four positively charged residues were identified which were required for spliceosome uptake. Molecular modelling suggests that three of these may form a binding site. We showed using a kinome array that NRG1-{beta}3 and a mutant exclusively localising to spliceosomes increased phosphorylation and/or expression of the HER4 and HER2 receptors. Using a transcriptomic analysis the same two constructs induced expression of several messenger RNAs and we confirmed the increased expression at the protein level of the most highly induced, Heat Shock Protein 70B'. These results suggest that Neuregulin activates receptor signalling in spliceosomes leading to altered gene expression.

  7. Interaction of disintegrins with the alpha IIb beta 3 receptor on resting and activated human platelets.

    PubMed Central

    McLane, M A; Kowalska, M A; Silver, L; Shattil, S J; Niewiarowski, S

    1994-01-01

    Viper venom disintegrins contain the RGD/KGD motif. They inhibit platelet aggregation and cell adhesion, but show structural and functional heterogeneity. We investigated the interaction of four prototypic disintegrins with alpha IIb beta 3 expressed on the surface of resting and activated platelets. The binding affinity (Kd) of 125I-albolabrin, 125I-echistatin, 125I-bitistatin and 125I-eristostatin toward resting platelets was 294, 153, 48 and 18 nM respectively. The Kd value for albolabrin decreased 3-fold and 6-fold after ADP- or thrombin-induced activation. The Kd values for bitistatin and echistatin also decreased with ADP, but there was no further decrease with thrombin. In contrast, eristostatin bound with the same high affinity to resting and activated platelets. The pattern of fluorescein isothiocyanate (FITC)-eristostatin and FITC-albolabrin binding to resting and activated platelets was consistent with observations using radiolabelled material. Eristostatin showed faster and more irreversible binding to platelets, and greater potency compared with albolabrin in inducing conformational neo-epitopes in beta 3. The anti-alpha IIb beta 3 monoclonal antibody OP-G2 that is RGD-dependent inhibited disintegrin binding to activated platelets more strongly than binding to resting platelets and it inhibited the binding to platelets of albolabrin more strongly than eristostatin. The specificity of disintegrin interaction with alpha IIb beta 3 was confirmed by demonstrating cross-linking of these peptides to alpha IIb beta 3 on normal platelets, but not to thrombasthenic platelets deficient in alpha IIb beta 3. Images Figure 6 PMID:8042985

  8. Etomidate, propofol and the neurosteroid THDOC increase the GABA efficacy of recombinant alpha4beta3delta and alpha4beta3 GABA A receptors expressed in HEK cells.

    PubMed

    Meera, Pratap; Olsen, Richard W; Otis, Thomas S; Wallner, Martin

    2009-01-01

    General anesthetics, once thought to exert their effects through non-specific membrane effects, have highly specific ion channel targets that can silence neuronal populations in the nervous system, thereby causing unconsciousness and immobility, characteristic of general anesthesia. Inhibitory GABA(A) receptors (GABA(A)Rs), particularly highly GABA-sensitive extrasynaptic receptor subtypes that give rise to sustained inhibitory currents, are uniquely sensitive to GABA(A)R-active anesthetics. A prominent population of extrasynaptic GABA(A)Rs is made up of alpha4, beta2 or beta3, and delta subunits. Considering the demonstrated importance of GABA receptor beta3 subunits for in vivo anesthetic effects of etomidate and propofol, we decided to investigate the effects of GABA anesthetics on "extrasynaptic" alpha4beta3delta and also binary alpha4beta3 receptors expressed in human embryonic kidney (HEK) cells. Consistent with previous work on similar receptor subtypes we show that maximal GABA currents through "extrasynaptic" alpha4beta3delta receptors, receptors defined by sensitivity to EtOH (30mM) and the beta-carboline beta-CCE (1microM), are enhanced by the GABA(A)R-active anesthetics etomidate, propofol, and the neurosteroid anesthetic THDOC. Furthermore, we show that receptors formed by alpha4beta3 subunits alone also show high GABA sensitivity and that saturating GABA responses of alpha4beta3 receptors are increased to the same extent by etomidate, propofol, and THDOC as are alpha4beta3delta receptors. Therefore, both alpha4beta3 and alpha4beta3delta receptors show low GABA efficacy, and GABA is also a partial agonist on certain binary alphabeta receptor subtypes. Increasing GABA efficacy on alpha4/6beta3delta and alpha4beta3 receptors is likely to make an important contribution to the anesthetic effects of etomidate, propofol and the neurosteroid THDOC. PMID:18778723

  9. Integrin subunit beta3 plays a crucial role in the movement of osteoclasts from the periosteum to the bone surface.

    PubMed

    Holt, I; Marshall, M J

    1998-04-01

    We have shown that, when mouse parietal bones were incubated in culture medium containing indomethacin, the number of tartrate-resistant acid phosphatase-positive osteoclasts (TRAP + OCs) on the bone surface was drastically reduced (down-regulation), and the number on the periosteal membrane adjacent to the resorbing surface was increased. Subsequent incubation of bones with prostaglandin E2 (PGE2) rapidly reversed these changes (up-regulation). In the work reported here, the osteoclast-associated integrin subunit beta3 was stained by immunohistochemistry. The beta3-positive osteoclast (beta3 + OC) population on freshly isolated bone was comprised of about 67% TRAP + OCs and 33% TRAP OCs. Like TRAP + OCs, beta3 + OCs were reduced in number on the surface of bones incubated with indomethacin, but, in contrast to the TRAP + OCs, beta3 + OCs were not seen on the periosteal membrane. Following up-regulation of TRAP + OCs with PGE2, large numbers of beta3 + OCs appeared on the bone surface and, again, were not seen on the periosteal membrane. Echistatin, a peptide that binds to the alphavbeta3 integrin on osteoclasts, was found to inhibit the up-regulation of TRAP + OCs in a dose-dependent manner but had no effect on the down-regulation of TRAP + OCs. Similarly, echistatin inhibited the upregulation of beta3 + OCs on the bone surface, and, under these conditions, beta3 + OCs were observed on the periosteal membrane. The addition of anti-beta3 antibody also inhibited the up-regulation of TRAP + OCs in response to PGE2. The association of beta3 protein expression with the up-regulated osteoclast and the inhibition of up-regulation by echistatin and by anti-beta3 antibody provide strong evidence that beta3 plays an essential role in the movement of osteoclasts from the membrane to the bone. PMID:9491775

  10. Effect of adipocyte beta3-adrenergic receptor activation on the type 2 diabetic MKR mice.

    PubMed

    Kim, Hyunsook; Pennisi, Patricia A; Gavrilova, Oksana; Pack, Stephanie; Jou, William; Setser-Portas, Jennifer; East-Palmer, Joyce; Tang, Yan; Manganiello, Vincent C; Leroith, Derek

    2006-06-01

    The antiobesity and antidiabetic effects of the beta3-adrenergic agonists were investigated on nonobese type 2 diabetic MKR mice after injection with a beta3-adrenergic agonist, CL-316243. An intact response to acute CL-316243 treatment was observed in MKR mice. Chronic intraperitoneal CL-316243 treatment of MKR mice reduced blood glucose and serum insulin levels. Hyperinsulinemic euglycemic clamps exhibited improvement of the whole body insulin sensitivity and glucose homeostasis concurrently with enhanced insulin action in liver and adipose tissue. Treating MKR mice with CL-316243 significantly lowered serum and hepatic lipid levels, in part due to increased whole body triglyceride clearance and fatty acid oxidation in adipocytes. A significant reduction in total body fat content and epididymal fat weight was observed along with enhanced metabolic rate in both wild-type and MKR mice after treatment. These data demonstrate that beta3-adrenergic activation improves the diabetic state of nonobese diabetic MKR mice by potentiation of free fatty acid oxidation by adipose tissue, suggesting a potential therapeutic role for beta3-adrenergic agonists in nonobese diabetic subjects. PMID:16682489

  11. beta 3-Adrenergic-mediated suppression of leptin gene expression in rats.

    PubMed

    Li, H; Matheny, M; Scarpace, P J

    1997-06-01

    To investigate the role of beta 3-adrenergic receptors in the suppression of leptin gene expression, we fasted F-344 rats to decrease leptin mRNA levels, refed the rats to stimulate leptin mRNA production, and examined the ability of the beta 3-adrenergic agonist CGP-12177 to prevent the rise in leptin mRNA levels. In the initial 2 h after CGP-12177 (0.75 mg/kg), there were significant reductions in both food consumption and leptin mRNA levels in epididymal, perirenal, and interscapular white adipose tissue. We were unable to detect leptin mRNA in interscapular brown adipose tissue (IBAT), whereas there was a significant increase in uncoupling protein mRNA levels in IBAT after CGP-12177. The suppression of leptin mRNA and food intake by CGP-12177 was confirmed in a second experiment using another rat strain, the F-344 x BN. Furthermore, refeeding after a period of fasting increased leptin mRNA, which was prevented by CGP-12177. These data indicate a role for beta 3-adrenergic-mediated regulation of leptin gene expression in nonmutant rodents and are consistent with other reports suggesting that beta 3-adrenergic agonists suppress food intake. PMID:9227448

  12. Expression of beta 3-adrenoceptor mRNA in rat brain.

    PubMed Central

    Summers, R. J.; Papaioannou, M.; Harris, S.; Evans, B. A.

    1995-01-01

    The reverse transcription/polymerase chain reaction was used to demonstrate beta 3-adrenoceptor mRNA in rat brain regions. Levels were highest in hippocampus, cerebral cortex and striatum and lower in hypothalamus, brainstem and cerebellum. Images Figure 1 PMID:8590968

  13. Pharmacological evidence for the presence of functional beta(3)-adrenoceptors in rat retinal blood vessels.

    PubMed

    Mori, Asami; Miwa, Tomoyo; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2010-08-01

    The aim of this study was to examine whether stimulation of beta(3)-adrenoceptors dilates rat retinal blood vessels and how diabetes affects the vasodilator responses. Images of ocular fundus were captured with an original high-resolution digital fundus camera in vivo. The retinal vascular responses were evaluated by measuring diameter of retinal blood vessels contained in the digital images. Both systemic blood pressure and heart rate (HR) were continuously recorded. The beta(3)-adrenoceptor agonist CL316243 (0.3-10 microg/kg/min, i.v.) increased diameter of retinal arterioles (at 10 microg/kg/min, a 31% increase) and decreased mean blood pressure (at 10 microg/kg/min, a 21% decrease) in a dose-dependent manner. CL316243 produced a small but significant increase in HR (at 10 microg/kg/min, a 9% increase). Both SR59230A (1 mg/kg, i.v.) and L-748337 (50 microg/kg, i.v.), beta(3)-adrenoceptor antagonists, significantly prevented CL316243-induced retinal vasodilator responses. Similar observations were made with another beta(3)-adrenoceptor agonist, BRL37344. The beta(2)-adrenoceptor agonist salbutamol also increased diameter of retinal arterioles (at 10 microg/kg/min, a 43% increase), whereas the drug produced greater decrease in blood pressure (at 10 microg/kg/min, a 46% decrease) and increase in HR (at 10 microg/kg/min, a 16% increase), compared with beta(3)-adrenoceptor agonists. The retinal vasodilator responses to CL316243 and BRL37344 observed under blockade of beta(1)/beta(2)-adrenoceptors with propranolol (2 mg/kg, i.v. bolus followed by 100 microg/kg/min infusion) were unaffected 2 weeks after induction of diabetes by the combination of streptozotocin treatment and D: -glucose feeding. On the other hand, the vasodilator responses to salbutamol of retinal arterioles were significantly reduced in diabetic rats. These results suggest that stimulation of beta(3)-adrenoceptors causes the vasodilation of retinal arterioles in vivo and the vasodilator responses are

  14. Incorporation of TGF-beta 3 within collagen-hyaluronic acid scaffolds improves their chondrogenic potential.

    PubMed

    Matsiko, Amos; Levingstone, Tanya J; Gleeson, John P; O'Brien, Fergal J

    2015-06-01

    Incorporation of therapeutics in the form of growth factors within biomaterials can enhance their biofunctionality. Two methods of incorporating transforming growth factor-beta 3 within collagen-hyaluronic acid scaffolds are described, markedly improving mesenchymal stem cell-mediated chondrogenic differentiation and matrix production. Such scaffolds offer control over the release of therapeutics, demonstrating their potential for repair of complex chondral defects requiring additional stimuli. PMID:25800862

  15. Demonstration of an in vivo functional beta 3-adrenoceptor in man.

    PubMed

    Enocksson, S; Shimizu, M; Lönnqvist, F; Nordenström, J; Arner, P

    1995-05-01

    Although it is well established in several mammalian species that beta 3-adrenoceptors play a major role in regulating lipolysis and thermogenesis in adipose tissue, the functional existence and role of this receptor subtype in man has been controversial. We investigated whether the beta 3-adrenoceptor functionally co-exists with beta 1- and beta 2-adrenoceptors in vivo in human adipose tissue. Subcutaneous abdominal adipose tissue of healthy non-obese subjects was microdialyzed with equimolar concentrations of dobutamine (selective beta 1-adrenoceptor agonist), terbutaline (selective beta 2-adrenoceptor agonist), or CGP 12177 (selective beta 3-adrenoceptor agonist). All three agents caused a rapid, sustained, concentration-dependent and significant elevation of the glycerol level in the microdialysate (lipolysis index). However, only terbutaline stimulated the nutritive blood flow in adipose tissue, as measured by an ethanol escape technique. Dobutamine and CGP 12177 was equally effective in elevating the glycerol level (maximum effect 150% above baseline). Terbutaline was significantly more effective than the other two beta-agonists (maximum effect 200% above baseline). When adipose tissue was pretreated with the beta 1/beta 2-selective adrenoceptor blocker propranolol the glycerol increasing effect of dobutamine or terbutaline was inhibited by 80-85% but the glycerol response to CGP 12177 was not influenced. It is concluded that a functional beta 3-adrenoceptor is present in vivo in man. It co-exists with beta 1- and beta 2-adrenoceptors in adipose tissue and may therefore play a role in lipolysis regulation. It appears, however, that the beta 2-adrenoceptor is the most important beta-adrenoceptor subtype for the mobilization of lipids from abdominal subcutaneous adipose tissue because of its concomitant stimulatory effect on lipolysis and blood flow. PMID:7738189

  16. Obesity-related phenotypes and the beta3-adrenoceptor gene variant in postmenopausal women.

    PubMed

    Tchernof, A; Starling, R D; Walston, J D; Shuldiner, A R; Dvorak, R V; Silver, K; Matthews, D E; Poehlman, E T

    1999-07-01

    We examined the hypothesis that postmenopausal women with the beta3-adrenoceptor gene variant (Trp64Arg) have reduced total daily energy expenditure (TEE), altered free fatty acid kinetics, and increased intra-abdominal fat. A secondary objective was to examine whether the obese state masks the effect of the variant on resting metabolic rate (RMR). There were 23 obese heterozygous women with the genetic variant (age 58 +/- 6 years; BMI 36 +/- 7 kg/m2) who were compared with 19 homozygous obese women with the normal allele (age 56 +/- 4 years; BMI 36 +/- 3 kg/m2). Daily energy expenditure was determined from doubly labeled water and indirect calorimetry, lipolysis from infusion of [1-13C]palmitate, and body fat distribution from computed tomography. No significant differences were found in TEE, RMR, energy expenditure of physical activity, the thermic effect of a meal, fat oxidation as estimated by fasting and postprandial respiratory quotients (RQs), or rate of lipolysis. Similarly, no difference was found in visceral adipose tissue and abdominal subcutaneous fat areas. When RMR was compared between obese (n = 23) and never-obese women with the Trp64Arg variant (n = 16), we found a 317 kcal/day lower RMR in never-obese women after controlling for fat mass, fat-free mass, and age (P < 0.0017). These results do not support the hypothesis that already obese women with the Trp64Arg polymorphism of the beta3-adrenergic receptor gene have lower daily energy expenditure, altered lipolysis, and increased abdominal obesity. On the other hand, the lower RMR in never-obese women suggests that the obese state may mask a moderate effect of the Trp64Arg variant on energy expenditure. Although these results need to be confirmed in other populations, the obese state may have been a confounding factor in previous studies of the beta3-adrenoceptor Trp64Arg variant and energy expenditure. PMID:10389848

  17. Expression of transforming growth factors beta-1, beta 2 and beta 3 in human bladder carcinomas.

    PubMed Central

    Eder, I. E.; Stenzl, A.; Hobisch, A.; Cronauer, M. V.; Bartsch, G.; Klocker, H.

    1997-01-01

    We previously detected elevated transforming growth factor beta-1 (TGF-beta1) serum levels in patients with invasive bladder carcinomas. In this study, we therefore investigated whether elevated serum levels correlate with enhanced TGF-beta expression in human bladder tumours. mRNA levels of TGF-beta1, -beta2 and -beta3 were reduced in bladder tumour tissue to 86%, 68% and 56%, respectively, of the levels in normal urothelium. On the other hand, TGF-beta1 protein levels were found to be higher in superficial tumours (Ta-T1) (mean level of 0.153 ng mg(-1)) and in invasive T2/T3 tumours (mean level of 0.104 ng mg(-1)) compared with normal urothelium (mean level of 0.065 ng mg(-1)). Invasive T4 tumours, however, contained only low amounts of TGF-beta1 (mean level of 0.02 ng mg(-1)). Neither in mean nor in individual patients were serum and tissue TGF-beta levels correlated with each other. Cell culture experiments on primary bladder cells revealed a 57% decrease in TGF-beta1 mRNA levels in tumour compared with normal epithelial cells. Tumour epithelial cells contained about two times higher levels of TGF-beta2 and TGF-beta3 mRNA than normal epithelial cells. Fibroblasts expressed about the same amount of TGF-beta1 or TGF-beta2 as epithelial cells. Yet, fibroblasts released only 19% and 13% of the amount secreted by tumour epithelial cells into the supernatant. TGF-beta3, on the other hand, was expressed by fibroblasts with higher levels than by epithelial cells. TGF-beta1 was the predominent isoform in bladder tissue and cells at protein as well as on mRNA levels indicating that TGFs-beta2 and -beta3 are of minor importance in bladder cancer. In summary, there is a lack of correlation between TGF-beta serum levels and TGF-beta expression in tumour tissue in bladder cancer. Images Figure 1 PMID:9192977

  18. Tumor suppressor KAI1 affects integrin {alpha}v{beta}3-mediated ovarian cancer cell adhesion, motility, and proliferation

    SciTech Connect

    Ruseva, Zlatna; Geiger, Pamina Xenia Charlotte; Hutzler, Peter; Kotzsch, Matthias; Luber, Birgit; Schmitt, Manfred; Gross, Eva; Reuning, Ute

    2009-06-10

    The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin {alpha}v{beta}3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin {alpha}v{beta}3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with {beta}1-integrins, also colocalizes with integrin {alpha}v{beta}3. Functionally, elevated KAI1 levels drastically increased integrin {alpha}v{beta}3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin {alpha}v{beta}3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin {alpha}v{beta}3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.

  19. Long-term effects of diazepam treatment of epileptic GABAA receptor beta3 subunit knockout mouse in early life.

    PubMed

    Liljelund, Patricia; Ferguson, Carolyn; Homanics, Gregg; Olsen, Richard W

    2005-01-01

    The knockout mouse for the beta3 subunit of the GABAA receptor exhibits spontaneous epilepsy and hyperactivity, and has been proposed as a model for the severe developmental disorder, Angelman's syndrome, which is known to be of genetic origin. We have used this mutant to test an approach of therapeutic intervention prior to seizure onset by daily injection with diazepam during either the first or second postnatal week. Results showed differences between postnatal week 1 and week 2 injections both acutely, with respect to sedative effects, and in long-term outcome, with respect to EEG and behavioral tests measured at 12-14 weeks of age. The EEG of control mice remained unaffected under all conditions, but the EEG of beta3 (-/-) injected with diazepam in week 1 was worsened, showing increased oscillatory activity at 5-6Hz, and more myoclonic jerks, particularly among males. For beta3 (-/-) injected with diazepam in week 2, the EEG was normalized in half the mice but worsened similarly to week 1 in the other half. Neonatal diazepam injection had a long-term normalizing effect on behavior of beta3 (-/-) mice injected in week 1, but diazepam treatment in week 2 did not affect the hyperactive and circling behavior characteristic of the beta3 knockout mouse. Diazepam treatment in postnatal week 2 significantly decreased anxiety in the adult beta3 group. Diazepam treatment in both postnatal weeks 1 and 2 improved the motor coordination of beta3 (-/-) on the rotarod, although performance of control mice injected with diazepam in postnatal week 2 was significantly impaired. The observed long-term outcome of neonatal diazepam injections may result from interference with developmental processes, and shows that enhancing GABAergic activity with diazepam during the period where GABA can be excitatory can produce narrow stage-related effects on brain development. PMID:16168624

  20. Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

    PubMed Central

    Liggett, S B; Freedman, N J; Schwinn, D A; Lefkowitz, R J

    1993-01-01

    The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In

  1. Beta3 adrenergic receptor is involved in vascular injury in deoxycorticosterone acetate-salt hypertensive mice.

    PubMed

    Sheng, Li-Juan; Ruan, Cheng-Chao; Ma, Yu; Chen, Dong-Rui; Kong, Ling-Ran; Zhu, Ding-Liang; Gao, Ping-Jin

    2016-03-01

    Beta3 adrenergic receptor (ADRB3) mediates vessel relaxation in the endothelium while it modulates lipolysis in the adipose tissue. However, the function and regulation mechanism of ADRB3 in the perivascular adipose tissue (PVAT), especially in hypertension, is still unclear. We show that ADRB3 protein is upregulated in the PVAT of deoxycorticosterone acetate-salt (DOCA-salt) hypertensive mice, with the characteristics of PVAT browning and increased uncoupling protein 1 (UCP1) expression. Inhibition of ADRB3 with selective antagonist SR59230A caused serious vascular injury in vivo, even though UCP1 expression was downregulated. ADRB3 protein was regulated by let-7b, which was decreased in the PVAT of the DOCA-salt group. These data reveal that ADRB3 in PVAT contributes to vascular function in the progression of hypertension. PMID:26910302

  2. Karyopherin {beta}3: A new cellular target for the HPV-16 E5 oncoprotein

    SciTech Connect

    Krawczyk, Ewa; Hanover, John A.; Schlegel, Richard; Suprynowicz, Frank A.

    2008-07-11

    Epidemiological and experimental studies have shown that high-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer worldwide, and that HPV-16 is associated with more than half of these cases. In addition to the well-characterized E6 and E7 oncoproteins of HPV-16, recent evidence increasingly has implicated the HPV-16 E5 protein (16E5) as an important mediator of oncogenic transformation. Since 16E5 has no known intrinsic enzymatic activity, its effects on infected cells are most likely mediated by interactions with various cellular proteins and/or its documented association with lipid rafts. In the present study, we describe a new cellular target that binds to 16E5 in COS cells and in stable human ectocervical cell lines. This target is karyopherin {beta}3, a member of the nuclear import receptor family with critical roles in the nuclear import of ribosomal proteins and in the secretory pathway.

  3. In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

    PubMed Central

    Miller, Peter G.; Al-Shahrour, Fatima; Hartwell, Kimberly A.; Chu, Lisa P.; Järås, Marcus; Puram, Rishi V.; Puissant, Alexandre; Callahan, Kevin P.; Ashton, John; McConkey, Marie E.; Poveromo, Luke P.; Cowley, Glenn S.; Kharas, Michael G.; Labelle, Myriam; Shterental, Sebastian; Fujisaki, Joji; Silberstein, Lev; Alexe, Gabriela; Al-Hajj, Muhammad A.; Shelton, Christopher A.; Armstrong, Scott A.; Root, David E.; Scadden, David T.; Hynes, Richard O.; Mukherjee, Siddhartha; Stegmaier, Kimberly; Jordan, Craig T.; Ebert, Benjamin L.

    2013-01-01

    SUMMARY We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML. PMID:23770013

  4. The contribution of IL-6 to beta 3 adrenergic receptor mediated adipose tissue remodeling.

    PubMed

    Buzelle, Samyra L; MacPherson, Rebecca E K; Peppler, Willem T; Castellani, Laura; Wright, David C

    2015-02-01

    The chronic activation of beta 3 adrenergic receptors results in marked alterations in adipose tissue morphology and metabolism, including increases in mitochondrial content and the expression of enzymes involved in lipogenesis and glyceroneogenesis. Acute treatment with CL 316,243, a beta 3 adrenergic agonist, induces the expression of interleukin 6. Interestingly, IL-6 has been shown to induce mitochondrial genes in cultured adipocytes. Therefore, the purpose of this paper was to examine the role of interleukin 6 in mediating the in vivo effects of CL 316,243 in white adipose tissue. Circulating IL-6, and markers of IL-6 signaling in white adipose tissue were increased 4 h following a single injection of CL 316,243 in C57BL6/J mice. Once daily injections of CL 316,243 for 5 days increased the protein content of a number of mitochondrial proteins including CORE1, Cytochrome C, PDH, MCAD, and Citrate Synthase to a similar extent in adipose tissue from WT and IL-6(-/-) mice. Conversely, CL 316,243-induced increases in COXIV and phosphorylated AMPK were attenuated in IL-6(-/-) mice. Likewise, the slight, but significant, CL 316,243-induced increases in ATGL, PEPCK, and PPARγ, were reduced or absent in adipose tissue IL-6(-/-) mice. The attenuated response to CL 316,243 in white adipose tissue in IL-6(-/-) mice was associated with reductions in whole-body oxygen consumption and energy expenditure in the light phase. Our findings suggest that IL-6 plays a limited role in CL 316,243-mediated adipose tissue remodeling. PMID:25713332

  5. Integrin-beta3 clusters recruit clathrin-mediated endocytic machinery in the absence of traction force

    PubMed Central

    Yu, Cheng-han; Rafiq, Nisha Bte Mohd; Cao, Fakun; Zhou, Yuhuan; Krishnasamy, Anitha; Biswas, Kabir Hassan; Ravasio, Andrea; Chen, Zhongwen; Wang, Yu-Hsiu; Kawauchi, Keiko; Jones, Gareth E.; Sheetz, Michael P.

    2015-01-01

    The turnover of integrin receptors is critical for cell migration and adhesion dynamics. Here we find that force development at integrins regulates adaptor protein recruitment and endocytosis. Using mobile RGD (Arg-Gly-Asp) ligands on supported lipid membranes (RGD membranes) and rigid RGD ligands on glass (RGD-glass), we find that matrix force-dependent integrin signals block endocytosis. Dab2, an adaptor protein of clathrin-mediated endocytosis, is not recruited to activated integrin-beta3 clusters on RGD-glass; however, it is recruited to integrin-mediated adhesions on RGD membranes. Further, when force generation is inhibited on RGD-glass, Dab2 binds to integrin-beta3 clusters. Dab2 binding to integrin-beta3 excludes other adhesion-related adaptor proteins, such as talin. The clathrin-mediated endocytic machinery combines with Dab2 to facilitate the endocytosis of RGD-integrin-beta3 clusters. From these observations, we propose that loss of traction force on ligand-bound integrin-beta3 causes recruitment of Dab2/clathrin, resulting in endocytosis of integrins. PMID:26507506

  6. Studies on the interactions between glycosylated beta3-peptides and the lectin Vicia villosa by saturation transfer difference NMR spectroscopy.

    PubMed

    Kaszowska, Marta; Norgren, Anna S; Arvidson, Per I; Sandström, Corine

    2009-12-14

    Saturation transfer difference (STD) NMR spectroscopy was used to study the interaction of the lectin Vicia villosa (VVLB(4)) with alpha-D-GalNAc glycosylated beta(3)-peptides. The data were compared to those obtained with the monosaccharides D-Gal, D-GalNAc, and D-Glc as well as with those obtained with the Tn antigen alpha-glycopeptide (D-GalNAc-alpha-O-Ser/Thr), molecule naturally recognized by V. villosa. Evidence that the lectin also recognizes glycosylated beta(3)-peptides and has close contact with both the sugar and amino acid moieties was obtained. PMID:19863951

  7. Lateral mobility of integrin alpha IIb beta 3 (glycoprotein IIb/IIIa) in the plasma membrane of a human megakaryocyte.

    PubMed

    Schootemeijer, A; van Willigen, G; van der Vuurst, H; Tertoolen, L G; De Laat, S W; Akkerman, J W

    1997-01-01

    The migration of integrins to sites of cell-cell and cell-matrix contact is thought to be important for adhesion strengthening. We studied the lateral diffusion of integrin alpha IIb beta 3 (glycoprotein IIb/IIIa) in the plasma membrane of a cultured human megakaryocyte by fluorescence recovery after photobleaching of FITC-labelled monovalent Fab fragments directed against the beta 3 subunit. The diffusion of beta 3 on the unstimulated megakaryocyte showed a lateral diffusion coefficient (D) of 0.37 x 10(-9) cm2/s and a mobile fraction of about 50%. Stimulation with ADP (20 microM) or alpha-thrombin (10 U/ml) at 22 degrees C induced transient decreases in both parameters reducing D to 0.21 x 10(-9) cm2/s and the mobile fraction to about 25%. The fall in D was observed within 1 min after stimulation but the fall in mobile fraction showed a lag phase of 5 min. The lag phase was absent in the presence of Calpain I inhibitor, where-as cytochalasin D completely abolished the decreased in mobile fraction. The data are compatible with the concept that cell activation induces anchorage of 50% of the mobile alpha IIb beta 3 (25% of the whole population of receptor) to the cytoplasmic actin filaments, although, as discussed, other rationals are not ruled out. PMID:9031465

  8. Pretreatment with transforming growth factor beta-3 protects small intestinal stem cells against radiation damage in vivo.

    PubMed Central

    Potten, C. S.; Booth, D.; Haley, J. D.

    1997-01-01

    The gastrointestinal tract, with its rapid cell replacement, is sensitive to cytotoxic damage and can be a site of dose-limiting toxicity in cancer therapy. Here, we have investigated the use of one growth modulator to manipulate the cell cycle status of gastrointestinal stem cells before cytotoxic exposure to minimize damage to this normal tissue. Transforming growth factor beta-3 (TGF-beta3), a known inhibitor of cell cycle progression through G1, was used to alter intestinal crypt stem cell sensitivity before 12-16 Gy of gamma irradiation, which was used as a model cytotoxic agent. Using a crypt microcolony assay as a measure of functional competence of gastrointestinal stem cells, it was shown that the administration of TGF-beta3 over a 24-h period before irradiation increased the number of surviving crypts by four- to six-fold. To test whether changes in crypt survival are reflected in the well-being of the animal, survival time analyses were performed. After 14.5 Gy of radiation, only 35% of the animals survived within a period of about 12 days, while prior treatment with TGF-beta3 provided significant protection against this early gastrointestinal animal death, with 95% of the treated animals surviving for greater than 30 days. PMID:9166937

  9. Transforming growth factor beta 3 involved in the pathogenesis of synovial chondromatosis of temporomandibular joint

    PubMed Central

    Li, Yingjie; El.Mozen, Loaye Abdelaziz; Cai, Hengxing; Fang, Wei; Meng, Qinggong; Li, Jian; Deng, Mohong; Long, Xing

    2015-01-01

    Synovial chondromatosis (SC) of temporomandibular joint is rare proliferative disorder featured by the formation of cartilaginous nodules in synovium and joint space. Transforming growth factor beta 3 (TGF-β3) is closely related to chondrogenic differentiation, and might participate in pathogenesis of SC. We discovered that increased quantity of synoviocytes and blood vessels were observed in SC synovium. The vessel wall and sublining fibroblasts were stained positively by the antibodies against TGF-β3, fibroblast growth factor 2 (FGF-2), and CD34. In loose bodies (LBs), TGF-β3 was mainly expressed in chondrocytes and FGF-2 was expressed in chondrocytes, fibroblasts, and vessel walls. Expressions of TGF-β1, TGF-β3, FGF-2, Sox9, Wnt-4, Foxc2, and VEGF-A mRNA were significantly higher in SC synovium. Stimulation of TGF-β3 on synoviocytes increased alkaline phosphatase (ALP) activity and expressions of chondrogenic genes (Sox9, Col2α1, Aggrecan, Wnt-4, and Wnt-11), osteogenic genes (Runx2, Foxc2, osteocalcin, and Col1α1), and VEGF-A, but failed to influence FGF-2 expression. However, the addition of FGF-2 increased TGF-β3 expression. In conclusion, TGF-β3 existed in synovium and LBs of SC, and was responsible for the pathogenesis of SC. PMID:25742744

  10. Transforming growth factor beta 3 involved in the pathogenesis of synovial chondromatosis of temporomandibular joint.

    PubMed

    Li, Yingjie; El Mozen, Loaye Abdelaziz; Cai, Hengxing; Fang, Wei; Meng, Qinggong; Li, Jian; Deng, Mohong; Long, Xing

    2015-01-01

    Synovial chondromatosis (SC) of temporomandibular joint is rare proliferative disorder featured by the formation of cartilaginous nodules in synovium and joint space. Transforming growth factor beta 3 (TGF-β3) is closely related to chondrogenic differentiation, and might participate in pathogenesis of SC. We discovered that increased quantity of synoviocytes and blood vessels were observed in SC synovium. The vessel wall and sublining fibroblasts were stained positively by the antibodies against TGF-β3, fibroblast growth factor 2 (FGF-2), and CD34. In loose bodies (LBs), TGF-β3 was mainly expressed in chondrocytes and FGF-2 was expressed in chondrocytes, fibroblasts, and vessel walls. Expressions of TGF-β1, TGF-β3, FGF-2, Sox9, Wnt-4, Foxc2, and VEGF-A mRNA were significantly higher in SC synovium. Stimulation of TGF-β3 on synoviocytes increased alkaline phosphatase (ALP) activity and expressions of chondrogenic genes (Sox9, Col2α1, Aggrecan, Wnt-4, and Wnt-11), osteogenic genes (Runx2, Foxc2, osteocalcin, and Col1α1), and VEGF-A, but failed to influence FGF-2 expression. However, the addition of FGF-2 increased TGF-β3 expression. In conclusion, TGF-β3 existed in synovium and LBs of SC, and was responsible for the pathogenesis of SC. PMID:25742744

  11. Modulation of beta2- and beta3-adrenoceptor-mediated relaxation of rat oesophagus smooth muscle by protein kinase C.

    PubMed

    Oostendorp, Jaap; Obels, Peter Ph; Terpstra, A Rene; Nelemans, S Adriaan; Zaagsma, Johan

    2004-07-01

    Although a prominent role for protein kinase C (PKC) in the cross-talk between the phosphoinositide pathway and beta2-adrenoceptor signalling has been indicated, modulation of beta3-adrenoceptor function by PKC has not been studied thus far. In the present study, we have compared the relative capacity of PKC in modulating beta2- and beta3-adrenoceptor-mediated relaxation of methacholine-contracted rat oesophagus smooth muscle. To this purpose the effects of the PKC-inhibitor GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) on relaxation induced by fenoterol, formoterol, (-)-noradrenaline, BRL 35135 (4-[2-[(2-hydroxy-2-(chlorophenyl)ethyl)amino]-propyl]-phenoxyacetic-acidmethylester) and IBMX (3-isobutyl-1-methyl-xanthine) were studied, in the absence and presence of the selective beta2-adrenoceptor antagonist ICI 118,551 (erythro-1(7-methylindan-4-yloxy)-3-(isopropylamin)-butan-2-ol). Our results show that inhibition of PKC resulted in differential augmentation of both beta2- and beta3-adrenoceptor-mediated relaxation. In contrast, relaxation induced by IBMX was not influenced at all by GF 109203X. The beta2-adrenoceptor bears phosphorylation sites for several kinases, including PKC. Since the beta3-adrenoceptor lacks these consensus sites, the results may also indicate that PKC-mediated Galphas phosphorylation is involved in the cross-talk between the muscarinic receptor-mediated phosphoinositide pathway and beta2- and, particularly, beta3-adrenoceptor signalling. PMID:15219823

  12. Apparent lack of beta 3-adrenoceptors and of insulin regulation of glucose transport in brown adipose tissue of guinea pigs.

    PubMed

    Himms-Hagen, J; Triandafillou, J; Begin-Heick, N; Ghorbani, M; Kates, A L

    1995-01-01

    Norepinephrine-induced thermogenesis was substantial in adipocytes from brown adipose tissue (BAT) of cold-acclimated guinea pigs but absent in adipocytes from BAT of warm-acclimated guinea pigs. There was no thermogenic response to any beta 3-adrenergic agonist (CL-316,243, ZD-7114, BRL-28410, CGP-12177). The receptor was characterized as a beta 1-adrenoceptor. Adrenergic agonists stimulated adenylate cyclase in membranes from BAT of both warm- and cold-acclimated guinea pigs also via a beta 1-adrenoceptor; beta 3-adrenergic agonists had no effect. Glucose transport by brown adipocytes from warm-acclimated guinea pigs was not stimulated by either norepinephrine or insulin. Cold acclimation induced the appearance of stimulation of glucose transport by norepinephrine in association with the appearance of a large capacity for thermogenesis, but there was little improvement in response to insulin. GLUT4 was present in membranes from BAT of both warm- and cold-acclimated guinea pigs. Insulin is known to have an antilipolytic effect on both BAT and white adipose tissue of guinea pigs. Thus there is a selective lack of insulin-regulated glucose transport that is not improved by cold acclimation. Guinea pigs may have a mutated component of the translocation mechanism for GLUT4. beta 3-Adrenoceptors appear to be absent in brown adipocytes of adult guinea pigs, as in white adipocytes of guinea pigs, yet are known to be present in the gut. Tissue-specific expression of beta 3-adrenergic receptors in guinea pigs may differ from that in rats, in which receptors are expressed in the adipose tissues and gut. PMID:7840345

  13. Beta-3 adrenergic receptors could be significant factors for overactive bladder-related symptoms

    PubMed Central

    Yamamichi, Fukashi; Shigemura, Katsumi; Behnsawy, Hosny M; Yamashita, Masuo; Shirakawa, Toshiro; Fujisawa, Masato

    2015-01-01

    The treatment failure often happens in overactive bladder (OAB) partly owing to its unknown pathogenesis. The purpose of this study is to find significant receptors or biological markers for OAB-related symptoms for establishment of potential order-made therapeutic strategies. The overactive bladder symptom scores (OABSS) and international prostate symptom scores (IPSS)/quality of life (QOL) were questioned in all the 18 patients with OAB diagnosis. Their bladder mucosal tissues were taken from the random biopsy of bladder cancer suspected patients without any finding such as inflammation or carcinoma in situ. They were investigated quantitatively by immunohistochemical (IHC) stainings for inflammatory or immune-system (Interleukin (IL)-6 and cyclooxygenase-2 (Cox-2)), Caspase-3 apoptosis markers, angiogenesis (CD-31), epithelial-mesenchymal transition (E-cadherin) and muscarinic receptor (Muscarine-2 (M)-2), adrenergic receptors (ARs) (alpha 1-d (α1-d) and beta-3 (β-3)). The statistical correlation between the expressions of these 5 markers and 3 receptors and these symptom scores were examined under the comparison between OAB patients and control patients who had urgency score with less than 2 in OABSS. The OABSS and IPSS/QOL was 7.39 ± 2.69 and 21.2 ± 6.59/4.33 ± 1.33, respectively but those of control patients were 2.00 ± 1.41 and 10.1 ± 9.52/2.14 ± 1.46, respectively (P<0.05). Regarding the correlation of those markers’ expressions and symptom scores, in OAB patients, OABSS total significantly correlated with β-3 AR expressions (P=0.0457). IPSS post-voiding significantly correlated with β-3 AR expressions (P=0.0308) but no significant relationship in control patients (P>0.05). In conclusion, this study demonstrated that β-3 AR in our tested 8 markers or receptors was correlated strongly with OAB-related symptoms. These data may help elucidate the pathophysiology of OAB and offer possible strategy for its order-made therapies. PMID:26617941

  14. The selectivity of beta-adrenoceptor antagonists at the human beta1, beta2 and beta3 adrenoceptors.

    PubMed

    Baker, Jillian G

    2005-02-01

    Beta-adrenoceptor antagonists ("beta-blockers") are one of the most widely used classes of drugs in cardiovascular medicine (hypertension, ischaemic heart disease and increasingly in heart failure) as well as in the management of anxiety, migraine and glaucoma. Where known, the mode of action in cardiovascular disease is from antagonism of endogenous catecholamine responses in the heart (mainly at beta1-adrenoceptors), while the worrisome side effects of bronchospasm result from airway beta2-adrenoceptor blockade. The aim of this study was to determine the selectivity of beta-antagonists for the human beta-adrenoceptor subtypes. (3)H-CGP 12177 whole cell-binding studies were undertaken in CHO cell lines stably expressing either the human beta1-, beta2- or the beta3-adrenoceptor in order to determine the affinity of ligands for each receptor subtype in the same cell background. In this study, the selectivity of well-known subtype-selective ligands was clearly demonstrated: thus, the selective beta1 antagonist CGP 20712A was 501-fold selective over beta2 and 4169-fold selective over beta3; the beta2-selective antagonist ICI 118551 was 550- and 661-fold selective over beta1 and beta3, respectively, and the selective beta3 compound CL 316243 was 10-fold selective over beta2 and more than 129-fold selective over beta1. Those beta2-adrenoceptor agonists used clinically for the treatment of asthma and COPD were beta2 selective: 29-, 61- and 2818-fold for salbutamol, terbutaline and salmeterol over beta1, respectively. There was little difference in the affinity of these ligands between beta1 and beta3 adrenoceptors. The clinically used beta-antagonists studied ranged from bisoprolol (14-fold beta1-selective) to timolol (26-fold beta2-selective). However, the majority showed little selectivity for the beta1- over the beta2-adrenoceptor, with many actually being more beta2-selective. This study shows that the beta1/beta2 selectivity of most clinically used beta-blockers is

  15. The transforming growth factor-beta 3 knock-out mouse: an animal model for cleft palate.

    PubMed

    Koo, S H; Cunningham, M C; Arabshahi, B; Gruss, J S; Grant, J H

    2001-09-15

    The recent report of a transforming growth factor-beta 3 (TGF-beta 3) knock-out mouse in which 100 percent of the homozygous pups have cleft palate raised the question as to the potential usefulness of these animals as a model for cleft palate research. The specific aim in this study was to carefully document the anatomy of the cleft palate in the TGF-beta 3 knock-out mice as compared with wild type controls. Special attention was paid to the levator veli palatini muscle, the tensor veli palatini muscle, and their respective innervation. Because the TGF-beta 3 knock-out is lethal in the early perinatal period and because the heterozygotes are phenotypically normal, polymerase chain reaction was required to genotype the animals before mating. Time-mated pregnancies between proven heterozygotes were then delivered by cesarean section at gestational day 18.5 to prevent maternal cannibalism of homozygote pups. All delivered pups were killed and their tails processed by polymerase chain reaction to verify genotype. The heads were then fixed and sectioned in axial, coronal, or sagittal planes. Sections were stained with hematoxylin and eosin or processed for immunohistochemistry with nerve specific protein gene product 9.5 and calcitonin gene-related peptide antibodies. Sections were analyzed in a serial fashion. Nine wild type control animals were analyzed along with nine TGF-beta 3 knock-out homozygotes. Time matings between proven heterozygotes yielded wild type pups, heterozygote pups, and homozygote knock-out pups in the expected mendelian ratios (28 percent to 46 percent to 26 percent; n = 43). The results demonstrated 100 percent clefting in the homozygous TGF-beta 3 knock-out pups. Complete clefting of the secondary palate was seen in four of nine and incomplete clefting was seen in five of nine. The levator veli palatini and tensor veli palatini muscles were demonstrated coursing parallel to the cleft margin in all cleft mice. The orientation of these muscles

  16. Molecular characterization and expression analysis of the gene coding for the porcine beta(3) integrin subunit (CD61).

