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Sample records for crecimiento transformante beta-3

  1. Expression of beta 3-adrenoceptor mRNA in rat tissues.

    PubMed Central

    Evans, B. A.; Papaioannou, M.; Bonazzi, V. R.; Summers, R. J.

    1996-01-01

    1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with

  2. Mutated human beta3-adrenergic receptor (Trp64Arg) lowers the response to beta3-adrenergic agonists in transfected 3T3-L1 preadipocytes.

    PubMed

    Kimura, K; Sasaki, N; Asano, A; Mizukami, J; Kayahashi, S; Kawada, T; Fushiki, T; Morimatsu, M; Yoshida, T; Saito, M

    2000-03-01

    Wild-type or mutated human beta3-adrenergic receptor (Trp64Arg) cDNAs were stably expressed in mouse 3T3-L1 cells. Saturation binding study using a beta-adrenergic ligand revealed that there was no significant difference in the receptor density and the equilibrium dissociation constant between the two cell lines. However, the ability of the mutant beta3-adrenergic receptor to accumulate cyclic AMP (cAMP) in response to isoproterenol was much reduced and Kact for cAMP accumulation was lowered as compared to the wild type receptor. The amount of alpha subunit of stimulatory GTP-binding protein (GSalpha) and adenylyl cyclase activity in response to forskolin were not different in the two cell lines. The responses of the mutant receptor to epinephrine, norepinephrine and L-755,507, a highly specific agonist for human beta3-adrenergic receptor, were also reduced, but the reduction of Kact for L-755,507 was more evident than other agonists tested. The cAMP accumulation in response to some conventional beta3 agonists was less than 10% of that to isoproterenol even in the cells expressing the wild type receptor. These results suggest that the Trp64Arg mutant beta3-adrenergic receptor has less ability to stimulate adenylyl cyclase, and that lipolytic activity through the beta3-adrenergic receptor by catecholamines in subjects carrying this mutation might be suppressed. PMID:10786926

  3. Role of human GABA(A) receptor beta3 subunit in insecticide toxicity.

    PubMed

    Ratra, G S; Kamita, S G; Casida, J E

    2001-05-01

    The gamma-aminobutyric acid type A (GABA(A)) receptor is the target for the major insecticides alpha-endosulfan, lindane, and fipronil and for many analogs. Their action as chloride channel blockers is directly measured by binding studies with [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB). This study tests the hypothesis that GABA(A) receptor subunit composition determines the sensitivity and selectivity of insecticide toxicity. Human receptor subtypes were expressed individually (alpha1, alpha6, beta1, beta3, and gamma2) and in combination in insect Sf9 cells. Binding parameters were similar for [(3)H]EBOB in the beta3 homooligomer, alpha1beta3gamma2 heterooligomer, and native brain membranes, but toxicological profiles were very different. Surprisingly, alpha-endosulfan, lindane, and fipronil were all remarkably potent on the recombinant beta3 homooligomeric receptor (IC50 values of 0.5-2.4 nM), whereas they were similar in potency on the alpha1beta3gamma2 subtype (IC50 values of 16-33 nM) and highly selective on the native receptor (IC50 values of 7.3, 306, and 2470 nM, respectively). The selectivity order for 29 insecticides and convulsants as IC50 ratios for native/beta3 or alpha1beta3gamma2/beta3 was as follows: fipronil > lindane > 19 other insecticides including alpha-endosulfan and picrotoxinin > 4 trioxabicyclooctanes and dithianes (almost nonselective) > tetramethylenedisulfotetramine, 4-chlorophenylsilatrane, or alpha-thujone. Specificity between mammals and insects at the target site (fipronil > lindane > alpha-endosulfan) paralleled that for toxicity. Potency at the native receptor is more predictive for inhibition of GABA-stimulated chloride uptake than that at the beta3 or alpha1beta3gamma2 receptors. Therefore, the beta3 subunit contains the insecticide target and other subunits differentially modulate the binding to confer compound-dependent specificity and selective toxicity.