    PubMed

    Jiménez-Marín, Angeles; Yubero, Noemí; Esteso, Gloria; Moreno, Angela; de las Mulas, Juana Martín; Morera, Luis; Llanes, Diego; Barbancho, Manuel; Garrido, Juan J

    2008-01-31

    Integrins are heterodimeric cell adhesion molecules with major roles in a variety of biological processes ranging from cell migration to tissue organization, immune and non-immune defense mechanisms and oncogenic transformation. Members of the beta(3) integrin subfamily are composed of a beta(3) subunit (CD61) non-covalently associated with two alpha subunits, alpha(IIb) (CD41) and alpha(v) (CD51), to constitute a group of transmembrane glycoproteins that participate in many physiologically important events. This investigation has focused on the molecular characterization of the cDNA encoding the porcine beta(3) integrin subunit. The deduced 762-amino acid sequence was 93, 92, 91, 89, 79 and 73% homologous to human, dog, rabbit, mouse, chicken and Xenopus laevis CD61 protein, respectively. Porcine CD61 molecule shares many structural features with human CD61, including a region containing a metal ion-dependent adhesion site (MIDAS) folding into an I domain-like structure. Through PCR-SSCP analysis and sequencing, six polymorphic positions were detected in the cDNA sequence of porcine CD61, and their frequencies were observed from a collection of 47 pigs. Expression analysis was done at two different levels: expression of the CD61 mRNA by RT-PCR and localization of the protein by immunohistochemistry. Our results show that CD61 transcripts were detected mainly in platelets and hematopoietic tissues. The immunohistochemical tissue localization of CD61 protein by a specific monoclonal antibody against CD61 recombinant protein showed that CD61 was expressed on vascular and non-vascular smooth muscle, epithelium and myeloid cells, being undetectable in cells of the lymphoid lineage. Furthermore, pulmonary intravascular macrophages (PIM), a subpopulation of macrophages which seem to play an important role in blood clearance, expressed much more CD61 when compared to pulmonary alveolar macrophages (PAM). The knowledge of the structure and distribution of the CD61 provides

  17. The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin alpha(IIb)beta3 in platelets.

    PubMed

    Wee, Janet L; Jackson, Denise E

    2005-12-01

    Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect. PMID:16081692

  18. Barbiturates require the N terminus and first transmembrane domain of the delta subunit for enhancement of alpha1beta3delta GABAA receptor currents.

    PubMed

    Feng, Hua-Jun; Macdonald, Robert L

    2010-07-30

    GABA(A) receptors are composed predominantly of alphabetagamma receptors, which mediate primarily synaptic inhibition, and alphabetadelta receptors, which mediate primarily extrasynaptic inhibition. At saturating GABA concentrations, the barbiturate pentobarbital substantially increased the amplitude and desensitization of the alpha1beta3delta receptor but not the alpha1beta3gamma2L receptor currents. To explore the structural domains of the delta subunit that are involved in pentobarbital potentiation and increased desensitization of alpha1beta3delta currents, chimeric cDNAs were constructed by progressive replacement of gamma2L subunit sequence with a delta subunit sequence or a delta subunit sequence with a gamma2L subunit sequence, and HEK293T cells were co-transfected with alpha1 and beta3 subunits or alpha1 and beta3 subunits and a gamma2L, delta, or chimeric subunit. Currents evoked by a saturating concentration of GABA or by co-application of GABA and pentobarbital were recorded using the patch clamp technique. By comparing the extent of enhancement and changes in kinetic properties produced by pentobarbital among chimeric and wild type receptors, we concluded that although potentiation of alpha1beta3delta currents by pentobarbital required the delta subunit sequence from the N terminus to proline 241 in the first transmembrane domain (M1), increasing desensitization of alpha1beta3delta currents required a delta subunit sequence from the N terminus to isoleucine 235 in M1. These findings suggest that the delta subunit N terminus and N-terminal portion of the M1 domain are, at least in part, involved in transduction of the allosteric effect of pentobarbital to enhance alpha1beta3delta currents and that this effect involves a distinct but overlapping structural domain from that involved in altering desensitization. PMID:20525684

  19. Glioblastoma expression of vitronectin and the alpha v beta 3 integrin. Adhesion mechanism for transformed glial cells.

    PubMed Central

    Gladson, C L; Cheresh, D A

    1991-01-01

    Glioblastoma multiforme, the most malignant astroglial-derived tumor, grows as an adherent mass and locally invades normal brain. An examination of adult cerebral glioblastoma biopsy material for the expression of adhesive proteins that might potentiate adhesion and invasion demonstrated tumor cell-associated vitronectin (5/5). In contrast, vitronectin was not detected associated with glial cells in low grade astroglial tumors (0/4), reactive astrogliosis (0/4), or in normal adult cortex and cerebral white matter (0/5). Also, a wide variety of other adhesive ligands were absent from the glioblastoma tumor parenchyma. The alpha v beta 3 integrin was the only vitronectin receptor identified in glioblastoma tumors in situ, and was also not expressed on low grade astroglial-derived tumors, reactive astrogliosis, or on glia or neurons in normal adult cortex and cerebral white matter. In a cell attachment assay, cultured glioblastoma cells attached to the parenchyma of glioblastoma tumor cryostat sections at the sites of vitronectin expression, but failed to attach to normal brain. This adhesion was inhibited by antibodies directed against vitronectin, the alpha v beta 3 integrin, and with an Arg-Gly-Asp-containing peptide. These data provide evidence for a cell adhesion mechanism in glioblastoma tumors that might potentiate glioblastoma cell invasion of normal brain. Images PMID:1721625

  20. Mapping of the human nicotinic acetylcholine receptor [beta]3 gene (CHRNB3) within chromosome 8p11. 2

    SciTech Connect

    Koyama, Koyama; Sudo, Kazunori; Nakamura, Yusuke )

    1994-05-15

    The authors have used an exon amplification method to construct a transcriptional map for human chromosome 8. With this method, transcribed sequences from defined regions of genomic DNA can be efficiently isolated using cosmid clones mapped to human chromosome 8. Cosmid DNAs were digested with BglII and BamHI and ligated into a BamHI site of an exon trapping vector, pSPL1. Transfection of the subcloned DNAs into Cos7 cells, isolation of cytoplasmic RNA, synthesis of cDNA by reverse transcriptase, and amplification of spliced fragments were performed according to the method described by Buckler et al. Amplified fragments were subcloned into a plasmid, pBluescriptII, and sequenced by the dideoxy chain termination method. Sequence analysis to search for similarity or identity to known genes with the program FASTA detected complete identity of one (ET634-2) of these exon amplification fragments, 227 bp in length, to the nucleotide sequence at 1138-1364 of the cDNA for the human nicotinic acetylcholine receptor (nAChR) [beta]3 gene. This transcribed fragment, containing a part of the human nAChR [beta]3 gene, was isolated from cosmid clone cCI8-328, which was previously mapped to 8p11.2 by fluorescence in situ hybridization. Localization of this gene to chromosome agreed with the results of previous mapping experiments using somatic hybrid cell lines.

  1. Differences between the third cardiac beta-adrenoceptor and the colonic beta 3-adrenoceptor in the rat.

    PubMed Central

    Kaumann, A. J.; Molenaar, P.

    1996-01-01

    1. The heart of several species including man contains atypical beta-adrenoceptors, in addition to coexisting beta 1- and beta 2-adrenoceptors. We now asked the question whether or not the third cardiac beta-adrenoceptor is identical to the putative beta 3-adrenoceptor. We compared the properties of the third cardiac beta-adrenoceptor with those of beta 3-adrenoceptors in isolated tissues of the rat. To study the third cardiac beta-adrenoceptor we used spontaneously beating right atria, paced left atria and paced left ventricular papillary muscles. As a likely model for putative beta 3-adrenoceptors we studied atypical beta-adrenoceptors of the colonic longitudinal muscle precontracted with 30 mM KCl. We used beta 3-adrenoceptor-selective agonists, antagonists and non-conventional partial agonists (ie high-affinity blockers of both beta 1- and beta 2-adrenoceptors know to exert also stimulant effects through beta 3-adrenoceptors). 2. The non-conventional partial agonist (-)-CGP 12177 caused positive chronotropic effects in right atria (pD2 = 7.3) and positive inotropic effects in left atria (pD2 = 7.5). The stimulant effects of (-)-CGP 12177 were resistant to blockade by 200 nM-2 microM (-)-propranolol and 3 microM ICI 118551 (a beta 2-selective antagonist) but antagonized by 1 microM (-)-bupranolol (pKB = 6.4-6.8), 3 microM CGP 20712A (a beta 1-selective antagonist) (pKB = 6.3-6.4) and 6.6 microM SR 59230A (a beta 3-selective antagonist, pKB = 5.1-5.4). 3. The non-conventional partial agonist cyanopindolol caused positive chronotropic effects in right atria (pD2 = 7.7) and positive inotropic effects in left atria (pD2 = 7.1). The stimulant effects of cyanopindolol were resistant to blockade by 200 nM (-)-propranolol but antagonized by 1 microM (-)-bupranolol (pKB = 6.8-7.1). 4. Neither (-)-CGP 12177 nor cyanopindolol caused stimulant effects in papillary muscles at concentrations between 0.2 nM and 20 microM. 5. In the presence of 200 nM (-)-propranolol the beta 3

  2. A homozygous nonsense mutation in the {beta}3 chain gene of laminin 5 (LAMB3) in herlitz junctional epidermolysis bullosa

    SciTech Connect

    Pulkkinen, L.; Christiano, A.M.; Uitto, J.

    1994-11-15

    Herlitz junctional epidermolysis bullosa (H-JEB) is a severe autosomal recessive disorder characterized by blister formation within the dermal-epidermal basement membrane. Based on immunofluorescence analysis recognizing laminin 5 epitopes (previously known as nicein/kalinin), the genes for this lamina lucida protein have been proposed as candidate genes in H-JEB. Amplification of mRNA by RT-PCR, followed by direct nucleotide sequencing, revealed a homozygous C-to T transition resulting in a premature termination codon (CGA{r_arrow}TGA) on both alleles. This mutation was verified at the genomic DNA level, and both parents were shown to be heterozygous carriers of the same mutation. This is the first description of a mutation in the laminin {beta}3 chain gene (LAMB3) of laminin 5 in an H-JEB patient. 15 refs., 2 figs.

  3. Crystal structure of the extracellular segment of integrin {alpha}V{beta}3 in complex with an Arg-Gly-Asp ligand.

    SciTech Connect

    Xiong, J.-P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Goodman, S.; Arnaout, M. A.; Biosciences Division; Massachusetts General Hospital; Harvard Medical School

    2002-04-05

    The structural basis for the divalent cation-dependent binding of heterodimeric alpha beta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alpha Vbeta 3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alpha V and beta 3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in beta A, the ligand-binding domain of beta 3; in the complex, beta A acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alpha V relative to beta 3.

  4. Binding of the 68-kilodalton protein of Mycobacterium avium to alpha(v)beta3 on human monocyte-derived macrophages enhances complement receptor type 3 expression.

    PubMed Central

    Hayashi, T; Rao, S P; Catanzaro, A

    1997-01-01

    Attachment to and uptake by host cells are important early events in the pathogenesis of intracellular organisms such as Mycobacterium avium. Monocyte-derived macrophages (MDM) are known to express multiple surface receptors that play a role in binding to and uptake of M. avium. These include complement receptor type 3 (CR3), fibronectin receptor, mannose receptor, and transferrin receptor. In addition to these, we have previously reported that the integrin receptor alpha(v)beta3 also plays a role in binding to M. avium in a nonopsonic environment. Further, we have shown that a 68-kDa surface protein of M. avium binds to human monocytes and plays a role in attachment of M. avium to MDM. The present study provides direct evidence that this protein mediates attachment of M. avium to MDM by binding to alpha(v)beta3. Using the technique of cell surface enzyme-linked immunosorbent assay, we have shown that the M. avium 68-kDa protein inhibits the binding of monoclonal antibodies (MAb) against alpha(v)beta3 to MDM compared to control proteins such as ovalbumin and laminin (P < 0.05). Dual-labeling studies were performed to demonstrate that after phagocytosis, alpha(v)beta3 is present along with M. avium in phagosomes of M. avium-infected MDM. In addition, we have demonstrated that this interaction between alpha(v)beta3 and the M. avium 68-kDa protein resulted in enhancement of CR3 expression, which is known to play a role in complement-mediated uptake of M. avium. Attachment of MDM to wells coated with the M. avium 68-kDa protein resulted in a twofold increase in CR3 expression compared to attachment of MDM to wells coated with ovalbumin. This enhancement was completely inhibited by pretreatment of MDM with MAb against alpha(v)beta3. In summary, M. avium binds to MDM via alpha(v)beta3 with the help of the M. avium 68-kDa protein, and this ligation enhanced the expression of CR3 on MDM. Since CR3 has been known to play a role in M. avium uptake, enhanced expression of

  5. Homology modeling of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABA receptor channels and Surflex-docking of fipronil.

    PubMed

    Cheng, Jin; Ju, Xiu-Lian; Chen, Xiang-Yang; Liu, Gen-Yan

    2009-09-01

    To further explore the mechanism of selective binding of the representative gamma-aminobutyric acid receptors (GABARs) noncompetitive antagonist (NCA) fipronil to insect over mammalian GABARs, three-dimensional models of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABAR were generated by homology modeling, using the cryo-electron microscopy structure of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata as a template. Fipronil was docked into the putative binding site of the human alpha 1 beta 2 gamma 2 and house fly beta 3 receptors by Surflex-docking, and the calculated docking energies are in agreement with experimental results. The GABA receptor antagonist fipronil exhibited higher potency with house fly beta 3 GABAR than with human alpha 1 beta 2 gamma 2 GABAR. Furthermore, analyses of Surflex-docking suggest that the H-bond interaction of fipronil with Ala2 and Thr6 in the second transmembrane segment (TM2) of these GABARs plays a relatively important role in ligand selective binding. The different subunit assemblies of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABARs may result in differential selectivity for fipronil. PMID:19238461

  6. Detection of sequence variants in the gene encoding the beta 3 chain of laminin 5 (LAMB3).

    PubMed

    Pulkkinen, L; McGrath, J A; Christiano, A M; Uitto, J

    1995-01-01

    Laminin 5, a candidate gene/protein system for mutations in the junctional forms of epidermolysis bullosa (JEB), consists of three polypeptides encoded by the LAMA3, LAMB3, and LAMC2 genes. In this study, primer pairs for the amplification of the complete cDNA as well as 22 exons of the LAMB3 gene encoding the entire beta 3 chain of laminin 5, were established. The primers for amplification of individual exons from genomic DNA were placed at least 50 bp away from the exon-intron borders in the flanking intronic sequences. For amplification of cDNA generated by RT-PCR, eight primer pairs covering overlapping segments of mRNA were used. The amplified sequences were used to study sequence variations of the LAMB3 gene in patients with JEB and unrelated individuals using heteroduplex analysis. Nine out of 13 JEB patients examined showed heteroduplexes in at least one of the PCR products, indicating the existence of two variable alleles in their DNA. Sequence analyses revealed putative pathogenetic mutations in seven of the JEB patients, while four of the heteroduplexes resulted from polymorphisms, reflecting a single basepair substitution. The results demonstrate that this method is useful in the detection of JEB mutations, as well as polymorphisms in the LAMB3 gene. PMID:7550237

  7. Cloning of the beta 3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa.

    PubMed

    Pulkkinen, L; Gerecke, D R; Christiano, A M; Wagman, D W; Burgeson, R E; Uitto, J

    1995-01-01

    Laminin 5 consists of three polypeptides, alpha 3, beta 3, and gamma 2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. In this study, we have elucidated the exon-intron organization of the human LAMB3 gene. Characterization of five overlapping lambda phage DNA clones revealed that the gene was approximately 29 kb in size. Subsequent sequence data revealed that the gene consisted of 23 exons that varied from 64 to 379 bp in size, accounting for the full-length cDNA with an open reading frame of 3516 bp encoding 1172 amino acids. Comparison of the LAMB3 gene structure with the previously characterized LAMB1 gene revealed that LAMB3 was considerably more compact. Knowledge of the exon-intron organization of the LAMB3 gene will facilitate elucidation of mutations in patients with the junctional forms of epidermolysis bullosa, some of which have been associated with mutations in the laminin 5 genes. PMID:7774918

  8. Cloning of the {beta}3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa

    SciTech Connect

    Pulkkinen, L.; Christiano, A.M.; Uitto, J.

    1995-01-01

    Laminin 5 consists of three polypeptides, {alpha}3, {beta}3, and {gamma}2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. In this study, we have elucidated the exon-intron organization of the human LAMB3 gene. Characterization of five overlapping {lambda} phage DNA clones revealed that the gene was approximately 29 kb in size. Subsequent sequence data revealed that the gene consisted of 23 exons that varied from 64 to 379 bp in size, accounting for the full-length cDNA with an open reading frame of 3516 hp encoding 1172 amino acids. Comparison of the LAMB3 gene structure with the previously characterized LAMB1 gene revealed that LAMB3 was considerably more compact. Knowledge of the exon-intron organization of the LAMB3 gene will facilitate elucidation of mutations in patients with the junctional forms of epidermolysis bullosa, some of which have been associated with mutations in the laminin 5 genes. 33 refs., 3 figs., 2 tabs.

  9. Serum-free, chemically defined medium with TGF-beta(3) enhances functional properties of nucleus pulposus cell-laden carboxymethylcellulose hydrogel constructs.

    PubMed

    Reza, Anna T; Nicoll, Steven B

    2010-02-01

    Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum-containing medium formulation was compared to a chemically defined, serum-free medium to determine the effect on matrix elaboration and functional properties of NP cell-laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor-beta 3 (TGF-beta(3)). Glycosaminoglycan (GAG) content increased in both TGF-beta(3)-treated groups and was highest for treated, serum-free constructs (9.46 +/- 1.51 microg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum-containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum-free constructs. Type II collagen content was greater in both serum-free groups and highest in treated, serum-free constructs. The equilibrium Young's modulus was highest in serum-free samples supplemented with TGF-beta(3) (18.54 +/- 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 +/- 0.46 vs. 19.94 +/- 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum-free, TGF-beta(3) supplemented medium, indicating the importance of medium formulation in NP construct development. PMID:19777586

  10. Transforming growth factors-beta 1, -beta 2, and -beta 3 stimulate fibroblast procollagen production in vitro but are differentially expressed during bleomycin-induced lung fibrosis.

    PubMed Central

    Coker, R. K.; Laurent, G. J.; Shahzeidi, S.; Lympany, P. A.; du Bois, R. M.; Jeffery, P. K.; McAnulty, R. J.

    1997-01-01

    Transforming growth factor (TGF)-beta 1 may potentiate wound healing and fibrosis by stimulating fibroblast collagen deposition. TGF-beta 1 is implicated in the pathogenesis of pulmonary fibrosis, but the role of TGF-beta 2 and TGF-beta 3 remains unclear. We examined their effects on lung fibroblast procollagen metabolism in vitro and localized their gene expression during bleomycin-induced lung fibrosis using in situ hybridization with digoxigenin-labeled riboprobes. All three isoforms stimulated fibroblast procollagen production. TGF-beta 3 was the most potent and also reduced procollagen degradation. In normal mouse lung, TGF-beta 1 and TGF-beta 3 mRNA transcripts were abundant in bronchiolar epithelium. After bleomycin, TGF-beta 1 gene expression was maximally enhanced at 10 days, with the signal being predominant in macrophages. Signal was also enhanced in mesenchymal, pulmonary endothelial, and mesothelial cells. After 35 days, the pattern of TGF-beta 1 gene expression returned to that of control lung. TGF-beta 3 gene expression remained unchanged throughout compared with controls. TGF-beta 2 mRNA was not detected with the antisense probe, but signal obtained with the sense probe suggests the presence of a naturally occurring antisense. This study demonstrates that TGF-beta 1, -beta 2, and -beta 3 all exert profibrotic effects in vitro. However, TGF-beta isoform gene expression is differentially controlled during experimental pulmonary fibrosis with TGF-beta 1 the predominant isoform expressed during pathogenesis. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:9060836

  11. [The Trp64Arg polymorphism of beta3-adrenoreceptor gene study in persons with overweight and obesity].

    PubMed

    Baturin, A K; Pogozheva, A V; Sorokina, E Iu; Makurina, O N; Tutel'ian, V A

    2012-01-01

    The development of obesity is determined by lifestyle and genetic mechanisms. In particular, the polymorphisms in the adrenergic receptor genes (ADRB) have been extensively studied for association with obesity-related phenotypes. ADRB3 is an obvious candidate gene given its involvement in the regulation of lipolysis and thermogenesis. ADRB3 Trp64Arg polymorphism, a missense mutation in the first transmembrane domain of the R3-adrenergic receptor is associated with visceral obesity and insulin resistance in the Pima Indian, French, and Finnish populations. The recent meta-analysis that combined data of 6582 individuals from Japanese populations showed significant association the Arg64 allele with increased BMI. There are tested the polymorphisms in the beta3-Adrenoreceptor (ADRB3) gene in associated with body mass index (BMI), fat mass and biochemical parameters.We have been examined 91 persons from Moscow region with BMI >25 kg/m2. The Trp64Arg polymorphism of ADRB3 genes were genotyped with the use of an allelic discrimination assay. The TaqMan-based real-time PCR method was applied. There have been estimated of anthropometric and biochemicalparameters. The frequencies of the Trp64Trp and Trp64Arggenotypes of ADRB3 gene were 82% and 12%, respectively, the frequencies of mutant allele was 6%. Trp64Arg genotypes of ADRB3 compared to Trp64Trp genotypes had significantly higher body fat percentage (respectively 48,6 +/- 0,96% and 43,8 +/- 1,72%, p<0,05), serum glucose (6,51 +/- 0,18 mmol/l and 5,67 +/- 0,09 mmol/l, p<0,01) and uric acid concentrations (0,46 +/- 0,02 mmol/l and 0,38 +/- 0,01 mmol/l, p<0,05). The test of the ADRB3 gene polymorphisms can be used for the personalization of diet in persons with obesity. PMID:22774474

  12. The activation mechanism of alpha1beta2gamma2S and alpha3beta3gamma2S GABAA receptors.

    PubMed

    Keramidas, Angelo; Harrison, Neil L

    2010-01-01

    The alpha1beta2gamma2 and alpha3beta3gamma2 are two isoforms of gamma-aminobutyric acid type A (GABAA) receptor that are widely distributed in the brain. Both are found at synapses, for example in the thalamus, where they mediate distinctly different inhibitory postsynaptic current profiles, particularly with respect to decay time. The two isoforms were expressed in HEK293 cells, and single-channel activity was recorded from outside-out patches. The kinetic characteristics of both isoforms were investigated by analyzing single-channel currents over a wide range of GABA concentrations. Alpha1beta2gamma2 channels exhibited briefer active periods than alpha3beta3gamma2 channels over the entire range of agonist concentrations and had lower intraburst open probabilities at subsaturating concentrations. Activation mechanisms were constructed by fitting postulated schemes to data recorded at saturating and subsaturating GABA concentrations simultaneously. Reaction mechanisms were ranked according to log-likelihood values and how accurately they simulated ensemble currents. The highest ranked mechanism for both channels consisted of two sequential binding steps, followed by three conducting and three nonconducting configurations. The equilibrium dissociation constant for GABA at alpha3beta3gamma2 channels was approximately 2.6 microM compared with approximately 19 microM for alpha1beta2gamma2 channels, suggesting that GABA binds to the alpha3beta3gamma2 channels with higher affinity. A notable feature of the mechanism was that two consecutive doubly liganded shut states preceded all three open configurations. The lifetime of the third shut state was briefer for the alpha3beta3gamma2 channels. The longer active periods, higher affinity, and preference for conducting states are consistent with the slower decay of inhibitory currents at synapses that contain alpha3beta3gamma2 channels. The reaction mechanism we describe here may also be appropriate for the analysis of other

  13. Macrophage migration inhibitory factor up-regulates alpha(v)beta(3) integrin and vascular endothelial growth factor expression in endometrial adenocarcinoma cell line Ishikawa.

    PubMed

    Bondza, Patrick Kibangou; Metz, Christine N; Akoum, Ali

    2008-04-01

    Human endometrium undergoes a series of dynamic physiological changes during the menstrual cycle of reproductive age women. Many factors, including hormones, cytokines, growth factors, matrix metalloproteinases and integrins, are essential for the success of embryonic implantation into endometrial tissue. Herein, we used a well-differentiated endometrial adenocarcinoma cell line, Ishikawa, to investigate in vitro the role played by macrophage migration inhibitory factor (MIF) in the regulation of endometrial receptivity markers. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that MIF induced a slight increase in alpha(v) (alphav) mRNA integrin subunit expression during the first 12h, but reached a significant difference after 24h MIF treatment compared to control, whereas beta(3) (beta3) integrin subunit displayed significant increase in mRNA 2h following treatment. Immunocytofluorescence showed strong alphav and beta3 immunostaining at 25 ng/ml MIF, and Western blotting clearly indicated increased alphav and beta3 protein expression. MIF treatment significantly stimulated vascular endothelial growth factor (VEGF) mRNA expression in a dose- and time-dependent manner after 24 h treatment. Moreover, immunocytofluorescence revealed positive VEGF immunostaining compared to control, and analysis by ELISA of VEGF release in culture supernatants demonstrated that MIF (25 ng/ml) significantly induced VEGF secretion at 12 and 24 h. In conclusion, this study provides evidence that MIF directly up-regulates alphavbeta3 integrin and VEGF expression in human endometrial Ishikawa cells and may advance our understanding of factors involved in the establishment of endometrial receptivity and successful implantation. PMID:17854909

  14. beta3-Adrenergic-dependent and -independent mechanisms participate in cold-induced modulation of insulin signal transduction in brown adipose tissue of rats.

    PubMed

    Gasparetti, Alessandra L; Alvarez-Rojas, Fernanda; de Araujo, Eliana P; Hirata, Aparecida E; Saad, Mário J A; Velloso, Lício A

    2005-03-01

    During cold exposure, homeothermic animals mobilize glucose with higher efficiency than at thermoneutrality. An interaction between the insulin signal transduction machinery and high sympathetic tonus is thought to play an important role in this phenomenon. In the present study, rats were exposed to cold during 8 days and treated, or not, with a beta3-adrenergic agonist, BRL37344 sodium 4-2-2-(3-chlorophenyl)-2-hydroxyethyl amino propyl phenoxy-acetic acid sodium (BRL37344), or antagonist, SR59230A 3-(2-ethylphenoxy)-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate (SR59230A), to evaluate the cross-talk between insulin and beta3-adrenergic intracellular signaling in brown adipose tissue. The drugs did not modify food ingestion, body temperature, and body weight in control and cold-exposed rats. Treatment of control rats with BRL37344 led to higher insulin-induced tyrosine phosphorylation of the insulin receptors, insulin receptor substrate (IRS)-1 and ERK, higher insulin-induced IRS-1/PI3-kinase association, and higher [Ser(473)] phosphorylation of Akt. Cold exposure alone promoted higher insulin-induced tyrosine phosphorylation of the insulin receptors, IRS-1, IRS-2, and ERK, and higher insulin-induced IRS-1 and IRS-2/PI3-kinase association. Except for the regulation of ERK, SR59230A abolished all the cold-induced effects upon the insulin signal transduction pathway. However, this antagonist only partially inhibited the cold-induced increase of glucose uptake. Thus, the sympathetic tonus generated during cold-exposure acts, in brown adipose tissue, through the beta3-adrenergic receptor and modulates insulin signal transduction, with the exception of ERK. However, insulin-independent mechanisms other than beta3-adrenergic activation participate in cold-induced glucose uptake in brown adipose tissue of rats. PMID:15750837

  15. An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3- mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation

    PubMed Central

    1991-01-01

    The synthetic peptide Gly-Arg-Gly-Asp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts. Well- defined adhesion substrates were prepared with GRGDY between 10(-3) fmol/cm2 and 10(4) fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized. The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 10(4) fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin alpha V beta 3 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface- bound GRGDY, while fibronectin receptor antiserum specific for alpha 5 beta 1 did not. Furthermore, alpha V beta 3 was observed to cluster into focal contacts in spread cells, but alpha 5 beta 1 did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for alpha V beta 3-mediated cellular spreading, while 140 nm was required for alpha V beta 3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts; these spacings represent much fewer ligands than were previously thought to be required. PMID:1714913

  16. Agglucetin, a tetrameric C-type lectin-like venom protein, regulates endothelial cell survival and promotes angiogenesis by activating integrin {alpha}v{beta}3 signaling

    SciTech Connect

    Wang, W.-J.

    2008-05-02

    Agglucetin, a platelet glycoprotein (GP)Ib binding protein from Formosan Agkistrodon acutus (A. acutus) venom, could sustain human umbilical vein endothelial cell (HUVEC) proliferation and HUVEC adhering to immobilized agglucetin showed extensive spreading, which was strongly abrogated by integrin antagonists 7E3 and triflavin. Flow cytometric analyses confirmed the expression of GPIb complex on HUVEC is absent and fluorescein isothiocyanate (FITC)-agglucetin binds to HUVEC in a dose-dependent and saturable manner. Furthermore, native agglucetin specifically and dose-dependently inhibited the binding of FITC-23C6, an anti-{alpha}v{beta}3 monoclonal antibody (mAb), but not antibodies against {alpha}2 and {alpha}5, toward HUVEC and purified {alpha}v{beta}3 also bound to immobilized agglucetin-{beta} in a dose-dependent manner. Moreover, agglucetin exhibited a pro-angiogenic effect in vitro, as well as the focal adhesion kinase (FAK)-associated signaling molecules responsible for HUVEC activation were initiated by agglucetin. In conclusion, agglucetin, acting as a survival factor, promotes endothelial adhesion and angiogenesis by triggering {alpha}v{beta}3 signaling through FAK/phosphatidylinositol 3-kinase (PI3K)/Akt pathway.

  17. Growth factor expression during rat development: a comparison of TGF-beta 3, TGF-alpha, bFGF, PDGF and PDGF-R.

    PubMed Central

    Burton, P. B.; Quirke, P.; Sorensen, C. M.; Nehlsen-Cannarella, S. L.; Bailey, L. L.; Knight, D. E.

    1993-01-01

    At least part of the mechanism underlying fetal development appears to be the production of a number of growth factors considered important in the process of tumour formation. Using immunocytochemistry, we have investigated the temporal and spatial pattern of expression of some of the important growth factors, by the fetus. We describe here the cellular localization of transforming growth factor beta 3 (TGF-beta 3), platelet derived growth factor (PDGF) and its receptor (PDGF-R), TGF-alpha and basic fibroblast growth factor (bFGF) in the fetal rat from day 13 to 21 of gestation. Using antisera raised against an N-terminal portion of TGF-beta 3, immunoreactivity peaked around day 16 and was seen predominantly within epithelial cells. However, using antisera raised against the C-terminal of this molecule immunoreactivity was seen exclusively within the extracellular matrix underlying adjacent epithelia, and was maintained up until day 21 of gestation. Strong expression of TGF-alpha was seen in cells of most organs throughout the gestation period studied. Immunoreactivity for bFGF, PDGF and PDGF-R peaked around day 18 in both epithelial and mesenchymal cells of all major organ systems and then declined by day 21. These data suggest distinct roles for each factor during embryogenesis and tumorigenesis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8471538

  18. Adhesion and migration of avian neural crest cells on fibronectin require the cooperating activities of multiple integrins of the (beta)1 and (beta)3 families.

    PubMed

    Testaz, S; Delannet, M; Duband, J

    1999-12-01

    Based on genetic, functional and histological studies, the extracellular matrix molecule fibronectin has been proposed to play a key role in the migration of neural crest cells in the vertebrate embryo. In the present study, we have analyzed in vitro the repertoire and function of integrin receptors involved in the adhesive and locomotory responses of avian truncal neural crest cells to fibronectin. Immunoprecipitation experiments showed that neural crest cells express multiple integrins, namely (alpha)3(beta)1, (alpha)4(beta)1, (alpha)5(beta)1, (alpha)8(beta)1, (alpha)v(beta)1, (alpha)v(beta)3 and a (beta)8 integrin, as potential fibronectin receptors, and flow cytometry analyses revealed no major heterogeneity among the cell population for expression of integrin subunits. In addition, the integrin repertoire expressed by neural crest cells was found not to change dramatically during migration. At the cellular level, only (alpha)v(beta)1 and (alpha)v(beta)3 were concentrated in focal adhesion sites in connection with the actin microfilaments, whereas the other integrins were predominantly diffuse over the cell surface. In inhibition assays with function-perturbing antibodies, it appeared that complete abolition of cell spreading and migration could be achieved only by blocking multiple integrins of the (beta)1 and (beta)3 families, suggesting possible functional compensations between different integrins. In addition, these studies provided evidence for functional partitioning of integrins in cell adhesion and migration. While spreading was essentially mediated by (alpha)v(beta)1 and (alpha)8(beta)1, migration involved primarily (alpha)4(beta)1, (alpha)v(beta)3 and (alpha)8(beta)1 and, more indirectly, (alpha)3(beta)1. (alpha)5(beta)1 and the (beta)8 integrin were not found to play any major role in either adhesion or migration. Finally, consistent with the results of inhibition experiments, recruitment of (alpha)4(beta)1 and (alpha)v(beta)3, individually or in

  19. Arg-Tyr-Asp (RYD) and Arg-Cys-Asp (RCD) motifs in dendroaspin promote selective inhibition of beta1 and beta3 integrins.

    PubMed Central

    Wattam, B; Shang, D; Rahman, S; Egglezou, S; Scully, M; Kakkar, V; Lu, X

    2001-01-01

    Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence that is found within several glycoprotein ligands. This sequence has also been found in snake-venom anti-platelet proteins, including the disintegrins and dendroaspin, a natural variant of short-chain neurotoxins isolated from the venom of Dendroaspis jamesonii. In the present study, the motifs RYD and RCD were introduced into the dendroaspin scaffold to replace RGD. Both motifs in dendroaspin caused inhibition of ADP-induced platelet aggregation with IC(50) values of 200 and 300 nM respectively, similar to that of the wild-type RGD motif (170 nM). In comparison with wild-type dendroaspin, both RYD- and RCD-containing dendroaspins were more selective in the inhibition of the adhesion of K562 cells to laminin rather than to fibrinogen and fibronectin, even though they were 10-30-fold less potent at inhibiting K562 cell (containing alpha(5)beta(1) integrin) adhesion to laminin compared with wild-type. Interestingly, the RYD motif produced a similar IC(50) value to the RGD motif at inhibiting A375-SM cell (beta(3) integrin) adhesion to collagen, whereas the RCD motif was approx. 2-6-fold less potent compared with either RGD or RYD. These findings show that the selectivity of dendroaspin binding to beta(1) and beta(3) integrins can be modulated by the introduction of alternative cell recognition sequences. PMID:11336631

  20. Shear stress-induced release of basic fibroblast growth factor from endothelial cells is mediated by matrix interaction via integrin alpha(v)beta3.

    PubMed

    Gloe, Torsten; Sohn, Hae Young; Meininger, Gerald A; Pohl, Ulrich

    2002-06-28

    Considering that chronic elevation of shear stress results in remodeling of the vasculature, we analyzed whether mechanical load could mediate basic fibroblast growth factor (bFGF) release and whether bFGF would act as mediator of shear stress-induced endothelial proliferation and differentiation. Supernatant media of shear stress-exposed endothelial cells (EC) contained significantly higher amounts of bFGF than medium from static cells. Released bFGF was fully intact with regard to its function as an inductor of proliferation and differentiation. Shear stress-conditioned media induced capillary-like structure formation, whereas static control medium did not. Likewise, only shear stress-conditioned medium induced proliferation of serum starved EC. Both capillary-like structure formation and proliferation could be inhibited by neutralization of bFGF or its receptor. The release of bFGF was subject to specific, integrin-mediated control, since inhibition of alpha(v)beta(3) integrin prevented it, whereas inhibition of alpha(5)beta(1) integrin had no effect. We conclude that shear stress induces the release of bFGF from EC in a tightly controlled manner. The release is dependent on specific cell-matrix interactions via alpha(v)beta(3) integrins. The effects on cell proliferation and differentiation suggest that release of bFGF is functionally significant and may represent a necessary initial step in adaptive remodeling processes induced by shear stress. PMID:11976347

  1. Membrane type-1-matrix metalloproteinase expressed by prostate carcinoma cells cleaves human laminin-5 beta3 chain and induces cell migration.

    PubMed

    Udayakumar, Thirupandiyur S; Chen, Man Ling; Bair, Elisabeth L; Von Bredow, Dorothea C; Cress, Anne E; Nagle, Raymond B; Bowden, G Timothy

    2003-05-01

    Degradation of the extracellular matrix by proteolytic enzymes is a central aspect of physiological and pathologic tissue-remodeling processes such as trophoblastic implantation, wound healing, and tumor invasion. We have hypothesized that prostate adenocarcinoma cell invasion through the normal basal lamina is attributable in part to metalloproteinase-induced cleavage of laminin-5 (Ln-5) and enhanced motility of the cancer cells. We studied the role of membrane type-1-matrix metalloproteinase (MT1-MMP) expressed on the surface of prostate tumor cells in cleaving Ln-5 and enhancing the migration of prostate tumor cells. We also determined the nature of the MT1-MMP cleavage of human Ln-5 and how this altered Ln-5 changes the migration of prostate carcinoma cells. We found that human MT1-MMP cleaves purified human Ln-5 to an 80-kDa fragment. Mass spectrometry analyses of the 80-kDa cleaved product by trypsin and chymotrypsin gave 14 and 9 different peptide sequences, respectively, that were identical to the expected amino acid sequence of the Ln-5-beta3 chain. The recovered peptides represent 14.4% (trypsin) and 10.3% (chymotrypsin) of Ln-5-beta3 chain by amino acid count. Both trypsin and chymotrypsin digestion of MT1-MMP-cleaved product of Ln-5 did not show any other peptides that were identical to the other chains of Ln-5. Using a linear migration assay we found that the Ln-5 cleaved by MT1-MMP enhanced the migration of DU-145 prostate carcinoma cells by 2-fold compared with uncleaved Ln-5. The use of blocked antisense MT1-MMP oligonucleotides inhibited the migration of DU-145 cells on Ln-5. We also found that the prostate carcinoma cells expressing high levels of MT1-MMP, such as PC3N and PPC, demonstrated enhanced migration on human Ln-5-coated substrate, and this migration was inhibited using blocked antisense MT1-MMP oligonucleotides. In conclusion, this is a novel and important finding where we have shown that beta3-chain is cleaved by MT1-MMP, and this

  2. Effect of the adrenergic beta 3-agonist, BRL37344, on heat production by brown adipocytes in obese and in older rats.

    PubMed

    Yamakawa, A; Tanaka, E; Nakano, S

    1994-12-01

    Heat production by isolated brown adipocytes, from hypothalamic obese rats and from older rats, was investigated by microcalorimetry. The obese and older rats were 12 and 40 weeks-old, respectively. The basal heat production by the brown adipocytes was significantly less in the obese and older rats than in control rats 12 weeks of age. Isoproterenol and a novel adrenergic beta 3-agonist, BRL37344, increased heat production in a concentration-dependent manner in all rats. The effects of isoproterenol were significantly less in the obese group than in the controls, while BRL37344 stimulated heat production in all rats almost identically. These results suggest that (1) the heat producing capacity of brown adipocytes is reduced by hypothalamic obesity and aging, and (2) BRL37344 might be useful as an anti-obesity drug. PMID:7570685

  3. alpha1-Adrenergic stimulation potentiates the thermogenic action of beta3-adrenoreceptor-generated cAMP in brown fat cells.

    PubMed

    Zhao, J; Cannon, B; Nedergaard, J

    1997-12-26

    The relationship between cAMP levels and thermogenesis was investigated in brown fat cells from Syrian hamsters. Irrespective of whether the selective beta3-, beta2-, and beta1-agonists BRL 37344, salbutamol, and dobutamine or the physiological agonist norepinephrine was used to stimulate the cells, increases in cAMP levels were mediated via the beta3-receptor, as were the thermogenic effects. However, the relationship "thermogenesis per cAMP" was much lower for agents other than norepinephrine. Similarly, forskolin, although more potent than norepinephrine in elevating cAMP, was less potent in inducing thermogenesis. The selective alpha1-agonist cirazoline was in itself without effect on cAMP levels or thermogenesis, but when added to forskolin-stimulated cells, potentiated thermogenesis, up to the norepinephrine level, without affecting cAMP. This potentiation could not be inhibited by chelerythrine, but could be mimicked by Ca2+ ionophores. It was apparently not mediated via calmodulin-dependent protein kinase and was not an effect on mitochondrial respiratory control. The ability of all cAMP-elevating agents to induce thermogenesis in brown fat cells has earlier been interpreted to mean that it is only through the beta-receptors and the resulting increase in cAMP levels that thermogenesis is induced. However, it is here concluded that the thermogenic response to norepinephrine involves two interacting parts, one mediated via beta-receptors and cAMP and the other via alpha1-receptors and increases in cytosolic Ca2+ levels. PMID:9407062

  4. Low-energy laser irradiation facilitates the velocity of tooth movement and the expressions of matrix metalloproteinase-9, cathepsin K, and alpha(v) beta(3) integrin in rats.

    PubMed

    Yamaguchi, Masaru; Hayashi, Masami; Fujita, Shouji; Yoshida, Takamasa; Utsunomiya, Tadahiko; Yamamoto, Hirotsugu; Kasai, Kazutaka

    2010-04-01

    It has previously been reported that low-energy laser irradiation stimulated the velocity of tooth movement via the receptor activator of nuclear factor kappa B (RANK)/RANK ligand and the macrophage colony-stimulating factor/its receptor (c-Fms) systems. Matrix metalloproteinase (MMP)-9, cathepsin K, and alpha(v) beta(3) [alpha(v)beta3] integrin are essential for osteoclastogenesis; therefore, the present study was designed to examine the effects of low-energy laser irradiation on the expression of MMP-9, cathepsin K, and alpha(v)beta3 integrin during experimental tooth movement. Fifty male, 6-week-old Wistar strain rats were used in the experiment. A total force of 10g was applied to the rat molars to induce tooth movement. A Ga-Al-As diode laser was used to irradiate the area around the moving tooth and, after 7 days, the amount of tooth movement was measured. To determine the amount of tooth movement, plaster models of the maxillae were made using a silicone impression material before (day 0) and after tooth movement (days 1, 2, 3, 4, and 7). The models were scanned using a contact-type three-dimensional (3-D) measurement apparatus. Immunohistochemical staining for MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3 was performed. Intergroup comparisons of the average values were conducted with a Mann-Whitney U-test for tooth movement and the number of tartrate-resistant acid phosphatase (TRAP), MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3-positive cells. In the laser-irradiated group, the amount of tooth movement was significantly greater than that in the non-irradiated group at the end of the experiment (P < 0.05). Cells positively stained with TRAP, MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3 were found to be significantly increased in the irradiated group on days 2-7 compared with those in the non-irradiated group (P < 0.05). These findings suggest that low-energy laser irradiation facilitates the velocity of tooth

  5. Dinámica y crecimiento de los granos de polvo en la nebulosa protoplanetaria

    NASA Astrophysics Data System (ADS)

    de La Fuente Marcos, Carlos

    2001-06-01

    En el escenario estándar de la formación planetaria, los planetesimales (cuerpos de tamaño kilométrico) crecen a partir de granos de polvo, similares a los interestelares, embebidos en un disco gaseoso denominado nebulosa protoplanetaria. Durante esta etapa, los movimientos del gas pueden tener gran influencia en la dinámica y el crecimiento de los granos de polvo, dado que el flujo kepleriano del gas frena el movimiento de los mismos haciendo que caigan hacia el Sol, y la turbulencia inhibe la inestabilidad gravitacional de la capa de polvo. Aunque se acepta que los planetesimales fueron los elementos constituyentes de los planetas, todavía se desconoce cómo se produjo la formación de los mismos. Por esta razón, en los estudios más recientes, existe un renovado interés por comprender mejor la evolución de la capa de polvo inmersa en el disco gaseoso de la Nebulosa. El gas que fluye en el disco puede engendrar estructuras carentes de simetría axial, como por ejemplo ondas espirales y vórtices, a partir de gran variedad de mecanismos de excitación e inestabilidad. En 1995, Barge y Sommeria pusieron de manifiesto que la existencia de vórtices gaseosos persistentes en la nebulosa solar tendría importantes consecuencias sobre la formación de los planetesimales y el posterior crecimiento de los planetas gigantes. La investigación desarrollada en esta Tesis analiza la relación entre el polvo y el gas debida al acoplamiento por fricción dinámica entre ambos; en concreto, se estudia el efecto del flujo medio del gas sobre la dinámica de las partículas de polvo. El primer objetivo es investigar en profundidad los procesos de captura y crecimiento de los granos de polvo dentro de un vórtice y su posible relevancia en cuanto a la formación de los planetesimales. El segundo objetivo es la exploración de los efectos de ondas espirales propagándose en el disco gaseoso sobre la dinámica y el crecimiento de las partículas. La presencia de líneas de

  6. Human-Specific SNP in Obesity Genes, Adrenergic Receptor Beta2 (ADRB2), Beta3 (ADRB3), and PPAR γ2 (PPARG), during Primate Evolution

    PubMed Central

    Takenaka, Akiko; Nakamura, Shin; Mitsunaga, Fusako; Inoue-Murayama, Miho; Udono, Toshifumi; Suryobroto, Bambang

    2012-01-01

    Adrenergic-receptor beta2 (ADRB2) and beta3 (ADRB3) are obesity genes that play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. The Glu27 allele in ADRB2 and the Arg64 allele in ADRB3 are associated with abdominal obesity and early onset of non-insulin-dependent diabetes mellitus (NIDDM) in many ethnic groups. Peroxisome proliferator-activated receptor γ (PPARG) is required for adipocyte differentiation. Pro12Ala mutation decreases PPARG activity and resistance to NIDDM. In humans, energy-expense alleles, Gln27 in ADRB2 and Trp64 in ADRB3, are at higher frequencies than Glu27 and Arg64, respectively, but Ala12 in PPARG is at lower frequency than Pro12. Adaptation of humans for lipolysis, thermogenesis, and reduction of fat accumulation could be considered by examining which alleles in these genes are dominant in non-human primates (NHP). All NHP (P. troglodytes, G. gorilla, P. pygmaeus, H. agilis and macaques) had energy-thrifty alleles, Gly16 and Glu27 in ADRB2, and Arg64 in ADRB3, but did not have energy-expense alleles, Arg16, Gln27 and Trp64 alleles. In PPARG gene, all NHP had large adipocyte accumulating type, the Pro12 allele. Conclusions These results indicate that a tendency to produce much more heat through the energy-expense alleles developed only in humans, who left tropical rainforests for savanna and developed new features in their heat-regulation systems, such as reduction of body hair and increased evaporation of water, and might have helped the protection of entrails from cold at night, especially in glacial periods. PMID:22937051

  7. Beta-3 adrenergic agonists reduce pulmonary vascular resistance and improve right ventricular performance in a porcine model of chronic pulmonary hypertension.

    PubMed

    García-Álvarez, Ana; Pereda, Daniel; García-Lunar, Inés; Sanz-Rosa, David; Fernández-Jiménez, Rodrigo; García-Prieto, Jaime; Nuño-Ayala, Mario; Sierra, Federico; Santiago, Evelyn; Sandoval, Elena; Campelos, Paula; Agüero, Jaume; Pizarro, Gonzalo; Peinado, Víctor I; Fernández-Friera, Leticia; García-Ruiz, José M; Barberá, Joan A; Castellá, Manuel; Sabaté, Manel; Fuster, Valentín; Ibañez, Borja

    2016-07-01

    Beta-3 adrenergic receptor (β3AR) agonists have been shown to produce vasodilation and prevention of ventricular remodeling in different conditions. Given that these biological functions are critical in pulmonary hypertension (PH), we aimed to demonstrate a beneficial effect of β3AR agonists in PH. An experimental study in pigs (n = 34) with chronic PH created by pulmonary vein banding was designed to evaluate the acute hemodynamic effect and the long-term effect of β3AR agonists on hemodynamics, vascular remodeling and RV performance in chronic PH. Ex vivo human experiments were performed to explore the expression of β3AR mRNA and the vasodilator response of β3AR agonists in pulmonary arteries. Single intravenous administration of the β3AR agonist BRL37344 produced a significant acute reduction in PVR, and two-weeks treatment with two different β3AR selective agonists, intravenous BRL37344 or oral mirabegron, resulted in a significant reduction in PVR (median of -2.0 Wood units/m(2) for BRL37344 vs. +1.5 for vehicle, p = 0.04; and -1.8 Wood units/m(2) for mirabegron vs. +1.6 for vehicle, p = 0.002) associated with a significant improvement in magnetic resonance-measured RV performance. Histological markers of pulmonary vascular proliferation (p27 and Ki67) were significantly attenuated in β3AR agonists-treated pigs. β3AR was expressed in human pulmonary arteries and β3AR agonists produced vasodilatation. β3AR agonists produced a significant reduction in PVR and improved RV performance in experimental PH, emerging as a potential novel approach for treating patients with chronic PH. PMID:27328822

  8. Thrombin enhances the adhesion and migration of human colon adenocarcinoma cells via increased beta 3-integrin expression on the tumour cell surface and their inhibition by the snake venom peptide, rhodostomin.

    PubMed Central

    Chiang, H. S.; Yang, R. S.; Huang, T. F.

    1996-01-01

    The interactions between tumour cells and the microvasculature, including the adhesion of tumour cells to endothelium and extracellular matrix (ECM) as well as their migratory ability, are prerequisites for metastasis to occur. In this study we showed that thrombin is capable of enhancing in vitro tumour cell metastatic potential in terms of adhesive properties and migratory response. Following exposure to subclotting concentrations of thrombin, SW-480 human colon adenocarcinoma cells exhibited increased adhesion to both the endothelium and ECM component (i.e. fibronectin). Likewise, the pretreatment of thrombin enhanced the migratory ability of SW-480 cells. The enhanced adhesion was significantly inhibited by complexing of thrombin with its inhibitor hirudin, or by serine proteinase inhibition with 3,4-DCI, but was unaffected by pretreatment of tumour cells with actinomycin D or cycloheximide. The effect of thrombin resulted in an upregulated cell-surface expression of beta 3 integrins, a group of receptors mediating interactions between tumour cells and endothelial cells, and between tumour cells and ECM. Antibodies against beta 3 integrins effectively blocked both the enhanced adhesion and migration. This thrombin-mediated up-regulation of beta 3 integrins involved the activation of protein kinase C (PKC) as thrombin-enhanced adhesion was diminished by PKC inhibition. Rhodostomin, an Arg-Gly-Asp-containing antiplatelet snake venom peptide that antagonises the binding of ECM toward beta 3 integrins on SW-480 cells, was about 600 and 500 times, more potent that RGDS in inhibiting thrombin-enhanced adhesion and migration respectively. Our data suggest that PKC inhibitors as well as rhodostomin may serve as inhibitory agents in the prevention of thrombin-enhanced metastasis. PMID:8611404

  9. Transforming growth factor-beta 3 stimulates cartilage matrix elaboration by human marrow-derived stromal cells encapsulated in photocrosslinked carboxymethylcellulose hydrogels: potential for nucleus pulposus replacement.

    PubMed

    Gupta, Michelle S; Cooper, Elana S; Nicoll, Steven B

    2011-12-01

    Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies using marrow-derived stromal cells (MSCs) have been used to develop cartilaginous tissue constructs, which may serve as viable NP replacements. Supplementation with growth factors, such as transforming growth factor-beta 3 (TGF-β3), has been shown to enhance the differentiation of MSCs and promote functional tissue development of such constructs. A potential candidate material that may be useful as a scaffold for NP tissue engineering is carboxymethylcellulose (CMC), a biocompatible, cost-effective derivative of cellulose. Photocrosslinked CMC hydrogels have been shown to support NP cell viability and promote phenotypic matrix deposition capable of maintaining mechanical properties when cultured in serum-free, chemically defined medium (CDM) supplemented with TGF-β3. However, MSCs have not been characterized using this hydrogel system. In this study, human MSCs (hMSCs) were encapsulated in photocrosslinked CMC hydrogels and cultured in CDM with and without TGF-β3 to determine the effect of the growth factor on the differentiation of hMSCs toward an NP-like phenotype. Constructs were evaluated for matrix elaboration and functional properties consistent with native NP tissue. CDM supplemented with TGF-β3 resulted in significantly higher glycosaminoglycan content (762.69±220.79 ng/mg wet weight) and type II collagen (COL II) content (6.25±1.64 ng/mg wet weight) at day 21 compared with untreated samples. Immunohistochemical analyses revealed uniform, pericellular, and interterritorial staining for chondroitin sulfate proteoglycan and COL II in growth factor-supplemented constructs compared with faint, strictly pericellular staining in untreated constructs at 21 days. Consistent with matrix deposition, mechanical properties of hydrogels treated with TGF-β3 increased over time and exhibited the highest peak stress in stress-relaxation (

  10. In situ assessment of the role of the beta 1-, beta 2- and beta 3-adrenoceptors in the control of lipolysis and nutritive blood flow in human subcutaneous adipose tissue.

    PubMed Central

    Barbe, P.; Millet, L.; Galitzky, J.; Lafontan, M.; Berlan, M.

    1996-01-01

    1. The involvement of beta 1-, beta 2- and beta 3-adrenoceptors in the control of lipolysis and nutritive blood flow was investigated in abdominal subcutaneous adipose tissue of healthy young adults by use of an in situ microdialysis technique. 2. Dialysis probes were infused either with isoprenaline (non-selective beta-adrenoceptor agonist), CGP 12,177 (selective beta 3-adrenoceptor agonist having beta 1-/beta 2-antagonist properties), dobutamine (selective beta 1-adrenoceptor agonist) or terbutaline (selective beta 2-adrenoceptor agonist). The recovery of each probe used for perfusion was calculated by an in vivo calibration method. The local blood flow was estimated through the measurement of the escape of ethanol infused simultaneously with the drugs included in the probe. 3. Isoprenaline infusion at 0.01 microM had a weak effect while higher concentrations of isoprenaline (0.1 and 1 microM) caused a rapid, sustained and concentration-dependent increase of glycerol outflow; the maximum increase was 306 +/- 34% with 1 microM. Isoprenaline also increased the nutritive blood flow in adipose tissue; a significant effect appeared at 0.1 microM isoprenaline and was greater at 1 microM. 4. CGP 12,177 (10 and 100 microM) increased the glycerol concentration in the dialysate (128 +/- 8 and 149 +/- 12%, respectively) and nutritive blood flow. Terbutaline and dobutamine (100 microM) both provoked rapid and similar increases in glycerol outflow (252 +/- 18 and 249 +/- 18%, respectively). Both, terbutaline and dobutamine increased nutritive blood flow. 5. It is concluded that beta 1- and beta 2-adrenoceptor subtypes are both mainly involved in the mobilization of lipids and in the control of nutritive blood flow. beta 3-Adrenoceptors play a weaker role in the control of lipolysis and nutritive blood flow in human subcutaneous abdominal adipose tissue. PMID:8851509

  11. Differentiation of some positional and diastereomeric isomers of Boc-carbo-[beta]3 dipeptides containing galactose, xylose and mannose sugars by electrospray ionization tandem mass spectrometry (ESI MS/MS)

    NASA Astrophysics Data System (ADS)

    Reddy, P. Nagi; Ramesh, V.; Srinivas, R.; Sharma, G. V. M.; Nagendar, Pendem; Subash, V.

    2006-02-01

    Electrospray ionization (ESI) tandem mass spectrometry has been used to distinguish the positional and diastereomeric isomers of Boc-C-linked carbo-[beta]3 dipeptides (1-38) synthesized from glycine (Gly), [beta]-h-glycine ([beta]-hGly), [beta]-h-alanine ([beta]-hAla) and C-linked carbo-[beta]3-amino acid (Caa) that contain galactose, xylose and mannose sugars as side chains with "R" and "S" configurations at the amine center. The major fragmentation of [M + H]+ of the dipeptides (1-38) yields mainly two ions: (i) [M + H-C(CH3)3 + H]+ ([`]a') and (ii) [M + H-Boc + H]+ ([`]b') corresponding to losses of 2-methyl-prop-1-ene and -Boc moiety from [M + H]+ ions, respectively. The diastereomeric dipeptide isomers with Caa (R) and (S) configurations at the N-terminus can easily be distinguished by the difference in the abundance of ion [`]a' and [`]b'. The isomeric peptides with Caa (R) at the N-terminus gives prominent [M + H-C(CH3)3 + H]+ ([`]a') where as it is insignificant or totally absent for peptides which have Caa (S) at the N-terminus. This is presumably due to the different steric crowdings around the Boc-group in the different diastereomers. The positional isomers of dipeptides can also be differentiated by the difference in the abundance of ion [`]a' and [`]b' in the CID of [M + H]+ ions. The CID of [M + H-Boc + H]+ ions of the isomeric peptides also show y1+ ions at different m/z values. All these results suggest that the CID of [M + H]+ ions is highly useful for distinguishing the Boc-NH-Caa-[beta]3 dipeptide isomers with Caa of "S" configuration from the isomers with Caa of "R" configuration and the positional isomers.

  12. Differential cytokine modulation of the genes LAMA3, LAMB3, and LAMC2, encoding the constitutive polypeptides, alpha 3, beta 3, and gamma 2, of human laminin 5 in epidermal keratinocytes.

    PubMed

    Korang, K; Christiano, A M; Uitto, J; Mauviel, A

    1995-07-24

    Laminin 5, an anchoring filament protein previously known as nicein/kalinin/epiligrin, consists of three polypeptide chains, alpha 3, beta 3, and gamma 2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. The expression of laminin 5 was detected by Northern hybridization with specific cDNA probes in various epidermal keratinocyte cultures, whereas no expression of any of the three genes could be detected in foreskin fibroblast cultures. Transforming growth factor-beta (TGF-beta) enhanced LAMA3, LAMB3, and LAMC2 gene expression in human epidermal keratinocytes, as well as in HaCaT and Balb/K cells in culture, although the extent of enhancement was greater for LAMA3 and LAMC2 genes than for LAMB3. Interestingly, tumor necrosis factor-alpha, (TNF-alpha) alone did not alter the expression of LAMB3 and LAMC2 genes in human epidermal keratinocytes, whereas it inhibited the expression of LAMA3. These results suggest that the expression of the three genes encoding the laminin 5 subunits is not coordinately regulated by the cytokines tested. PMID:7635220

  13. Effects of lifestyle intervention on weight and metabolic parameters in patients with impaired glucose tolerance related to beta-3 adrenergic receptor gene polymorphism Trp64Arg(C/T): Results from the Japan Diabetes Prevention Program.

    PubMed

    Sakane, Naoki; Sato, Juichi; Tsushita, Kazuyo; Tsujii, Satoru; Kotani, Kazuhiko; Tominaga, Makoto; Kawazu, Shoji; Sato, Yuzo; Usui, Takeshi; Kamae, Isao; Yoshida, Toshihide; Kiyohara, Yutaka; Sato, Shigeaki; Tsuzaki, Kokoro; Takahashi, Kaoru; Kuzuya, Hideshi

    2016-05-01

    The beta-3 adrenergic receptor (ADRB3), primarily expressed in adipose tissue, is involved in the regulation of energy metabolism. The present study hypothesized that ADRB3 (Trp64Arg, rs4994) polymorphisms modulate the effects of lifestyle intervention on weight and metabolic parameters in patients with impaired glucose tolerance. Data were analyzed from 112 patients with impaired glucose tolerance in the Japan Diabetes Prevention Program, a lifestyle intervention trial, randomized to either an intensive lifestyle intervention group or usual care group. Changes in weight and metabolic parameters were measured after the 6-month intervention. The ADRB3 polymorphisms were determined using the polymerase chain reaction restriction fragment length polymorphism method. Non-carriers showed a greater weight reduction compared with the carriers in both the lifestyle intervention group and usual care group, and a greater increase of high-density lipoprotein cholesterol levels than the carriers only in the lifestyle intervention group. ADRB3 polymorphisms could influence the effects of lifestyle interventions on weight and lipid parameters in impaired glucose tolerance patients. PMID:27330719

  14. High-resolution mapping of the [gamma]-aminobutyric acid receptor subunit [beta]3 and [alpha]5 gene cluster on chromosome 15q11-q13, and localization of breakpoints in two Angelman syndrome patients

    SciTech Connect

    Sinnett, D.; Wagstaff, J.; Woolf, E. Harvard Medical School, Boston, MA ); Glatt, K. ); Kirkness, E.J. )Lalande, M. Harvard Medical School, Boston, MA Howard Hughes Medical Inst., Boston, MA )

    1993-06-01

    The [gamma]-aminobutyric acid (GABA[sub A]) receptors are a family of ligand-gated chloride channels constituting the major inhibitory neurotransmitter receptors in the nervous system. In order to determine the genomic organization of the GABA[sub A] receptor [beta]3 subunit gene (GABRB3) and [alpha]5 subunit gene (GABRA5) in chromosome 15q11-q13, the authors have constructed a high-resolution physical map using the combined techniques of field-inversion gel electrophoresis and phage genomic library screening. This map, which covers nearly 1.0 Mb, shows that GABRB3 and GABRA5 are separated by less than 100 kb and are arranged in a head-to-head configuration. GABRB3 encompasses approximately 250 kb, while GABRA5 is contained within 70 kb. This difference in size is due in large part to an intron of 150 kb within GABRB3. The authors have also identified seven putative CpG islands within a 600-kb interval. Chromosomal rearrangement breakpoints -- in one Angelman syndrome (AS) patient with an unbalanced translocation and in another patient with a submicroscopic deletion -- are located within the large GABRB3 intron. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the DNA rearrangements in these AS patients and will be valuable for mapping new genes to the AS chromosomal region. 64 refs., 6 figs., 2 tabs.

  15. Preferential antagonism of the interactions of the integrin alpha IIb beta 3 with immobilized glycoprotein ligands by snake-venom RGD (Arg-Gly-Asp) proteins. Evidence supporting a functional role for the amino acid residues flanking the tripeptide RGD in determining the inhibitory properties of snake-venom RGD proteins.

    PubMed Central

    Lu, X; Williams, J A; Deadman, J J; Salmon, G P; Kakkar, V V; Wilkinson, J M; Baruch, D; Authi, K S; Rahman, S

    1994-01-01

    The inhibitory properties of a panel of snake-venom-derived RGD (Arg-Gly-Asp) proteins, including the disintegrins kistrin, elegantin and albolabrin, and the neurotoxin homologue dendroaspin, were investigated in a platelet-adhesion assay using three immobilized ligands of the glycoprotein IIb-IIIa complex (alpha IIb beta 3), namely fibrinogen, fibronectin and von Willebrand factor (vWF). The snake-venom proteins preferentially inhibited the adhesion of ADP-treated platelets to one or more of the immobilized ligands. Kistrin and dendroaspin exhibited similar inhibitory characteristics, abrogating platelet adhesion to fibrinogen and vWF at nanomolar concentrations, but poorly inhibiting adhesion to fibronectin. Kistrin and dendroaspin share little overall amino-acid-sequence identity, but a considerable level of sequence similarity exists around the RGD tripeptide. Synthetic cyclic peptides corresponding to these regions of kistrin and dendroaspin inhibited platelet adhesion to both fibrinogen and fibronectin with approximately equal potency, but were 100-fold weaker antagonists of the interactions of the alpha IIb beta 3 complex with fibrinogen than their parent proteins. The disintegrins elegantin and albolabrin, which share approx. 60% overall amino-acid-sequence similarity with kistrin but have different residues around the RGD tripeptide, exhibited different antagonistic preferences. Elegantin inhibited platelet adhesion to immobilized vWF and fibronectin, but was significantly less effective at disrupting adhesion to fibrinogen. Albolabrin selectively inhibited platelet adhesion to immobilized vWF and was less effective with fibrinogen and fibronectin as adhesive ligands. In contrast with the behaviour of these venom proteins, the adhesion of ADP-treated platelets to immobilized fibrinogen, fibronectin and vWF was inhibited non-selectively by a range of monoclonal antibodies with specificity for the alpha IIb beta 3 complex. These observations, therefore

  16. The integrin alpha IIb beta 3 contains distinct and interacting binding sites for snake-venom RGD (Arg-Gly-Asp) proteins. Evidence that the receptor-binding characteristics of snake-venom RGD proteins are related to the amino acid environment flanking the sequence RGD.

    PubMed Central

    Rahman, S; Lu, X; Kakkar, V V; Authi, K S

    1995-01-01

    We have previously demonstrated [Lu, Williams, Deadman, Salmon, Kakkar, Wilkinson, Baruch, Authi and Rahman (1994) Biochem. J. 304, 929-936] the preferential antagonism of the interactions of the integrin alpha IIb beta 3 on activated platelets with three immobilized glycoprotein ligands (fibrinogen, fibronectin and von Willebrand factor) by a selected panel of snake-venom RGD (Arg-Gly-Asp)-containing proteins including the disintegrins kistrin and elegantin, and the neurotoxin variant dendroaspin. Kistrin and dendroaspin, although structurally unrelated, contain similar amino acids flanking the tripeptide RGD and behaved as identical antagonists preferentially inhibiting platelet adhesion to immobilized fibrinogen as opposed to fibronectin. In contrast, elegantin, which shares extensive sequence similarity with kistrin but has different amino acids around the tripeptide RGD, preferentially inhibited platelet adhesion to immobilized fibronectin as opposed to fibrinogen. To develop further insights into the mechanisms underlying the preferential antagonism shown by the venom proteins in the adhesion studies, we, in the present study, sought to determine the binding properties of kistrin, elegantin and dendroaspin to the alpha IIb beta 3 complex by radioligand kinetic and competition studies. In direct binding experiments, both kistrin and dendroaspin were observed to bind to a single class of binding site on ADP-activated platelets with apparent equilibrium dissociation constant (Kdapp) values of 42 +/- 2 nM and 21 +/- 6 nM respectively. In competition studies, dendroaspin blocked the binding of 125I-labelled kistrin to ADP-activated platelets in a simple competitive manner, with an apparent equilibrium inhibition constant (Kiapp) of 143 +/- 14 nM, from which an indirect Kdapp = 22 nM for dendroaspin was determined. This result suggests that kistrin and dendroaspin bind to the same site on the integrin alpha IIb beta 3 consistent with their similar inhibitory

  17. Synthesis of 25-hydroxyvitamin D sub 3 3. beta. -3 prime -(N-(4-azido-2-nitrophenyl)amino)propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D sub 3 : Photoaffinity labeling of rat serum vitamin D binding protein

    SciTech Connect

    Ray, R.; Holick, M.F. ); Bouillon, R.; Van Baelen, H. )

    1991-05-14

    Vulnerability of 25-hydroxy-(26,27-{sup 3}H)vitamin D{sub 3} 3{beta}-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D{sub 3} (25-OH-D{sub 3}) toward standard conditions of carboxymethylationin promoted the authors to synthesize 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D{sub 3}, and 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE), the radiolabeled counterpart of 25-ANE competes for the 25-OH-D{sub 3} binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with {sup 3}H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D{sub 3}. These results provide strong evidence for the covalent labeling of the 25-OH-D{sub 3} binding site in rDPB by {sup 3}H-25-ANE.

  18. Variabilidad de la Estación de Crecimiento en la Región Sur de Tamaulipas en condiciones climaticas actuales y futuras.

    NASA Astrophysics Data System (ADS)

    Medina-Barrios, M.; Conde-Alvarez, C.; Gay-Garcia, C.

    2007-05-01

    El impacto de la variabilidad y cambio climáticos, afectan el potencial agrícola de la Región Sur de Tamaulipas. Además de los cambios estacionales, bajos rendimientos agrícolas, el manejo de los cultivos y las políticas locales de producción, existe la incertidumbre del mercado regional para los pequeños y grandes productores. La diversificación agrícola ha sido una alternativa para enfrentar las condiciones imperantes en esta región. Pero ésta ha provocado la fragmentación territorial, por lo que sólo algunos productores logran competir en un mercado nacional. Existe una preocupación generalizada por buscar soluciones que permitan que la población que es afectada por la inseguridad de la producción agrícola y económica, pueda adaptarse a las variaciones climáticas que afectan el proceso productivo. La seguridad alimentaría queda sujeta a la fluctuación de las importaciones para los sectores básicos y a las estrategias mercantiles de empresas trasnacionales. La percepción local sugiere un número creciente de eventos climatológicos extremos, constantes y severos en los últimos 20 años, con el aumento creciente de pérdidas económicas. El análisis se centra en la disponibilidad de agua, agregándose un aspecto de capital importancia como es la variabilidad interanual de la lluvia, que condiciona muy fuertemente el riesgo agrícola en el trópico seco, siendo ésta la que determina el momento de inicio de la estación favorable para el crecimiento y su duración. En este trabajo se han obtenido modelos de la distribución espacial de la precipitación y temperaturas, para el escenario base 1961-1990, el escenario actual 1971-2000, para algunos años El Niño y La Niña, así como para los escenarios de Cambio Climático HADLEY, ECHAM y GFDL, con escenarios A2 y B2, para las décadas de los 20s y 50s, para establecer el inicio y duración de la Estación de Crecimiento, utilizando Sistemas de Información Geográfica (ArcView). Estos

  19. Multiple Signals Regulate PLC beta 3 in Human Myometrial Cells

    PubMed Central

    Zhong, Miao; Murtazina, Dilyara A.; Phillips, Jennifer; Ku, Chun-Ying; Sanborn, Barbara M.

    2008-01-01

    Summary The regulation of PLCB3-Serine1105 phosphorylation by both negative feedback and negative crosstalk facilitates the integration of multiple signaling pathways in myometrial cells. Phospholipase CB3 (PLCB3) Serine1105, a substrate for multiple protein kinases, represents a potential point of convergence of several signaling pathways in the myometrium. To explore this hypothesis, the regulation of PLCB3-Serine1105 phosphorylation (P-S1105) was studied in immortalized and primary human myometrial cells. CPT-cAMP and calcitonin gene-related peptide (CALCA) transiently increased P-S1105. Relaxin also stimulated P-S1105; this effect was partially blocked by the protein kinase A (PRKA) inhibitor Rp-8-CPT-cAMPS. Oxytocin, which stimulates Gαq-mediated pathways, also rapidly increased P-S1105, as did PGF2α and ATP. Oxytocin-stimulated phosphorylation was blocked by the protein kinase C (PRKC) inhibitor Go6976 and by pretreatment overnight with a phorbol ester. Cypermethrin, a PP2B phosphatase inhibitor, but not okadaic acid, a PP1/PP2A inhibitor, prolonged the effect of CALCA on P-S1105, whereas the reverse was the case for the oxytocin-stimulated increase in P-S1105. PLCB3 was the predominant PLC isoform expressed in the myometrial cells and PLCB3 shRNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. Oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid but enhanced by pretreatment with Go6976. CPT-cAMP inhibited oxytocin-stimulated PI turnover in the presence of overexpressed PLCB3, but not overexpressed PLCB3-S1105A. These data demonstrate that both negative crosstalk from the cAMP/PRKA pathway and a negative feedback loop in the oxytocin/G protein/PLCB pathway involving PRKC operate in myometrial cells and suggest that different protein phosphatases predominate in mediating P-S1105 dephosphorylation in these pathways. The integration of multiple signal components at the level of PLCB3 may be important to its function in the myometrium. PMID:18322273

  20. Cardiac salvage by tweaking with beta-3-adrenergic receptors.

    PubMed

    Balligand, Jean-Luc

    2016-07-15

    Overstimulation of the orthosympathetic system leads to cardiovascular cell and tissue damage through prolonged activation of β-1-2 adrenergic receptors (BARs). The more recent identification of the third isotype of BAR (B3AR) in cardiac myocytes and endothelial cells with a distinctive coupling and effect on cardiac function and remodelling introduced a new facet to this paradigm. In particular, B3AR is up-regulated in cardiac disease and less prone to homologous desensitization, which may reinforce its influence on the diseased myocardium. Mice with transgenic cardiac-specific expression of the human B3AR are protected from cardiac hypertrophy and fibrosis in response to neurohormonal stimulation. B3AR has also been implicated in cardiac protection after ischaemia-reperfusion and the benefits of exercise on the heart. Many of these salvage mechanisms are mediated by B3AR coupling to nitric oxide synthase (eNOS and nNOS) and downstream cGMP/protein kinase G signalling. Notably, B3AR exerts antioxidant protective effects on these and other signalling elements, which may subserve its protective properties in the setting of chronic heart failure. Additional vasorelaxing properties and paracrine NO-mediated signalling by B3AR in endothelium, together with systemic metabolic effects on beige/brown fat complete the pleiotropic protective properties of this new therapeutic target. PMID:27001422

  1. Mirabegron: A Beta-3 Agonist for Overactive Bladder

    PubMed Central

    Bragg, Rebecca; Hebel, Danielle; Vouri, Scott Martin

    2015-01-01

    Objective To review the literature regarding the efficacy and safety of mirabegron for the treatment of overactive bladder (OAB). Data Sources A literature search was performed using MEDLINE (PubMed) prior to 12/31/2013 using the terms “mirabegron” and “randomized-controlled trial.” Study Selection/Data Extraction All published, double-blind, randomized controlled trials assessing mirabegron were included. Articles were reviewed and included if mirabegron was used as monotherapy and if the primary outcome analyzed drug efficacy. Data Synthesis The efficacy of mirabegron for the treatment of OAB has been demonstrated in the selected five randomized, placebo-controlled trials. The majority of these trials lasted 12 weeks in duration and compared various doses of mirabegron to placebo and/or tolterodine extended release (ER). Primary efficacy outcomes for the trials included mean number of micturitions per 24 hours and mean number of incontinence episodes per 24 hours. Included trials showed statistically significant reductions in both efficacy outcomes for various doses of mirabegron when compared to placebo. Conclusion Based on the trials reviewed, mirabegron has been efficacious in reducing mean number of micturitions and incontinence episodes per 24 hours, as well as improved other secondary outcomes like OAB symptoms and quality of life measures. Common adverse drug events seen with mirabegron include: hypertension, nasopharyngitis, urinary tract infections, headache, constipation, upper respiratory tract infection, arthralgia, diarrhea, tachycardia, abdominal pain, and fatigue. Given the efficacy and safety data currently available, mirabegron represents a reasonable alternative to antimuscarinics for patients with OAB.Future studies are needed to determine the utility of mirabegron for OAB in a variety of demographics. PMID:25521658

  2. The Absence of Beta-3 Integrin Accelerates Early Skeletal Repair

    PubMed Central

    Hu, Diane; Lu, Chuanyong; Sapozhnikova, Anna; Barnett, Michael; Sparrey, Carolyn; Miclau, Theodore; Marcucio, Ralph S.

    2010-01-01

    Integrins are heterodimeric transmembrane proteins that mediate cell-matrix interactions and modulate cell behavior. β3 subunit is a component of αII β 3 and αv β3 integrins. In this study, we first determined that β3 transcripts are expressed by cells within fracture calluses at 7 and 10 days after injury in a mouse model. We then analyzed fracture healing in mice deficient of β3 integrin with molecular, histomorphometric, and biomechanical techniques. We found that lack ofβ3 integrin results in an extended bleeding time and leads to more bone formation and accelerated cartilage maturation at 7 days after injury. However, β3 deficiency does not appear to affect later fracture healing. At days 14 and 21, histological appearance or biomechanical properties of fracture calluses are similar between wild type and mutant mice. We also found that altered fracture healing in β3 null mice is not associated with accelerated angiogenesis, since no significant difference of length density and surface density of blood vessels in fracture limbs was detected at 3 days after injury between wild type and β3 null mice. In conclusion, our findings demonstrate that β3 integrin plays an important role during early fracture healing. Further research is required to determine the underlying mechanisms. PMID:19637214

  3. Modulation of RGD sequence motifs regulates disintegrin recognition of alphaIIb beta3 and alpha5 beta1 integrin complexes. Replacement of elegantin alanine-50 with proline, N-terminal to the RGD sequence, diminishes recognition of the alpha5 beta1 complex with restoration induced by Mn2+ cation.

    PubMed Central

    Rahman, S; Aitken, A; Flynn, G; Formstone, C; Savidge, G F

    1998-01-01

    Several recent studies have demonstrated that the amino acid residues flanking the RGD sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. The present study has addressed the role of the residues flanking the RGD sequence in regulating the recognition by disintegrin of the alphaIIb beta3 and alpha5beta1 complexes by construction of a panel of recombinant molecules of Elegantin (the platelet aggregation inhibitor from the venom of Trimerasurus elegans) expressing specific RGD sequence motifs. Wild-type Elegantin (ARGDNP) and several variants including Eleg. AM (ARGDMP), Eleg. PM (PRGDMP) and Eleg. PN (PRGDNP) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The inhibitory efficacies of the panel of Elegantin variants were analysed in platelet adhesion assays with substrates immobilized with fibrinogen and fibronectin. Elegantin molecules containing an Ala residue N-terminal to the RGD sequence (wild-type Elegantin and Eleg. AM) showed strong inhibitory activity towards alphaIIbbeta3-dependent platelet adhesion on fibronectin, whereas a Pro residue in this position (Eleg. PM and Kistrin, the inhibitor from the venom of Calloselasma rhodostoma) engendered lower activity. The decreased activity could not be attributed to a decrease in the affinity of the disintegrin for the alphaIIb beta3 complex because both Eleg. AM and Eleg. PM had similar Kd (app) values. In contrast, Elegantin molecules into which a Met residue was introduced in place of the Asn residue C-terminal to the RGD sequence showed 10-13-fold elevated inhibitory activity towards platelet adhesion on fibrinogen and this was maintained with either a Pro or Ala residue N-terminal to the RGD sequence. In experiments with the alpha5 beta1 complex on K562 cells, the inhibitory efficacies of the panel of Elegantin molecules were analysed under two different cation conditions. First, in the presence of Ca2+/Mg2+, K562 cell

  4. Closed headpiece of integrin [alpah]IIb[beta]3 and its complex with an [alpha]IIb[beta]3-specific antagonist that does not induce opening

    SciTech Connect

    Zhu, Jieqing; Zhu, Jianghai; Negri, Ana; Provasi, Davide; Filizola, Marta; Coller, Barry S.; Springer, Timothy A.

    2011-08-24

    The platelet integrin {alpha}{sub IIb}{beta}{sub 3} is essential for hemostasis and thrombosis through its binding of adhesive plasma proteins. We have determined crystal structures of the {alpha}{sub IIb}{beta}{sub 3} headpiece in the absence of ligand and after soaking in RUC-1, a novel small molecule antagonist. In the absence of ligand, the {alpha}{sub IIb}{beta}{sub 3} headpiece is in a closed conformation, distinct from the open conformation visualized in presence of Arg-Gly-Asp (RGD) antagonists. In contrast to RGD antagonists, RUC-1 binds only to the {alpha}{sub IIb} subunit. Molecular dynamics revealed nearly identical binding. Two species-specific residues, {alpha}{sub IIb} Y190 and {alpha}{sub aIIb} D232, in the RUC-1 binding site were confirmed as important by mutagenesis. In sharp contrast to RGD-based antagonists, RUC-1 did not induce {alpha}{sub IIb}{beta}{sub 3} to adopt an open conformation, as determined by gel filtration and dynamic light scattering. These studies provide insights into the factors that regulate integrin headpiece opening, and demonstrate the molecular basis for a novel mechanism of integrin antagonism.

  5. Cardioprotective Effect of Beta-3 Adrenergic Receptor Agonism: Role of Neuronal Nitric Oxide Synthase

    PubMed Central

    Niu, Xiaolin; Watts, Vabren L.; Cingolani, Oscar H.; Sivakumaran, Vidhya; Leyton-Mange, Jordan S.; Ellis, Carla L.; Miller, Karen L.; Vandegaer, Konrad; Bedja, Djahida; Gabrielson, Kathleen L.; Paolocci, Nazareno; Kass, David A.; Barouch, Lili A.

    2012-01-01

    Objective The aim of this study is to determine if activation of β3-adrenoceptor (β3-AR) and downstream signaling of NOS isoforms protects the heart from failure and hypertrophy induced by pressure overload. Background β3-AR and its downstream signaling pathways are recognized as novel modulators of heart function. Unlike _1- and _2-ARs, _3-ARs are stimulated at high catecholamine concentrations and induce negative inotropic effects, serving as a “brake” to protect the heart from catecholamine overstimulation. Methods C57BL/6J and nNOS knock-out mice were assigned to receive transverse aortic constriction (TAC), BRL37344 (β3-agonist, BRL0.1 mg/kg/hour), or both. Results Three weeks of BRL treatment in wild type mice attenuated left ventricular dilation and systolic dysfunction, and partially reduced cardiac hypertrophy induced by TAC. This effect was associated with increased nitric oxide (NO) production and superoxide suppression. TAC decreased endothelial NO synthase (eNOS) dimerization, indicating eNOS uncoupling, which was not reversed by BRL treatment. However, nNOS protein expression was up-regulated 2-fold by BRL, and the suppressive effect of BRL on superoxide generation was abrogated by acute neuronal NO synthase (nNOS) inhibition. Furthermore, BRL cardioprotective effects were actually detrimental in nNOS−/− mice. Conclusion These results are the first to show in vivo cardioprotective effects of β3-AR specific agonism in pressure overload hypertrophy and heart failure, and support nNOS as the primary downstream NOS isoform in maintaining NO and reactive oxygen species (ROS) balance in the failing heart. PMID:22624839

  6. Generation of Mice With a Conditional Allele for the Transforming Growth Factor Beta3 Gene

    PubMed Central

    Doetschman, Thomas; Georgieva, Teodora; Li, Hongqi; Reed, Thomas D.; Grisham, Christina; Friel, Jacqueline; Estabrook, Mark A.; Gard, Connie; Sanford, L.P.; Azhar, Mohamad

    2013-01-01

    Summary The transforming growth factor beta (TGFβ) pathway is involved in embryonic development and several inherited and acquired human diseases. The gene for TGFβ3 (Tgfb3) encodes one of the three ligands for TGF b receptors. It is widely expressed in the embryo and its mutation or misexpression is found in human diseases. Tgfb3−/− mice die at birth from cleft palate, precluding functional studies in adults. Here, we generated mice in which exon 6 of Tgfb3 was flanked with LoxP sites (Tgfb3flox/flox). The adult mice were normal and fertile. EIIa-Cre-mediated deletion of exon 6 in Tgfb3flox/flox mice efficiently generated Tgfb3 conditional knockout (Tgfb3cko/cko) mice which died at birth from the same cleft palate defect as Tgfb3−/− mice, indicating that the conditional and knockout alleles are functionally equivalent. This Tgfb3cko allele will now enable studies of TGFβ3 function in different cell or tissue types in embryonic development and during adulthood. genesis 50:59-66, 2012. PMID:22223248

  7. Platelet activating factor antagonist design. 2. X-ray structure of dimethyl 2,3,4,5-tetrahydro-5 beta-(3,4-methylenedioxyphenyl)-2-oxo-3 beta-(3,4,5-trimethoxybenzoyl)-3 alpha,4 alpha-furandicarboxylate.

    PubMed

    Peterson, J R; Do, H D; Rogers, R D

    1989-07-15

    C25H24O12, Mr = 516.46, triclinic, P-1, a = 8.780 (3), b = 11.298 (4), c = 13.271 (6) A, alpha = 71.77 (4), beta = 70.31 (3), gamma = 72.66 (3) degrees, V = 1189 A3, Z = 2, Dx = 1.44 g cm-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.74 cm-1, F(000) = 540, T = 293 K, final R = 0.046 for 2495 observed [Fo greater than or equal to 5 sigma (Fo)] reflections. The observed structure reveals a trans disposition for the methoxycarbonyl and aryl substituents at positions 4 and 5 of the heterocycle and a cis-3,4-bis(methoxycarbonyl) relationship. There is no crystallographically imposed symmetry. Several intermolecular van der Waals interactions occur in the cell lattice of this compound. PMID:2610989

  8. Association analysis of the beta-3 adrenergic receptor Trp64Arg (rs4994) polymorphism with urate and gout.

    PubMed

    Fatima, Tahzeeb; Altaf, Sara; Phipps-Green, Amanda; Topless, Ruth; Flynn, Tanya J; Stamp, Lisa K; Dalbeth, Nicola; Merriman, Tony R

    2016-02-01

    The Arg64 allele of variant rs4994 (Trp64Arg) in the β3-adrenergic receptor gene has been associated with increased serum urate and risk of gout. Our objective was to investigate the relationship of rs4994 with serum urate and gout in New Zealand European, Māori and Pacific subjects. A total of 1730 clinically ascertained gout cases and 2145 controls were genotyped for rs4994 by Taqman(®). Māori and Pacific subjects were subdivided into Eastern Polynesian (EP) and Western Polynesian (WP) sample sets. Publicly available genotype data from the Atherosclerosis Risk in Communities Study and the Framingham Heart Study were utilized for serum urate association analysis. Multivariate logistic and linear regression adjusted for potential confounders was carried out using R version 2.15.2. No significant association of the minor Arg64 (G) allele of rs4994 with gout was found in the combined Polynesian cohorts (OR = 0.98, P = 0.88), although there was evidence, after adjustment for renal disease, for association in both the WP (OR = 0.53, P = 0.03) and the lower Polynesian ancestry EP sample sets (OR = 1.86, P = 0.05). There was no evidence for association with gout in the European sample set (OR = 1.11, P = 0.57). However, the Arg64 allele was positively associated with urate in the WP data set (β = 0.036, P = 0.004, P Corrected = 0.032). Association of the Arg64 variant with increased urate in the WP sample set was consistent with the previous literature, although the protective effect of this variant with gout in WP was inconsistent. This association provides an etiological link between metabolic syndrome components and urate homeostasis. PMID:26410617

  9. The integrin alphav beta3 increases cellular stiffness and cytoskeletal remodeling dynamics to facilitate cancer cell invasion

    NASA Astrophysics Data System (ADS)

    Mierke, Claudia Tanja

    2013-01-01

    The process of cancer cell invasion through the extracellular matrix (ECM) of connective tissue plays a prominent role in tumor progression and is based fundamentally on biomechanics. Cancer cell invasion usually requires cell adhesion to the ECM through the cell-matrix adhesion receptors integrins. The expression of the αvβ3 integrin is increased in several tumor types and is consistently associated with increased metastasis formation in patients. The hypothesis was that the αvβ3 integrin expression increases the invasiveness of cancer cells through increased cellular stiffness, and increased cytoskeletal remodeling dynamics. Here, the invasion of cancer cells with different αvβ3 integrin expression levels into dense three-dimensional (3D) ECMs has been studied. Using a cell sorter, two subcell lines expressing either high or low amounts of αvβ3 integrins (αvβ3high or αvβ3low cells, respectively) have been isolated from parental MDA-MB-231 breast cancer cells. αvβ3high cells showed a threefold increased cell invasion compared to αvβ3low cells. Similar results were obtained for A375 melanoma, 786-O kidney and T24 bladder carcinoma cells, and cells in which the β3 integrin subunit was knocked down using specific siRNA. To investigate whether contractile forces are essential for αvβ3 integrin-mediated increased cellular stiffness and subsequently enhanced cancer cell invasion, invasion assays were performed in the presence of myosin light chain kinase inhibitor ML-7 and Rho kinase inhibitor Y27632. Indeed, cancer cell invasiveness was reduced after addition of ML-7 and Y27632 in αvβ3high cells but not in αvβ3low cells. Moreover, after addition of the contractility enhancer calyculin A, an increase in pre-stress in αvβ3low cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase, STAT3 or Rac1 strongly reduced the invasiveness of αvβ3high cells, whereas the invasiveness of β3 specific knock-down cells and αvβ3low cells was not altered. In summary, these results suggest that the αvβ3 integrin enhances cancer cell invasion through increased cellular stiffness and enhanced cytoskeletal remodeling dynamics, which enables the cells to generate and transmit contractile forces to overcome the steric hindrance of 3D ECMs.