  4. Mediation of most atypical effects by species homologues of the beta 3-adrenoceptor.

    PubMed Central

    Blin, N.; Nahmias, C.; Drumare, M. F.; Strosberg, A. D.

    1994-01-01

    1. A wide panel of compounds acting on beta-adrenoceptors active either in mammalian heart or in rodent digestive tract and adipose tissues, were investigated for their effects on Chinese hamster ovary cells transfected with the human or murine beta 3-adrenoceptor gene. 2. The beta 3-agonists, bucindolol, CGP 12177A and pindolol exhibited the highest binding affinities; BRL 37344, LY 79771, ICI 201651 and SR 58611A presented high potencies in stimulating adenylyl cyclase; bupranolol appeared as the most efficient beta 3-antagonist. 3. This pharmacological analysis further established that the beta 3-adrenoceptor is the prototype of the adipose tissue atypical beta-adrenoceptor, since these receptors share a number of pharmacological properties which differ strikingly from those of beta 1- and beta 2-adrenoceptors: low affinities for conventional beta-adrenoceptor agonists and antagonists, high potencies for novel compounds active in adipose tissues, partial agonistic activities for several beta 1/beta 2-antagonists. 4. Although the pharmacological profiles of the human and murine beta 3-receptor were very similar, some quantitative or even qualitative differences were observed for particular compounds such as propranolol, which exhibited weak and partial agonistic effects at the human beta 3-receptors and antagonistic effects at the murine beta 3-receptors. These differences may result from key amino-acid substitutions between the human and the murine beta 3-receptor sequences, which may alter the binding site or signal processing.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7921620

  5. Embryonic expression of the divergent Drosophila beta3-tubulin isoform is required for larval behavior.

    PubMed Central

    Dettman, R W; Turner, F R; Hoyle, H D; Raff, E C

    2001-01-01

    We have sought to define the developmental and cellular roles played by differential expression of distinct beta-tubulins. Drosophila beta3-tubulin (beta3) is a structurally divergent isoform transiently expressed during midembryogenesis. Severe beta3 mutations cause larval lethality resulting from failed gut function and consequent starvation. However, mutant larvae also display behavioral abnormalities consistent with defective sensory perception. We identified embryonic beta3 expression in several previously undefined sites, including different types of sensory organs. We conclude that abnormalities in foraging behavior and photoresponsiveness exhibited by prelethal mutant larvae reflect defective beta3 function in the embryo during development of chordotonal and other mechanosensory organs and of Bolwig's organ and nerve. We show that microtubule organization in the cap cells of chordotonal organs is altered in mutant larvae. Thus transient zygotic beta3 expression has permanent consequences for the architecture of the cap cell microtubule cytoskeleton in the larval sensilla, even when beta3 is no longer present. Our data provide a link between the microtubule cytoskeleton in embryogenesis and the behavioral phenotype manifested as defective proprioreception at the larval stage. PMID:11333234

  6. [Beta-3 adrenergic receptor--structure and role in obesity and metabolic disorders].

    PubMed

    Wiejak, J; Wyroba, E

    1999-01-01

    Structure and essential motifs of beta 3-adrenergic receptor (known previously as atypical beta-AR), which plays a central role in regulation of lipid metabolism have been described. Obesity results from an imbalance between caloric intake and energy expenditure. The consequence of catecholamine activation of beta 3-AR is increased mobilization of fatty acids from triglyceride stores (lipolysis) in brown and white adipose tissue as well as increased fatty acid beta-oxidation and heat-production via UCP-1 (thermogenesis) in brown adipose tissue. A pharmacokinetic effects of beta 3-agonists and putative involvement of Trp/Arg mutation in beta 3-AR gene in obesity and another metabolic disorders have been discussed.