  10. Staphylococcus epidermidis serine--aspartate repeat protein G (SdrG) binds to osteoblast integrin alpha V beta 3.

    PubMed

    Claro, T; Kavanagh, N; Foster, T J; O'Brien, F J; Kerrigan, S W

    2015-06-01

    Staphylococcus epidermidis is the leading etiologic agent of orthopaedic implant infection. Contamination of the implanted device during insertion allows bacteria gain entry into the sterile bone environment leading to condition known as osteomyelitis. Osteomyelitis is characterised by weakened bones associated with progressive bone loss. The mechanism through which S. epidermidis interacts with bone cells to cause osteomyelitis is poorly understood. We demonstrate here that S. epidermidis can bind to osteoblasts in the absence of matrix proteins. S. epidermidis strains lacking the cell wall protein SdrG had a significantly reduced ability to bind to osteoblasts. Consistent with this, expression of SdrG in Lactococcus lactis resulted in significantly increased binding to the osteoblasts. Protein analysis identified that SdrG contains a potential integrin recognition motif. αVβ3 is a major integrin expressed on osteoblasts and typically recognises RGD motifs in its ligands. Our results demonstrate that S. epidermidis binds to recombinant purified αVβ3, and that a mutant lacking SdrG failed to bind. Blocking αVβ3 on osteoblasts significantly reduced binding to S. epidermidis. These studies are the first to identify a mechanism through which S. epidermidis binds to osteoblasts and potentially offers a mechanism through which implant infection caused by S. epidermidis leads to osteomyelitis. PMID:25749709

  11. microPET Imaging of Glioma Integrin (alpha-v, beta-3) Expression Using Cu-64-Labeled Tetrameric RGD Peptide

    SciTech Connect

    Wu, Yun; Zhang, , Xianzhong; Xiong, , Zhengming; Cheng, Zhen; Fisher, Darrell R.; Liu, Shu-hong; Gambhir, Sanjiv S.; Chen, Xiaoyuan

    2005-10-01

    Integrins ?v?3 and ?v?5 play a critical role in tumor-induced angiogenesis and metastasis, and have become promising diagnostic indicators and therapeutic targets of tumors. Radiolabeled RGD peptides that are integrin-specific may be used for non-invasive imaging of integrin expression level as well as for integrin-targeted radionuclide therapy. We previously conjugated a series of mono- and dimeric RGD peptides with 1,4,7,10-tetraazacyclododecane-N, N?,N??,N???-tetraacetic acid (DOTA) and labeled these with copper-64 for microPET imaging in various mouse xenograft models. The copper-64 tracers showed ?v?3-selective tumor uptake, but the magnitude of tumor uptake was relatively low, the tumor washout was rapid, and non-target organ/tissue retention was high. In this study we developed a tetrameric RGD peptide tracer 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 for positron emission tomography (PET) imaging of integrin ?v?3 expression in a subcutaneous U87MG glioma xenograft model in female athymic nude mice. The RGD tetramer showed significantly higher integrin binding affinity than the corresponding mono- and dimeric RGD analogs, most likely due to polyvalency effect. The radiolabeled peptide showed rapid blood clearance (0.61 ? 0.01%ID/g at 30 min and 0.21 ? 0.01 %ID/g at 4 h postinjection (p.i.), respectively) and predominantly renal excretion. Tumor uptake was rapid and high and the tumor washout was slow (9.93 ? 1.05 %ID/g at 30 min p.i. and 4.56 ? 0.51 %ID/g at 24 h post-injection). The metabolic stability of 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 was determined in mouse blood, urine, and liver and kidney homogenates at different times after tracer injection. The average fractions of intact tracer in these organs at 1 h were approximately 70, 58, 51 and 26 percent, respectively. Non-invasive microPET imaging studies showed significant tumor uptake and good contrast in the subcutaneous tumor-bearing mice, which agreed well with the biodistribution results. Integrin ?v?3 specificity was demonstrated by successful blocking of tumor uptake of 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 in the presence of excess amount of c(RGDyK) at 1 h postinjection. The highest absorbed radiation doses determined for the human reference adult were received by the urinary bladder wall (0.263 mGy/MBq), kidneys (0.0298 mGy/MBq), and liver (0.0244 mGy/MBq). Assuming 0.5-g U87MG glioma tumors in man, we calculated an absorbed dose of 65.3 mGy/MBq (242 rad/mCi) following a single injection of 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2. In conclusion, the high integrin avidity and favorable biokinetics make 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 a promising agent for peptide receptor radionuclide imaging therapy of integrin-positive tumors.

  12. [Molecular cloning of tubulin beta 3 (TUBB3) in Gekko japonicus and preparation of its polyclonal antibody].

    PubMed

    Li, Jing; Qin, Yong; Gu, Yun; Liu, Yan; Liu, Mei

    2012-08-01

    The tubulin beta III (TUBB3) gene encodes a class III member of the beta tubulin protein family that is primarily expressed in neurons and is considered to play a critical role in proper axon guidance and maintenance. This protein is generally used as a specific marker of neurons in the central nervous system. We obtained the full length cDNA sequence of TUBB3 by using the RACE method based on the EST fragment from the brain and spinal cord cDNA library of Gekko japonicus. We further investigated the multi-tissue expression pattern by RT-PCR and identified one transcript of TUBB3 about 1.8 kb in the central nervous system of Gekko japonicus by Northern blotting. The completed cDNA of gecko TUBB3 is 1 790 bp with an open reading frame of 1 350 bp, encoding a 450 amino-acid protein. The recombinant plasmid of pET-32a-TUBB3 was constructed and induced to express His-tagged TUBB3 protein in prokaryotic BL21 cells. The purified TUBB3 protein was then used to immunize rabbits to generate polyclonal antisera. The titer of the antiserum was more than 1:65 536 determined by ELISA. The result of western blotting showed that the TUBB3 antibody could specifically recognize the recombinant TUBB3 protein and endogenous TUBB3 protein. Our findings provide the tools to further understand the TUBB3 gene and investigate the regeneration of the central nervous system in Gekko japonicas. PMID:22855447

  13. Anti-microRNA-378a Enhances Wound Healing Process by Upregulating Integrin Beta-3 and Vimentin

    PubMed Central

    Li, Haoran; Chang, Leslie; Du, William W; Gupta, Shaan; Khorshidi, Azam; Sefton, Michael; Yang, Burton B

    2014-01-01

    Delayed or impaired wound healing is a major public health issue worldwide, especially in patients with diabetes mellitus and vascular atherosclerosis. MicroRNAs have been identified as key regulators of wound healing. Here, we show that miR-Pirate378a transgenic mice (and thus have inhibited miR-378a-5p function) display enhanced wound healing. Expression of vimentin and β3 integrin, two important modulators of wound healing, is markedly elevated in the transgenic mice. MiR-Pirate378a-transfected cells display greater mobility during migration assays, which was hypothesized to be due to the upregulation of vimentin and β3 integrin. Both molecules were confirmed to be targets of miR-378a, and thus their expression could be rescued by miR-Pirate378a. Overexpression of vimentin also contributed to fibroblast differentiation, and upregulation of β3 integrin was responsible for increased angiogenesis. Mice treatment with miR-Pirate378a-conjugated nanoparticles displayed enhanced wound healing. Thus, we have demonstrated that knockdown of miR-378a increased the expression of its target proteins, vimentin, and β3 integrin, which accelerated fibroblast migration and differentiation in vitro and enhanced wound healing in vivo. PMID:24954475

  14. Imaging integrin alpha-v-beta-3 expression in tumors with an 18F-labeled dimeric RGD peptide

    PubMed Central

    Dijkgraaf, Ingrid; Terry, Samantha; McBride, William J.; Goldenberg, David M.; Laverman, Peter; Franssen, Gerben M.; Oyen, Wim J. G.; Boerman, Otto C.

    2014-01-01

    Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine-glycine-aspartatic acid (RGD) peptide and integrin αvβ3, but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of 18F. In this study, the dimeric RGD peptide, E-[c(RGDfK)]2, was conjugated with NODAGA and radiolabeled with 18F in a simple one-pot process with a radiolabeling yield of 20%; the whole process lasting only 45 min. NODAGA-E-[c(RGDfK)]2 labeled with 18F at a specific activity of 1.8 MBq/nmol and a radiochemical purity of 100% could be achieved. Log P value of 18F-labeled NODAGA-E-[c(RGDfK)]2 was −4.26 ± 0.02. In biodistribution studies, 18F-NODAGA-E-[c(RGDfK)]2 cleared rapidly from the blood with 0.03 ± 0.01 %ID/g in the blood at 2 h p.i., mainly via the kidneys and showed good in vivo stability. Tumor uptake of 18F-NODAGA-E-[c(RGDfK)]2 (3.44 ± 0.20 %ID/g, 2 h p.i.) was significantly lower than that of reference compounds 68Ga-labeled NODAGA-E-[c(RGDfK)]2 (6.26 ± 0.76 %ID/g; P <0.001) and 111In-labeled NODAGA-E-[c(RGDfK)]2 (4.99 ± 0.64 %ID/g; P < 0.01). Co-injection of an excess of unlabeled NODAGA-E-[c(RGDfK)]2 along with 18F-NODAGA-E-[c(RGDfK)]2 resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID/g). The αvβ3 integrin-expressing SK-RC-52 tumor could be successfully visualized by microPET with 18F-labeled NODAGA-E-[c(RGDfK)]2. In conclusion, NODAGA-E-[c(RGDfK)]2 could be labeled rapidly with 18F using a direct aqueous, one-pot method and it accumulated specifically in αvβ3 integrin-expressing SK-RC-52 tumors, allowing for visualization by microPET. PMID:23606427

  15. Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to alpha4/6beta3delta GABAA receptors.

    PubMed

    Hanchar, H Jacob; Chutsrinopkun, Panida; Meera, Pratap; Supavilai, Porntip; Sieghart, Werner; Wallner, Martin; Olsen, Richard W

    2006-05-30

    Although GABA(A) receptors have long been implicated in mediating ethanol (EtOH) actions, receptors containing the "nonsynaptic" delta subunit only recently have been shown to be uniquely sensitive to EtOH. Here, we show that delta subunit-containing receptors bind the imidazo-benzodiazepines (BZs) flumazenil and Ro15-4513 with high affinity (K(d) < 10 nM), contrary to the widely held belief that these receptors are insensitive to BZs. In immunopurified native cerebellar and recombinant delta subunit-containing receptors, binding of the alcohol antagonist [(3)H]Ro15-4513 is inhibited by low concentrations of EtOH (K(i) approximately 8 mM). Also, Ro15-4513 binding is inhibited by BZ-site ligands that have been shown to reverse the behavioral alcohol antagonism of Ro15-4513 (i.e., flumazenil, beta-carbolinecarboxylate ethyl ester (beta-CCE), and N-methyl-beta-carboline-3-carboxamide (FG7142), but not including any classical BZ agonists like diazepam). Experiments that were designed to distinguish between a competitive and allosteric mechanism suggest that EtOH and Ro15-4513 occupy a mutually exclusive binding site. The fact that only Ro15-4513, but not flumazenil, can inhibit the EtOH effect, and that Ro15-4513 differs from flumazenil by only a single group in the molecule (an azido group at the C7 position of the BZ ring) suggest that this azido group in Ro15-4513 might be the area that overlaps with the alcohol-binding site. Our findings, combined with previous observations that Ro15-4513 is a behavioral alcohol antagonist, suggest that many of the behavioral effects of EtOH at relevant physiological concentrations are mediated by EtOH/Ro15-4513-sensitive GABA(A) receptors. PMID:16581914

  16. Phospholipase C beta3 is a key component in the Gbetagamma/PKCeta/PKD-mediated regulation of trans-Golgi network to plasma membrane transport.

    PubMed

    Díaz Añel, Alberto M

    2007-08-15

    The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gbetagamma (GTP-binding protein betagamma subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCeta (protein kinase Ceta) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCeta, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gbetagamma, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that betagamma-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCbeta3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCbeta3, which is necessary to activate PKCeta and PKD in that Golgi compartment, via DAG production. PMID:17492941

  17. Association of beta3-adrenergic receptor (ADRB3) Trp64Arg gene polymorphism with obesity and metabolic syndrome in the Balinese: a pilot study

    PubMed Central

    2011-01-01

    Background Prevalence of obesity is increasing all over the world. ADRB3 Trp64Arg gene polymorphism was proposed to be associated with obesity, although inconsistent findings and differences of the Arg64 allele frequency among various ethnics were reported. Westernization was reported to increase the prevalence of obesity in developing world. In this study we determined the prevalence of obesity and metabolic syndrome among urban and rural Balinese, and studied the association of ADRB3 Trp64Arg polymorphism with obesity and MetS. Findings A total of 528 Balinese (urban 282, rural 246) were recruited. Body mass index (BMI) and waist circumference (WC) were determined; high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), systolic and diastolic blood pressure (SBP and DBP), and fasting plasma glucose (FPG) were measured using standard procedures. BMI and WC classifications were based on WHO classifications for Asian. Metabolic syndrome (MetS) was defined as described in the Joint Interim Statement. Chi-square test was employed to test the association between the ADRB3 Trp64Arg genotype and disease traits. Urban have higher BMI (p = 2.8 × 10-13), WC ( p < 2.2 × 10-16), TG (p = 0.0028), DBP (p = 1.8 × 10-5), and lower HDL-C (p = 0.0376) when compared to rural. Abdominal obesity and MetS prevalence were significantly higher in urban as compared to rural (both p < 0.001). The Arg64 allele frequency was similar between urban (0.06) and rural (0.05). The Arg64 rural female carriers have higher BMI and WC as compared to their Trp64 counterparts (p = 0.041 for BMI and p = 0.012 for WC), and consequently higher abdominal obesity prevalence (p = 0.007). Comparison between male and female, as well as urban and rural, showed different prevalence of MetS co-morbidities. Abdominal obesity and hypertriglyceridaemia were consistently appeared in all groups, suggesting to play a role as determinant of MetS in both urban and rural. Conclusions Prevalence of obesity and MetS in urban were two times higher when compared to rural. Abdominal obesity and hypertriglyceridaemia appears to be the key determinant of MetS in both urban and rural Balinese. Our results indicated an association of the ADRB3 Trp64Arg gene polymorphism with obesity in the rural female. PMID:21619577

  18. PDE5A suppression of acute β-adrenergic activation requires modulation of myocyte beta-3 signaling coupled to PKG-mediated troponin I phosphorylation

    PubMed Central

    Lee, Dong I.; Vahebi, Susan; Tocchetti, Carlo Gabriele; Barouch, Lili A.; Solaro, R. John; Takimoto, Eiki

    2010-01-01

    Phosphodiesterase type 5A (PDE5A) inhibitors acutely suppress beta-adrenergic receptor (β-AR) stimulation in left ventricular myocytes and hearts. This modulation requires cyclic GMP synthesis via nitric oxide synthase (NOS)-NO stimulation, but upstream and downstream mechanisms remain un-defined. To determine this, adult cardiac myocytes from genetically engineered mice and controls were studied by video microscopy to assess sarcomere shortening (SS) and fura2-AM fluorescence to measure calcium transients (CaT). Enhanced SS from isoproterenol (ISO, 10 nM) was suppressed ≥50% by the PDE5A inhibitor sildenafil (SIL, 1 µM), without altering CaT. This regulation was unaltered despite co-inhibition of either the cGMP-stimulated cAMP-esterase PDE2 (Bay 60-7550), or cGMP-inhibited cAMP-esterase PDE3 (cilostamide). Thus, the SIL response could not be ascribed to cGMP interaction with alternative PDEs. However, genetic deletion (or pharmacologic blockade) of β3-ARs, which couple to NOS signaling, fully prevented SIL modulation of ISO-stimulated SS. Importantly, both PDE5A protein expression and activity were similar in β3-AR knockout (β3-AR−/−) myocytes as in controls. Downstream, cGMP stimulates protein kinase G (PKG), and we found contractile modulation by SIL required PKG activation and enhanced TnI phosphorylation at S23, S24. Myocytes expressing the slow skeletal TnI isoform which lacks these sites displayed no modulation of ISO responses by SIL. Non-equilibrium isoelectric focusing gel electrophoresis showed SIL increased TnI phosphorylation above that from concomitant ISO in control but not β3-AR−/− myocytes. These data support a cascade involving β3-AR stimulation, and subsequent PKG-dependent TnI S23, S24 phosphorylation as primary factors underlying the capacity of acute PDE5A inhibition to blunt myocardial β-adrenergic stimulation. PMID:20107996

  19. Structural basis for distinctive recognition of fibrinogen [gamma]C peptide by the platelet integrin [alpha][subscript IIb][beta]3

    SciTech Connect

    Springer, Timothy A.; Zhu, Jianghai; Xiao, Tsan

    2009-01-12

    Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin {alpha}{sub IIb}{beta}{sub 3} on platelets, resulting in platelet aggregation. {alpha}{sub v}{beta}{sub 3} binding fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's {alpha} subunit. {alpha}{sub IIb}{beta}{sub 3} also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the {gamma} subunit ({gamma}C peptide). These distinct modes of fibrinogen binding enable {alpha}{sub IIb}{beta}{sub 3} and {alpha}{sub v}{beta}{sub 3} to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin {alpha}{sub IIb}{beta}{sub 3}-{gamma}C peptide interface, and, for comparison, integrin {alpha}{sub IIb}{beta}{sub 3} bound to a lamprey {gamma}C primordial RGD motif. Compared with RGD, the GAKQAGDV motif in {gamma}C adopts a different backbone configuration and binds over a more extended region. The integrin metal ion-dependent adhesion site (MIDAS) Mg{sup 2+} ion binds the {gamma}C Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca{sup 2+} ion binds the {gamma}C C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered {gamma}C peptide enhances our understanding of the involvement of {gamma}C peptide and integrin {alpha}{sub IIb}{beta}{sub 3} in hemostasis and thrombosis.

  20. Polymorphism of the ovine beta3-adrenergic receptor gene (ADRB3) and its association with wool mean staple strength and yield.

    PubMed

    Forrest, R H; Itenge-Mweza, T O; McKenzie, G W; Zhou, H; Frampton, C M; Hickford, J G H

    2009-12-01

    We investigated the possibility that variation in ovine ADRB3 is associated with various wool traits, in particular mean staple strength (MSS). Polymerase chain reaction-single strand conformational polymorphism analysis of part of the ADRB3 intron was used to genotype 695 Merino lambs born on three farms in the South Island of New Zealand and which were shorn as 2-tooths. For each fleece, MSS, mean fibre diameter, mean staple length and yield were measured. The results from mixed-effects models and half-sib analyses suggest that ADRB3 alleles A and D have a negative impact on some wool traits, whereas ADRB3 alleles C and E appear to have a positive impact, with allele C potentially having a greater impact than allele E on MSS. This variation in the ADRB3 may assist in the genetic selection for increased MSS and yield in Merino sheep. PMID:19531115

  1. Beta3-adrenergic receptors modulate vascular endothelial growth factor release in response to hypoxia through the nitric oxide pathway in mouse retinal explants.

    PubMed

    Dal Monte, Massimo; Filippi, Luca; Bagnoli, Paola

    2013-04-01

    Beta-adrenergic receptors (β-ARs) play a role in angiogenic processes that characterize neovascularization-associated retinal diseases, but the role of β3-ARs has not been disclosed yet. We used ex vivo retinal explants to investigate the role of β3-ARs in regulating vascular endothelial growth factor (VEGF) release associated with hypoxia. Whether nitric oxide (NO) mediates β3-AR regulation of VEGF release was also investigated. β3-AR activation was obtained using BRL 37344, whereas SR59230A, L-748,337, or specific siRNAs were used to block β3-ARs. Pharmacological approaches were used to interfere with the NO pathway. Western blot was used to determine β-AR levels. Enzyme-linked immunosorbent assay was used to measure VEGF release. NO production was assessed by a colorimetric assay. We found that hypoxia upregulates β3-ARs. In addition, we observed that β3-AR activation with BRL 37344 increases VEGF release in response to hypoxia. Either β3-AR blocker or β3-AR silencing downregulates drastically hypoxic levels of VEGF. With experiments using NO synthase (NOS) blockade with L-NAME, NOS activation with fluvastatin or NO supplementation with SNAP, we demonstrated that β3-ARs and VEGF are functionally coupled via the NO pathway. In summary, the data presented here support the assumption that β3-ARs are involved in the regulation of angiogenic responses to hypoxia through the NO signalling, a key pathway in hypoxic/ischemic diseases. Although extrapolation of these data to the human situation is difficult, these findings may help to explore the possible role of β3-ARs in vascularization-associated disorders. PMID:23283573

  2. Crecimiento adiabático de un agujero negro en el centro de un sistema estelar

    NASA Astrophysics Data System (ADS)

    Arrigoni, M.; Carpintero, D. D.

    We present preliminary results on the dynamical influence of an adiabatically grown central black hole upon its surroundings. Young (1980), in his pioneer work, found an r-3/2 density profile for the neighborhood of a black hole, a result not confirmed by observation. In this work, we extend Young's result to a more realistic case, namely an inhomogeneous galactic center, although keeping the spherical symmetry hypothesis, in order to verify whether the final density profile changes.

  3. Etomidate blocks LTP and impairs learning but does not enhance tonic inhibition in mice carrying the N265M point mutation in the beta3 subunit of the GABA(A) receptor.

    PubMed

    Zarnowska, E D; Rodgers, F C; Oh, I; Rau, V; Lor, C; Laha, K T; Jurd, R; Rudolph, U; Eger, E I; Pearce, R A

    2015-06-01

    Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with β3 subunits, we tested the hypothesis that etomidate acts through β3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR β3-subunit (β3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by β3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5β1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 μM and below), whereas α5β2γ2L and α5β3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5β3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5β2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons). PMID:25680234

  4. The human [gamma]-aminobutyric acid receptor subunit [beta]3 and [alpha]5 gene cluster in chromosome 15q11-q13 is rich in highly polymorphic (CA)[sub n] repeats

    SciTech Connect

    Glatt, K.; Lalande, M. ); Sinnett, D. )

    1994-01-01

    The [gamma]-aminobutyric acid (GABA[sub A]) receptor [beta]33 (GABRB3) and [alpha]5 (GABRA5) subunit genes have been localized to the Angelman and Prader-Willi syndrome region of chromosome 15q11-q13. GABRB3, which encompasses 250 kb, is located 100 kb proximal of GABRA5, with the two genes arranged in head-to-head transcriptional orientation. In screening 135 kb of cloned DNA within a 260-kb interval extending from within GABRB3 to the 5[prime] end of GABRA5, 10 new (CA), repeats have been identified. Five of these have been analyzed in detail and found to be highly polymorphic, with the polymorphism information content (PIC) ranging from 0.7 to 0.85 and with heterozygosities of 67 to 94%. In the clones from GABRB3/GABRA5 region, therefore, the frequency of (CA)[sub n] with PICs [ge] 0.7 is 1 per 27 kb. Previous estimates of the density of (CA)[sub n] with PICs [ge] 0.7 in the human genome have been approximately 10-fold lower. The GABRB3/GABRA5 region appears, therefore, to be enriched for highly informative (CA)[sub n]. This set of closely spaced, short tandem repeat polymorphisms will be useful in the molecular analyses of Prader-Willi and Angelman syndromes and in high-resolution studies of genetic recombination within this region. 21 refs., 2 figs., 1 tab.

  5. GABA{sub A} receptor open-state conformation determines non-competitive antagonist binding

    SciTech Connect

    Chen Ligong; Xue Ling; Giacomini, Kathleen M.; Casida, John E.

    2011-02-01

    The {gamma}-aminobutyric acid (GABA) type A receptor (GABA{sub A}R) is one of the most important targets for insecticide action. The human recombinant {beta}3 homomer is the best available model for this binding site and 4-n-[{sup 3}H]propyl-4'-ethynylbicycloorthobenzoate ([{sup 3}H]EBOB) is the preferred non-competitive antagonist (NCA) radioligand. The uniquely high sensitivity of the {beta}3 homomer relative to the much-less-active but structurally very-similar {beta}1 homomer provides an ideal comparison to elucidate structural and functional features important for NCA binding. The {beta}1 and {beta}3 subunits were compared using chimeragenesis and mutagenesis and various combinations with the {alpha}1 subunit and modulators. Chimera {beta}3/{beta}1 with the {beta}3 subunit extracellular domain and the {beta}1 subunit transmembrane helices retained the high [{sup 3}H]EBOB binding level of the {beta}3 homomer while chimera {beta}1/{beta}3 with the {beta}1 subunit extracellular domain and the {beta}3 subunit transmembrane helices had low binding activity similar to the {beta}1 homomer. GABA at 3 {mu}M stimulated heteromers {alpha}1{beta}1 and {alpha}1{beta}3 binding levels more than 2-fold by increasing the open probability of the channel. Addition of the {alpha}1 subunit rescued the inactive {beta}1/{beta}3 chimera close to wildtype {alpha}1{beta}1 activity. EBOB binding was significantly altered by mutations {beta}1S15'N and {beta}3N15'S compared with wildtype {beta}1 and {beta}3, respectively. However, the binding activity of {alpha}1{beta}1S15'N was insensitive to GABA and {alpha}1{beta}3N15'S was stimulated much less than wildtype {alpha}1{beta}3 by GABA. The inhibitory effect of etomidate on NCA binding was reduced more than 5-fold by the mutation {beta}3N15'S. Therefore, the NCA binding site is tightly regulated by the open-state conformation that largely determines GABA{sub A} receptor sensitivity. - Graphical Abstract: Display Omitted Research Highlights

  6. Coupling of nuclear quadrupole and octupole degrees of freedom in an angular momentum dependent potential of two deformation variables

    SciTech Connect

    Minkov, N.; Yotov, P.; Drenska, S.; Scheid, W.; Bonatsos, Dennis; Lenis, D.; Petrellis, D.

    2006-04-26

    We propose a collective rotation-vibration Hamiltonian of nuclei in which the axial quadrupole {beta}2 and octupole {beta}3 variables are coupled through the centrifugal interaction. We consider that the system oscillates between positive and negative {beta}3-values by rounding a potential core in the ({beta}2,{beta}3)- space. We examine the effect of the 'rounding' in the structure of the spectrum.

  7. Bioavailabilty of beta-amino acid and C-terminally derived PK/PBAN analogs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of linear beta amino-acid-substituted peptides (PK-betaA-1: Ac-YFT[beta3-P]RLa; PK-betaA-2: Ac-Y[beta2-homoF]TPRLa; PK-betaA-3: Ac-Y[beta3-F]TPRLa and PK-betaA-4: Ac-[beta3-F]FT[beta3-P]RLa) and unsubstituted analogs (Ac-YFTPRLa and YFTPRLa) of the pyrokinin(PK)/pheromone biosynthesis-ac...

  8. Terapias dirigidas contra el cáncer

    Cancer.gov

    Hoja informativa que trata de los fármacos que impiden el crecimiento y la diseminación del cáncer al interferir en las moléculas específicas que participan en la carcinogénesis y el crecimiento de tumores.

  9. ...And That's How It All Began: Putting Information about Your Child's Growth, Health and Safety All within Your Reach...Because the First Years Last Forever! = ...Y asi es como empezo todo: Ponemos a su alcance en forma conjunta la informacion sobre el crecimiento, la salud y la seguridad de su hijo...Porque los primeros anos duran para siempre!

    ERIC Educational Resources Information Center

    North Carolina Partnership for Children, Raleigh.

    Smart Start is North Carolina's model early childhood initiative to help all North Carolina children enter school healthy and prepared for success. This resource guide, in Spanish and English versions, is designed to provide parents with information on infant and child development, health care, and resources for further assistance. Presented in an…

  10. Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa.

    PubMed

    Dellambra, E; Vailly, J; Pellegrini, G; Bondanza, S; Golisano, O; Macchia, C; Zambruno, G; Meneguzzi, G; De Luca, M

    1998-06-10

    Laminin-5 is composed of three distinct polypeptides, alpha3, beta3, and gamma2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta3 polypeptide. In vitro, beta3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta3 cDNA was used to transduce primary beta3-null keratinocytes. Clonogenic beta3-null keratinocytes were transduced with an efficiency of 100%. Beta3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses. PMID:9650620

  11. The heparin binding domain of vitronectin is the region that is required to enhance insulin-like growth factor-I signaling.

    PubMed

    Maile, Laura A; Busby, Walker H; Sitko, Kevin; Capps, Byron E; Sergent, Tiffany; Badley-Clarke, Jane; Ling, Yan; Clemmons, David R

    2006-04-01

    We have shown that vitronectin (Vn) binding to a cysteine loop sequence within the extracellular domain of the beta3-subunit (amino acids 177-184) of alphaVbeta3 is required for the positive effects of Vn on IGF-I signaling. When Vn binding to this sequence is blocked, IGF-I signaling in smooth muscle cells is impaired. Because this binding site is distinct from the site on beta3 to which the Arg-Gly-Asp sequence of extracellular matrix ligands bind (amino acids 107-171), we hypothesized that the region of Vn that binds to the cysteine loop on beta3 is distinct from the region that contains the Arg-Gly-Asp sequence. The results presented in this study demonstrate that this heparin binding domain (HBD) is the region of Vn that binds to the cysteine loop region of beta3 and that this region is sufficient to mediate the positive effects of Vn on IGF-I signaling. We provide evidence that binding of the HBD of Vn to alphaVbeta3 has direct effects on the activation state of beta3 as measured by beta3 phosphorylation. The increase in beta3 phosphorylation associated with exposure of cells to this HBD is associated with enhanced phosphorylation of the adaptor protein Src homology 2 domain-containing transforming protein C and enhanced activation MAPK, a downstream mediator of IGF-I signaling. We conclude that the interaction of the HBD of Vn binding to the cysteine loop sequence of beta3 is necessary and sufficient for the positive effects of Vn on IGF-I-mediated effects in smooth muscle cells. PMID:16322097

  12. Enzyme-catalyzed formation of beta-peptides: beta-peptidyl aminopeptidases BapA and DmpA acting as beta-peptide-synthesizing enzymes.

    PubMed

    Heck, Tobias; Kohler, Hans-Peter E; Limbach, Michael; Flögel, Oliver; Seebach, Dieter; Geueke, Birgit

    2007-09-01

    In recent studies, we discovered that the three beta-peptidyl aminopeptidases, BapA from Sphingosinicella xenopeptidilytica 3-2W4, BapA from S. microcystinivorans Y2, and DmpA from Ochrobactrum anthropi LMG7991, possess the unique feature of cleaving N-terminal beta-amino acid residues from beta- and alpha/beta-peptides. Herein, we investigated the use of the same three enzymes for the reverse reaction catalyzing the oligomerization of beta-amino acids and the synthesis of mixed peptides with N-terminal beta-amino acid residues. As substrates, we employed the beta-homoamino acid derivatives H-beta hGly-pNA, H-beta3 hAla-pNA, H-(R)-beta3 hAla-pNA, H-beta3 hPhe-pNA, H-(R)-beta3 hPhe-pNA, and H-beta3 hLeu-pNA. All three enzymes were capable of coupling the six beta-amino acids to oligomers with chain lengths of up to eight amino acid residues. With the enzyme DmpA as the catalyst, we observed very high conversion rates, which correspond to dimer yields of up to 76%. The beta-dipeptide H-beta3 hAla-beta3 hLeu-OH and the beta/alpha-dipeptide H-beta hGly-His-OH (carnosine) were formed with almost 50% conversion, when a five-fold excess of beta3-homoleucine or histidine was incubated with H-beta3 hAla-pNA and H-beta hGly-pNA, respectively, in the presence of the enzyme BapA from S. microcystinivorans Y2. BapA from S. xenopeptidilytica 3-2W4 turned out to be a versatile catalyst capable of coupling various beta-amino acid residues to the free N-termini of beta- and alpha-amino acids and even to an alpha-tripeptide. Thus, these aminopeptidases might be useful to introduce a beta-amino acid residue as an N-terminal protecting group into a 'natural' alpha-peptide, thereby stabilizing the peptide against degradation by other proteolytic enzymes. PMID:17886858

  13. Role of phosphatidylinositol 3-kinase-gamma in mediating lung neutrophil sequestration and vascular injury induced by E. coli sepsis.

    PubMed

    Ong, Evan; Gao, Xiao-Pei; Predescu, Dan; Broman, Michael; Malik, Asrar B

    2005-12-01

    We addressed the in vivo role of phosphatidylinositol 3-kinase-gamma (PI3K-gamma) in signaling the sequestration of polymorphonuclear leukocytes (PMNs) in lungs and in the mechanism of inflammatory lung vascular injury. We studied mice with deletion of the p110 catalytic subunit of PI3K-gamma (PI3K-gamma(-/-) mice). We measured lung tissue PMN sequestration, microvascular permeability, and edema formation after bacteremia induced by intraperitoneal Escherichia coli challenge. PMN infiltration into the lung interstitium in PI3K-gamma(-/-) mice as assessed morphometrically was increased 100% over that in control mice within 1 h after bacterial challenge. PI3K-gamma(-/-) mice also developed a greater increase in lung microvascular permeability after E. coli challenge, resulting in edema formation. The augmented lung tissue PMN sequestration in PI3K-gamma(-/-) mice was associated with increased expression of the PMN adhesive proteins CD47 and beta(3)-integrins. We observed increased association of CD47 and beta(3)-integrins with the extracellular matrix protein vitronectin in lungs of PI3K-gamma(-/-) mice after E. coli challenge. PMNs from these mice also showed increased beta(3)-integrin expression and augmented beta(3)-integrin-dependent PMN adhesion to vitronectin. These results point to a key role of PMN PI3K-gamma in negatively regulating CD47 and beta(3)-integrin expression in gram-negative sepsis. PI3K-gamma activation in PMNs induced by E. coli may modulate the extent of lung tissue PMN sequestration secondary to CD47 and beta(3)-integrin expression. Therefore, the level of PI3K-gamma activation may be an important determinant of PMN-dependent lung vascular injury. PMID:16183669

  14. Conformation specific spectroscopy in the complexity gap: beta-peptides and flexible bichromophores

    NASA Astrophysics Data System (ADS)

    Baquero, Esteban Edwin

    Flexible biomolecules with many degrees of freedom have the ability to sample a great number of structural minima. The intrinsic structural preferences of these molecules are driven by stabilizations due to intramolecular interactions such as hydrogen bonding and interatomic attractive forces. These interactions become increasingly important as the atoms in the molecule come in close proximity to one another. The present work describes the conformational preferences of several model bio-relevant molecules of different sizes where different kinds of intramolecular interactions can occur. Conformation specific ultraviolet and infrared spectroscopies were utilized to obtain the spectroscopic signatures of different conformations of the jet cooled biomolecules. These experiments allowed for the assignment of conformational families and the development of a general protocol to identify the infrared signatures of amide NH stretches, which were found to vary in frequency due to their immediate chemical and structural environment. Specific examples of systems studied include molecules containing unnatural polypeptide chains and a flexible bichromophore. The unnatural polypetides (Ac-beta3-hPhe-NHMe, Ac-beta 3-hTyr-NHMe Ac-beta3-hPhe-beta3-hAla-NHMe and Ac-beta3-hAla-beta3-hPhe-NHMe) were found to have a rich conformational potential energy landscape that contained many kinds of intramolecular hydrogen bonded minima. The flexible bichromophore (HNBPA), containing two spectroscopically distinguishable chromophores (Phenol and Phenyl), was found to be an interesting case study for conformational specific electronic energy transfer and testing how well different theoretical methods manage non-covalent interactions, such as those between side-chain atoms and aromatic pi-clouds.

  15. Effect of ligand radicals on vibrational IR, Raman and vibronic spectra of europium beta-diketonates.

    PubMed

    Tsaryuk, V; Zolin, V; Legendziewicz, J; Szostak, R; Sokolnicki, J

    2005-01-01

    Vibrational IR, Raman spectra and vibronic sidebands of Eu(3+) electronic transitions of europium tris-beta-diketonates Eu(beta)(3).Ph (beta-dipyvaloylmethane (DPM), acetylacetone (AA), benzoylacetone (BA), thenoyltrifluoroacetone (TTFA) and other beta-diketones; Ph-methyl-, phenyl-, and nitro-derivatives of 1,10-phenanthroline (Phen)) as well as Eu(beta)(3).Bpy and Eu(beta)(3).D-Bpy (Bpy- and D-Bpy-H- and D-2,2'-bipyridine) were studied. Effect of ligand radical properties on spectra and manifestation of the reciprocal influence of non-equivalent ligands in spectra are discussed. Dependence of the spectra on electronic density distribution in both ligands as well as on the strength of M-O and M-N bonds at the variation of radicals of one of the ligands, beta or Ph, was examined. Shape of vibronic sidebands was analysed. Behaviour of bands in the middle and far regions of IR spectra of the series Eu(beta)(3).Phen and Eu(TTFA)(3).Ph was investigated. Increase of the polarising influence of Eu(3+) ions on Phen and Bpy molecules and strengthening the Eu-N bonds in TTFA compounds in comparison with DPM compounds were disclosed from the Raman spectra of Eu(beta)(3).Phen and Eu(beta)(3).Bpy, that is in accordance with properties of beta-diketone radicals. Conclusion about weaker Eu-N bonds in europium beta-diketonates with heterocyclic diimines in comparison with corresponding nitrates was derived from the spectra. Spectral data concerning the relative strength of Eu-ligand bonds are in agreement with available X-ray data. PMID:15556438

  16. Cucurbitacins from Cayaponia racemosa: isolation and total assignment of 1H and 13C NMR spectra.

    PubMed

    Chaves, Davina C; Assunção, João Carlos C; Braz-Filho, Raimundo; Lemos, Telma L G; Monte, Francisco J Q

    2007-05-01

    Two new cucurbitane-type triterpenoids, 2beta,3beta,16alpha,20(R),25-pentahydroxy-9-methyl-19-norlanost-5-en-7,22-dione and 2beta,3beta,16alpha,20(R),25-pentahydroxy-9-methyl-19-norlanost-5-en-7,11,22-trione, were isolated from fruits of Cayaponia racemosa. The total (1)H and (13)C chemical shift assignment of these two closely related compounds is described, making use of one- and two-dimensional NMR techniques. PMID:17372957

  17. The effect of microstructure on the fracture toughness of titanium alloys

    NASA Technical Reports Server (NTRS)

    Vanstone, R. H.; Low, J. R., Jr.; Shannon, J. L., Jr.

    1974-01-01

    The microstructure of the alpha titanium alloy Ti-5Al-2.5Sn and the metastable beta titanium alloy Beta 3 was examined. The material was from normal and extra low interstitial grade plates which were either air-cooled or furnace-cooled from an annealing treatment. Beta 3 was studied in alpha-aged and omega-aged plates which were heat treated to similar strength levels. Tensile and plane strain fracture toughness tests were conducted at room temperature on the alpha-aged material. The microstructure and fracture mechanisms of alloys were studied using optical metallography, electron microscopy, microprobe analyses, and texture pole figures. Future experiments are described.

  18. Anticuerpos derivados de pacientes se dirigen a células tumorales

    Cancer.gov

    Artículo del blog Temas y relatos de un anticuerpo derivado de pacientes que destruye células tumorales en estirpes celulares de varios tipos de cáncer e hizo lento el crecimiento en modelos murinos de cáncer de cerebro y pulmón sin signo de efectos.

  19. Book Reviews.

    ERIC Educational Resources Information Center

    International Journal of Early Childhood, 1994

    1994-01-01

    Reviews "La Educacion Preescolar: Desafio y Aventura" (Lavanchy Bobsien); "Working towards Better Childcare" (Peeters and Vandenbroeck, editors); "Children's Savings: A Study in the Development of Economic Behavior" (Sonuga-Barke and Webley); "Curvas de Crecimiento Estaturo-ponderal en Escolares" (Saez Crespo and others); and "Helping Bereaved…

  20. Role of Jumonji C-domain containing protein 6 (JMJD6) in infectivity of foot-and-mouth disease virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-mouth disease virus (FMDV) can utilize as many as three distinct groups of receptor molecules to attach and enter a susceptible host cell. Four integrin heterodimers (alphavBeta1, alphavBeta3, alphavBeta6, and alphavBeta8) can function as the primary receptor for FMDV field strains. FMDV ...

  1. A beta-amino acid pyrokinin analog induces irregular pupariation behavior in larvae of the flesh fly Sarcophaga bullata

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The developmental process of pupariation is accelerated by members of the pyrokinin class of neuropeptides in larvae of the flesh fly Sarcophaga bullata. A pyrokinin analog (Ac-Y[beta-3-Phe]TPRLamide), in which a Phe residue is replaced with a beta-amino acid, accelerates pupariation in this fly at...

  2. Role of Jumonji c-domain containing protein 6 (JMJD6) in infectivity of foot-and-mouth disease virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-mouth disease virus (FMDV) can utilize as many as three distinct groups of receptor molecules to attach and enter a susceptible host cell. Four integrin heterodimers (alphavBeta1, alphavBeta3, alphavBeta6, and alphavBeta8) can function as the primary receptor for FMDV field strains. FMDV ...

  3. CL316243 induces phosphatidylinositol 3,4,5-triphosphate production in rat adipocytes in an adenosine deaminase-, pertussis toxin-, or wortmannin-sensitive manner.