  7. Affinity modulation of the alpha IIb beta 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor.

    PubMed Central

    O'Toole, T E; Loftus, J C; Du, X P; Glass, A A; Ruggeri, Z M; Shattil, S J; Plow, E F; Ginsberg, M H

    1990-01-01

    To analyze the basis of affinity modulation of integrin function, we studied cloned stable Chinese hamster ovary cell lines expressing recombinant integrins of the beta 3 family (alpha IIb beta 3 and alpha v beta 3). Antigenic and peptide recognition specificities of the recombinant receptors resembled those of the native receptors found in platelets or endothelial cells. The alpha IIb beta 3-expressing cell line (A5) bound RGD peptides and immobilized fibrinogen (Fg) but not soluble fibrinogen or the activation-specific monoclonal anti-alpha IIb beta 3 (PAC1), indicating that it was in the affinity state found on resting platelets. Several platelet agonists failed to alter the affinity state of ("activate") recombinant alpha IIb beta 3. The binding of soluble Fg and PAC1, however, was stimulated in both platelets and A5 cells by addition of IgG papain-digestion products (Fab) fragments of certain beta 3-specific monoclonal antibodies. These antibodies stimulated PAC1 binding to platelets fixed under conditions rendering them unresponsive to other agonists. Addition of these antibodies to detergent-solubilized alpha IIb beta 3 also stimulated specific Fg binding. These data demonstrate that certain anti-beta 3 antibodies activate alpha IIb beta 3 by acting directly on the receptor, possibly by altering its conformation. Furthermore, they indicate that the activation state of alpha IIb beta 3 is a property of the receptor itself rather than of the surrounding cell membrane microenvironment. Images PMID:2100193

  8. 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.

  9. Adrenergic lipolysis in guinea pig is not a beta 3-adrenergic response: comparison with human adipocytes.

    PubMed

    Carpéné, C; Castan, I; Collon, P; Galitzky, J; Moratinos, J; Lafontan, M

    1994-03-01

    beta 3-Adrenoceptor agonists are potent lipolytic activators in rats, but they are only weak stimulators in human adipocytes, indicating interspecies differences in the adrenergic regulation of lipid mobilization. Like human but not rat adipocytes, guinea pig fat cells were poorly responsive to the beta 3-agonists BRL-37344, CGP-12177, SR-58611, and ICI-215001, acid metabolite of ICI-D7114. In guinea pigs, the beta 1-agonist dobutamine was more lipolytic than the beta 2-agonist procaterol. Anatomic location of fat deposits was without major influence on the beta-adrenergic responsiveness. Weak responses to beta 3-agonists were found whatever the sex or the age (from 2 days to 16 mo) of the animals. Even in the interscapular brown adipose tissue, which is well known in rats for its beta 3-adrenergic responsiveness, a blunted response to BRL-37344 was observed. The alpha 2-adrenergic antilipolytic effect and receptor number were smaller in guinea pig than in human adipocytes, but the beta-adrenergic receptor number was similar in the two species. Thus guinea pig adipocytes resemble human fat cells when their weak beta 3-adrenergic responsiveness is considered. PMID:7909205

  10. Nonshivering thermogenesis in marsupials: absence of thermogenic response to beta 3-adrenergic agonists.

    PubMed

    Nicol, S C; Pavlides, D; Andersen, N A

    1997-07-01

    The status of nonshivering thermogenesis (NST) in marsupials remains controversial. Although morphological studies have failed to find evidence for the presence of brown adipose tissue (BAT) in adults or juveniles of species from all extant families of marsupial, a number of studies have investigated the metabolic response of marsupials to noradrenaline (NA) and yielded conflicting results. In eutherian mammals, NA stimulates NST in BAT by acting on beta 3-receptors, and in the experiments reported here we investigated the response of adult and juvenile brush tail possums (Trichosurus vulpecula), a Brazilian opossum (Monodelphis domestica), adult and juvenile red-necked (Bennett's) wallabies (Macropus rufogriseus) and the laboratory rat to selective beta 3-agonists (ICI D7114 and BRL 35135) and to NA. Wallabies were tested with the beta 3-agonists only. Although NA and both beta 3-agonists caused an 85% increase in oxygen consumption in rats, there was no significant effect on any of the marsupials. These results clearly indicate no beta 3-stimulated NST in these marsupials. All reports of metabolic responses to NA are from macropods, and a recent study demonstrates that NA and other alpha-adrenergic agonists stimulate thermogenesis in a small macropod, the bettong (Bettongia gaimardi), by acting on alpha 1-receptors. Thermogenic responses to NA seems to be restricted to macropods, showing the danger of characterising the response of any one marsupial species as being representative of marsupials as a group. PMID:9172391