    PubMed

    Ohsaka, Y; Nomura, Y

    2016-07-18

    The effect of beta(3)-adrenoceptor (beta(3)-AR) agonists on adipocytes treated or not treated with signaling modulators has not been sufficiently elucidated. Using rat epididymal adipocytes (adipocytes) labeled with [(32)P]orthophosphate, we found that treatment with the selective beta(3)-AR agonist CL316243 (CL; 1 microM) induces phosphatidylinositol (PI) 3,4,5-triphosphate (PI[3,4,5]P(3)) production and that this response is inhibited by adenosine deaminase (ADA, an adenosine-degrading enzyme; 2 U/ml), pertussis toxin (PTX, an inactivator of inhibitory guanine-nucleotide-binding protein; 1 microg/ml), or wortmannin (WT, a PI-kinase inhibitor; 3 microM). The results showed that CL induced PI(3,4,5)P(3) production in intact adipocytes and that this production was affected by signaling modulators. Taken together, our findings indicate that CL produces PI(3,4,5)P(3) in an ADA-sensitive, PTX-sensitive, or WT-sensitive manner and will advance understanding of the effect of beta(3)-AR agonists on adipocytes. PMID:26988163

  4. A new triterpenoid, madhunolic acid, from the seeds of Madhuca latifolia.

    PubMed

    Siddiqui, B S; Khan, S; Kardar, M N; Perwaiz, S

    2007-01-01

    A new triterpenoid, madhunolic acid, along with the three known constituents stigmasterol, 4-hydroxymethyl benzoate, and hydroquinone were isolated from the fruit seeds of Madhuca latifolia. The structure of the new compound was elucidated as 2beta,3beta,23-trihydroxyurs-5,12,20-trien-28-oic acid (1) on the basis of spectral data and chemical evidence. PMID:17566924

  5. GENE-ENVIRONMENT INTERACTION AND THE GNB3 GENE IN THE ATHEROSCLEROSIS RISK IN COMMUNITIES STUDY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of this study was to investigate the interaction between the G-protein beta-3 (GNB3) 825C>T polymorphism and physical activity in relation to prevalent obesity and hypertension. The GNB3 825C>T genotype was measured in a sample of 14 716 African Americans (AAs) and whites from the Athero...

  6. Extracellular matrix receptors and the differentiation of human megakaryocytes in vitro.

    PubMed

    Molla, A; Mossuz, P; Berthier, R

    1999-03-01

    We investigated the expression and functions of extracellular matrix receptors (or integrins) in the course of the differentiation of human megakaryocytes (Mks) leading to the formation of platelets. Integrins beta1 or Very Late Antigens (VLA) are specialized transmembrane receptors allowing the attachment of the cells to collagen (VLA-2), fibronectin (VLA-4 and -5) and laminin (VLA-6). A proportion of committed megakaryocytic progenitor cells (CFU-MK) adhere to fibronectin but not to collagen or laminin. The early immature Mks are retained on fibronectin (30%) and laminin (12%) but not on collagen whereas large mature Mks are still adherent to fibronectin and laminin and also acquired the capacity to adhere to collagen. The expression of the different VLA in the maturation of Mks correlates well with their adhesive properties. Hence, VLA-2 is not expressed on immature Mks but is present on the mature polyploid cells. VLA-4 is detected only on immature Mks which do not seem to bear VLA-5, while this last integrin appears on late Mks. VLA-6 showed a broad distribution from the early to late stages of Mks differentiation. Integrins beta3 of the cytoadhesin family are represented by alphaIIb beta3 that is the receptor for fibrinogen and alphaV beta3 which mediates adhesion to vitronectin. AlphaIIb beta3 is present on the CFU-MK and highly expressed throughout the Mks maturation stages while alphaV beta3 expression is much lower and seems to be detected only on the late Mks. The regulation of the expression of these receptors by cytokines and their respective roles in the maturation of Mks and the final production of platelets, are discussed. The development of efficient culture systems of human Mks in the presence of the recently cloned thrombopoietin will undoubtedly help to shed more light on the molecular mechanisms of their interactions via integrins with the BM microenvironment. PMID:10194117

  7. Two-hybrid analysis reveals multiple direct interactions for thrombospondin 1.

    PubMed

    Aho, S; Uitto, J

    1998-10-01

    The yeast two-hybrid system was used to reveal the interactions between proteins residing within the cutaneous basement membrane zone and other gene products expressed in cultured human keratinocytes. The proteins of interest included type VII collagen, the predominant component of anchoring fibrils, and laminin 5, a component of anchoring filaments. Although the two-hybrid system was not able to verify a direct interaction between the type VII collagen NC1 domain and the short arm of Lam(beta)3, the type VII collagen NC1 domain (tVII/NC1) and the laminin 5 beta3 chain globular domain VI (lam5/beta3) cDNAs, when used as baits, detected four overlapping cDNA clones encoding thrombospondin 1 (TSP1). The overlapping region of these cDNAs encodes amino acids 400-459, a segment included within a 70 kDa chymotryptic fragment known to bind type V collagen, laminin-1 and other matrix components. The type VII collagen NC1/TSP1 interaction was confirmed by exchanging the vectors, and the interacting domain was mapped by testing a set of both 5' and 3' deletion constructs. The central region of TSP1, when used as a bait in two-hybrid system, showed strong binding to the fibronectin (FN) type III-like repeats 4-7 of type VII collagen NC1 domain. The TSP1 bait also interacted with laminin 5 beta3 chain domain V/III, and the TSP1/laminin 5 beta3 chain interaction was verified by a GST-fusion protein interaction assay. The transcripts encoding TSP1, TSP2, Lam(beta)3 and type VII collagen were abundant in cultured foreskin keratinocytes, and the expression of TSP1 and TSP2 in a wide variety of adult and fetal tissues was confirmed by PCR analysis of multiple tissue cDNA panels. Furthermore, TSP1 type I repeats showed self interaction, and recognized a clone for extracellular matrix protein fibrillin-2. In addition, clones encoding angiogenesis related protein Jagged1 and a platelet enzyme phospholipase scramblase were identified. Thus, the results indicate several previously

  8. Dysregulation of autocrine TGF-beta isoform production and ligand responses in human tumour-derived and Ha-ras-transfected keratinocytes and fibroblasts.

    PubMed Central

    Fahey, M. S.; Paterson, I. C.; Stone, A.; Collier, A. J.; Heung, Y. L.; Davies, M.; Patel, V.; Parkinson, E. K.; Prime, S. S.

    1996-01-01

    This study examined the autocrine production of TGF-beta 1, -beta 2 and -beta 3 in culture supernatants from tumour-derived (H series, n = 7; BICR series, n = 5), Ha-ras-transfected (n = 4) and normal (n = 2) human keratinocytes using a sandwich enzyme-linked immunosorbent assay (ELISA). Detection limits were 39.0 pg ml-1 for TGF-beta 1, 78.0 pg ml-1 for TGF-beta 2 and 1.9 ng ml-1 for TGF-beta 3. Tumour-derived oral keratinocytes predominantly produced less TGF-beta 1 than normal oral epithelial cells; the expression of endogenous TGF-beta 2 was variable. In keratinocytes containing mutant Ha-ras, TGF-beta 1 production was enhanced and TGF-beta 2 was undetectable. TGF-beta 3 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) but the protein was not detected in conditioned media, most probably because of the low detection limits of the ELISA for this isoform. Neutralisation experiments indicated that the latent TGF-beta peptide was secreted in keratinocyte conditioned medium. Seven tumour-derived keratinocyte cell lines (H series) and fibroblasts separated from normal (n = 1) and tumour-derived (n = 2) keratinocyte cultures were examined for their response to exogenous TGF-beta 1, -beta 2 and -beta 3. Six of seven tumour-derived keratinocyte cell lines were inhibited by TGF-beta 1 and TGF-beta 2 (-beta 1 > -beta 2); one cell line was refractory to both TGF-beta 1 and TGF-beta 2. Keratinocytes were inhibited (4 of 7), stimulated (1 of 7) or failed to respond (2 of 7) to TGF-beta 3, TGF-beta 1, -beta 2 and -beta 3 stimulated both normal and tumour-associated fibroblasts, but the tumour-associated fibroblasts showed less response to the ligands than their normal counterparts following prolonged treatment with each isoform. The results demonstrate variable autocrine production of TGF-beta isoforms by malignant keratinocytes, with loss of TGF-beta 1 generally associated with the tumour-derived phenotype and modification of endogenous isoform

  9. Se calcula que los costos del cáncer ascenderán po

    Cancer.gov

    Con base en el crecimiento y en el avance de edad de la población de EE. UU., se proyecta que los gastos médicos por cáncer en el año 2020 ascenderán por lo menos a $158.000 millones (en dólares de 2010); lo que significa un aumento de 27% sobre 2010. Si

  10. Modulation of adhesion-dependent cAMP signaling by echistatin and alendronate

    NASA Technical Reports Server (NTRS)

    Fong, J. H.; Ingber, D. E.

    1996-01-01

    We measured intracellular cAMP levels in cells during attachment and spreading on different extracellular matrix (ECM) proteins. Increases in cAMP were observed within minutes when cells attached to fibronectin, vitronectin, and a synthetic RGD-containing fibronectin peptide (Petite 2000), but not when they adhered to another integrin alpha nu beta 3 ligand, echistatin. Because echistatin also inhibits bone resorption, we measured the effects of adding another osteoporosis inhibitor, alendronate, in this system. Alendronate inhibited the cAMP increase induced by ligands that primarily utilize integrin alpha nu beta 3 (vitronectin, Peptite 2000), but not by fibronectin which can also use integrin alpha 5 beta 1. These results show that cell adhesion to ECM can increase intracellular cAPM levels and raise the possibility that inhibitors of osteoporosis may act, in part, by preventing activation of this pathway by integrins.

  11. Elevated plasma levels of transforming growth factor (TGF)-beta1 and TGF-beta2 in patients with disseminated malignant melanoma.

    PubMed Central

    Krasagakis, K.; Thölke, D.; Farthmann, B.; Eberle, J.; Mansmann, U.; Orfanos, C. E.

    1998-01-01

    Overexpression of transforming growth factor-beta isoforms (TGF-beta1, -beta2, -beta3) has been previously reported in human melanoma cell lines and tumours. The aim of the present study was to evaluate the plasma levels of TGF-beta isoforms in melanoma patients. Significantly elevated levels of TGF-beta1 (4.2 x the controls, P = 0.0094) and of TGF-beta2 (1.5 x the controls, P = 0.012) but not of TGF-beta3 were measured in patients with disseminated but not locoregional melanoma. These results indicate systemic circulation of potentially immunosuppressive peptides of the TGF-beta family in end-stage melanoma patients. PMID:9652767

  12. Phosphorylation of a neuronal-specific beta-tubulin isotype

    SciTech Connect

    Diaz-Nido, J.; Serrano, L.; Lopez-Otin, C.; Vandekerckhove, J.; Avila, J. )

    1990-08-15

    Adult rats were intracraneally injected with ({sup 32}P) phosphate and brain microtubules isolated. The electrophoretically purified, in vivo phospholabeled, beta-tubulin was digested with the V8-protease and the labeled peptide purified by reversed-phase liquid chromatography. Its amino acid sequence corresponds to the COOH-terminal sequence of a minor neuronal beta 3-tubulin isoform from chicken and human. The phosphorylation site was at serine 444. A synthetic peptide with sequence EMYEDDEEESESQGPK, corresponding to that of the COOH terminus of beta 3-tubulin, was efficiently phosphorylated in vitro by casein kinase II at the same serine 444. The functional meaning of tubulin phosphorylation is still unclear. However, the modification of the protein takes place after microtubule assembly, and phosphorylated tubulin is mainly present in the assembled microtubule protein fraction.

  13. GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs.

    PubMed Central

    Ymer, S; Schofield, P R; Draguhn, A; Werner, P; Köhler, M; Seeburg, P H

    1989-01-01

    Cloned cDNAs encoding two new beta subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The beta 2 and beta 3 subunits share approximately 72% sequence identity with the previously characterized beta 1 polypeptide. Northern analysis showed that both beta 2 and beta 3 mRNAs are more abundant in the brain than beta 1 mRNA. All three beta subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each beta subunit, when co-expressed in Xenopus oocytes with an alpha subunit, forms functional GABAA receptors. These results, together with the known alpha subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations. Images PMID:2548852

  14. Felbamate is a subunit selective modulator of recombinant gamma-aminobutyric acid type A receptors expressed in Xenopus oocytes.

    PubMed

    Simeone, Timothy A; Otto, James F; Wilcox, Karen S; White, H Steve

    2006-12-15

    Felbamate (2-phenyl-1,3-propanediol dicarbamate) is clinically available for the treatment of refractory epileptic seizures, and is known to modulate several ion channels including gamma-aminobutyric acid type A (GABA(A)) receptors. To determine felbamate subunit selectivity for GABA(A) receptors we expressed 15 different GABA(A) receptor combinations in Xenopus laevis oocytes. Felbamate positively modulated GABA-currents of alpha(1)beta(2)gamma(2S), alpha(1)beta(3)gamma(2S), alpha(2)beta(2)gamma(2S) and alpha(2)beta(3)gamma(2S), whereas felbamate was either ineffective or negatively modulated the other 11 receptor combinations. Regional distributions of GABA(A) receptor subunits suggest that felbamate may differentially modulate distinct inhibitory circuits, a possibility that may have relevance to felbamate efficacy in refractory epilepsies. PMID:17056029

  15. The molecular structure of the Na(+)-translocating F1F0-ATPase of Acetobacterium woodii, as revealed by electron microscopy, resembles that of H(+)-translocating ATPases.

    PubMed

    Reidlinger, J; Mayer, F; Müller, V

    1994-12-12

    The Na(+)-translocating F1F0-ATPase of Acetobacterium woodii was examined by electron microscopy. After reconstitution into proteoliposomes, knobs typical for the F1 domain were visible on the outside of the membrane. The F1-part of the isolated enzyme showed a hexagonal symmetry suggesting an alpha 3 beta 3 structure, and the F1F0 complex had molecular dimensions very similar to those of H(+)-translocating ATPases of E. coli, chloroplasts, and mitochondria. PMID:7988711

  16. Ternary Complex of Transforming Growth Factor-[beta]1 Reveals Isoform-specific Ligand Recognition and Receptor Recruitment in the Superfamily

    SciTech Connect

    Radaev, Sergei; Zou, Zhongcheng; Huang, Tao; Lafer, Eileen M.; Hinck, Andrew P.; Sun, Peter D.

    2010-11-03

    Transforming growth factor (TGF)-{beta}1, -{beta}2, and -{beta}3 are 25-kDa homodimeric polypeptides that play crucial nonoverlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. Here we report the 3.0-{angstrom} resolution crystal structure of the ternary complex between human TGF-{beta}1 and the extracellular domains of its type I and type II receptors, T{beta}RI and T{beta}RII. The TGF-{beta}1 ternary complex structure is similar to previously reported TGF-{beta}3 complex except with a 10{sup o} rotation in T{beta}RI docking orientation. Quantitative binding studies showed distinct kinetics between the receptors and the isoforms of TGF-{beta}. T{beta}RI showed significant binding to TGF-{beta}2 and TGF-{beta}3 but not TGF-{beta}1, and the binding to all three isoforms of TGF-{beta} was enhanced considerably in the presence of T{beta}RII. The preference of TGF-{beta}2 to T{beta}RI suggests a variation in its receptor recruitment in vivo. Although TGF-{beta}1 and TGF-{beta}3 bind and assemble their ternary complexes in a similar manner, their structural differences together with differences in the affinities and kinetics of their receptor binding may underlie their unique biological activities. Structural comparisons revealed that the receptor-ligand pairing in the TGF-{beta} superfamily is dictated by unique insertions, deletions, and disulfide bonds rather than amino acid conservation at the interface. The binding mode of T{beta}RII on TGF-{beta} is unique to TGF-{beta}s, whereas that of type II receptor for bone morphogenetic protein on bone morphogenetic protein appears common to all other cytokines in the superfamily. Further, extensive hydrogen bonds and salt bridges are present at the high affinity cytokine-receptor interfaces, whereas hydrophobic interactions dominate the low affinity receptor-ligand interfaces.

  17. Human soluble guanylate cyclase: functional expression and revised isoenzyme family.

    PubMed Central

    Zabel, U; Weeger, M; La, M; Schmidt, H H

    1998-01-01

    Soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to the second messenger cGMP, functions as the receptor for nitric oxide (NO) and nitrovasodilator drugs. Three distinct cDNA species of each subunit (alpha1-alpha3, beta1-beta3) have been reported from various species. From human sources, none of these have been expressed as functionally active enzyme. Here we describe the expression of human alpha/beta heterodimeric sGC in Sf9 cells yielding active recombinant enzyme that was stimulated by the nitrovasodilator sodium nitroprusside or the NO-independent activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1). At the protein level, both alpha and beta subunits were detected in human tissues, suggesting co-expression also in vivo. Moreover, resequencing of the human cDNA clones [originally termed alpha3 and beta3; Giuili, Scholl, Bulle and Guellaen (1992) FEBS Lett. 304, 83-88] revealed several sequencing errors in human alpha3; correction of these eliminated major regions of divergence from rat and bovine alpha1. As human beta3 also displays more than 98% similarity to rat and bovine beta1 at the amino acid level, alpha3 and beta3 represent the human homologues of rat and bovine alpha1 and beta1, and the isoenzyme family is decreased to two isoforms for each subunit (alpha1, alpha2; beta1, beta2). Having access to the human key enzyme of NO signalling will now permit the study of novel sGC-modulating compounds with therapeutic potential. PMID:9742212

  18. ENG — EDRN Public Portal

    Cancer.gov

    ENG, or endoglin, is a cell surface adhesion protein involved the regulation of angiogenesis. ENG is a major glycoprotein of the vascular endothelium and is also a component of the transforming growth factor beta receptor complex. ENG binds to the beta1 and beta3 peptides with high affinity. ENG may also be involved in preeclampsia and several types of cancer. There are several isoforms encoded by transcript variants.

  19. Acute effect of ephedrine on 24-h energy balance

    NASA Technical Reports Server (NTRS)

    Shannon, J. R.; Gottesdiener, K.; Jordan, J.; Chen, K.; Flattery, S.; Larson, P. J.; Candelore, M. R.; Gertz, B.; Robertson, D.; Sun, M.

    1999-01-01

    Ephedrine is used to help achieve weight control. Data on its true efficacy and mechanisms in altering energy balance in human subjects are limited. We aimed to determine the acute effect of ephedrine on 24-h energy expenditure, mechanical work and urinary catecholamines in a double-blind, randomized, placebo-controlled, two-period crossover study. Ten healthy volunteers were given ephedrine (50 mg) or placebo thrice daily during each of two 24-h periods (ephedrine and placebo) in a whole-room indirect calorimeter, which accurately measures minute-by-minute energy expenditure and mechanical work. Measurements were taken of 24-h energy expenditure, mechanical work, urinary catecholamines and binding of (+/-)ephedrine in vitro to human beta1-, beta2- and beta3-adrenoreceptors. Twenty-four-hour energy expenditure was 3.6% greater (8965+/-1301 versus 8648+/-1347 kJ, P<0.05) with ephedrine than with placebo, but mechanical work was not different between the ephedrine and placebo periods. Noradrenaline excretion was lower with ephedrine (0.032+/-0.011 microg/mg creatinine) compared with placebo (0.044+/-0.012 microg/mg creatinine) (P<0.05). (+/-)Ephedrine is a relatively weak partial agonist of human beta1- and beta2-adrenoreceptors, and had no detectable activity at human beta3-adrenoreceptors. Ephedrine (50 mg thrice daily) modestly increases energy expenditure in normal human subjects. A lack of binding of ephedrine to beta3-adrenoreceptors and the observed decrease in urinary noradrenaline during ephedrine treatment suggest that the thermogenic effect of ephedrine results from direct beta1-/beta2-adrenoreceptor agonism. An indirect beta3-adrenergic effect through the release of noradrenaline seems unlikely as urinary noradrenaline decreased significantly with ephedrine.

  20. Fibrochondrogenesis of hESCs: growth factor combinations and cocultures.

    PubMed

    Hoben, Gwendolyn M; Willard, Vincent P; Athanasiou, Kyriacos A

    2009-03-01

    The successful differentiation of human embryonic stem cells (hESCs) to fibrochondrocyte-like cells and characterization of these differentiated cells is a critical step toward tissue engineering of musculoskeletal fibrocartilages (e.g., knee meniscus, temporomandibular joint disc, and intervertebral disc). In this study, growth factors and primary cell cocultures were applied to hESC embryoid bodies (EBs) for 3 weeks and evaluated for their effect on the synthesis of critical fibrocartilage matrix components: glycosaminoglycans (GAG) and collagens (types I, II, and VI). Changes in surface markers (CD105, CD44, SSEA, PDGFR alpha) after the differentiation treatments were also analyzed. The study was conducted in three phases: (1) examination of growth factors (TGF-beta 3, BMP-2, BMP-4, BMP-6, PDGF-BB, sonic hedgehog protein); (2) comparison of two cocultures (primary chondrocytes or fibrochondrocytes); and (3) the combination of the most effective growth factor and coculture regimen. TGF-beta 3 with BMP-4 yielded EBs positive for collagens I, II, and VI, with up to 6.7- and 4.8-fold increases in GAG and collagen, respectively. Analysis of cell surface markers showed a significant increase in CD44 with the TGF-beta 3 + BMP-4 treatment compared to the controls. Coculture with fibrochondrocytes resulted in up to a 9.8-fold increase in collagen II production. The combination of the growth factors BMP-4 + TGF-beta 3 with the fibrochondrocyte coculture led to an increase in cell proliferation and GAG production compared to either treatment alone. This study determined two powerful treatments for inducing fibrocartilaginous differentiation of hESCs and provides a foundation for using flow cytometry to purify these differentiated cells. PMID:18454697

  1. Insect molting hormone and sterol biosynthesis in spinach

    SciTech Connect

    Grebenok, R.J.; Adler, J.H. )

    1990-05-01

    Insect molting hormones, which are produced by plants and are effective molecules in the control of insect crop pests, are biosynthesized in developing spinach leaves (Spinacia oleracea L.). The major sterols biosynthesized by spinach are avenasterol (24{alpha}-ethyl-5{alpha}-cholesta-7,24(28)-dien-3{beta}-ol), spinasterol (24{alpha}-ethyl-5{alpha}-cholesta-7,22-dien-3{beta}-ol), and 22-dihydrospinasterol (24{alpha}-ethyl-5{alpha}-cholest-7-en-3{beta}-ol). The major ecdysteroids biosynthesized are ecdysterone (2{beta},3{beta},14{alpha},20R,22R,25-hexahydroxy-5{beta}-cholest-7-en-6-one) and polypodine B (2{beta},3{beta},5{beta},14{alpha},20R,22R,25-heptahycroxycholest-7-en-6-one) and polypodine B (2{beta},3{beta},5{beta},14{alpha},20R,22R,25-heptahydroxycholest-7-en-6-one). When labeled 2-{sup 14}C-mevalonic acid was incorporated into young leaves isolated squalene, sterols and ecdysteroids contained the label. During a short (16 h) incorporation period in intact young leaves of 100 day old plants, the avenasterol has the highest specific activity in counts per minute per {mu}g of sterol followed by 22-dihydrospinasterol which is more highly labeled than spinasterol. The ecdysteroids synthesized, on an entire plant basis, account for 20% of the total steroid (sterol and ecdysteroid) isolated from the plant.

  2. The adrenergic receptor subtypes present in frog (Rana esculenta) skin.

    PubMed

    Bellantuono, Vito; Cassano, Giuseppe; Lippe, Claudio

    2008-08-01

    Frog skin transports ions and water under hormonal control. In spite of the fundamental role played by adrenergic stimulation in maintaining the water balance of the organism, the receptor subtype(s) present in the skin have not been identified yet. We measured the increase in short-circuit current (ISC, an estimate of ion transport) induced by cirazoline, clonidine, xamoterol, formoterol, or BRL 37344, in order to verify the presence of alpha1, alpha2, beta1, beta2, or beta3 receptor subtypes, respectively. Only after treatment with formoterol, BRL 37344 and, to a lesser extent, cirazoline was measured a significant increase in ISC (57%, 33.2%, and 4.7%, respectively). The formoterol and BRL 37344 concentrations producing half-maximal effect (EC50) were 1.12 and 70.1 nM, respectively. Moreover, the formoterol effect was inhibited by treatment with ICI 118551 (antagonist of beta2 receptors) while SR 59230A (antagonist of beta3 receptors) had no effect; opposite findings were obtained when the BRL 37344 stimulation was investigated. Finally, by measuring the transepithelial fluxes of 22Na+ and 36Cl-, we demonstrated that Na+ absorption is increased by activation of beta2 and beta3 and is cAMP-sensitive, whereas the Cl- secretion is only increased by activation of beta2 receptors and is cAMP- and calmodulin-sensitive. PMID:18544474

  3. Immunohistochemical analysis of the skin in junctional epidermolysis bullosa using laminin 5 chain specific antibodies is of limited value in predicting the underlying gene mutation.

    PubMed

    McMillan, J R; McGrath, J A; Pulkkinen, L; Kon, A; Burgeson, R E; Ortonne, J P; Meneguzzi, G; Uitto, J; Eady, R A

    1997-06-01

    The anchoring filament protein laminin 5 is composed of three polypeptide chains (alpha 3, beta 3 and gamma 2) each encoded by separate genes (LAMA3, LAMB3 and LAMC2, respectively). Mutations in any of these three genes may give rise to the autosomal recessive blistering skin disease, junctional epidermolysis bullosa. At present, there is no easy way of predicting which of these three genes might harbour the pathogenetic laminin 5 mutations in a case of junctional epidermolysis bullosa. In this study, we assessed whether immunohistochemistry might be helpful in this regard. We performed immunohistochemical labelling of the dermal-epidermal junction using alpha 3, beta 3 and gamma 2 chain-specific antibodies in 11 patients with junctional epidermolysis bullosa, in whom the laminin 5 mutations had been previously delineated. Although, labelling for the laminin 5 chain bearing the mutations was attenuated or undetectable in all cases, a complete absence of labelling or a reduction in the staining intensity for the other two chains was also seen in all cases. The results showed that immunohistochemical labelling of the dermal-epidermal junction using alpha 3, beta 3 and gamma 2 chain-specific antibodies is not a specific indicator for which of the laminin 5 chain genes contains the pathogenetic mutations, and is therefore unreliable in screening for individual laminin 5 gene mutations in cases of junctional epidermolysis bullosa. PMID:9217810

  4. Chemical cross-linking of the urease complex from Helicobacter pylori and analysis by Fourier transform ion cyclotron resonance mass spectrometry and molecular modeling

    NASA Astrophysics Data System (ADS)

    Carlsohn, Elisabet; Ångström, Jonas; Emmett, Mark R.; Marshall, Alan G.; Nilsson, Carol L.

    2004-05-01

    Chemical cross-linking of proteins is a well-established method for structural mapping of small protein complexes. When combined with mass spectrometry, cross-linking can reveal protein topology and identify contact sites between the peptide surfaces. When applied to surface-exposed proteins from pathogenic organisms, the method can reveal structural details that are useful in vaccine design. In order to investigate the possibilities of applying cross-linking on larger protein complexes, we selected the urease enzyme from Helicobacter pylori as a model. This membrane-associated protein complex consists of two subunits: [alpha] (26.5 kDa) and [beta] (61.7 kDa). Three ([alpha][beta]) heterodimers form a trimeric ([alpha][beta])3 assembly which further associates into a unique dodecameric 1.1 MDa complex composed of four ([alpha][beta])3 units. Cross-linked peptides from trypsin-digested urease complex were analyzed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and molecular modeling. Two potential cross-linked peptides (present in the cross-linked sample but undetectable in [alpha], [beta], and native complex) were assigned. Molecular modeling of urease [alpha][beta] complex and trimeric urease units ([alpha][beta])3 revealed a linkage site between the [alpha]-subunit and the [beta]-subunit, and an internal cross-linkage in the [beta]-subunit.

  5. Structural insights into how the MIDAS ion stabilizes integrin binding to an RGD peptide under force.

    PubMed

    Craig, David; Gao, Mu; Schulten, Klaus; Vogel, Viola

    2004-11-01

    Integrin alpha(V)beta(3) binds to extracellular matrix proteins through the tripeptide Arg-Gly-Asp (RGD), forming a shallow crevice rather than a deep binding pocket. A dynamic picture of how the RGD-alpha(V)beta(3) complex resists dissociation by mechanical force is derived here from steered molecular dynamic (SMD) simulations in which the major force peak correlates with the breaking of the contact between Asp(RGD) and the MIDAS ion. SMD predicts that the RGD-alpha(V)beta(3) complex is stabilized from dissociation by a single water molecule tightly coordinated to the divalent MIDAS ion, thereby blocking access of free water molecules to the most critical force-bearing interaction. The MIDAS motif is common to many other proteins that contain the phylogenetically ancient von Willebrand A (vWA) domain. The functional role of single water molecules tightly coordinated to the MIDAS ion might reflect a general strategy for the stabilization of protein-protein adhesion against cell-derived forces through divalent cations. PMID:15530369

  6. Transforming growth factor-{beta}2 enhances differentiation of cardiac myocytes from embryonic stem cells

    SciTech Connect

    Kumar, Dinender . E-mail: Dinender.Kumar@uvm.edu; Sun, Baiming

    2005-06-24

    Stem cell therapy holds great promise for the treatment of injured myocardium, but is challenged by a limited supply of appropriate cells. Three different isoforms of transforming growth factor-{beta} (TGF-{beta}) -{beta}1, -{beta}2, and -{beta}3 exhibit distinct regulatory effects on cell growth, differentiation, and migration during embryonic development. We compared the effects of these three different isoforms on cardiomyocyte differentiation from embryonic stem (ES) cells. In contrast to TGF-{beta}1, or -{beta}3, treatment of mouse ES cells with TGF-{beta}2 isoform significantly increased embryoid body (EB) proliferation as well as the extent of the EB outgrowth that beat rhythmically. At 17 days, 49% of the EBs treated with TGF-{beta}2 exhibited spontaneous beating compared with 15% in controls. Cardiac myocyte specific protein markers sarcomeric myosin and {alpha}-actin were demonstrated in beating EBs and cells isolated from EBs. In conclusion, TGF-{beta}2 but not TGF-{beta}1, or -{beta}3 promotes cardiac myocyte differentiation from ES cells.

  7. Suppression of laminin-5 expression leads to increased motility, tumorigenicity, and invasion

    SciTech Connect

    Yuen Hengwai; Ziober, Amy F.; Gopal, Pallavi; Nasrallah, Ilya; Falls, Erica M.; Meneguzzi, Guerrino; Ang, Hwee-Quan; Ziober, Barry L. . E-mail: bziober@mail.med.upenn.edu

    2005-09-10

    Laminin-5 (Ln-5) is expressed in several human carcinomas and hypothesized to contribute to tumor invasion. To understand the role of Ln-5 in human cancers, we stably delivered small interfering RNAs (siRNAs) directed against the Ln-5 {gamma}2 chain into JHU-022-SCC cells (022), a non-invasive oral squamous cell carcinoma (OSCC) cell line which secretes Ln-5. Lysates from {gamma}2 siRNA cells (022-si{gamma}2) had nearly undetectable levels of the {gamma}2 chain while the {alpha}3 and {beta}3 subunits of Ln-5 remained unchanged compared to parental and control. In conditioned medium from 022-si{gamma}2 cells, the {gamma}2 chain and the Ln-5 heterotrimer were barely detectable, similar to an invasive OSCC cell line. Conditioned medium from 022-si{gamma}2 cells contained less {alpha}3 and {beta}3 subunits than both parental and control. Although the proliferation and adhesive properties of the 022-si{gamma}2 cells remained similar to parental and control cells, 022-si{gamma}2 cells showed increased detachment and a fibroblastic morphology similar to invasive cells. Moreover, migration, in vitro invasion, and in vivo tumorigenicity were enhanced in 022-si{gamma}2 cells. Our results suggest that the Ln-5 {gamma}2 chain regulates the secretion of the {alpha}3 and {beta}3 subunits. More importantly, suppression of Ln-5 results in a phenotype that is representative of invasive tumor cells.

  8. TGF-{beta}'s delay skeletal muscle progenitor cell differentiation in an isoform-independent manner

    SciTech Connect

    Schabort, Elske J.; Merwe, Mathilde van der; Loos, Benjamin; Moore, Frances P.; Niesler, Carola U.

    2009-02-01

    Satellite cells are a quiescent heterogenous population of mononuclear stem and progenitor cells which, once activated, differentiate into myotubes and facilitate skeletal muscle repair or growth. The Transforming Growth Factor-{beta} (TGF-{beta}) superfamily members are elevated post-injury and their importance in the regulation of myogenesis and wound healing has been demonstrated both in vitro and in vivo. Most studies suggest a negative role for TGF-{beta} on satellite cell differentiation. However, none have compared the effect of these three isoforms on myogenesis in vitro. This is despite known isoform-specific effects of TGF-{beta}1, -{beta}2 and -{beta}3 on wound repair in other tissues. In the current study we compared the effect of TGF-{beta}1, -{beta}2 and -{beta}3 on proliferation and differentiation of the C2C12 myoblast cell-line. We found that, irrespective of the isoform, TGF-{beta} increased proliferation of C2C12 cells by changing the cellular localisation of PCNA to promote cell division and prevent cell cycle exit. Concomitantly, TGF-{beta}1, -{beta}2 and -{beta}3 delayed myogenic commitment by increasing MyoD degradation and decreasing myogenin expression. Terminal differentiation, as measured by a decrease in myosin heavy chain (MHC) expression, was also delayed. These results demonstrate that TGF-{beta} promotes proliferation and delays differentiation of C2C12 myoblasts in an isoform-independent manner.

  9. Integrin receptors play a role in the internalin B-dependent entry of Listeria monocytogenes into host cells.

    PubMed

    Auriemma, Clementina; Viscardi, Maurizio; Tafuri, Simona; Pavone, Luigi Michele; Capuano, Federico; Rinaldi, Laura; Della Morte, Rossella; Iovane, Giuseppe; Staiano, Norma

    2010-09-01

    Listeria monocytogenes enters non-phagocytic cells by binding its surface proteins inlA (internalin) and inlB to the host's E-cadherin and Met, respectively. The two internalins play either separate or cooperative roles in the colonization of infected tissues. Here, we studied bacterial uptake into HeLa cells using an L. monocytogenes mutant strain (DeltainlA) carrying a deletion in the gene coding for inlA. The DeltainlA mutant strain showed the capability to invade HeLa cells. The monoclonal anti-beta(3)- and anti-beta(1)-integrin subunit antibodies prevented bacterial uptake into the cells, while the anti-beta(2)- and anti-beta(4)-integrin subunit antibodies failed to affect L. monocytogenes entry into HeLa cells. Three structurally distinct disintegrins (kistrin, echistatin and flavoridin) also inhibited bacterial uptake, showing different potencies correlated to their selective affinity for the beta(3)- and beta(1)-integrin subunits. In addition to inducing Met phosphorylation, infection of cells by the L. monocytogenes DeltainlA mutant strain promoted the tyrosine phosphorylation of the focal adhesion-associated proteins FAK and paxillin. Our findings provide the first evidence that beta(3)- and beta(1)-integrin receptors play a role in the inlB-dependent internalization of L. monocytogenes into host cells. PMID:20526749

  10. Mouse egg integrin alpha 6 beta 1 functions as a sperm receptor.

    PubMed

    Almeida, E A; Huovila, A P; Sutherland, A E; Stephens, L E; Calarco, P G; Shaw, L M; Mercurio, A M; Sonnenberg, A; Primakoff, P; Myles, D G; White, J M

    1995-06-30

    Binding between sperm and egg plasma membranes is an essential step in fertilization. Whereas fertilin, a mammalian sperm surface protein, is involved in this crucial interaction, sperm receptors on the egg plasma membrane have not been identified. Because fertilin contains a predicted integrin ligand domain, we investigated the expression and function of integrin subunits in unfertilized mouse eggs. Polymerase chain reactions detected mRNAs for alpha 5, alpha 6, alpha v, beta 1, beta 3, and beta 5. Immunofluorescence revealed alpha 6 beta 1 and alpha v beta 3 on the plasma membrane. GoH3, a function-blocking anti-alpha 6 monoclonal antibody, abolished sperm binding, but a nonfunction-blocking anti-alpha 6 monoclonal antibody, a function-blocking anti-alpha v beta 3 polyclonal antibody, and an RGD peptide had no effect. Somatic cells bound sperm avidly, but only if they expressed alpha 6 beta 1. A peptide analog of the fertilin integrin ligand domain inhibited sperm binding to eggs and alpha 6 beta 1+ cells and diminished GoH3 staining of eggs. Our results indicate a novel role for the integrin alpha 6 beta 1 as a cell-cell adhesion receptor that mediates sperm-egg binding. PMID:7600577

  11. Molecular interaction studies of hemostasis: fibrinogen ligand-human platelet receptor interactions.

    PubMed

    Lee, Imshik; Marchant, Roger E

    2003-01-01

    The interactions between fibrinogen ligands and platelet receptor alpha(IIb)beta(3) were studied under physiological conditions by atomic force microscopy (AFM). Two linear peptide sequences in fibrinogen, RGD and HHLGGAKQAGDV, play central roles in the regulation of hemostasis and thrombosis by facilitating adhesion and aggregation of platelets. In order to measure the interactions (i.e., debonding force), oligopeptides, GSSSGaaa, where aaa is -RGDSPA or -HHLGGAKQAGDV, were synthesized and grafted on to the surface of AFM probe tips. The interaction forces between a peptide-modified AFM probe tip and platelet surface were determined from pN to nN levels using AFM force measurements. Our results show that the zero kinetic off-rate, K(off)(0), for RGDSPA is significantly smaller than that for HHLGGAKQAGDV, under the consideration of flexible receptor surfaces. From our analysis, the K(off)(0), the single molecular binding energy E(b), and the transition state x(b), were extracted from the data, and estimated to be 1.53s(-1), -2.64x10(-20)J and 1.03A for the RGD-alpha(IIb)beta(3) system, and 47.58s(-1), 2.67x10(-20), 1.09A for the HHLGGAKQAGDV-alpha(IIb)beta(3) system, respectively. PMID:12801687

  12. Effects of antenatal application of ambroxol and glucocorticoid on lung morphometry and signal transduction of bone morphogenetic protein in the fetal rat.

    PubMed

    Chen, Xiao-Qing; Wu, Sheng-Hua; Guo, Xi-Rong; Zhou, Xiao-Yu

    2012-07-01

    Antenatal ambroxol, dexamethasone (Dex) and betamethasone (Beta) are used to prevent neonate respiratory distress syndrome. The present study aimed to investigate the role of ambroxol, Dex and Beta administered antenatally on lung morphogenesis and signal transduction of bone morphogenetic protein (BMP) in rat embryo. Fetal lungs treated with ambroxol, 1-day Beta, 3-day Dex and 3-day Beta were more mature compared to the controls as determined by light microscopy and transmission electron microscopy. Expression of BMP4 and bone morphogenetic protein receptor II (BMPR‑II) mRNA was upregulated in the 1-day-Beta-, 3-day-Dex- and 3-day-Beta-treated animals. BMP4 and BMPR-II protein were significantly increased in the 1-day-Beta-, 3-day-Dex- and 3-day-Beta-treated animals. Ambroxol, Dex and Beta promoted the morphological development of rat fetal lung; Beta was more effective than Dex. A multi-dose of glucocorticoids exhited a more beneficial effect than a single dose. The effects of Beta and Dex may be mediated by regulation of BMP signal transduction in rat fetal lung. PMID:22552703

  13. Demonstration of tyrosinase in the vitiligo skin of human beings by a sensitive fluorometric method as well as by 14C(U)-L-tyrosine incorporation into melanin

    SciTech Connect

    Husain, I.; Vijayan, E.; Ramaiah, A.; Pasricha, J.S.; Madan, N.C.

    1982-03-01

    Tyrosinase activity (Monophenol, dihydroxyphenylalanine: oxygen oxidoreductase EC 1.14.18.1) in vitiligo and normal epidermal homogenates of skin from human beings was measured by estimating beta 3,4-dihydroxyphenylalanine (dopa) by a highly sensitive fluorometric method described in this paper. The tyrosine activity in the vitiligo skin was about 4 to 37% of corresponding normal skin. The activity of tyrosinase in normal human skin from different individuals and from different regions of the body was in the range of 4 to 140 picomoles of beta 3,4-dihydroxyphenylalanine formed per min/mg protein of epidermal homogenate. The enzyme from vitiligo and normal skin was severely inhibited by substance(s) of low molecular weight. The enzyme exhibits a lag of about 4 hr in the absence of added beta 3,4-dihydroxyphenylalanine and 1 hr in presence of 5 microM dopa. Tyrosinase from the normal and vitiligo skin was inhibited by excess concentration of tyrosine. The homogenates from vitiligo skin could synthesize melanin from C14(U)-L-Tyrosine. The rate of tyrosine incorporation into melanin by the epidermal homogenates is increased by 3,4-dihydroxyphenylalanine (dopa) disproportionate to its effect on tyrosinase activity. Based on the data presented in this paper it is concluded that melanocytes are present in the vitiligo skin. A tentative hypothesis is put forward to explain the lack of melanin synthesis by the vitiligo skin under in vivo conditions, although melanocytes are present.

  14. Regulation of heat production of brown adipocytes via typical and atypical beta-adrenoceptors in the rat.

    PubMed

    Tanaka, E; Yamakawa, A; Yamamura, M; el Borai, N; Ito, K; Nakano, S; Nakazawa, H

    1995-01-01

    Previous studies in our laboratory demonstrated that microcalorimetry is an appropriate method for estimating the physiological function of isolated rat brown adipocytes. In the present study, to elucidate the mode of action of typical and atypical beta-adrenoceptors on heat production of this cell, the effect of novel adrenergic beta 3-agonists was compared with that of other typical adrenergic reagents by direct microcalorimetry. Isoproterenol and beta 3-agonists, BRL37344, ICI215001, and CGP12177, increased heat production in a dose-dependent manner, however, phenylephrine had no effect. Propranolol and pindolol did not increase the heat production but attenuated the effect of isoproterenol and BRL37344 in a dose-dependent manner. Molar IC50 values of propranolol and pindolol for BRL37344 were about 10(-5) and 3 x 10(-6) M, respectively, whereas those of the two antagonists for isoproterenol were about 3 x 10(-7)M. The pA2 values by Schild analysis of propranolol vs. isoproterenol and BRL37344 were 7.91 and 6.13, respectively. These results suggest that heat production may be regulated via both beta 3- and typical beta-adrenoceptors in brown adipocytes. PMID:8676573

  15. Low temperature deposition and characterization of n- and p-type silicon carbide thin films and associated ohmic and Schottky contacts. Annual report, 1 January-31 December 1992

    SciTech Connect

    Davis, R.F.; Nemanich, R.J.; Kern, R.S.; Patterson, R.; Rowland, L.B.

    1992-01-01

    Single-crystal epitaxial films of cubic Beta(3C)-SiC(111) and AlN(0001) have been deposited on alpha(6H)-SiC(OOO1) substrates oriented 3-4 deg towards 1120 at 1050 deg C via gas-source molecular beam epitaxy using disilane (Si2H6), ethylene (C2H4), thermal evaporation of Al and activated N species derived from an ECR plasma. High resolution transmission electron microscopy revealed that the nucleation and growth of the Beta(3C)-SiC regions occurred primarily on terraces between closely spaced steps. Pseudomorphic bilayer structures containing Beta(3C)-SiC and 2H-AlN have been grown under the same conditions for the first time. HREED and cross-sectional HRTEM showed all layers to be monocrystalline. Initial high temperature chemical interdiffusion studies between SiC and AIN show that all components diffuse very slowly across the interface. AHRTEM and SAS are being used to determine the concentration profiles. Thin film solid solutions of AIN and SiC have been deposited using similar techniques and conditions as the individual compounds. Metal contacts of Ti, Pt and Hf deposited at RT on n-type alpha(6H)-SiC(OOO1) exhibit rectifying behavior with ideality factors between 1.01 and 1.09. The Pt and Hf contacts had leakage currents of 5xl0-8 A/cm2 at -10V. Values of barrier heights for all contacts were within a few tenths of 1.0eV which is indicative that the Fermi level is pinned at the SiC surface.... Films, SiC, AlN, Gas source molecular beam epitaxy, Transmission electron microscopy, Chemical interdiffusion, Metal contacts, Ti, Pt, Hf, Ideality factors, Fermi level pinning.

  16. Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

    PubMed Central

    Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

    2001-01-01

    Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding. PMID:11350033

  17. Limited T-cell receptor beta-chain heterogeneity among interleukin 2 receptor-positive synovial T cells suggests a role for superantigen in rheumatoid arthritis.

    PubMed Central

    Howell, M D; Diveley, J P; Lundeen, K A; Esty, A; Winters, S T; Carlo, D J; Brostoff, S W

    1991-01-01

    Rheumatoid arthritis (RA) is a disease affecting the synovial membranes of articulating joints that is thought to result from T-cell-mediated autoimmune phenomena. T cells responsible for the pathogenesis of RA are likely present in that fraction of synovial T cells that expresses the interleukin 2 receptor (IL-2R), one marker of T-cell activation. We report herein an analysis of T-cell receptor (TCR) beta-chain gene expression by IL-2R-positive synovial T cells. These T cells were isolated from uncultured synovial tissue specimens by using IL-2R-specific monoclonal antibodies and magnetic beads, and TCR beta-chain transcription was analyzed by PCR-catalyzed amplification using a panel of primers specific for the human TCR beta-chain variable region (V beta). Multiple V beta gene families were found to be transcribed in these patients samples; however, three gene families, V beta 3, V beta 14, and V beta 17, were found in a majority of the five synovial samples analyzed, suggesting that T cells bearing these V beta s had been selectively retained in the synovial microenvironment. In many instances, the V beta 3, V beta 14, or V beta 17 repertoires amplified from an individual patient were dominated by a single rearrangement, indicative of clonal expansion in the synovium and supportive of a role for these T cells in RA. Of note is a high sequence similarity between V beta 3, V beta 14, and V beta 17 polypeptides, particularly in the fourth complementarity-determining region (CDR). Given that binding sites for superantigens have been mapped to the CDR4s of TCR beta chains, the synovial localization of T cells bearing V beta s with significant CDR4 homology indicates that V beta-specific T-cell activation by superantigen may play a role in RA. PMID:1660155

  18. Autoimmune diabetes-prone NOD mice express the Lyt2{sup a} (Lyt2.1) and Lyt3{sup a} (Lyt3.1) alleles of CD8

    SciTech Connect

    Johnson-Tardieu, J.M.; Cornelius, J.G.; Ye, X.

    1996-06-01

    Predisposition to Type I insulin-dependent diabetes (IDD) has a strong underlying genetic basis involving class II major histocompatibility complex (MHC) genes as well as several non-MHC genetic systems. In the non-obese diabetic (NOD) mouse, a model for human IDD, genes associated with the appearance of immune cell infiltrates in the pancreatic islets (insulitis) and/or overt IDD have been mapped to chromosomes 1, 3, 6, 11, and 17. A recent report has suggested that CD8+ lymphocytes of the NOD mouse might be deficient in the expression of the CD8{Beta} molecule, a protein encoded by a gene on chromosome 6. The CD8{Beta} molecule is a T-cell surface marker, the lack of which could affect selection in the thymus, possibly permitting auto-reactive T-cell clones to populate the peripheral lymphoid tissues. For this reason, we examined the expression of the CD8 molecule by lymphocytes in the NOD mouse. Results indicate that the NOD mouse is not deficient in its transcription of detectable mRNA encoding either the CD8{alpha} or {Beta} subunits. However, the NOD mouse expresses the Lyt2{sup a} and Lyt3{sup a} alleles, suggesting that a portion of chromosome 6 centromeric to the diabetes-susceptibility genetic region is derived from an ancestry common to AKR and, like AKR, the CD8{alpha} and CD8{Beta}3.1 (but not CD8{Beta}3.2) subunits are detected on the cell surface of T lymphocytes of the NOD mouse. Interestingly, though, the CD8{Beta}3.1 molecule may not be expressed in the NOD mouse to the same extent as it is expressed in the AKR/J mouse, suggesting the possibility that the NOD mouse possesses a defect somewhere between transcription and cell surface expression of the CD8{Beta} molecule. 36 refs., 5 figs.

  19. Temperature dependence of estrogen binding: importance of a subzone in the ligand binding domain of a novel piscine estrogen receptor.

    PubMed

    Tan, N S; Frecer, V; Lam, T J; Ding, J L

    1999-11-11

    The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones beta1 and beta3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a approximately 3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37 degrees C, OaER requires approximately 80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37 degrees C to 25 degrees C or 20 degrees C resulted in a 4-fold increase in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25 degrees C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactivation capability. Amino acid replacements of OaER beta3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRalpha apo, RARgamma holo and ERalpha LBDs. Unliganded and 17beta-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone beta3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties. PMID:10559464

  20. Single nucleotide polymorphism and lifestyle-related diseases in the Asia-Pacific region: comparative study in Okinawa, Palau and Thailand.

    PubMed

    Kagawa, Y; Dever, G J; Otto, C T Y; Charupoonphol, P; Supannatas, S; Yanagisawa, Y; Sakuma, M; Hasegawa, K

    2003-01-01

    Genetic differences between Asians and Caucasians may be involved in the rapid increase in lifestyle-related diseases in the Asia-Pacific region that has coincided with Westernisation of diets in the region. In the present study, we assessed correlation between 10 single nucleotide polymorphisms (SNPs) and chronic disease risk factors in age-matched and population-based groups in four Asian-Pacific locations: Okinawa, Palau and Thailand (two areas). The following allelic SNP profiles significantly differed (p<0.01) among the four populations, in both men and women: uncoupling protein 2 (UCP2), uncoupling protein 3 promoter (UCP3p), leptin receptor (LEPR) exon 6, and angiotensinogen (AGTa-20c). Multiple regression analyses showed significant associations between SNPs and clinical data. For men, these associations were between beta3 adrenergic receptor (beta3AR) and diastolic blood pressure (DBP) (p<0.01), UCP3p and total cholesterol (p<0.01), UCP2 and age (p<0.05), and AGTa-20c and age (p<0.01). For women, these associations were between LEPR exon 14 and body mass index (BMI) (p<0.05), UCP2 and systolic blood pressure (p<0.05), UCP3p and DBP (p<0.05), UCP2 and DBP (p<0.01), apolipoprotein E (ApoE)nd total cholesterol (p<0.01), beta3AR and triglyceride (p<0.05), AGTa-20c and triglyceride (p<0.05), and UCP2 and age (p<0.05). These results illustrate the interrelationships among SNPs and risk factors in the Asia-Pacific including China and Japan. PMID:18924534

  1. Solution NMR and X-ray Crystal Structures of Membrane-associated Lipoprotein-17 Domain Reveal a Novel Fold

    SciTech Connect

    R Mani; S Vorobiev; G Swapna; H Neely; H Janjua; C Ciccosanti; D Xiao; J Hunt; G Montelione; et al.

    2011-12-31

    The conserved Lipoprotein-17 domain of membrane-associated protein Q9PRA0{_}UREPA from Ureaplasma parvum was selected for structure determination by the Northeast Structural Genomics Consortium, as part of the Protein Structure Initiative's program on structure-function analysis of protein domains from large domain sequence families lacking structural representatives. The 100-residue Lipoprotein-17 domain is a 'domain of unknown function' (DUF) that is a member of Pfam protein family PF04200, a large domain family for which no members have characterized biochemical functions. The three-dimensional structure of the Lipoprotein-17 domain of protein Q9PRA0{_}UREPA was determined by both solution NMR and by X-ray crystallography at 2.5 {angstrom}. The two structures are in good agreement with each other. The domain structure features three {alpha}-helices, {alpha}1 through {alpha}3, and five {beta}-strands. Strands {beta}1/{beta}2, {beta}3/{beta}4, {beta}4/{beta}5 are anti-parallel to each other. Strands {beta}1 and {beta}2 are orthogonal to strands {beta}3, {beta}4, {beta}5, while helix {alpha}3 is formed between the strands {beta}3 and {beta}4. One-turn helix {alpha}2 is formed between the strands {beta}1 and {beta}2, while helix {alpha}1 occurs in the N-terminal polypeptide segment. Searches of the Protein Data Bank do not identify any other protein with significant structural similarity to Lipoprotein-17 domain of Q9PRA0{_}UREPA, indicating that it is a novel protein fold.

  2. Reticular nucleus-specific changes in alpha3 subunit protein at GABA synapses in genetically epilepsy-prone rats.

    PubMed

    Liu, Xiao-Bo; Coble, Jeffrey; van Luijtelaar, Gilles; Jones, Edward G

    2007-07-24

    Differential composition of GABA(A) receptor (GABA(A)R) subunits underlies the variability of fast inhibitory synaptic transmission; alteration of specific GABA(A)R subunits in localized brain regions may contribute to abnormal brain states such as absence epilepsy. We combined immunocytochemistry and high-resolution ImmunoGold electron microscopy to study cellular and subcellular localization of GABA(A)R alpha1, alpha3, and beta2/beta3 subunits in ventral posterior nucleus (VP) and reticular nucleus (RTN) of control rats and WAG/Rij rats, a genetic model of absence epilepsy. In control rats, alpha1 subunits were prominent at inhibitory synapses in VP and much less prominent in RTN; in contrast, the alpha3 subunit was highly evident at inhibitory synapses in RTN. beta2/beta3 subunits were evenly distributed at inhibitory synapses in both VP and RTN. ImmunoGold particles representing all subunits were concentrated at postsynaptic densities with no extrasynaptic localization. Calculated mean number of particles for alpha1 subunit per postsynaptic density in nonepileptic VP was 6.1 +/- 3.7, for alpha3 subunit in RTN it was 6.6 +/- 3.4, and for beta2/beta3 subunits in VP and RTN the mean numbers were 3.7 +/- 1.3 and 3.5 +/- 1.2, respectively. In WAG/Rij rats, there was a specific loss of alpha3 subunit immunoreactivity at inhibitory synapses in RTN, without reduction in alpha3 subunit mRNA or significant change in immunostaining for other markers of RTN cell identity such as GABA or parvalbumin. alpha3 immunostaining in cortex was unchanged. Subtle, localized changes in GABA(A)R expression acting at highly specific points in the interconnected thalamocortical network lie at the heart of idiopathic generalized epilepsy. PMID:17630284

  3. Identification of the sites for CaMK-II-dependent phosphorylation of GABA(A) receptors.

    PubMed

    Houston, Catriona M; Lee, Henry H C; Hosie, Alastair M; Moss, Stephen J; Smart, Trevor G

    2007-06-15

    Phosphorylation can affect both the function and trafficking of GABA(A) receptors with significant consequences for neuronal excitability. Serine/threonine kinases can phosphorylate the intracellular loops between M3-4 of GABA(A) receptor beta and gamma subunits thereby modulating receptor function in heterologous expression systems and in neurons (1, 2). Specifically, CaMK-II has been demonstrated to phosphorylate the M3-4 loop of GABA(A) receptor subunits expressed as GST fusion proteins (3, 4). It also increases the amplitude of GABA(A) receptor-mediated currents in a number of neuronal cell types (5-7). To identify which substrate sites CaMK-II might phosphorylate and the consequent functional effects, we expressed recombinant GABA(A) receptors in NG108-15 cells, which have previously been shown to support CaMK-II modulation of GABA(A) receptors containing the beta3 subunit (8). We now demonstrate that CaMK-II mediates its effects on alpha1beta3 receptors via phosphorylation of Ser(383) within the M3-4 domain of the beta subunit. Ablation of beta3 subunit phosphorylation sites for CaMK-II revealed that for alphabetagamma receptors, CaMK-II has a residual effect on GABA currents that is not mediated by previously identified sites of CaMK-II phosphorylation. This residual effect is abolished by mutation of tyrosine phosphorylation sites, Tyr(365) and Tyr(367), on the gamma2S subunit, and by the tyrosine kinase inhibitor genistein. These results suggested that CaMK-II is capable of directly phosphorylating GABA(A) receptors and activating endogenous tyrosine kinases to phosphorylate the gamma2 subunit in NG108-15 cells. These findings were confirmed in a neuronal environment by expressing recombinant GABA(A) receptors in cerebellar granule neurons. PMID:17442679

  4. Mercury's perihelion advance - Determination by radar.

    NASA Technical Reports Server (NTRS)

    Shapiro, I. I.; Pettengill, G. H.; Ash, M. E.; Ingalls, R. P.; Campbell, D. B.; Dyce, R. B.

    1972-01-01

    Measurement of echo delays of radar signals transmitted from earth to Mercury, yielding an accurate value for the advance of the latter's perihelion position. Given that the sun's gravitational quadrupole moment is negligible, the result in terms of the Eddington-Robertson parameters is (2 + 2 gamma-beta)/3 approximately equal to 1.005 plus or minus 0.007, where gamma = beta = 1 in general relativity, and where 0.007 represents the statistical standard error. Inclusion of the probable contribution of systematic errors raises the uncertainty to about 0.02.

  5. Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function.

    PubMed

    Jobling, Andrew Ian; Wan, Ran; Gentle, Alex; Bui, Bang Viet; McBrien, Neville Anthony

    2009-03-01

    A visually evoked signalling cascade, which begins in the retina, transverses the choroid, and mediates scleral remodelling, is considered to control eye growth. The ubiquitous cytokine TGF-beta has been associated with alterations in ocular growth, where alterations in scleral TGF-beta isoforms mediate the scleral remodelling that results in myopia. However, while the TGF-beta isoforms have been implicated in the scleral change during myopia development, it is unclear whether alterations in retinal and choroidal isoforms constitute part of the retinoscleral cascade. This study characterised the retinal and choroidal TGF-beta isoform profiles and TGF-beta2 activation during different stages of myopia development, as induced by form deprivation, in a mammalian model of eye growth. Using quantitative real-time PCR, the mRNA for all three mammalian isoforms of TGF-beta was detected in tree shrew retina and choroid. Distinct tissue-specific isoform profiles were observed for the retina (TGF-beta1:TGF-beta2:TGF-beta3=20:2085:1) and choroid (TGF-beta1:TGF-beta2:TGF-beta3=16:23:1), which remained constant over the development period under investigation. The active and latent pools of retinal TGF-beta2 were quantified using ELISA with the majority (>94%) of total TGF-beta2 found in the latent form. Unlike previous scleral data showing early and continuous decreases in TGF-beta isoform expression during myopia development, the levels of the three isoforms remained within normal ranges for retinal (TGF-beta1, -14 to +14%; TGF-beta2, -2 to +20%; TGF-beta3, -10 to +26%) and choroidal (TGF-beta1, -19 to +21%; TGF-beta2, -26 to +8%; TGF-beta3, -11 to +28%) tissues during myopia development (induction times of 3h, 7h, 11h, 24h, and 5 days). A 40% decrease in retinal TGF-beta2 activation was observed after 5 days of myopia development, however, there was no functional correlate of altered TGF-beta2 activity, as assessed by the retinal ERG response. Overall, these data highlight

  6. Mechanism of activation and functional role of protein kinase Ceta in human platelets.

    PubMed

    Bynagari, Yamini S; Nagy, Bela; Tuluc, Florin; Bhavaraju, Kamala; Kim, Soochong; Vijayan, K Vinod; Kunapuli, Satya P

    2009-05-15

    The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation. PMID:19286657

  7. Recent cyanobacterial Kai protein structures suggest a rotary clock.

    PubMed

    Wang, Jimin

    2005-05-01

    The cyanobacterial circadian oscillator consists of three Kai proteins, KaiA, KaiB, and KaiC, in its oscillation feedback loop. Structural comparison reveals that the Kai system resembles the F1-ATPase system in which KaiC is equivalent to alpha(3)beta(3), KaiA to gammadelta, and KaiB to its inhibitory factor. It also suggests that there exists a possible haemagglutinin-like spring-loaded mechanism for the activation of KaiA during the formation of Kai complexes. PMID:15893664

  8. [Ureter drugs].

    PubMed

    Raynal, G; Bellan, J; Saint, F; Tillou, X; Petit, J

    2008-03-01

    Many improvements have been made recently in the field of the ureteral smooth muscle pharmacology. After a brief summary on physiological basis, we review what is known about effects on ureter of different drugs class. In a second part, we review clinical applications for renal colic analgesia, calculi expulsive medical therapy, ESWL adjuvant treatment and preoperative treatment before retrograde access. There are now sufficient data on NSAID and alpha-blockers. beta-agonists, especially for beta3 selective ones, and topical drugs before retrograde access are interesting and should be further evaluated. PMID:18472067

  9. Production of amino acids by analog-resistant mutants of the cyanobacterium Spirulina platensis.

    PubMed Central

    Riccardi, G; Sora, S; Ciferri, O

    1981-01-01

    Mutants of Spirulina platensis resistant to 5-fluorotryptophan, beta-3-thienyl-alanine, ethionine, p-fluorophenylalanine, or azetidine-2-carboxylic acid were isolated. Some of these mutants appeared to be resistant to more than one analog and to overproduce the corresponding amino acids. A second group was composed of mutants that were resistant to one analog only. Of the latter mutants, one resistant to azetidine-2-carboxylic acid was found to overproduce proline only, whereas one resistant to fluorotryptophan and one resistant to ethionine did not overproduce any of the tested amino acids. PMID:6792182

  10. Hemoglobin D-beta (0) thalassemia. A case report and family study.

    PubMed

    Dawod, S T; Abulaban, M; Kamel, K; Huisman, T H

    1988-01-01

    Findings are presented on a Pakistani family in Doha, Qatar with hemoglobin D-Los Angeles (alpha 2 beta 3 121 (glutamine----glycine) and thalassemia trait. The propositus, a child, has hemoglobin D-beta(0) thalassemia, and suffers from moderately severe hemolytic anemia. The father has beta-thalassemia trait, and the mother is heterozygous for hemoglobin D-Los Angeles. This, the eighth confirmed case in the medical literature, is reported to emphasize its clinical manifestations and genetic basis. PMID:3239708

  11. Folding of proteins with diverse folds.

    PubMed

    Mohanty, Sandipan; Hansmann, Ulrich H E

    2006-11-15

    Using parallel tempering simulations with high statistics, we investigate the folding and thermodynamic properties of three small proteins with distinct native folds: the all-helical 1RIJ, the all-sheet beta3s, and BBA5, which has a mixed helix-sheet fold. In all three cases, simulations with our energy function find the native structures as global minima in free energy at experimentally relevant temperatures. However, the folding process strongly differs for the three molecules, indicating that the folding mechanism is correlated with the form of the native structure. PMID:16950845

  12. Streptococcus sanguis-induced platelet activation involves two waves of tyrosine phosphorylation mediated by FcgammaRIIA and alphaIIbbeta3.

    PubMed

    Pampolina, Caroline; McNicol, Archibald

    2005-05-01

    The low-affinity IgG receptor, FcgammaRIIA, has been implicated in Streptococcus sanguis-induced platelet aggregation. Therefore, it is likely that signal transduction is at least partly mediated by FcgammaRIIA activation and a tyrosine kinase-dependent pathway. In this study the signal transduction mechanisms associated with platelet activation in response to the oral bacterium, S. sanguis were characterised. In the presence of IgG, S. sanguis strain 2017-78 caused the tyrosine phosphorylation of FcgammaRIIA 30s following stimulation, which led to the phosphorylation of Syk, LAT, and PLCgamma2. These early events were dependent on Src family kinases but independent of either TxA(2) or the engagement of the alpha(IIb)beta(3) integrin. During the lag phase prior to platelet aggregation, FcgammaRIIA, Syk, LAT, and PLCgamma2 were each dephosphorylated, but were re-phosphorylated as aggregation occurred. Platelet stimulation by 2017-78 also induced the tyrosine phosphorylation of PECAM-1, an ITIM-containing receptor that recruits protein tyrosine phosphatases. PECAM-1 co-precipitated with the protein tyrosine phosphatase SHP-1 in the lag phase. SHP-1 was also maximally tyrosine phosphorylated during this phase, suggesting a possible role for SHP-1 in the observed dephosphorylation events. As aggregation occurred, SHP-1 was dephosphorylated, while FcgammaRIIA, Syk, LAT, and PLCgamma2 were rephosphorylated in an RGDS-sensitive, and therefore alpha(IIb)beta(3)-dependent, manner. Additionally, TxA(2) release, 5-hydroxytryptamine secretion and phosphatidic acid formation were all blocked by RGDS. Aspirin also abolished these events, but only partially inhibited alpha(IIb)beta(3) -mediated re-phosphorylation. Therefore, S. sanguis -bound IgG cross links FcgammaRIIA and initiates a signaling pathway that is down-regulated by PECAM-1-bound SHP-1. Subsequent engagement of alpha(IIb)beta(3) leads to SHP-1 dephosphorylation permiting a second wave of signaling leading to TxA(2

  13. Update on medical therapy for male LUTS

    PubMed Central

    Radomski, Sidney B.

    2014-01-01

    The medical management of lower urinary tract symptoms (LUTS) is aimed at addressing voiding and storage symptoms in patients with benign prostate hyperplasia (BPH) symptoms with or without an over-active bladder (OAB). Current available options for BPH include alpha-blockers, 5-alpha reductase inhibitors and phosphodiesterase type 5 inhibitors. For OAB, options include antimuscarinics, with or without an alpha-blocker, the beta-3-adrenergic agonist mirabegron and the synthetic diuretic desmopressin. With the availability of numerous options and combinations available for the treatment of LUTS, individual patient assessment is the key to optimal symptom control and management of adverse effects. PMID:25243039

  14. Phase I trial of a monoclonal antibody specific for alphavbeta3 integrin (MEDI-522) in patients with advanced malignancies, including an assessment of effect on tumor perfusion.

    PubMed

    McNeel, Douglas G; Eickhoff, Jens; Lee, Fred T; King, David M; Alberti, Dona; Thomas, James P; Friedl, Andreas; Kolesar, Jill; Marnocha, Rebecca; Volkman, Jennifer; Zhang, Jianliang; Hammershaimb, Luz; Zwiebel, James A; Wilding, George

    2005-11-01

    At present, a variety of agents targeting tumor angiogenesis are under clinical investigation as new therapies for patients with cancer. Overexpression of the alpha(v)beta(3) integrin on tumor vasculature has been associated with an aggressive phenotype of several solid tumor types. Murine models have shown that antibodies targeting the alpha(v)beta(3) integrin can affect tumor vasculature and block tumor formation and metastasis. These findings suggest that antibodies directed at alpha(v)beta(3) could be investigated in the treatment of human malignancies. The current phase I dose escalation study evaluated the safety of MEDI-522, a monoclonal antibody specific for the alpha(v)beta(3) integrin, in patients with advanced malignancies. Twenty-five patients with a variety of metastatic solid tumors were treated with MEDI-522 on a weekly basis with doses ranging from 2 to 10 mg/kg/wk. Adverse events were assessed weekly; pharmacokinetic studies were done; and radiographic staging was done every 8 weeks. In addition, dynamic computed tomography imaging was done at baseline and at 8 weeks in patients with suitable target lesions amenable to analysis, to potentially identify the effect of MEDI-522 on tumor perfusion. Treatment was well tolerated, and a maximum tolerated dose was not identified by traditional dose-limiting toxicities. The major adverse events observed were grade 1 and 2 infusion-related reactions (fever, rigors, flushing, injection site reactions, and tachycardia), low-grade constitutional and gastrointestinal symptoms (fatigue, myalgias, and nausea), and asymptomatic hypophosphatemia. Dynamic computed tomography imaging suggested a possible effect on tumor perfusion with an increase in contrast mean transit time from baseline to the 8-week evaluation with increasing doses of MEDI-522. No complete or partial responses were observed. Three patients with metastatic renal cell cancer experienced prolonged stable disease (34 weeks, >1 and >2 years) on

  15. Modulation of the Na,K-pump function by beta subunit isoforms

    PubMed Central

    1994-01-01

    To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K- pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+. PMID:8057080

  16. Comparison between the in vivo rate of metabolism of prostaglandin I2 and its blood-pressure-lowering response after intravenous administration in the rat.

    PubMed

    Pace-Asciak, C R; Rosenthal, A; Domazet, Z

    1979-07-27

    Intravenous bolus injection of prostaglandin I2 in the Inactin-anaesthetised rat produces a slow dose-dependant vasodepression which reaches maximum approximately 15 s. after injection. Administration of 9 beta-[3H1]-prostaglandin I2 by the same route followed by serial arterial sampling and TLC analysis revealed a slow conversion into one less polar metabolite starting after 20 s and reaching 40% by two minutes in the circulation. These experiments indicate that prostaglandin I2 survives pulmonary transit for a sufficiently long time to elicit a biological action. Thus its continuous systemic vascular synthesis could play an important role in the control of hypertension. PMID:383157

  17. Down-regulation of phospholipase C-beta1 following chronic muscarinic receptor activation.

    PubMed

    Sorensen, S D; Linseman, D A; Fisher, S K

    1998-04-01

    To determine whether prolonged activation of a phospholipase C-coupled receptor can lead to a down-regulation of its effector enzyme, SH-SY5Y neuroblastoma cells were incubated for 24 h with the muscarinic receptor agonist, oxotremorine-M. Under these conditions, significant reductions (46-53%) in muscarinic cholinergic receptor density, G(alphaq/11) and phospholipase C-beta1 (but not the beta3-or gamma1 isoforms) were observed. These results suggest that a selective down-regulation of phospholipase C-beta1 may play a role in adaptation to chronic muscarinic receptor activation. PMID:9617763

  18. TECNOLOGÍAS DE INFORMACIÓN Y COMUNICACIÓN PARA LA PREVENCIÓN Y CONTROL DE LA INFECCIÓN POR EL VIH Y OTRAS ITS*

    PubMed Central

    Curioso, Walter H.; Blas, Magaly M.; Kurth, Ann E.; Klausner, Jeffrey D.

    2010-01-01

    Avances tecnológicos innovadores como Internet, computadoras personales de bolsillo, teléfonos celulares y otros equipos son un arsenal en crecimiento en el esfuerzo de impedir y controlar el VIH y otras infecciones de transmisión sexual (ITS). A pesar que existe una diversidad de tecnologías de información y comunicación en diferentes etapas de desarrollo para la prevención del VIH e ITS, la investigación en esta área se encuentra aún en crecimiento, y el impacto en la incidencia de enfermedad, las evaluaciones con diseños rigurosos y los estudios económicos todavía son muy limitados. Sin embargo, algunas de estas evidencias son prometedoras y poseen un gran potencial para su uso en nuestro medio. En este artículo hemos realizado una revisión sistemática de la literatura relacionada con el uso de la tecnología aplicada a la prevención y control del VIH e ITS. De ser usada apropiadamente, esta tecnología podría mejorar la vigilancia del VIH y otras ITS, diagnóstico, notificación de parejas, prevención, manejo clínico, y capacitación de profesionales de la salud. PMID:26339254

  19. La guerra de los Estados Unidos contra la inmigración. Efectos paradójicos1

    PubMed Central

    Massey, Douglas S.; Pren, Karen A.

    2016-01-01

    Resumen A finales de la década de los cincuenta, Estados Unidos permitía la entrada de aproximadamente medio millón de inmigrantes mexicanos al año, de los cuales 450.000 entraban con visados de trabajo temporal y 50.000 llegaban con visados de residentes permanentes. A mediados de los años sesenta, los cambios en la política migratoria de Estados Unidos realizados en nombre de los derechos civiles redujeron drásticamente las oportunidades de entrada legal a Estados Unidos. Se eliminaron los visados de trabajo temporal y se limitaron los visados de residentes a 20.000 por año. Con las oportunidades de entrada legal restringidas, los flujos migratorios ya establecidos simplemente continuaron, fuera de los límites legales, dando comienzo a una inesperada reacción en cadena de eventos que culminaron en una guerra total contra los inmigrantes y el rápido crecimiento -sin precedentes- de población residente no autorizada en Estados Unidos. El presente artículo demuestra que el aumento de inmigración indocumentada en los Estados Unidos y el crecimiento de la población sin papeles son un producto de políticas migratorias y fronterizas mal concebidas. PMID:27076695

  20. CHOQUES AGREGADOS E INVERSIÓN EN CAPITAL HUMANO: EL LOGRO EDUCATIVO SUPERIOR DURANTE LA DÉCADA PERDIDA EN MÉXICO

    PubMed Central

    Peña, Pablo A.

    2014-01-01

    Este artículo documenta una respuesta agregada negativa del logro educativo superior (más de 12 años de escolaridad) en México a la recesión de 1982–83 y el estancamiento que le siguió. La respuesta no fue homogénea entre géneros, regiones y entornos familiares. Los hombres experimentaron una caída en el logro mientras que las mujeres experimentaron un crecimiento más lento. En promedio, los estados con un mayor logro antes del choque experimentaron mayores caídas. La respuesta entre distintos entornos familiares no presenta un patrón claro. Sin embargo, el efecto negativo en el logro se observa incluso entre hermanos. La evidencia sugiere una historia por el lado de la demanda: la caída en el ingreso de los hogares parece ser el determinante de la caída/desaceleración del logro educativo superior. La conclusión es que la recesión y la falta de crecimiento que le siguió tuvieron un efecto negativo importante y duradero en la formación de capacidades en México. PMID:25328251

  1. Osteopontin stimulates gelsolin-associated phosphoinositide levels and phosphatidylinositol triphosphate-hydroxyl kinase.

    PubMed Central

    Chellaiah, M; Hruska, K

    1996-01-01

    Based on previous studies demonstrating activation of phosphatidylinositol 3-hydroxyl kinase (PI3-kinase) and stimulation of a change in cell shape, we examined the effect of osteopontin on the association of phospholipids with gelsolin, an actin-capping/severing protein. Osteopontin stimulated a rapid increase in phosphatidylinositol bisphosphate and phosphatidylinositol triphosphate levels associated with gelsolin in Triton-soluble fractions of cell lysates. The increased levels of phosphatidylinositol triphosphate associated with gelsolin were due to stimulation of PI3-kinase activity associated with gelsolin in the Triton-soluble fractions, and they were blocked by the PI3-kinase inhibitor wortmannin. Osteopontin stimulated translocation of PI3-kinase from the Triton-insoluble to Triton-soluble gelsolin. Osteopontin also decreased Triton-soluble gelsolin/actin complexes consistent with actin uncapping, and increased F-actin levels, which were also blocked by wortmannin. The osteopontin effects were mediated through binding to the alpha(v)beta 3 integrin. Taken together, our studies indicate that osteopontin/alpha(v)beta 3-mediated changes in gelsolin-associated phosphoinositide levels and PI3-kinase activity are related to stimulation of F-actin formation in osteoclasts. Images PMID:8744948

  2. Quality analysis of in vivo near-infrared fluorescence and conventional gamma images acquired using a dual-labeled tumor-targeting probe.

    PubMed

    Houston, Jessica P; Ke, Shi; Wang, Wei; Li, Chun; Sevick-Muraca, Eva M

    2005-01-01

    The cyclic peptide, cyclopentapeptide cyclo(lys-Arg-Gly-Asp-phe) (c(KRGDf)), which is known to target alpha(v)beta3 integrin, is dual-labeled with a radiotracer, (111)indium, for gamma scintigraphy as well as with a near-infrared dye, IRDye800, for continuous-wave (cw) imaging of alpha(v)beta3 positive human M21 melanoma in xenografts. Twenty-four hours after administration of the dual-labeled peptide at a dose equivalent to 90 microCi of (111)In and 5 nmol of near-infrared (NIR) dye, whole-body gamma scintigraphy and cw imaging was conducted. Image acquisition time was 15 min for the gamma scintigraphy images and 800 ms for the optical images acquired using an NIR sensitive intensified charge-coupled device. The results show that while the target-to-background ratio (TBR) of nuclear and optical imaging were similar for surface regions of interest and consistent with the origin of gamma and NIR radiation from a common targeted peptide, the signal-to-noise ratio (SNR) was significantly higher for optical than nuclear imaging. Furthermore, an analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for the subcutaneous tumor targets. While tomographic reconstructions are necessary to probe TBR, SNR, and contrast for interior tissues, this work demonstrates for the first time the direct comparison of molecular optical and planar nuclear imaging for surface and subsurface cancers. PMID:16292970

  3. Stability constants determination of successive metal complexes by hyphenated CE-ICPMS.

    PubMed

    Petit, Jeremy; Aupiais, Jean; Topin, Sylvain; Geertsen, Valérie; Beaucaire, Catherine; Stambouli, Moncef

    2010-01-01

    The study of radionuclides speciation requires accurate evaluation of stability constants, which can be achieved by CE-ICPMS. We have previously described a method for 1:1 metal complexes stability constants determination. In this paper, we present its extension to the case of successive complexations and its application to uranyl-oxalate and lanthanum-oxalate systems. Several significant steps are discussed: analytical conditions choice, mathematical treatment by non-linear regression, ligand concentration and ionic strength corrections. The following values were obtained: at infinite dilution, log(beta(1) degrees (UO(2)Oxa))=6.93+/-0.05, log(beta(2) degrees (UO(2)(Oxa)(2) (2-)))=11.92+/-0.43 and log(beta(3) degrees (UO(2)(Oxa)(3) (4-)))=15.11+/-0.12; log(beta(1) degrees (LaOxa(+)))=5.90+/-0.07, log(beta(2) degrees (La(Oxa)(2) (-)))=9.18+/-0.19 and log(beta(3) degrees (La(Oxa)(3) (3-)))=9.81+/-0.33. These values are in good agreement with the literature data, even though we suggest the existence of a new lanthanum-oxalate complex: La(Oxa)(3) (3-). This study confirms the suitability of CE-ICPMS for complexation studies. PMID:20084632

  4. Quantitative analysis of laminin 5 gene expression in human keratinocytes.

    PubMed

    Akutsu, Nobuko; Amano, Satoshi; Nishiyama, Toshio

    2005-05-01

    To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms. PMID:15854126

  5. Assessing the influence of electrostatic schemes on molecular dynamics simulations of secondary structure forming peptides

    NASA Astrophysics Data System (ADS)

    Monticelli, Luca; Simões, Carlos; Belvisi, Laura; Colombo, Giorgio

    2006-04-01

    Electrostatic interactions play a fundamental role in determining the structure and dynamics of biomolecules in solution. However the accurate representation of electrostatics in classical mechanics based simulation approaches such as molecular dynamics (MD) is a challenging task. Given the growing importance that MD simulation methods are taking on in the study of protein folding, protein stability and dynamics, and in structure prediction and design projects, it is important to evaluate the influence that different electrostatic schemes have on the results of MD simulations. In this paper we performed long timescale simulations (500 ns) of two peptides, beta3 and RN24 forming different secondary structures, using for each peptide four different electrostatic schemes (namely PME, reaction field correction, and cut-off schemes with and without neutralizing counterions) for a total of eight 500 ns long MD runs. The structural and conformational features of each peptide under the different conditions were evaluated in terms of the time dependence of the flexibility, secondary structure evolution, hydrogen-bonding patterns, and several other structural parameters. The degree of sampling for each simulation as a function of the electrostatic scheme was also critically evaluated. Our results suggest that, while in the case of the short peptide RN24 the performances of the four methods are comparable, PME and RF schemes perform better in maintaining the structure close to the native one for the β-sheet peptide beta3, in which long range contacts are mostly responsible for the definition of the native structure.

  6. Isolation and characterization of testis-specific cDNAs for luteinizing hormone beta-subunit in the rat.

    PubMed

    Zhang, F P; Rannikko, A; Huhtaniemi, I

    1995-05-25

    To study further the unexpected expression of the luteinizing hormone (LH) beta-subunit in the rat testis, we identified in a rat testicular cDNA library three LH beta clones with lengths of 3.2, 2.4 and 0.86 kb (TLH beta 1, TLH beta 2 and TLH beta 3). The clones were identified using a 32P-labeled cDNA probe complimentary to the known rat pituitary LH beta mRNA. Clone TLH beta 2 corresponds in size to the main LH beta mRNA species (2.7 kb) detected by Northern hybridization in the rat testis. Sequence analysis indicated that the different sizes of the three clones are due to alternative RNA splicing and differences at the 5' ends of transcripts. The sequence of one open reading frame deduced from TLH beta 1 is almost identical with the pituitary LH beta peptide, differing only in three amino acids in the putative signal peptide. It might encode a functional testis-specific LH beta peptide. Shorter transcripts from clones TLH beta 2 and TLH beta 3 may correspond to short testicular LH beta peptides. The present findings provide further evidence in the rat for expression of testis-specific mRNA variants of the LH beta gene. Their translation products may form a novel class of testicular para/autocrine factors. PMID:7763258

  7. EGF AND TGF-{alpha} motogenic activities are mediated by the EGF receptor via distinct matrix-dependent mechanisms

    SciTech Connect

    Ellis, Ian R.; Schor, Ana M.; Schor, Seth L. . E-mail: s.l.schor@dundee.ac.uk

    2007-02-15

    EGF and TGF-{alpha} induce an equipotent stimulation of fibroblast migration and proliferation. In spite of their homologous structure and ligation by the same receptor (EGFR), we report that their respective motogenic activities are mediated by different signal transduction intermediates, with p70{sup S6K} participating in EGF signalling and phospholipase C{gamma} in TGF-{alpha} signalling. We additionally demonstrate that EGF and TGF-{alpha} motogenic activities may be resolved into two stages: (a) cell 'activation' by a transient exposure to either cytokine, and (b) the subsequent 'manifestation' of an enhanced migratory phenotype in the absence of cytokine. The cell activation and manifestation stages for each cytokine are mediated by distinct matrix-dependent mechanisms: motogenetic activation by EGF requires the concomitant functionality of EGFR and the hyaluronan receptor CD44, whereas activation by TGF-{alpha} requires EGFR and integrin {alpha}v{beta}3. Manifestation of elevated migration no longer requires the continued presence of exogenous cytokine and functional EGFR but does require the above mentioned matrix receptors, as well as their respective ligands, i.e., hyaluronan in the case of EGF, and vitronectin in the case of TGF-{alpha}. In contrast, the mitogenic activities of EGF and TGF-{alpha} are independent of CD44 and {alpha}v{beta}3 functionality. These results demonstrate clear qualitative differences between EGF and TGF-{alpha} pathways and highlight the importance of the extracellular matrix in regulating cytokine bioactivity.

  8. Melanoma-associated expression of transforming growth factor-beta isoforms.

    PubMed Central

    Van Belle, P.; Rodeck, U.; Nuamah, I.; Halpern, A. C.; Elder, D. E.

    1996-01-01

    Melanocytic neoplasia is characterized by the aberrant overproduction of multiple cytokines in vitro. However, it is largely unknown how cytokine expression relates to melanoma progression in vivo. Transforming growth factor beta (TGF-beta) is a multifunctional cytokine commonly produced by cultured melanoma cells. The in situ expression of all three TGF-beta isoforms (TGF-beta1, -2, and -3) was determined immunohistochemically in melanocytes and in 51 melanocytic lesions using isoform-specific antibodies. Significant linear trends of expression were observed from melanocytes through nevi and primary and metastatic melanomas for all three isoforms. TGF-beta1 was expressed by some melanocytes and almost uniformly by nevi and melanomas. TGF-beta2 and -3 were not expressed in normal melanocytes but were expressed in nevi and early and advanced primary (radial and vertical growth phase) and metastatic melanomas in a tumor-progression-related manner. TGF-beta2 was heterogeneously expressed in advanced primary and metastatic melanomas, whereas TGF-beta3 was uniformly and highly expressed in these lesions. Thus, expression of TGF-beta isoforms in melanocytes and melanocytic lesions is heterogeneous and related to tumor progression, and expression of TGF-beta2 and TGF-beta3 commences at critical junctures during progression of nevi to primary melanomas. Images Figure 2 Figure 3 PMID:8669474

  9. Mass spectral study of hybrid peptides derived from (R)-aminoxy ester and [beta]-amino acids: The influence of aminoxy peptide bond (CO-NH-O) on peptide fragmentation under electrospray ionization conditions

    NASA Astrophysics Data System (ADS)

    Ramesh, V.; Ramesh, M.; Srinivas, R.; Sharma, G. V. M.; Manohar, V.

    2009-04-01

    A new class of Boc-protected aminoxy hybrid peptides containing repeats of [beta]-hAla-(R)-Ama-, and [beta]-Caa-(R)-Ama- ([beta]-hAla = [beta]3-(S)-hAlanine, (R)-Ama = (R)-aminoxy ester, and [beta]-Caa = (R)-C-linked carbo-[beta]3-amino acid) have been studied by electrospray ionization (ESI) ion-trap and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) of their protonated, cationized, and negative ions. MS3 CID of protonated aminoxy peptides of [beta]-hAla-(R)-Ama- yield intense [beta]-amino acid characteristic retro-Mannich fragmentation. The bn+ and [bn-methyl imine]+ (n = 3, 5) ions formed by cleavage of aminoxy peptide bond (CO-NH-O) are more intense than bn+ (n = 2, 4) formed by that of peptide bond (CO-NH-C) cleavage. Another characteristic ion observed is due to loss of H3NO from yn+ ions. The cationized (Li+, and Na+) peptides dissociate differently compared to protonated peptides. Intense cationized cn and zn ions are formed due to the cleavage of N-O bond. The deprotonated peptides also show abundant cn- and zn- ions (n = 1, 3, 5) and do not form any yn- ions. All these results clearly indicate the influence of aminoxy peptide bond on fragmentation of these hybrid peptides.

  10. Properties of an entropy-based signal receiver with an application to ultrasonic molecular imaging.

    PubMed

    Hughes, M S; McCarthy, J E; Marsh, J N; Arbeit, J M; Neumann, R G; Fuhrhop, R W; Wallace, K D; Znidersic, D R; Maurizi, B N; Baldwin, S L; Lanza, G M; Wickline, S A

    2007-06-01

    Qualitative and quantitative properties of the finite part, H(f), of the Shannon entropy of a continuous waveform f(t) in the continuum limit are derived in order to illuminate its use for waveform characterization. Simple upper and lower bounds on H(f), based on features of f(t), are defined. Quantitative criteria for a priori estimation of the average-case variation of H(f) and log E(f), where E(f) is the signal energy of f(t) are also derived. These provide relative sensitivity estimates that could be used to prospectively choose optimal imaging strategies in real-time ultrasonic imaging machines, where system bandwidth is often pushed to its limits. To demonstrate the utility of these sensitivity relations for this application, a study designed to assess the feasibility of identification of angiogenic neovasculature targeted with perfluorocarbon nanoparticles that specifically bind to alpha(v)beta3-integrin expression in tumors was performed. The outcome of this study agrees with the prospective sensitivity estimates that were used for the two receivers. Moreover, these data demonstrate the ability of entropy-based signal receivers when used in conjunction with targeted nanoparticles to elucidate the presence of alpha(v)beta3 integrins in primordial neovasculature, particularly in acoustically unfavorable environments. PMID:17552706

  11. A novel genotype encoding a single amino acid insertion and five other substitutions between residues 64 and 74 of the HIV-1 reverse transcriptase confers high-level cross-resistance to nucleoside reverse transcriptase inhibitors. Abacavir CNA2007 International Study Group.