  11. Anti-obesity effects of selective agonists to the beta 3-adrenergic receptor in dogs. I. The presence of canine beta 3-adrenergic receptor and in vivo lipomobilization by its agonists.

    PubMed

    Sasaki, N; Uchida, E; Niiyama, M; Yoshida, T; Saito, M

    1998-04-01

    It is known that in rodents and humans the beta 3-adrenergic receptor (beta 3-AR) is present primarily in adipocytes and plays a significant role in the adrenergic stimulation of lipolysis. We examined the expression of beta 3-AR mRNA in the dog and the lipomobilizing effects of beta 3-AR-selective agonists in vivo. Reverse transcription polymerase chain reaction of RNA extracted from dog adipose tissue produced a cDNA fragment, the nucleotide sequence of which was highly homologous to the corresponding regions of human (86.4%) and mouse (79.5%) beta 3-AR cDNA. The beta 3-AR mRNA was present at high levels in subcutaneous and visceral adipose tissues, but undetectable in other organs. When a selective beta 3-AR agonist, CL316,243, was infused intravenously into beagle dogs, the plasma level of free fatty acid increased in 30 min and persisted at higher levels for several hours. ICI D7114, another beta 3-AR agonist, also showed a similar lipomobilizing effect, but with lower potency. beta 3-AR agonist infusion also increased the plasma insulin level. These results suggested that functional beta 3-AR is present in adipose tissues of the dog and that it is effective for in vivo lipomobilization. PMID:9592718

  12. 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.

  13. Beta 3-adrenoreceptor regulation of nitric oxide in the cardiovascular system.

    PubMed

    Moens, An L; Yang, Ronghua; Watts, Vabren L; Barouch, Lili A

    2010-06-01

    The presence of a third beta-adrenergic receptor (beta 3-AR) in the cardiovascular system has challenged the classical paradigm of sympathetic regulation by beta1- and beta2-adrenergic receptors. While beta 3-AR's role in the cardiovascular system remains controversial, increasing evidence suggests that it serves as a "brake" in sympathetic overstimulation - it is activated at high catecholamine concentrations, producing a negative inotropic effect that antagonizes beta1- and beta2-AR activity. The anti-adrenergic effects induced by beta 3-AR were initially linked to nitric oxide (NO) release via endothelial NO synthase (eNOS), although more recently it has been shown under some conditions to increase NO production in the cardiovascular system via the other two NOS isoforms, namely inducible NOS (iNOS) and neuronal NOS (nNOS). We summarize recent findings regarding beta 3-AR effects on the cardiovascular system and explore its prospective as a therapeutic target, particularly focusing on its emerging role as an important mediator of NO signaling in the pathogenesis of cardiovascular disorders.