    PubMed

    Rakik, A; Ait-Khaled, M; Griffin, P; Thomas, T A; Tisdale, M; Kleim, J P

    1999-10-01

    We investigated HIV-1 reverse transcriptase (RT) polymorphisms of plasma isolates from 98 HIV-1-infected study subjects with >2 years of antiretroviral therapy who were failing their current protease inhibitor (PI)-containing regimen. In 1 patient, we detected a virus with a heavily mutated beta3-beta4 connecting loop of the HIV-1 RT fingers subdomain, consisting of a single aspartate codon insertion between positions 69 and 70 and five additional variations: 64N, K65, K66, 67G, 68Y, T69, Ins D, 70R, W71, R72, K73, 74I. Mutants with the recently described 2-aa insertions between codons 68 and 70 of RT were detected in another 3 patients. Among the four isolates with the 1- or 2-aa insertions, the novel genotype was the most refractory to therapy and displayed the highest level of phenotypic resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Follow-up samples demonstrated that the novel mutant represents a stable genetic rearrangement and that the amino acid insertions can coexist with nonnucleoside analogue reverse transcriptase inhibitors (NNRTI) mutations resulting in phenotypic resistance to both NRTIs and NNRTIs. An increasing number of HIV-1 isolates containing various insertions in the beta3-beta4 hairpin of the HIV-1 RT fingers subdomain appear to emerge after prolonged therapy with different NRTIs, and these polymorphisms can confer multiple drug resistance against NRTIs. PMID:10843527

  12. Formation of a stable triplex incorporating a CG interrupting site by a new WNA derivative containing 3-aminopyrazole as a nucleobase.

    PubMed

    Uchida, Yuko; Taniguchi, Yosuke; Aoki, Eriko; Togo, Mieko; Sasaki, Shigeki

    2008-01-01

    Triplex-forming oligonucleotides (TFOs) bind within the major groove of duplex DNA in a sequence-specific manner, and have attracted much interest as genomic tools. However, as the triplex DNA is formed by the interaction between the TFOs and homopurine/homopyrimidine sequences of the target duplex DNA, the stable triplex formation is prevented by one pyrimidine base in the homopurine strand. Previously, we developed the nucleoside analogues (WNA: W-shaped nucleoside analogues) that furnish an aromatic ring as a stacking part and a nucleobase as a recognition part onto the bicyclic skeleton. Selective recognition of a TA and a CG interrupting site has been achieved by WNA-beta T and WNA-beta C, respectively. In the subsequent study, it was found that the triplex formation by the WNA analogues depend on its neighbouring bases within the TFO. In this paper, we describe the synthesis and the evaluation of the triplex forming ability of WNA-beta 3AP, having 3-aminopyrazole (3AP) as a nucleobase. It is remarkable that the TFO containing the WNA-beta 3AP recognizes the CG interrupting site with high selectivity in the TFO sequence of 3'-GZG-5', in which the previous WNA-beta C did not show the stabilizing effect. PMID:18776291

  13. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.

    PubMed

    Mavilio, Fulvio; Pellegrini, Graziella; Ferrari, Stefano; Di Nunzio, Francesca; Di Iorio, Enzo; Recchia, Alessandra; Maruggi, Giulietta; Ferrari, Giuliana; Provasi, Elena; Bonini, Chiara; Capurro, Sergio; Conti, Andrea; Magnoni, Cristina; Giannetti, Alberto; De Luca, Michele

    2006-12-01

    The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease. PMID:17115047

  14. Platelet adhesion signalling and the regulation of thrombus formation.

    PubMed

    Gibbins, Jonathan M

    2004-07-15

    Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) Ib-V-IX receptor complex, GPVI and integrin alpha(2)beta(1). These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.g. Src, Fyn, Lyn, Syk and Btk), adaptor proteins, phospholipase C and lipid kinases such as phosphoinositide 3-kinase. They are also coupled to an increase in cytosolic calcium levels and protein kinase C activation, leading to the secretion of paracrine/autocrine platelet factors and an increase in integrin receptor affinities. Through the binding of plasma fibrinogen and von Willebrand Factor to integrin alpha(IIb)beta(3), a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPVI and integrins alpha(2)beta(1) and alpha(IIb)beta(3) contribute to the signalling that regulates this process, the individual roles of each are only beginning to be dissected. By contrast, adhesion receptor signalling through platelet endothelial cell adhesion molecule 1 (PECAM-1) is implicated in the inhibition of platelet function and thrombus formation in the healthy circulation. Recent studies indicate that understanding of platelet adhesion signalling mechanisms might enable the development of new strategies to treat and prevent thrombosis. PMID:15252124

  15. P-selectin anchors newly released ultralarge von Willebrand factor multimers to the endothelial cell surface.

    PubMed

    Padilla, Arnoldo; Moake, Joel L; Bernardo, Aubrey; Ball, Chalmette; Wang, Yongtao; Arya, Maneesh; Nolasco, Leticia; Turner, Nancy; Berndt, Michael C; Anvari, Bahman; López, José A; Dong, Jing-Fei

    2004-03-15

    von Willebrand factor (VWF) released from endothelium is ultralarge (UL) and hyperreactive. If released directly into plasma, it can spontaneously aggregate platelets, resulting in systemic thrombosis. This disastrous consequence is prevented by the ADAMTS13 (ADisintegrin and Metalloprotease with ThromboSpondin motif) cleavage of ULVWF into smaller, less active forms. We previously showed that ULVWF, on release, forms extremely long stringlike structures. ADAMTS13 cleaves these strings under flow significantly faster than it does under static conditions. As ULVWF tethering to endothelium is important for its rapid proteolysis, we investigated 2 molecules for their potential to anchor the ULVWF strings: P-selectin and integrin alpha v beta 3. We demonstrated that P-selectin anchors ULVWF to endothelium by several means. First, Chinese hamster ovary (CHO) cells expressing P-selectin specifically adhered to immobilized ULVWF and ULVWF-coated beads to immobilized P-selectin. Second, an anti-VWF antibody coimmunoprecipitates P-selectin from the histamine-activated endothelial cells. Third, P-selectin antibody or soluble P-selectin, but not a alpha v beta 3 antibody, RGDS peptide, or heparin, blocked the formation of ULVWF strings. Fourth, P-selectin expression was in clusters predominantly along the ULVWF strings. Finally, the strength of the minimal ULVWF-P-selectin bond was measured to be 7.2 pN. We, therefore, conclude that P-selectin may anchor ULVWF strings to endothelial cells and facilitate their cleavage by ADAMTS13. PMID:14630802

  16. Allyl m-Trifluoromethyldiazirine Mephobarbital: An Unusually Potent Enantioselective and Photoreactive Barbiturate General Anesthetic

    SciTech Connect

    Savechenkov, Pavel Y.; Zhang, Xi; Chiara, David C.; Stewart, Deirdre S.; Ge, Rile; Zhou, Xiaojuan; Raines, Douglas E.; Cohen, Jonathan B.; Forman, Stuart A.; Miller, Keith W.; Bruzik, Karol S.

    2012-12-10

    We synthesized 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (14), a trifluoromethyldiazirine-containing derivative of general anesthetic mephobarbital, separated the racemic mixture into enantiomers by chiral chromatography, and determined the configuration of the (+)-enantiomer as S by X-ray crystallography. Additionally, we obtained the {sup 3}H-labeled ligand with high specific radioactivity. R-(-)-14 is an order of magnitude more potent than the most potent clinically used barbiturate, thiopental, and its general anesthetic EC{sub 50} approaches those for propofol and etomidate, whereas S-(+)-14 is 10-fold less potent. Furthermore, at concentrations close to its anesthetic potency, R-(-)-14 both potentiated GABA-induced currents and increased the affinity for the agonist muscimol in human {alpha}1{beta}2/3{gamma}2L GABA{sub A} receptors. Finally, R-(-)-14 was found to be an exceptionally efficient photolabeling reagent, incorporating into both {alpha}1 and {beta}3 subunits of human {alpha}1{beta}3 GABAA receptors. These results indicate R-(-)-14 is a functional general anesthetic that is well-suited for identifying barbiturate binding sites on Cys-loop receptors.

  17. Contributions of dysregulated energy metabolism to type 2 diabetes development in NZO/H1Lt mice with polygenic obesity.

    PubMed

    Koza, Robert A; Flurkey, Kevin; Graunke, Dawn M; Braun, Christopher; Pan, Huei-Ju; Reifsnyder, Peter C; Kozak, Leslie P; Leiter, Edward H

    2004-06-01

    New Zealand Obese (NZO) male mice develop a polygenic juvenile-onset obesity and maturity-onset hyperinsulinemia and hyperglycemia (diabesity). Here we report on metabolic and molecular changes associated with the antidiabesity action of CL316,243 (CL), a beta(3)-adrenergic receptor agonist. Dietary CL treatment initiated at weaning reduced the peripubertal rise in body weight and adiposity while promoting growth without suppressing hyperphagia. The changes in adiposity, in turn, suppressed development of hyperinsulinemia, hyperleptinemia, hyperlipidemia, and hyperglycemia. These CL-induced alterations were reflected by decreased adipose tissue mass, increased expression of transcripts for uncoupling protein-1 (UCP-1), peroxisome proliferator-activated receptor alpha (PPARalpha), peroxisome proliferater-activated receptor coactivator-1 (PGC-1), and robust development of brown adipocyte function in white fat. Increased drug-mediated energy dissipation elicited a 1.5 degrees C increase in whole body temperature under conditions of increased food intake but with no change in physical activity. Indirect calorimetry of mice treated with CL showed both increased energy expenditure and a restoration of a prominent diurnal pattern in the respiratory exchange ratio suggesting improved nutrient sensing. Our data suggest that CL promotes increased energy dissipation in white and brown fat depots by augmenting thermogenesis and by metabolic re-partitioning of energy in a diabesity-protective fashion. This is the first report demonstrating the effects of dietary beta(3)-agonist in preventing the onset of diabesity in a polygenic rodent model of type 2 diabetes. PMID:15164332

  18. Modes of heme binding and substrate access for cytochrome P450 CYP74A revealed by crystal structures of allene oxide synthase

    SciTech Connect

    Li, Lenong; Chang, Zhenzhan; Pan, Zhiqiang; Fu, Zheng-Qing; Wang, Xiaoqiang

    2009-01-12

    Cytochrome P450s exist ubiquitously in all organisms and are involved in many biological processes. Allene oxide synthase (AOS) is a P450 enzyme that plays a key role in the biosynthesis of oxylipin jasmonates, which are involved in signal and defense reactions in higher plants. The crystal structures of guayule (Parthenium argentatum) AOS (CYP74A2) and its complex with the substrate analog 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid have been determined. The structures exhibit a classic P450 fold but possess a heme-binding mode with an unusually long heme binding loop and a unique I-helix. The structures also reveal two channels through which substrate and product may access and leave the active site. The entrances are defined by a loop between {beta}3-2 and {beta}3-3. Asn-276 in the substrate binding site may interact with the substrate's hydroperoxy group and play an important role in catalysis, and Lys-282 at the entrance may control substrate access and binding. These studies provide both structural insights into AOS and related P450s and a structural basis to understand the distinct reaction mechanism.

  19. Mapping the binding surface of interleukin-8 complexed with an N-terminal fragment of the type 1 human interleukin-8 receptor.

    PubMed

    Clubb, R T; Omichinski, J G; Clore, G M; Gronenborn, A M

    1994-01-24

    Interleukin-8 and its receptors are key mediators of immune and inflammatory responses. Heteronuclear NMR spectroscopy has been utilized to map the binding surface on interleukin-8 (IL-8) for an N-terminal fragment of the human Type-1 IL-8 receptor. A peptide corresponding to residues 1-40 of the IL-8 type 1 receptor (IL8-r1) was titrated into a sample of uniformly 15N-labeled IL-8. IL8-r1 binds to IL-8 with a dissociation constant of 170 +/- 50 microM assuming the peptide binds with a stoichiometry of one peptide per IL-8 monomer, exchanges rapidly (> 900 s-1) between free and bound states, and selectively perturbs the chemical environment of several IL-8 residues. The binding surface on IL-8 suggested by our results is comprised of residues in strand beta 3 of the beta-sheet (Glu48 to Cys50), the turn preceding beta 3 (Ser44), the C-terminal alpha-helix (Val61) and the irregular N-terminal loop region (Thr12, Lys15, Phe17, His18, Lys20 and Phe21). The IL-8 dimer appears to present two symmetrical binding surfaces for the IL8-r1 peptide, suggesting two receptor peptides may bind per dimer. PMID:8307164

  20. Enhanced expression of TGF-betas and their receptors in human acute pancreatitis.

    PubMed Central

    Friess, H; Lu, Z; Riesle, E; Uhl, W; Bründler, A M; Horvath, L; Gold, L I; Korc, M; Büchler, M W

    1998-01-01

    OBJECTIVES: To determine which mechanisms are involved in pancreatic remodeling, repair, and fibrosis after acute necrotizing pancreatitis (NP) in humans. SUMMARY BACKGROUND DATA: Transforming growth factor betas (TGF-betas) are multifunctional polypeptides that have been implicated in the regulation and formation of extracellular matrix and fibrosis. They exert their functions by binding to specific receptors. In this study, we analyze the expression of TGF-beta1, TGF-beta2, and TGF-beta3 and their receptors type I (Tbeta-RI [ALK5]), type II (Tbeta-RII), and type III (Tbeta-RIII) in NP. PATIENTS: Pancreatic tissue samples were obtained from 6 female and 8 male patients with a median age of 65 years (range, 37 to 77 years) undergoing surgery for NP. The median Ranson score of the patients was 6 (range, 2 to 9). The operation was performed a median 5.5 days (range, 4 to 17 days) after the onset of acute pancreatitis. Pancreatic tissue obtained from 12 previously healthy organ donors (6 male, 6 female; median age of 43 years) served as controls. METHODS: The expression of TGF-beta1, TGF-beta2, TGF-beta3, Tbeta-RI (ALK5), Tbeta-RII, Tbeta-RIII, and collagen type I mRNA was analyzed by Northern blot analysis. In addition, immunohistochemical analysis using polyclonal antibodies was performed to detect TGF-beta1, TGF-beta2, TGF-beta3, Tbeta-RI (ALK5), and Tbeta-RII. RESULTS: Northern blot analysis showed an increase in TGF-betas and their receptors in NP tissue samples compared with samples from normal controls. The increase was 3.5-fold for TGF-beta1 (p < 0.05), 2.7-fold for TGF-beta2 (p < 0.05), 3.5-fold for TGF-beta3 (p < 0.05), 10-fold for Tbeta-RI (ALK5) (p < 0.05), 5.7-fold for Tbeta-RII (p < 0.05), and 1.4-fold for Tbeta-RIII (not significant). Collagen type I mRNA was also markedly increased in NP samples and correlated with the level of TGF-betas. Immunohistochemical analysis demonstrated intense TGF-beta1, TGF-beta2, TGF-beta3, Tbeta-RI (ALK5), and Tbeta

  1. GABAA receptor subtypes: the "one glass of wine" receptors.

    PubMed

    Olsen, Richard W; Hanchar, Harry J; Meera, Pratap; Wallner, Martin

    2007-05-01

    This review discusses evidence for and apparent controversy about, gamma-aminobutyric acid type A (GABAA) receptor (GABAAR) subtypes that mediate alcohol effects experienced during social drinking. GABAARs that contain the beta3 and delta subunits were shown to be enhanced by alcohol concentrations that mirror the concentration dependence of alcohol responses in humans. A mutation (alpha6R100Q) previously found in alcohol nontolerant rats in the cerebellar GABAAR alpha6 subunit is sufficient for increased alcohol-induced ataxia in rats homozygous for this mutation (alpha6-100QQ) and further increases alcohol sensitivity of tonic GABA currents (mediated by alpha6betadelta receptors) in cerebellar granule cells of alpha6-100QQ rats and in recombinant alpha6R100Qbeta3delta receptors. This provided the first direct evidence that these types of receptors mediate behavioral effects of ethanol. Furthermore, the behavioral alcohol antagonist Ro15-4513 specifically reverses ethanol enhancement on alpha4/6beta3delta receptors. Unexpectedly, native and recombinant alpha4/6beta3delta receptors bind the behavioral alcohol antagonist Ro15-4513 with high affinity and this binding is competitive with EtOH, suggesting a specific and mutually exclusive (competitive) ethanol/Ro15-4513 site, which explains the puzzling activity of Ro15-4513 as a behavioral alcohol antagonist. Our conclusion from these findings is that alcohol/Ro15-4513-sensitive GABAAR subtypes are important alcohol targets and that alcohol at relevant concentrations is more specific than previously thought. In this review, we discuss technical difficulties in expressing recombinant delta subunit-containing receptors in oocytes and mammalian cells that may have contributed to negative results and confusion. Not only because we have reproduced detailed positive results numerous times, and we and many others have built extensively on basic findings, but also because we explain and combine many previously puzzling

  2. Stage-specific expression of integrin alphaVbeta3 in neuroblastic tumors.

    PubMed Central

    Gladson, C. L.; Hancock, S.; Arnold, M. M.; Faye-Petersen, O. M.; Castleberry, R. P.; Kelly, D. R.

    1996-01-01

    The ligand specificity of the integrin cell adhesion receptors probably determines the ability of specific integrins to promote tumor cell proliferation and metastasis. Therefore, we compared the expression of integrin alphaVbeta3, a promiscuous receptor that binds with high affinity to numerous cell matrix proteins, with the expression of integrin alphaVbeta5 and the integrin beta 1 subunit (which pairs with multiple alpha subunits) in neuroblastic tumors at various stages of differentiation. Undifferentiated neuroblastoma tumors rapidly invade and metastasize, whereas ganglioneuroblastomas rarely metastasize. Differentiating neuroblastomas are associated with an intermediate prognosis. Paraffin sections of neuroblastic tumors at various stages of differentiation obtained at biopsy from 17 patients were hybridized with antisense integrin subunit-specific alphaV, beta3, beta1, and beta5 riboprobes. All neuroblastic tumors and seven adrenal glands obtained at autopsy were analyzed immunohistochemically with antibodies directed toward the alphaV, beta3, beta1, and beta5 subunits. The alphaV subunit was expressed in neuroblastic tumors independent of the stage of differentiation, although mRNA and protein expression were generally weak in ganglioneuroblastomas, and was also detected in adrenal gland medullae. The beta1 subunit was detected in most neuroblastic tumors independent of the stage of differentiation as well as in adrenal gland medullae. In contrast, the beta3 subunit, which was not expressed in adrenal gland medullae, was expressed at the protein and mRNA levels in undifferentiated neuroblastomas (six of seven and seven of seven, respectively) but was not expressed in neuroblasts or ganglion cells in ganglioneuroblastomas (one case weakly positive out of five). The beta 5 subunit was expressed at the protein (five of five) and mRNA (four of five) levels in the ganglion cells of ganglioneuroblastomas and, although mRNA for this subunit was detectable in

  3. Prediction of Treatment Outcome in Patients with Obsessive-Compulsive Disorder with Low-Resolution Brain Electromagnetic Tomography: A Prospective EEG Study.

    PubMed

    Krause, Daniela; Folkerts, Malte; Karch, Susanne; Keeser, Daniel; Chrobok, Agnieszka I; Zaudig, Michael; Hegerl, Ulrich; Juckel, Georg; Pogarell, Oliver

    2015-01-01

    The issue of predicting treatment response and identifying, in advance, which patient will profit from treating obsessive-compulsive disorder (OCD) seems to be an elusive goal. This prospective study investigated brain electric activity [using Low-Resolution Brain Electromagnetic Tomography (LORETA)] for the purpose of predicting response to treatment. Forty-one unmedicated patients with a DSM-IV diagnosis of OCD were included. A resting 32-channel EEG was obtained from each participant before and after 10 weeks of standardized treatment with sertraline and behavioral therapy. LORETA was used to localize the sources of brain electrical activity. At week 10, patients were divided into responders and non-responders (according to a reduction of symptom severity >50% on the Y-BOCS). LORETA analysis revealed that at baseline responders showed compared to non-responders a significantly lower brain electric activity within the beta 1 (t = 2.86, p < 0.05), 2 (t = 2.81, p < 0.05), and 3 (t = 2.76, p < 0.05) frequency bands and ROI analysis confirmed a reduced activity in alpha 2 (t = 2.06, p < 0.05) in the anterior cingulate cortex (ACC). When baseline LORETA data were compared to follow-up data, the analysis showed in the responder group a significantly lower brain electrical resting activity in the beta 1 (t = 3.17. p < 0.05) and beta 3 (t = 3.11. p < 0.05) frequency bands and equally for the ROI analysis of the orbitofrontal cortex (OFC) in the alpha 2 (t = 2.15. p < 0.05) frequency band. In the group of non-responders the opposite results were found. In addition, a positive correlation between frequency alpha 2 (rho = 0.40, p = 0.010), beta 3 (rho = 0.42, p = 0.006), delta (rho = 0.33, p = 0.038), theta (rho = 0.34, p = 0.031), alpha 1 (rho = 0.38, p = 0.015), and beta1 (rho = 0.34, p = 0.028) of the OFC and the bands delta (rho = 0.33, p = 0.035), alpha 1 (rho = 0.36, p = 0.019), alpha 2 (rho = 0.34, p = 0.031), and beta 3 (rho = 0.38, p = 0.015) of the ACC with a

  4. Prediction of Treatment Outcome in Patients with Obsessive-Compulsive Disorder with Low-Resolution Brain Electromagnetic Tomography: A Prospective EEG Study

    PubMed Central

    Krause, Daniela; Folkerts, Malte; Karch, Susanne; Keeser, Daniel; Chrobok, Agnieszka I.; Zaudig, Michael; Hegerl, Ulrich; Juckel, Georg; Pogarell, Oliver

    2016-01-01

    The issue of predicting treatment response and identifying, in advance, which patient will profit from treating obsessive-compulsive disorder (OCD) seems to be an elusive goal. This prospective study investigated brain electric activity [using Low-Resolution Brain Electromagnetic Tomography (LORETA)] for the purpose of predicting response to treatment. Forty-one unmedicated patients with a DSM-IV diagnosis of OCD were included. A resting 32-channel EEG was obtained from each participant before and after 10 weeks of standardized treatment with sertraline and behavioral therapy. LORETA was used to localize the sources of brain electrical activity. At week 10, patients were divided into responders and non-responders (according to a reduction of symptom severity >50% on the Y-BOCS). LORETA analysis revealed that at baseline responders showed compared to non-responders a significantly lower brain electric activity within the beta 1 (t = 2.86, p < 0.05), 2 (t = 2.81, p < 0.05), and 3 (t = 2.76, p < 0.05) frequency bands and ROI analysis confirmed a reduced activity in alpha 2 (t = 2.06, p < 0.05) in the anterior cingulate cortex (ACC). When baseline LORETA data were compared to follow-up data, the analysis showed in the responder group a significantly lower brain electrical resting activity in the beta 1 (t = 3.17. p < 0.05) and beta 3 (t = 3.11. p < 0.05) frequency bands and equally for the ROI analysis of the orbitofrontal cortex (OFC) in the alpha 2 (t = 2.15. p < 0.05) frequency band. In the group of non-responders the opposite results were found. In addition, a positive correlation between frequency alpha 2 (rho = 0.40, p = 0.010), beta 3 (rho = 0.42, p = 0.006), delta (rho = 0.33, p = 0.038), theta (rho = 0.34, p = 0.031), alpha 1 (rho = 0.38, p = 0.015), and beta1 (rho = 0.34, p = 0.028) of the OFC and the bands delta (rho = 0.33, p = 0.035), alpha 1 (rho = 0.36, p = 0.019), alpha 2 (rho = 0.34, p = 0.031), and beta 3 (rho = 0.38, p = 0.015) of the ACC with a

  5. Beta-aminopeptidase-catalyzed biotransformations of beta(2)-dipeptides: kinetic resolution and enzymatic coupling.

    PubMed

    Heck, Tobias; Reimer, Artur; Seebach, Dieter; Gardiner, James; Deniau, Gildas; Lukaszuk, Aneta; Kohler, Hans-Peter E; Geueke, Birgit

    2010-05-17

    We have previously shown that the beta-aminopeptidases BapA from Sphingosinicella xenopeptidilytica and DmpA from Ochrobactrum anthropi can catalyze reactions with non-natural beta(3)-peptides and beta(3)-amino acid amides. Here we report that these exceptional enzymes are also able to utilize synthetic dipeptides with N-terminal beta(2)-amino acid residues as substrates under aqueous conditions. The suitability of a beta(2)-peptide as a substrate for BapA or DmpA was strongly dependent on the size of the C(alpha) substituent of the N-terminal beta(2)-amino acid. BapA was shown to convert a diastereomeric mixture of the beta(2)-peptide H-beta(2)hPhe-beta(2)hAla-OH, but did not act on diastereomerically pure beta(2),beta(3)-dipeptides containing an N-terminal beta(2)-homoalanine. In contrast, DmpA was only active with the latter dipeptides as substrates. BapA-catalyzed transformation of the diastereomeric mixture of H-beta(2)hPhe-beta(2)hAla-OH proceeded along two highly S-enantioselective reaction routes, one leading to substrate hydrolysis and the other to the synthesis of coupling products. The synthetic route predominated even at neutral pH. A rise in pH of three log units shifted the synthesis-to-hydrolysis ratio (v(S)/v(H)) further towards peptide formation. Because the equilibrium of the reaction lies on the side of hydrolysis, prolonged incubation resulted in the cleavage of all peptides that carried an N-terminal beta-amino acid of S configuration. After completion of the enzymatic reaction, only the S enantiomer of beta(2)-homophenylalanine was detected (ee>99 % for H-(S)-beta(2)-hPhe-OH, E>500); this confirmed the high enantioselectivity of the reaction. Our findings suggest interesting new applications of the enzymes BapA and DmpA for the production of enantiopure beta(2)-amino acids and the enantioselective coupling of N-terminal beta(2)-amino acids to peptides. PMID:20340152

  6. The expression of transforming growth factor-beta by cultured chick growth plate chondrocytes: differential regulation by 1,25-dihydroxyvitamin D3.

    PubMed

    Farquharson, C; Law, A S; Seawright, E; Burt, D W; Whitehead, C C

    1996-05-01

    1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor-beta (TGF-beta) are both important regulators of chondrocyte growth and differentiation. We report here that 1,25(OH)2D3 differentially regulates the expression of the genes for TGF-beta 1 to -beta 3 and the secretion of the corresponding proteins in cultured chick chondrocytes. Confluent growth plate chondrocytes were serum-deprived and cultured in varying concentrations of 1,25(OH)2D3. Cells were assayed for TGF-beta mRNA and conditioned medium was assayed for TGF-beta activity and isoform composition. Active TGF-beta was only detected in 10(-8) M 1,25(OH)2D3-treated cultures (8.37 ng active TGF-beta/mg protein). There was a significant decrease in total (latent-active) TGF-beta activity in conditioned medium of 10(-12) M (23.4%; P < 0.05) and 10(-10) M (20.7%; P < 0.05) 1,25(OH)2D3-treated cultures but 10(-8) M 1,25(OH)2D3 significantly increased (30.9%; P < 0.01) TGF-beta activity. The amounts of TGF-beta 1, -beta 2 and -beta 3 isoforms produced were similar in control, 10(-10) or 10(-12) M 1,25(OH)2D3-treated cultures but the conditioned medium of 10(-8) M 1,25(OH)2D3-treated cultures contained significantly higher amounts of all three isoforms. Quantification of TGF-beta mRNA demonstrated differential control of TGF-beta gene expression with TGF-beta 1 and -beta 3 mRNA levels reduced by all concentrations of 1,25(OH)2D3 examined (10(-8), 10(-10) and 10(-12) M) whilst TGF-beta 2 mRNA concentrations were elevated. Our results indicated that 1,25(OH)2D3 regulates chick growth plate chondrocyte TGF-beta secretion and mRNA expression in a concentration-dependent and isoform-specific manner. This interaction may be important in the regulation of chondrocyte metabolism and endochondral bone growth. PMID:8708539

  7. GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry.

    PubMed

    June, H L; Harvey, S C; Foster, K L; McKay, P F; Cummings, R; Garcia, M; Mason, D; Grey, C; McCane, S; Williams, L S; Johnson, T B; He, X; Rock, S; Cook, J M

    2001-03-15

    GABA receptors within the mesolimbic circuitry have been proposed to play a role in regulating alcohol-seeking behaviors in the alcohol-preferring (P) rat. However, the precise GABA(A) receptor subunit(s) mediating the reinforcing properties of EtOH remains unknown. We examined the capacity of intrahippocampal infusions of an alpha5 subunit-selective ( approximately 75-fold) benzodiazepine (BDZ) inverse agonist [i.e., RY 023 (RY) (tert-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate)] to alter lever pressing maintained by concurrent presentation of EtOH (10% v/v) and a saccharin solution (0.05% w/v). Bilateral (1.5-20 microgram) and unilateral (0.01-40 microgram) RY dose-dependently reduced EtOH-maintained responding, with saccharin-maintained responding being reduced only with the highest doses (e.g., 20 and 40 microgram). The competitive BDZ antagonist ZK 93426 (ZK) (7 microgram) reversed the RY-induced suppression on EtOH-maintained responding, confirming that the effect was mediated via the BDZ site on the GABA(A) receptor complex. Intrahippocampal modulation of the EtOH-maintained responding was site-specific; no antagonism by RY after intra-accumbens [nucleus accumbens (NACC)] and intraventral tegmental [ventral tegmental area (VTA)] infusions was observed. Because the VTA and NACC contain very high densities of alpha1 and alpha2 subunits, respectively, we determined whether RY exhibited a "negative" or "neutral" pharmacological profile at recombinant alpha1beta3gamma2, alpha2beta3gamma2, and alpha5beta3gamma2 receptors expressed in Xenopus oocytes. RY produced "classic" inverse agonism at all alpha receptor subtypes; thus, a neutral efficacy was not sufficient to explain the failure of RY to alter EtOH responding in the NACC or VTA. The results provide the first demonstration that the alpha5-containing GABA(A) receptors in the hippocampus play an important role in regulating Et

  8. Down-regulation of laminin-5 in breast carcinoma cells.

    PubMed Central

    Martin, K. J.; Kwan, C. P.; Nagasaki, K.; Zhang, X.; O'Hare, M. J.; Kaelin, C. M.; Burgeson, R. E.; Pardee, A. B.; Sager, R.

    1998-01-01

    BACKGROUND: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. MATERIALS AND METHODS: We have studied ln-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. RESULTS: Ln-5 alpha 3, beta 3, and gamma 2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-10A immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln alpha 3 and beta 3 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in gamma 2 levels were elevated in specimens with increased amounts of

  9. Produccion Gaseosa del Cometa Halley: Erupciones Y Fotodisociacion del Radical OH

    NASA Astrophysics Data System (ADS)

    Silva, A. M.; Mirabel, I. F.

    1990-11-01

    RESUMEN:En este trabajo informamos la detecci6n de 20 erupciones en la li'nea de =18cm (1667MHz) del radical OH en el Cometa Halley.Las observaciones incluyen todos los monitoreos existentes y se extienden desde 120 dias antes del perihelio hasta 90 dias despues.Se detectan bruscos crecimientos en el flujo medido,hasta un factor 1O,seguidos por decaimientos lentos asociados con la fotodisociaci6n del OH. Se obtuvieron valores para el tiempo de vida fotoquimico del OH y del H2O basandose en el modelo desarrollado previamente por Silva(1988). Esos tiempos de vida estan de acuerdo con predicciones teoricas y con las observaciones en el Ultravioleta, y los resultados, los que son fuertemente dependientes de la velocidad heliocentrica del Coineta (variando hasta un factor 6), han sido calculados para varios rangos de velocidad entre +28 y -28 km/seg. Key wo'L :

  10. Fragrance contact dermatitis - a worldwide multicenter investigation (Part III).

    PubMed

    Larsen, Walter; Nakayama, Hideo; Fischer, Torkil; Elsner, Peter; Frosch, Peter; Burrows, Desmond; Jordan, William; Shaw, Stephanie; Wilkinson, John; Marks, James; Sugawara, M; Nethercott, Marc; Nethercott, James

    2002-03-01

    The purpose of this study was to determine the frequency of responses to selected fragrance materials in patients who were fragrance sensitive. 218 fragrance sensitive subjects were evaluated in eight centres worldwide with a fragrance mixture (FM) and 17 less well-studied fragrance materials. Reaction to the fragrance mixture (FM) occurred in 76% of the subjects. The (FM) detected all reactions to nerol and hydroxycitronellol and 93% of the reactions to clove bud oil. Ten fragrance materials were not detected by the FM and deserve further study: benzenepropanol, beta, beta, 3-trimethyl, hexyl-salicylate, dl-citronellol, synthetic ylang ylang oil, benzyl mixture, cyclohexyl-acetate, eugenyl methyl ether, isoeugenyl methyl ether, 3-phenyl-1-propanol, and 3, 7-dimethyl-7-methoxyoctan-2-ol. PMID:12000321

  11. Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Type 1: A Light on Molecular Mechanisms

    PubMed Central

    Vanderschuren, Koen L. A.; Sieverink, Tom; Wilders, Ronald

    2013-01-01

    Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited cardiomyopathy associated with cardiac arrhythmias originating in the right ventricle, heart failure, and sudden cardiac death. Development of ARVD/C type 1 has been attributed to differential expression of transforming growth factor beta 3 (TGFβ3). Several mechanisms underlying the molecular basis of ARVD/C type 1 have been proposed. Evaluating previously described mechanisms might elucidate how TGFβ3 contributes to disease progression in ARVD/C type 1. Here we review how TGFβ3 can induce fibrogenesis through Smad and/or β-catenin signaling. Moreover, the role of apoptosis is addressed. Finally the extent to which the immune system has been demonstrated to be a modulating and amplifying agent in the onset and progression of ARVD/C in general is discussed. PMID:24416594

  12. Airborne Coherent Lidar for Advanced In-Flight Measurements (ACLAIM) Flight Testing of the Lidar Sensor

    NASA Technical Reports Server (NTRS)

    Soreide, David C.; Bogue, Rodney K.; Ehernberger, L. J.; Hannon, Stephen M.; Bowdle, David A.

    2000-01-01

    The purpose of the ACLAIM program is ultimately to establish the viability of light detection and ranging (lidar) as a forward-looking sensor for turbulence. The goals of this flight test are to: 1) demonstrate that the ACLAIM lidar system operates reliably in a flight test environment, 2) measure the performance of the lidar as a function of the aerosol backscatter coefficient (beta), 3) use the lidar system to measure atmospheric turbulence and compare these measurements to onboard gust measurements, and 4) make measurements of the aerosol backscatter coefficient, its probability distribution and spatial distribution. The scope of this paper is to briefly describe the ACLAIM system and present examples of ACLAIM operation in flight, including comparisons with independent measurements of wind gusts, gust-induced normal acceleration, and the derived eddy dissipation rate.

  13. [Kinetics of Ca2+, NADH, and oxidized flavoproteins in the frog olfactory lining under the effect of odorants].

    PubMed

    Rudenko, Ia N; Bigdaĭ, E V; Samoĭlov, V O

    2007-01-01

    The kinetics of fluorescence of Ca(2+) - chlortertacyclin-cell membrane complex as well as of NADH and oxidized flavoproteins in receptor cells of the frog olfactory lining under the effect of odorants has been studied. Changes in the fluorescence of the olfactory lining upon stimulation by cineole and vanillin occurred more rapidly than under the effect of camphor and amyl alcohol. Differences in the kinetics of reactions of NADH and the Ca(2+)-CTC-CM complex to different odorants are apparently due to heterogeneity of molecular mechanisms associated with the involvement of different intracellular signal systems in the transduction of these odorants in the olfactory lining. In contrast to them, ammonia and beta3-mercaptoethanol penetrate into olfactory cells and inhibit the mitochondrial respiratory chain without the participation of second messengers. At the same time, the motor activity of olfactory cilia is depressed. PMID:17348402

  14. cDNA sequence of the horse (Equus caballus) LAMA3 gene and characterization of two intronic SNP markers.

    PubMed

    Milenkovic, Dragan; Mata, Xavier; Chadi, Sead; Guérin, Gérard

    2005-12-01

    Laminins are large heterotrimeric basement membrane glycoproteins composed of alpha, beta and gamma chains. The Laminin 5 isoform has an alpha3beta3gamma2 composition and is essential for the adhesion of basal keratinocytes to the underlying epithelial basement membrane where it is mainly located. Mutations in the genes coding for the 3 chains have been associated with a severe skin blistering disease, Herlitz's junctional epidermolysis bullosa (JEB), observed in different species as man, dog, cat and horse. In this study, we report the sequence of the 5.2 kb horse laminin alpha 3 cDNA (LAMA3) as well as the detection of two intronic SNPs. These data will be useful to further identify causal mutations for the disease in this gene. PMID:16287627

  15. A homozygous nonsense mutation in the alpha 3 chain gene of laminin 5 (LAMA3) in Herlitz junctional epidermolysis bullosa: prenatal exclusion in a fetus at risk.

    PubMed

    McGrath, J A; Kivirikko, S; Ciatti, S; Moss, C; Dunnill, G S; Eady, R A; Rodeck, C H; Christiano, A M; Uitto, J

    1995-09-01

    Mutations in the three genes (LAMA3, LAMB3, and LAMC2) that encode the three chains (alpha 3, beta 3, and gamma 2, respectively) of laminin 5, a protein involved in epidermal-dermal adhesion, have been established as the genetic basis for the inherited blistering skin disorder, Herlitz junctional epidermolysis bullosa (H-JEB). In this study, we performed mutational analysis on genomic DNA from a child with H-JEB and identified a nonsense mutation in the alpha 3 chain gene (LAMA3) consisting of a homozygous C-to-T transition resulting in a premature termination codon (CGA-->TGA) on both alleles. The parents were shown to be heterozygous carriers of the same mutation. Direct mutation analysis was used to perform DNA-based prenatal diagnosis from a chorionic villus biopsy at 10 weeks' gestation in a subsequent pregnancy. The fetus was predicted to be genotypically normal with respect to the LAMA3 mutation. PMID:8530087

  16. Partial deletion of the LAMA3 gene is responsible for hereditary junctional epidermolysis bullosa in the American Saddlebred Horse.

    PubMed

    Graves, K T; Henney, P J; Ennis, R B

    2009-02-01

    Laminin 5 is a heterotrimeric basement membrane protein integral to the structure and function of the dermal-epidermal junction. It consists of three glycoprotein subunits: the alpha3, beta3 and gamma2 chains, which are encoded by the LAMA3, LAMB3 and LAMC2 genes respectively. A mutation in any of these genes results in the condition known as hereditary junctional epidermolysis bullosa (JEB). A 6589-bp deletion spanning exons 24-27 was found in the LAMA3 gene in American Saddlebred foals born with the skin-blistering condition epitheliogenesis imperfecta. The deletion confirms that this autosomal recessive condition in the American Saddlebred Horse can indeed be classified as JEB and corresponds to Herlitz JEB in humans. A diagnostic test was developed and nine of 175 randomly selected American Saddlebred foals from the 2007 foal crop were found to be carriers of the mutation (frequency of 0.026). PMID:19016681

  17. [Atypical antipsychotics and metabolic syndrome].

    PubMed

    Baranyi, Andreas; Yazdani, Renè; Haas-Krammer, Alexandra; Stepan, Alexandra; Kapfhammer, Hans-Peter; Rothenhäusler, Hans-Bernd

    2007-01-01

    The introduction of atypical antipsychotics in psychopharmacology represented a major advance in the treatment of psychotic disorders. However, there have been numerous studies that certain atypical antipsychotics may be associated with a greater risk of metabolic abnormalities than others, including weight gain, hyperlipidemia and new-onset typ 2 diabetes mellitus. A G-Protein beta3 subunit Gen (C825T) polymorphism, an increased carbohydrate metabolism and dyshormonism are discussed as pathogenetic mechanisms. High risk patients (adiposity, hyperlipidaemia, hyperglycaemia, preexisting diabetes) should maintain an antipsychotic agent with a favourable side effect profile. In these cases a periodical diabetes screening and blood lipid controls are required. Clinicans must balance the significant benefits of atypical antipsychotics against the risk of metabolic disturbances. In this article recent findings are reviewed. PMID:17915438

  18. Lipid, polyamide, and flavonol phagostimulants for adult western corn rootworm from sunflower (Helianthus annuus L.) pollen.

    PubMed

    Lin, S; Mullin, C A

    1999-03-01

    Adult Diabroticites including western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, consume pollen of corn, squash, sunflower, and other species. Short-chain neutral amino acids in methanol-water extracts of pollen have been previously identified in our laboratory as strong phagostimulants for Diabrotica. Bioassay-driven fractionation was used to characterize the interacting lipid and midpolarity phagostimulants for adult WCR in Giant Gray Stripe sunflower, Helianthus annuus L., pollen. Lipids rich in omega3-linolenic acid including triglycerides, free fatty acids, phosphatidylethanolamines, phosphatidic acids, and phosphatidylcholines were highly phagostimulatory. Other important phagostimulatory components included a hydroxycinnamic acid-polyamine amide, N(1),N(5),N(10)-tri[(E)-p-coumaroyl]spermidine, and a flavonol, quercetin beta-3-O-glucoside. The structural characteristics of these phagoactive compounds and their role in the pollinivory specialization of rootworm beetles are discussed. PMID:10552441

  19. Chlorine adsorption on the InAs (001) surface

    SciTech Connect

    Bakulin, A. V.; Eremeev, S. V.; Tereshchenko, O. E.; Kulkova, S. E.

    2011-01-15

    Chlorine adsorption on the In-stabilized InAs(001) surface with {zeta}-(4 Multiplication-Sign 2) and {beta}3 Prime -(4 Multiplication-Sign 2) reconstructions and on the Ga-stabilized GaAs (001)-{zeta}-(4 Multiplication-Sign 2) surface has been studied within the electron density functional theory. The equilibrium structural parameters of these reconstructions, surface atom positions, bond lengths in dimers, and their changes upon chlorine adsorption are determined. The electronic characteristics of the clean surface and the surface with adsorbed chlorine are calculated. It is shown that the most energetically favorable positions for chlorine adsorption are top positions over dimerized indium or gallium atoms. The mechanism of chlorine binding with In(Ga)-stabilized surface is explained. The interaction of chlorine atoms with dimerized surface atoms weakens surface atom bonds and controls the initial stage of surface etching.

  20. Focally regulated endothelial proliferation and cell death in human synovium.

    PubMed Central

    Walsh, D. A.; Wade, M.; Mapp, P. I.; Blake, D. R.

    1998-01-01

    Angiogenesis and vascular insufficiency each may support the chronic synovial inflammation of rheumatoid arthritis. We have shown by quantitative immunohistochemistry and terminal uridyl deoxynucleotide nick end labeling that endothelial proliferation and cell death indices were each increased in synovia from patients with rheumatoid arthritis compared with osteoarthritic and noninflamed controls, whereas endothelial fractional areas did not differ significantly among disease groups. Markers of proliferation were associated with foci immunoreactive for vascular endothelial growth factor and integrin alpha(v)beta3, whereas cell death was observed in foci in which immunoreactivities for these factors were weak or absent. No association was found with thrombospondin immunoreactivity. The balance between angiogenesis and vascular regression in rheumatoid synovitis may be determined by the focal expression of angiogenic and endothelial survival factors. Increased endothelial cell turnover may contribute to microvascular dysfunction and thereby facilitate persistent synovitis. Images Figure 1 Figure 3 Figure 4 PMID:9502411

  1. ENDF/B-VII.0, ENDF/B-VI, JEFF-3.1, AND JENDL-3.3 RESULTS FOR UNREFLECTED PLUTONIUM SOLUTIONS AND MOX LATTICES (U)

    SciTech Connect

    MOSTELLER, RUSSELL D.