  14. The pharmacology of spontaneously open alpha 1 beta 3 epsilon GABA A receptor-ionophores.

    PubMed

    Maksay, Gábor; Thompson, Sally A; Wafford, Keith A

    2003-06-01

    Human alpha(1)beta(3) epsilon GABA(A) receptors were expressed in Xenopus oocytes and examined using the conventional two-electrode voltage-clamp technique and compared to alpha(1)beta(3)gamma(2) receptors. The effects of several GABA(A) agonists were studied, and the allosteric modulation of the channel by a number of GABAergic modulators investigated. The presence of the epsilon subunit increased the potency and efficacy of direct activation by partial GABA(A) agonists (piperidine-4-sulphonic acid and thio-4-PIOL), pentobarbital and neuro-steroids. Direct activation by 3-hydroxylated neurosteroids was restricted to 3alpha epimers, while chirality at C5 was indifferent. The 3beta-sulfate esters of pregnenolone and dehydroepiandrosterone inhibited the spontaneous currents with efficacies higher, while bicuculline methiodide and SR 95531 did so lower than picrotoxin and TBPS. Furosemide, fipronil, triphenylcyanoborate and Zn(2+) blocked the spontaneous currents of alpha(1)beta(3) epsilon receptors with different efficacies. Flunitrazepam and 4'-chlorodiazepam inhibited the spontaneous currents with micromolar potencies. In conclusion, spontaneously active alpha(1)beta(3) epsilon GABA(A) receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.

  15. Fipronil-based photoaffinity probe for Drosophila and human beta 3 GABA receptors.

    PubMed

    Sirisoma, N S; Ratra, G S; Tomizawa, M; Casida, J E

    2001-11-19

    Modification of the major insecticide fipronil (1) by replacing three pyrazole substituents (hydrogen for both cyano and amino and trifluoromethyldiazirinyl for trifluoromethylsulfinyl) gives a candidate photoaffinity probe (3) of high potency (IC(50) 2-28 nM) in blocking the chloride channel of Drosophila and human beta 3 GABA receptors.

  16. Inhibitory effect of beta3-adrenoceptor agonist in lower esophageal sphincter smooth muscle: in vitro studies.

    PubMed

    Sarma, D N K; Banwait, Kuldip; Basak, Ashim; DiMarino, Anthony J; Rattan, Satish

    2003-01-01

    We investigated the effects of (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl] - amino]propyl] - 1,3 - benzodioxole - 2, 2 - dicarboxylate (CL 316243) (a typical beta3-agonist) on the spontaneously tonic smooth muscle of the lower esophageal sphincter (LES). Studies were carried out in smooth muscle strips and smooth muscle cells (SMCs) of opossum LES. Isometric tension was recorded in the basal state and after CL 316243, and before and after beta3-antagonist (S)-N-[4-[2-[[3-[-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L 748337) and nonselective antagonist propranolol. In some experiments, the effects of nonadrenergic noncholinergic (NANC) nerve activation by electrical field stimulation (EFS) were also examined. The effects of CL 316243 were compared with those of nonselective beta-agonist isoproterenol. CL 316243 caused a concentration-dependent relaxation of the LES smooth muscle. The relaxant action of CL 316243 was determined to be directly at the smooth muscle because it remained unmodified by the neurotoxin tetrodotoxin and other neurohumoral antagonists, and also was observed in the SMCs. L 748337 selectively antagonized the relaxant effect of CL 316243 and, conversely, had no significant effect on the inhibitory actions of isoproterenol. CL 316243 (1 x 10(-8) M) caused an augmentation of NANC relaxation in the LES. Another beta3-agonist, (S)-4-[hydroxy-3-phenoxy-propylamino-ethoxy]-N-(2-methoxyethyl)-phenoxyacetamide (ZD 7114), also caused concentration-dependent full relaxation of the LES that was selectively antagonized by beta3-anatagonist 3-(2-ethylphenoxy)-1-[(1S)1,2,3,4-tetrahydronaphth-1-ylaminol]-(2S)-2-propanol oxalate (SR 59230A). These studies defined the effects of characteristic inhibitory beta3-adrenoceptors in the spontaneously tonic LES smooth muscle and suggested a potential therapeutic role in the esophageal motility disorders characterized by hypertensive LES. PMID:12490574

  17. 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

  18. Endometrial beta3 Integrin profile reflects endometrial receptivity defects in women with unexplained recurrent pregnancy loss