    2007-02-09

    Previous studies have indicated that ENDF/B-VII preliminary releases {beta}-2 and {beta}-3, predecessors to the recent initial release of ENDF/B-VII.0, produce significantly better overall agreement with criticality benchmarks than does ENDF/B-VI. However, one of those studies also suggests that improvements still may be needed for thermal plutonium cross sections. The current study substantiates that concern by examining criticality benchmarks for unreflected spheres of plutonium-nitrate solutions and for slightly and heavily borated mixed-oxide (MOX) lattices. Results are presented for the JEFF-3.1 and JENDL-3.3 nuclear data libraries as well as ENDF/B-VII.0 and ENDF/B-VI. It is shown that ENDF/B-VII.0 tends to overpredict reactivity for thermal plutonium benchmarks over at least a portion of the thermal range. In addition, it is found that additional benchmark data are needed for the deep thermal range.

  2. Search for Supersymmetry Using Final States with One Lepton, Jets, and Missing Transverse Momentum with the ATLAS Detector in {radical}(s)=7 TeV pp Collisions

    SciTech Connect

    Aad, G.; Ahles, F.; Beckingham, M.; Bernhard, R.; Bitenc, U.; Bruneliere, R.; Caron, S.; Carpentieri, C.; Christov, A.; Consorti, V.; Dahlhoff, A.; Dietrich, J.; Eckert, S.; Fehling-Kaschek, M.; Flechl, M.; Glatzer, J.; Hartert, J.; Heldmann, M.; Herten, G.; Horner, S.

    2011-04-01

    This Letter presents the first search for supersymmetry in final states containing one isolated electron or muon, jets, and missing transverse momentum from {radical}(s)=7 TeV proton-proton collisions at the LHC. The data were recorded by the ATLAS experiment during 2010 and correspond to a total integrated luminosity of 35 pb{sup -1}. No excess above the standard model background expectation is observed. Limits are set on the parameters of the minimal supergravity framework, extending previous limits. Within this framework, for A{sub 0}=0 GeV, tan{beta}=3, and {mu}>0 and for equal squark and gluino masses, gluino masses below 700 GeV are excluded at 95% confidence level.

  3. Studies on the phenylethanoid glycosides with anti-complement activity from Paulownia tomentosa var. tomentosa wood.

    PubMed

    Si, Chuan-Ling; Deng, Xiao-Juan; Liu, Zhong; Kim, Jin-Kyu; Bae, Young-Soo

    2008-01-01

    Four epimeric phenylethanoid glycosides, including a new one, R,S-beta-ethoxy-beta-(3,4-dihydroxyphenyl)-ethyl-O-alpha-L-rhamnopyranosyl(1-->3)-beta-D-(6-O-E-caffeoyl)-glucopyranoside named isoilicifolioside A (1), and three known compounds, ilicifolioside A (2), campneoside II (3), and isocampneoside II (4), were isolated from Paulownia tomentosa var. tomentosa wood. The structures of the four compounds were elucidated by the interpretation of 1D and 2D NMR and MS spectra. This is the first report of the chemical profile of this tree. Compounds 1-4 exhibited excellent anti-complement activity with IC(50) values less than 74 microM, compared with tiliroside (IC(50) = 104 microM) and rosmarinic acid (IC(50) = 182 microM) that were used as positive controls. PMID:19031237

  4. An autoinhibitory helix in the C-terminal region of phospholipase C-[beta] mediates G[alpaha subscript q] activation

    SciTech Connect

    Lyon, Angeline M.; Tesmer, Valerie M.; Dhamsania, Vishan D.; Thal, David M.; Gutierrez, Joanne; Chowdhury, Shoaib; Suddala, Krishna C.; Northup, John K.; Tesmer, John J.G.

    2012-03-16

    The enzyme phospholipase C-{beta} (PLC{beta}) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the G{sub q} family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLC{beta} (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLC{beta}3 considerably increase basal activity and diminish stimulation by G{alpha}{sub q}. G{alpha}{sub q} binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLC{beta}.

  5. Mirabegron – a selective β3-adrenoreceptor agonist for the treatment of overactive bladder

    PubMed Central

    Bhide, Alka A; Digesu, G Alessandro; Fernando, Ruwan; Khullar, Vik

    2012-01-01

    Overactive bladder is a common condition that significantly impacts overall quality of life. Antimuscarinics are the current main pharmacological option for treatment; however, many patients fail to adhere to therapy due to troublesome side effects. Mirabegron is a new beta-3 adrenoreceptor agonist which causes detrusor smooth muscle relaxation and has been proposed to be effective for treating overactive bladder symptoms. Mirabegron has been shown to be superior to placebo for reducing the mean number of incontinence episodes per 24 hours and the mean number of micturitions per 24 hours. Side effects such as dry mouth were observed at similar or lower rates than those seen for placebo and antimuscarinics. Higher doses of mirabegron were associated with minor increases in pulse rate and mean blood pressure. Mirabegron offers a new alternative for treating overactive bladder in patients for which antimuscarinics are either not tolerated or not appropriate. PMID:24199179

  6. Rotational partition functions for linear molecules

    NASA Astrophysics Data System (ADS)

    McDowell, Robin S.

    1988-01-01

    An accurate closed-form expression for the rotational partition function of linear polyatomic molecules in 1Sigma electronic states is derived, including the effect of nuclear spin (significant at very low temperatures) and of quartic and sextic centrifugal distortion terms (significant at moderate and high temperatures). The proper first-order quantum correction to the classical rigid-rotator partition function is shown to yield Qr = about 1/beta exp beta/3, where beta is defined as hcB / kT and B is the rotational constant in per cm; for beta of 0.2 or greater additional power-series terms in beta are necessary. Comparison between the results of this treatment and exact summations are made for HCN and C2H2 at temperatures from 2 to 5000 K, including separate evaluation of the conributions of nuclear spin and centrifugal distortion.

  7. A hantavirus causing hemorrhagic fever with renal syndrome requires gC1qR/p32 for efficient cell binding and infection

    SciTech Connect

    Choi, Yun; Kwon, Young-Chan; Kim, Soo-In; Park, Jung-Min; Lee, Kyung-Hee; Ahn, Byung-Yoon

    2008-11-25

    Hantaan virus (HTNV) is a pathogenic hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). HTNV infection is mediated by {alpha}v{beta}3 integrin. We used protein blots of Vero E6 cell homogenates to demonstrate that radiolabeled HTNV virions bind to gC1qR/p32, the acidic 32-kDa protein known as the receptor for the globular head domain of complement C1q. RNAi-mediated suppression of gC1qR/p32 markedly reduced HTNV binding and infection in human lung epithelial A549 cells. Conversely, transient expression of either simian or human gC1qR/p32 rendered non-permissive CHO cells susceptible to HTNV infection. These results suggest an important role for gC1qR/p32 in HTNV infection and pathogenesis.

  8. A novel de novo mutation in LAMB3 causes localized hypoplastic enamel in the molar region.

    PubMed

    Kim, Young-Jae; Shin, Teo J; Hyun, Hong-Keun; Lee, Sang-Hoon; Lee, Zang H; Kim, Jung-Wook

    2016-08-01

    Amelogenesis imperfecta (AI) is a collection of diseases characterized by hereditary enamel defects and is heterogeneous in genetic etiology and clinical phenotype. In this study, we recruited a nuclear AI family with a proband having unique irregular hypoplastic pits and grooves in all surfaces of the deciduous molar teeth but not in the deciduous anterior teeth. Based on the candidate gene approach, we screened the laminin subunit beta 3 (LAMB3) gene and identified a novel de novo mutation in the proband. The mutation was a frameshift mutation caused by a heterozygous 7-bp deletion in the last exon (c.3452_3458delAGAAGCG, p.Glu1151Valfs*57). This study not only expands the mutational spectrum of the LAMB3 gene causing isolated AI but also broadens the understanding of genotype-phenotype correlations. PMID:27220909

  9. Comprehensive profiling analysis of actively resorbing osteoclasts identifies critical signaling pathways regulated by bone substrate

    PubMed Central

    Purdue, P. Edward; Crotti, Tania N.; Shen, Zhenxin; Swantek, Jennifer; Li, Jun; Hill, Jonathan; Hanidu, Adedayo; Dimock, Janice; Nabozny, Gerald; Goldring, Steven R.; McHugh, Kevin P.

    2014-01-01

    As the only cells capable of efficiently resorbing bone, osteoclasts are central mediators of both normal bone remodeling and pathologies associates with excessive bone resorption. However, despite the clear evidence of interplay between osteoclasts and the bone surface in vivo, the role of the bone substrate in regulating osteoclast differentiation and activation at a molecular level has not been fully defined. Here, we present the first comprehensive expression profiles of osteoclasts differentiated on authentic resorbable bone substrates. This analysis has identified numerous critical pathways coordinately regulated by osteoclastogenic cytokines and bone substrate, including the transition from proliferation to differentiation, and sphingosine-1-phosphate signaling. Whilst, as expected, much of this program is dependent upon integrin beta 3, the pre-eminent mediator of osteoclast-bone interaction, a surprisingly significant portion of the bone substrate regulated expression signature is independent of this receptor. Together, these findings identify an important hitherto underappreciated role for bone substrate in osteoclastogenesis. PMID:25534583

  10. Busulfan Triggers Intrinsic Mitochondrial-Dependent Platelet Apoptosis Independent of Platelet Activation.

    PubMed

    Qiao, Jianlin; Wu, Yulu; Liu, Yun; Li, Xiaoqian; Wu, Xiaoqing; Liu, Na; Zhu, Feng; Qi, Kunming; Cheng, Hai; Li, Depeng; Li, Hongchun; Li, Zhenyu; Zeng, Lingyu; Ma, Ping; Xu, Kailin

    2016-09-01

    As a nonspecific alkylating antineoplastic agent, busulfan has been widely used in the treatment of patients with chronic myeloid leukemia. In vitro and in vivo studies demonstrated busulfan-induced cell apoptosis. Whether busulfan triggers platelet apoptosis remains unclear. This study aimed to evaluate the role of busulfan in platelet apoptosis. Isolated human platelets were incubated with busulfan followed by analysis of platelet apoptosis by flow cytometry or western blot, including mitochondrial depolarization, expression of Bcl-2, and Bax and caspase 3 activation. Meanwhile, platelet activation, expression of glycoprotein Ibα (GPIbα), glycoprotein VI (GPVI), and IIb<beta>3 and platelet aggregation in response to collagen and adenosine diphosphate (ADP) were measured. Additionally, busulfan was injected into mice with or without administration of caspase inhibitor QVD-Oph to investigate its effect on platelet lifespan. Our results showed that busulfan-treated platelets displayed increased mitochondrial membrane depolarization, decreased expression of Bcl-2, increased expression of Bax and caspase 3 activation in dose-dependent manner, which were inhibited by QVD-Oph. Platelet activation was not observed in busulfan-treated platelets as showed by no increased P-selectin expression and PAC-1 binding. However, busulfan reduced collagen- or ADP-induced platelet aggregation without affecting expression of GPIbα, GPVI, and IIb<beta>3. Furthermore, busulfan reduced circulating platelet lifespan which was ameliorated by QVD-Oph in mice. In conclusion, busulfan triggers mitochondrial-dependent platelet apoptosis and reduces platelet lifespan in mice. These data suggest targeting caspase activation might be beneficial in the prophylaxis of platelet apoptosis-associated thrombocytopenia after administration of busulfan. PMID:27292166

  11. Cementogenesis and the induction of periodontal tissue regeneration by the osteogenic proteins of the transforming growth factor-beta superfamily.

    PubMed

    Ripamonti, U; Petit, J-C; Teare, J

    2009-04-01

    The antiquity and severity of periodontal diseases are demonstrated by the hard evidence of alveolar bone loss in gnathic remains of the Pliocene/Pleistocene deposits of the Bloubank Valley at Sterkfontein, Swartkrans and Kromdrai in South Africa. Extant Homo has characterized and cloned a superfamily of proteins which include the bone morphogenetic proteins that regulate tooth morphogenesis at different stages of development as temporally and spatially connected events. The induction of cementogenesis, periodontal ligament and alveolar bone regeneration are regulated by the co-ordinated expression of bone morphogenetic proteins. Naturally derived and recombinant human bone morphogenetic proteins induce periodontal tissue regeneration in mammals. Morphological analyses on undecalcified sections cut at 3-6 mum on a series of mandibular molar Class II and III furcation defects induced in the non-human primate Papio ursinus show the induction of cementogenesis. Sharpey's fibers nucleate as a series of composite collagen bundles within the cementoid matrix in close relation to embedded cementocytes. Osteogenic protein-1 and bone morphogenetic protein-2 possess a structure-activity profile, as shown by the morphology of tissue regeneration, preferentially cementogenic and osteogenic, respectively. In Papio ursinus, transforming growth factor-beta(3) also induces cementogenesis, with Sharpey's fibers inserting into newly formed alveolar bone. Capillary sprouting and invasion determine the sequential insertion and alignment of individual collagenic bundles. The addition of responding stem cells prepared by finely mincing fragments of autogenous rectus abdominis muscle significantly enhances the induction of periodontal tissue regeneration when combined with transforming growth factor-beta(3) implanted in Class II and III furcation defects of Papio ursinus. PMID:18842117

  12. Targeted inactivation of murine laminin gamma2-chain gene recapitulates human junctional epidermolysis bullosa.

    PubMed

    Meng, Xianmin; Klement, John F; Leperi, Dominic A; Birk, David E; Sasaki, Takako; Timpl, Rupert; Uitto, Jouni; Pulkkinen, Leena

    2003-10-01

    Junctional forms of epidermolysis bullosa (JEB) are associated with mutations in six distinct genes expressed in the cutaneous basement membrane zone; these include LAMA3, LAMB3, and LAMC2, which encode laminin 5 subunit polypeptides, the alpha3-, beta3-, and gamma2-chains, respectively. Here we generated a mouse model for JEB by inactivating the laminin gamma2-chain gene by targeted frameshift deletion of exon 8 in Lamc2. Heterozygous mice were phenotypically normal, whereas the majority of Lamc2-/- mice showed blistering phenotype on days 1 to 2 and died within 5 days of birth. The Lamc2-/- mice demonstrated absent expression of laminin gamma2-chain on the basement membrane zone as well as attenuated expression of alpha3- and beta3-chains of laminin. Transmission electron microscopy revealed rudimentary, poorly developed hemidesmosomes. The epidermis of the Lamc2-/- mice revealed induced apoptosis in the basal cells of the blistered skin, suggesting that cell-matrix adhesion provided by laminin 5 plays a role in cell survival in vivo. Cultured Lamc2-/- keratinocytes demonstrated slightly positive staining with gamma2-chain-specific antibodies, which could be explained by the presence of a transcript with partial restoration of the reading frame owing to alternative splicing in vitro. These cells proliferated in different matrices and attached to type IV collagen and Matrigel as efficiently as the wild-type keratinocytes, whereas their attachment on plastic and laminin was significantly weaker. In summary, Lamc2-/- mouse recapitulates human JEB and provides novel insight into the role of laminin 5 in keratinocyte biology. PMID:14632187

  13. Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavage of the labeled protein: Identification of an 11. 5-kDa peptide containing the putative 25-hydroxyvitamin D sub 3 binding site

    SciTech Connect

    Ray, R.; Holick, M.F. ); Bouillon, R.; Baelen, H.V. )

    1991-07-30

    In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D{sub 3} for the binding site of the latter in hDBP and (2) {sup 3}H-25-ANE is capable of covalently labeling the hDBP molecule when exposed ot UV light. Treatment of a sample of purified hDBP, labeled with {sup 3}H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was assoicated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, the results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D{sub 3}.

  14. Applicability of the product isolation and the radiometric aromatase assays for the measurement of low levels of aromatase: lack of aromatase activity in the human endometrium.

    PubMed

    Prefontaine, M; Shih, C; Pan, C C; Bhavnani, B R

    1990-12-01

    The purpose of this investigation was to assess the applicability of two well established procedures: (i) the product isolation assay and (ii) the radiometric 3H2O assay for the determination of very low levels of aromatase activity. The methods were validated and used to assess the capacity of normal and neoplastic human endometrium to synthesize oestrogens from androgens. Using the product isolation assay, various specimens (n = 27) of normal and neoplastic endometrium were incubated with [1,2,6,7-3H]testosterone either by a standard incubation procedure or by a superfusion technique. Following the incubation, carrier oestrone and oestradiol or [14C]oestrone and [14C]oestradiol were added, and the oestrogens were isolated and purified by paper chromatography and high-performance liquid chromatography. The radiochemical purity of oestrone and oestradiol was checked by the isotope dilution technique. In all samples, the 3H associated with oestrone and oestradiol failed to recrystallize as oestrone and oestradiol. No radioactivity was detectable in the oestrone and oestradiol crystals after acetylation. Similarly, 16 endometrial samples were tested for aromatase activity by the 3H2O release assay using [1 beta-3H]androstenedione as substrate. The results indicate that 3H2O was indeed released during these incubations, but this activity could not be inhibited by the aromatase inhibitor 4-hydroxyandrostenedione, by excess substrate or by heat inactivation of the tissue. Furthermore, the release of 3H2O from [1 beta-3H]androstenedione under the incubation conditions used (Dulbecco's modified Eagle's medium or RPMI-1640 containing fetal bovine serum and NADPH) also occurred in the absence of any tissue. This activity was not inhibited by 4-hydroxyandrostenedione nor by excess substrate. The results demonstrate that the human endometrium does not contain detectable levels of aromatase activity and that the radiometric assay can give rise to false-positive results if used

  15. Efficacy of Korean Red Ginseng by Single Nucleotide Polymorphism in Obese Women: Randomized, Double-blind, Placebo-controlled Trial

    PubMed Central

    Kwon, Dong Hyun; Bose, Shambhunath; Song, Mi Young; Lee, Myeong Jong; Lim, Chi Yeon; Kwon, Bum Sun; Kim, Ho Jun

    2012-01-01

    This study examined the effects of Korean red ginseng (KRG) on obese women and aimed to confirm that the effects of KRG on obesity differ dependently on a gene. Fifty obese women were recruited and randomized to receive KRG (n=24) or placebo (n=26) for 8 wk. Measurements of blood pressure, height, weight, waist circumference, waist-hip ratio (WHR), total fat mass, percentage of body fat, resting metabolic rate, basal body temperature, and daily food intake (FI), blood test (serum lipid, liver and renal function), Korean version of obesity-related quality of life scale (KOQOL), and a gene examination were performed. Comparisons of subjects before and after the administration of KRG revealed significant improvements of obesity in terms of weight, body mass index (BMI), WHR, FI, and KOQOL. However, in the comparison between KRG group and placebo group, only KOQOL was significantly different. KRG displayed significant efficacy on BMI and KOQOL in the CT genotype of the G protein beta 3 gene, but not in the CC genotype, on blood sugar test in the Trp64/Arg genotype of the beta 3 adrenergic receptor gene, but not in Trp64/ Trp genotype, on KOQOL in the DD genotype of the angiotensin I converting enzyme gene, but not in the ID and DD genotypes. The effects of KRG on obesity were confirmed to some extent. However, a distinct effect compared to placebo was not confirmed. KRG is more effective for improving the secondary issues of the quality of life derived from obesity rather than having direct effects on the obesity-related anthropometric assessment and blood test indices. PMID:23717118

  16. Lower reinforcing strength of the phenyltropane cocaine analogs RTI-336 and RTI-177 compared to cocaine in nonhuman primates.

    PubMed

    Czoty, Paul W; Martelle, Jennifer L; Carroll, F Ivy; Nader, Michael A

    2010-09-01

    Drugs that inhibit brain dopamine transporters (DAT) have been developed as potential agonist medications for cocaine abuse and dependence. Because the mechanism of action of such drugs is similar to cocaine, one concern regarding their use is the abuse potential of the medications themselves. The present study compared the reinforcing strength of cocaine (0.003-0.3mg/kg) and two 3-phenyltropane analogs of cocaine, RTI-336 (3beta-(4-chlorophenyl)-2beta-[3-(4'-methylphenyl)isoxazol-5-yl]tropane hydrochloride; 0.003-0.1mg/kg) and RTI-177 (3beta-(4-chlorophenyl)-2beta-[3-phenylisoxazol-5-yl]tropane hydrochloride; 0.003-0.1mg/kg), using a progressive-ratio (PR) schedule in rhesus monkeys (n=4). PR schedules of reinforcement are frequently used to measure reinforcing strength of drugs. Earlier research using limited-access conditions reported that cocaine was a stronger reinforcer than either RTI-336 or RTI-177. Because the 3-phenyltropanes have longer durations of action, one purpose of the present study was to examine reinforcing strength using longer experimental sessions. Under these conditions, cocaine functioned as a reinforcer in all monkeys, and RTI-336 and RTI-177 functioned as a reinforcer in three of four subjects. Consistent with their documented slower onset of neurochemical and pharmacological effects, RTI-336 and RTI-177 were weaker reinforcers, resulting in fewer injections than cocaine. On average, the potencies of the two RTI compounds were not different than that of cocaine. These results support the view that slow-onset DA-selective uptake inhibitors have lower abuse liability than cocaine. In addition, the present findings suggest that changes in PR session length can influence potency comparisons between drugs, but not measures of reinforcing strength. PMID:20580733

  17. The role of integrin alpha D beta2 (CD11d/CD18) in monocyte/macrophage migration.

    PubMed

    Yakubenko, Valentin P; Belevych, Nataly; Mishchuk, Daria; Schurin, Aleksey; Lam, Stephen C-T; Ugarova, Tatiana P

    2008-08-15

    Integrin alpha(D)beta(2) (CD11d/CD18) is a multiligand macrophage receptor with recognition specificity identical to that of the major myeloid cell-specific integrin alpha(M)beta(2) (CD11b/CD18, Mac-1). Despite its prominent upregulation on inflammatory macrophages, the role of alpha(D)beta(2) in monocyte and macrophage migration is unknown. In this study, we have generated model and natural cell lines expressing different densities of alpha(D)beta(2) and examined their migration to various extracellular matrix proteins. When expressed at a low density, alpha(D)beta(2) on the surface of recombinant HEK293 cells and murine IC-21 macrophages cooperates with beta(1)/beta(3) integrins to support cell migration. However, its increased expression on the alpha(D)beta(2)-expressing HEK293 cells and its upregulation by PMA on the IC-21 macrophages result in increased cell adhesiveness and inhibition of cell migration. Furthermore, ligation of alpha(D)beta(2) with anti-alpha(D) blocking antibodies restores beta(1)/beta(3)-driven cell migration by removing the excess alpha(D)beta(2)-mediated adhesive bonds. Consistent with in vitro data, increased numbers of inflammatory macrophages were recovered from the inflamed peritoneum of mice after the administration of anti-alpha(D) antibody. These results demonstrate that the density of alpha(D)beta(2) is critically involved in modulating macrophage adhesiveness and their migration, and suggest that low levels of alpha(D)beta(2) contribute to monocyte migration while alpha(D)beta(2) upregulation on differentiated macrophages may facilitate their retention at sites of inflammation. PMID:18621369

  18. Acquisition of double-stranded DNA-binding ability in a hybrid protein between Escherichia coli CspA and the cold shock domain of human YB-1.

    PubMed

    Wang, N; Yamanaka, K; Inouye, M

    2000-11-01

    Escherichia coli CspA, a major cold shock protein, is dramatically induced upon temperature downshift. As it binds co-operatively to single-stranded DNA (ssDNA) and RNA without apparent sequence specificity, it has been proposed that CspA acts as an RNA chaperone to facilitate transcription and translation at low temperature. CspA consists of a five-stranded beta-barrel structure containing two RNA-binding motifs, RNP1 and RNP2. Eukaryotic Y-box proteins, such as human YB-1, are a family of nucleic acid-binding proteins that share a region of high homology with CspA (43% identity), termed the cold shock domain (CSD). Their cellular functions are very diverse and are associated with growth-related processes. Here, we replaced the six-residue loop region of CspA between the beta3 and beta4 strands with the corresponding region of the CSD of human YB-1 protein. The resulting hybrid protein became capable of binding to double-stranded DNA (dsDNA) in addition to ssDNA and RNA. The dsDNA-binding ability of an RNP1 point mutant (F20L) of the hybrid was almost unchanged. On the other hand, the dsDNA-binding ability of the hybrid protein was abolished in high salt concentrations in contrast to its ssDNA-binding ability. These results indicate that the loop region between the beta3 and beta4 strands of Y-box proteins, which is a little longer and more basic than that of CspA, plays an important role in their binding to dsDNA. PMID:11069676

  19. Analysis of integrins and vascular endothelial growth factor isoforms mRNA expression in the canine uterus during perimplantation period.

    PubMed

    Bukowska, D; Kempisty, B; Jackowska, M; Woźna, M; Antosik, P; Piotrowska, H; Jaśkowski, J M

    2011-01-01

    Integrins are the major receptors within the extracellular matrix (ECM) that mediate several functions connected with cell life and metabolism, such as cell adhesion, migration, cytoskeletal organization, proliferation, survival, and differentiation. A vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors. It has been suggested that the expression of this gene may play crucial physiological roles in reproductive organs. All investigated endometrial tissues were isolated on day 10-12 after mating. Control bitches, used in this study, were in metestrus, which was determined according to the vaginal cytology and progesterone level in blood. Early pregnancy was verified by flushing the uterine horns with PBS. Total RNA was isolated from the bitches endometrium by means of the Chomczyński and Sacchi method, treated by DNase I, and reverse-transcribed into cDNA. A quantitative analysis of integrins alpha2b, beta2 and beta3, VEGF 164, 182 and 188 cDNA was performed by RT-PCR. In results we have shown an increased expression of all investigated genes (integrins alpha2b, beta2 and beta3, VEGF 164, 182, and 188) in pregnant bitches uterus as compared to non-pregnant females (P < 0.001). Our results indicated that the expression of genes encoding integrins and vascular endothelial growth factors is different in relation to the time of the embryo implantation and it is increased in the first period of this process. This may be associated with the induction of specific mechanisms responsible for receptivity of uterus following the embryo attachment. In addition, all of investigated genes are up-regulated in a pregnancy-specific manner and the increased expression of these genes may regulate the uterus function during the implantation of canine embryos. PMID:21721410

  20. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    SciTech Connect

    Taguchi, J.; Kuriyama, K. )

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  1. Common variants in the CRP promoter are associated with a high C-reactive protein level in Kawasaki disease.

    PubMed

    Kim, Jae-Jung; Yun, Sin Weon; Yu, Jeong Jin; Yoon, Kyung Lim; Lee, Kyung-Yil; Kil, Hong-Ryang; Kim, Gi Beom; Han, Myung Ki; Song, Min Seob; Lee, Hyoung Doo; Byeon, Jung Hye; Sohn, Saejung; Hong, Young Mi; Jang, Gi Young; Lee, Jong-Keuk

    2015-02-01

    Kawasaki disease (KD) is an acute self-limiting form of vasculitis that afflicts infants and children and manifests as fever and signs of mucocutaneous inflammation. Children with KD show various laboratory inflammatory abnormalities, such as elevations in their white blood cell (WBC) count, C-reactive protein (CRP) level, and erythrocyte sedimentation rate (ESR). We here performed a genome-wide association study (GWAS) of 178 KD patients to identify the genetic loci that influence 10 important KD laboratory markers: WBC count, neutrophil count, platelet count, CRP, ESR, hemoglobin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, and total protein. A total of 165 loci passed our arbitrary stage 1 threshold for replication (p < 1 × 10(-5)). Of these, only 2 SNPs (rs12068753 and rs4786091) demonstrated a significant association with the CRP level in replication study of 473 KD patients (p < 0.05). The SNP located at the CRP locus (rs12068753) demonstrated the most significant association with CRP in KD patients (beta = 4.73 and p = 1.20 × 10(-6) according to the stage 1 GWAS; beta = 3.65 and p = 1.35 × 10(-8) according to the replication study; beta = 3.97 and p = 1.11 × 10(-13) according to combined analysis) and explained 8.1% of the phenotypic variation observed. However, this SNP did not demonstrate any significant association with CRP in the general population (beta = 0.37 and p = 0.1732) and only explained 0.1% of the phenotypic variation in this instance. Furthermore, rs12068753 did not affect the development of coronary artery lesions or intravenous immunoglobulin resistance in KD patients. These results indicate that common variants in the CRP promoter can play an important role in the CRP levels in KD. PMID:25266886

  2. The molecular genetic basis of Glanzmann thrombasthenia in the Iraqi-Jewish and Arab populations in Israel.

    PubMed Central

    Newman, P J; Seligsohn, U; Lyman, S; Coller, B S

    1991-01-01

    Glanzmann thrombasthenia is an autosomal recessive bleeding disorder characterized by a decrease or absence of functional platelet glycoprotein (GP) IIb-IIIa (alpha IIb beta 3) integrin receptors. Although thrombasthenia is a rare disorder, its occurrence is increased in some regions of the world where intracommunity marriage and consanguinity are commonplace, resulting in increased expression of autosomal recessive traits. We have been studying two populations having an unusually high frequency of Glanzmann disease, Iraqi Jews and Arabs living in Israel, and were able to distinguish the populations on the basis of immunodetectable GPIIIa and platelet surface vitronectin receptor (alpha v beta 3) expression. In this article, we describe molecular genetic studies based on use of the PCR that have allowed us to characterize platelet mRNA sequences encoding GPIIb and GPIIIa from patients in these populations. In six of six Iraqi-Jewish families studied, cDNA sequence analysis identified an 11-base deletion within exon 12 of the GPIIIa gene. This mutation produces a frameshift leading to protein termination shortly before the transmembrane domain of GPIIIa. In contrast, a 13-base deletion encompassing the splice acceptor site of exon 4 of the GPIIb gene was found in three of five Arab kindreds studied. This deletion results in forced alternative splicing to a downstream AG acceptor, producing a 6-amino acid deletion in the GPIIb protein, including a single cysteine residue. These nucleotide sequence variations were exploited to design a rapid, PCR-based oligonucleotide dot-blot hybridization test for both pre- and postnatal diagnosis of Glanzmann disease. These studies demonstrate the heterogeneity of Glanzmann thrombasthenia in different populations, and its homogeneity within geographically restricted populations, and offer insight into the requirements for integrin surface expression. Images PMID:2014236

  3. Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors.

    PubMed Central

    Guidotti, A; Forchetti, C M; Corda, M G; Konkel, D; Bennett, C D; Costa, E

    1983-01-01

    A brain polypeptide termed diazepam-binding inhibitor (DBI) and thought to be chemically and functionally related to the endogenous effector of the benzodiazepine recognition site was purified to homogeneity. This peptide gives a single band of protein on NaDodSO4 and acidic urea gel electrophoresis. A single UV-absorbing peak was obtained by HPLC using three different columns and solvent systems. DBI has a molecular mass of approximately equal to 11,000 daltons. Carboxyl-terminus analysis shows that tyrosine is the only residue while the amino-terminus was blocked. Cyanogen bromide treatment of DBI yields three polypeptide fragments, and the sequences of two of them have been determined for a total of 45 amino acids. DBI is a competitive inhibitor for the binding of [3H]diazepam, [3H]flunitrazepam, beta-[3H]carboline propyl esters, and 3H-labeled Ro 15-1788. The Ki for [3H]-diazepam and beta-[3H]carboline binding were 4 and 1 microM, respectively. Doses of DBI that inhibited [3H]diazepam binding by greater than 50% fail to change [3H]etorphine, gamma-amino[3H]butyric acid, [3H]-quinuclidinyl benzilate, [3H]dihydroalprenolol, [3H]adenosine, and [3H]imipramine binding tested at their respective Kd values. DBI injected intraventricularly at doses of 5-10 nmol completely reversed the anticonflict action of diazepam on unpunished drinking and, similar to the anxiety-inducing beta-carboline derivative FG 7142 (beta-carboline-3-carboxylic acid methyl ester), facilitated the shock-induced suppression of drinking by lowering the threshold for this response. Images PMID:6304714

  4. Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis.

    PubMed

    Gonzalez, Anjelica L; El-Bjeirami, Wafa; West, Jennifer L; McIntire, Larry V; Smith, C Wayne

    2007-03-01

    Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with specific peptide sequences relevant to extracellular matrix proteins. We evaluated fMLP-stimulated human neutrophil motility on peptides Arg-Gly-Asp-Ser (RGDS) and TMKIIPFNRTLIGG (P2), alone and in combination. RGDS is a bioactive sequence found in a number of proteins, and P2 is a membrane-activated complex-1 (Mac-1) ligand located in the gamma-chain of the fibrinogen protein. We evaluated, via video microscopy, cell motility by measuring cell displacement from origin and total accumulated distance traveled and then calculated average velocity. Results indicate that although adhesion and shape change were supported by hydrogels containing RGD alone, motility was not. Mac-1-dependent motility was supported on hydrogels containing P2 alone. Motility was enhanced through combined presentation of RGD and P2, engaging Mac-1, alpha(V)beta(3), and beta(1) integrins. Naïve neutrophil motility on combined peptide substrates was dependent on Mac-1, and alpha(4)beta(1) while alpha(6)beta(1) contributed to speed and linear movement. Transmigrated neutrophil motility was dependent on alpha(v)beta(3) and alpha(5)beta(1), and alpha(4)beta(1), alpha(6)beta(1), and Mac-1 contributed to speed and linear motion. Together, the data demonstrate that efficient neutrophil migration, dependent on multi-integrin interaction, is enhanced after transendothelial migration. PMID:17164427

  5. Effects of polyphenol-rich extract from berries of Aronia melanocarpa on the markers of oxidative stress and blood platelet activation.

    PubMed

    Olas, Beata; Kedzierska, Magdalena; Wachowicz, Barbara; Stochmal, Anna; Oleszek, Wieslaw

    2010-01-01

    Bioactive substances found in numerous foods can be successfully and safely used to modify various cellular functions and affect the oxidative stress. Aronia melanocarpa fruits (Rosaceae) are one of the richest plant sources of phenolic substances shown to have anti-inflammatory, antitumor, antioxidative and antiplatelet activities. We investigated antioxidant properties of the extract from berries of A. melanocarpa by the estimation of the selected and other biomarkers of oxidative stress, i.e. the level of 8-epi-prostaglandin F(2) (8-EPI) (by immunoassay kit) and the amount of glutathione (by HPLC method) in control platelets and platelets treated with H(2)O(2). The expression of alpha(IIb)beta(3) (a marker of platelet activation) was measured by flow cytometer. The antioxidative and antiplatelet properties of the tested extract were also compared with the action of a well characterized antioxidative and antiplatelet commercial monomeric polyphenol-resveratrol. The extract from berries of A. melanocarpa (at the highest tested concentration -100 microg/ml) decreased the production of 8-EPI (a marker of lipid peroxidation) in control blood platelets and platelets treated with H(2)O(2) (2 mM). A combined action of the tested plant extract and H(2)O(2) evoked a significant increase of reduced form of glutathione in platelets compared with cells treated with H(2)O(2) only. Moreover, the tested plant extract (at the highest used concentration -100 microg/ml) reduced the expression of alpha(IIb)beta(3) on blood platelets. Comparative studies indicate that the tested plant extract was found to be more reactive in blood platelets than the solution of pure resveratrol. PMID:20218910

  6. Emergence of the persistent spin helix in semiconductor quantum wells.

    PubMed

    Koralek, J D; Weber, C P; Orenstein, J; Bernevig, B A; Zhang, Shou-Cheng; Mack, S; Awschalom, D D

    2009-04-01

    According to Noether's theorem, for every symmetry in nature there is a corresponding conservation law. For example, invariance with respect to spatial translation corresponds to conservation of momentum. In another well-known example, invariance with respect to rotation of the electron's spin, or SU(2) symmetry, leads to conservation of spin polarization. For electrons in a solid, this symmetry is ordinarily broken by spin-orbit coupling, allowing spin angular momentum to flow to orbital angular momentum. However, it has recently been predicted that SU(2) can be achieved in a two-dimensional electron gas, despite the presence of spin-orbit coupling. The corresponding conserved quantities include the amplitude and phase of a helical spin density wave termed the 'persistent spin helix'. SU(2) is realized, in principle, when the strengths of two dominant spin-orbit interactions, the Rashba (strength parameterized by alpha) and linear Dresselhaus (beta(1)) interactions, are equal. This symmetry is predicted to be robust against all forms of spin-independent scattering, including electron-electron interactions, but is broken by the cubic Dresselhaus term (beta(3)) and spin-dependent scattering. When these terms are negligible, the distance over which spin information can propagate is predicted to diverge as alpha approaches beta(1). Here we report experimental observation of the emergence of the persistent spin helix in GaAs quantum wells by independently tuning alpha and beta(1). Using transient spin-grating spectroscopy, we find a spin-lifetime enhancement of two orders of magnitude near the symmetry point. Excellent quantitative agreement with theory across a wide range of sample parameters allows us to obtain an absolute measure of all relevant spin-orbit terms, identifying beta(3) as the main SU(2)-violating term in our samples. The tunable suppression of spin relaxation demonstrated in this work is well suited for application to spintronics. PMID:19340077

  7. Improvement in verbal memory following SSRI augmentation of antipsychotic treatment is associated with changes in the expression of mRNA encoding for the GABA-A receptor and BDNF in PMC of schizophrenic patients.

    PubMed

    Silver, Henry; Mandiuk, Nina; Einoch, Reef; Susser, Ehud; Danovich, Lena; Bilker, Warren; Youdim, Moussa; Weinreb, Orly

    2015-05-01

    Verbal memory impairment in schizophrenia is associated with abnormalities in gamma-aminobutyric acid (GABA)-ergic and brain-derived neurotrophic factor (BDNF) systems. Recent evidence from animal and clinical studies that adding fluvoxamine to antipsychotics alters the expression of transcripts encoding for the GABA-A receptor and BDNF led us to postulate that fluvoxamine augmentation may improve memory in schizophrenia. To test this, we examined the effect of add-on fluvoxamine on verbal memory and other cognitive functions and related it to the expression of mRNA coding for the GABA-A receptor and BDNF in peripheral mononuclear cells (PMC) of schizophrenic patients. Twenty-nine patients completed a 6-week study in which fluvoxamine (100 mg/day) was added to ongoing antipsychotic treatment. Verbal memory, abstraction working memory, object and face recognition, and psychomotor speed and clinical symptoms were assessed at baseline and after 3 and 6 weeks of treatment. Blood samples were taken at baseline and weeks 1, 3, and 6 and PMC was assayed for the GABA-A beta3 receptor and BDNF mRNA by quantitative real-time reverse transcription-PCR. Associative and logical verbal memory improved significantly and showed a significant correlation with changes in PMC BDNF and GABA-A beta3 receptor mRNA, which increased during treatment. Abstraction and object recognition improved, but this did not correlate with PMC measures. Negative and positive symptoms improved significantly; the latter showed significant correlations with changes in PMC measures. Addition of fluvoxamine to antipsychotics improves verbal memory. It is postulated that the mechanism involves enhanced GABA-A receptor/BDNF-dependent synaptic plasticity in the hippocampus. PMID:25756551

  8. Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla.

    PubMed

    Mousavi, M; Hellström-Lindahl, E; Guan, Z Z; Bednar, I; Nordberg, A

    2001-12-21

    Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent. PMID:11811902

  9. Intimate Partner Violence Associated with Postpartum Depression, Regardless of Socioeconomic Status.

    PubMed

    Kothari, Catherine L; Liepman, Michael R; Shama Tareen, R; Florian, Phyllis; Charoth, Remitha M; Haas, Suzanne S; McKean, Joseph W; Moe, Angela; Wiley, James; Curtis, Amy

    2016-06-01

    Objective This study examined whether socioeconomic status moderated the association between intimate partner violence (IPV) and postpartum depression among a community-based sample of women. Defining the role of poverty in the risk of postpartum depression for IPV victims enables prioritization of health promotion efforts to maximize the effectiveness of existing maternal-infant resources. Methods This cross-sectional telephone-survey study interviewed 301 postpartum women 2 months after delivery, screening them for IPV and depression [using Edinburgh Postnatal Depression Scale (EPDS)]. Socioeconomic status was defined by insurance (Medicaid-paid-delivery or not). This analysis controlled for the following covariates, collected through interview and medical-record review: demographics, obstetric history, prenatal health and additional psychosocial risk factors. After adjusting for significant covariates, multiple linear regression was conducted to test whether socioeconomic status confounded or moderated IPV's relationship with EPDS-score. Results Ten percent of participants screened positive for postpartum depression, 21.3 % screened positive for current or previous adult emotional or physical abuse by a partner, and 32.2 % met poverty criteria. IPV and poverty were positively associated with each other (χ(2) (1) = 11.76, p < .001) and with EPDS score (IPV: beta 3.2 (CI 2.0, 4.5) p < .001, poverty: beta 1.3 (CI 0.2, 2.4) p = .017). In the multiple linear regression, IPV remained significantly associated, but poverty did not (IPV: adjusted beta 3.1 (CI 1.8, 4.3) p < .001, poverty: adjusted beta 0.8 (CI -0.3, 1.9) p = .141), and no statistically significant interaction between IPV and poverty was found. Conclusions Study findings illustrated that IPV was strongly associated with postpartum depression, outweighing the influence of socioeconomic status upon depression for postpartum women. PMID:26955998

  10. Crystal structure of schistatin, a disintegrin homodimer from saw-scaled viper (Echis carinatus) at 2.5 A resolution.

    PubMed

    Bilgrami, Sameeta; Tomar, Shailly; Yadav, Savita; Kaur, Punit; Kumar, Janesh; Jabeen, Talat; Sharma, Sujata; Singh, Tej P

    2004-08-13

    This is the first structure of a biological homodimer of disintegrin. Disintegrins are a class of small (4-14 kDa) proteins that bind to transmembrane integrins selectively. The present molecule is the first homodimer that has been isolated from the venom of Echis carinatus. The monomeric chain contains 64 amino acid residues. The three-dimensional structure of schistatin has been determined by the multiple isomorphous replacement method. It has been refined to an R-factor of 0.190 using all the data to 2.5 A resolution. The two subunits of the disintegrin homodimer are related by a 2-fold crystallographic symmetry. Thus, the crystallographic asymmetric unit contains a monomer of disintegrin. The monomer folds into an up-down topology with three sets of antiparallel beta-strands. The structure is well ordered with four intramolecular disulfide bonds. the two monomers are firmly linked to each other through two intermolecular disulfide bridges at their N termini together with several other interactions. This structure has corrected the error in the disulfide bond pattern of the two intermolecular disulfide bridges that was reported earlier using chemical methods. Unique sequence and structural features of the schistatin monomers suggest that they have the ability to bind well with both alphaIIb beta3 and alphav beta3 integrins. The N termini anchored two chains of the dimer diverge away at their C termini exposing the Arg-Gly-Asp motif into opposite directions thus enhancing their binding efficiency to integrins. This is one of the unique features of the present disintegrin homodimer and seems to be responsible for the clustering of integrin molecules. The homodimer binds to integrins apparently with a higher affinity than the monomers and also plays a role in the signaling pathway. PMID:15317139