    PubMed Central

    2014-01-01

    Background The pathophysiology of recurrent pregnancy loss (RPL) is still unknown in 50% of the cases. Herein we measure the expression of beta3 integrin subunit, a well-known implantation marker, in women with or without RPL and correlate it with the histological dating of the endometrial tissue. Methods LH-timed endometrial biopsies were obtained from cases (RPL; n = 21, age 33.9+/-4.7) and healthy controls (n = 29; age 29.8+/-4.1) during the mid-secretory phase (post ovulatory day: 8 to 10). Endometrial samples were timed histologically according to Noyes’ criteria and underwent immunohistochemical staining for beta3 integrin expression. For statistical analysis the semi-quantitative HSCORE was assessed. Type I (beta3 negative in an out-of-phase endometrium) and Type II defect (beta3 negative in an in-phase endometrium) were also analysed. Statistical analysis was done with Student t-test, Mann Whitney U test, ANCOVA and chi square for trend. Significance was set as P < 0.05. Results The mean (SD) age in controls was lower compared to cases [(29.8 (4.1) vs. 33.9 (4.7) – P = 0.001; Student t-test)]. The median (range) expression of beta3 integrin in controls and cases was 1.94 (0 to 3.5) vs. 0.82 (0 to 3.6), respectively (P = 0.001; Mann Whitney U test). Significance was still significant after adjusting for age (P = 0.03;ANCOVA). The normal positive staining > =0.7 of beta3 integrin subunit and in-phase endometrium was seen in 24 out of 29 (82.8%) controls, but in only 6 out of 21 (28.6%) of cases with RPL; Type I and II defects were seen in 10.3 and 6.9% of controls, while present in 52.4 and 19.1% of cases, respectively (P = 0.0005; chi-square). Conclusions Women with unexplained RPL had significantly reduced integrin expression compared to controls. Our findings underline the need for further molecular analysis of endometrial tissue in affected women. PMID:24950982

  19. Structure of alpha6 beta3 delta GABA(A) receptors and their lack of ethanol sensitivity.

    PubMed

    Baur, Roland; Kaur, Kuldeep H; Sigel, Erwin

    2009-12-01

    Delta (delta) subunit containing GABA(A) receptors are expressed extra-synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with alpha(6) subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABA(A) receptor pentamers by subunit concatenation. These receptors (composed of alpha(6), beta(3) and delta subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3alpha, 21-dihydroxy-5alpha-pregnan-20-one and to 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. alpha(6)-beta(3)-alpha(6)/delta receptors showed a substantial response to GABA alone. Three receptors, beta(3)-alpha(6)-delta/alpha(6)-beta(3), alpha(6)-beta(3)-alpha(6)/beta(3)-delta and beta(3)-delta-beta(3)/alpha(6)-beta(3), were only uncovered in the combined presence of the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one with GABA. All four receptors were activated by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the delta subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the delta subunit can assume multiple positions in a receptor pentamer composed of alpha(6), beta(3) and delta subunits.

  20. 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.

  1. Crystal structure of the extracellular segment of integrin {alpha}V{beta}3.

    SciTech Connect

    Xiong, J.-P.; Stehle, T.; Diefenbach, B.; Zhang, R.; Dunker, R.; Scott, D. L.; Joachimiak, A.; Goodman, S. L.; Arnaout, M. A.; Biosciences Division; Massachusetts General Hospital; Harvard Medical School; Merck KGaA

    2001-10-12

    Integrins are {alpha}{beta} heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin {alpha}V{sup {beta}}3 at 3.1 Angstroms resolution. Its 12 domains assemble into an ovoid 'head' and two 'tails.' In the crystal, {alpha}V{sup {beta}}3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed {beta}-propeller from {alpha}V and an A domain from {beta}3, and bears a striking resemblance to the G{alpha} /G{sup {beta}} interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the {beta}A domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and {beta}A domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.

  2. Hyperplasia of brown adipose tissue after chronic stimulation of beta 3-adrenergic receptor in rats.

    PubMed

    Nagase, I; Sasaki, N; Tsukazaki, K; Yoshida, T; Morimatsu, M; Saito, M

    1994-12-01

    When mammals are exposed to a cold environment for a long time, the capacity of nonshivering thermogenesis by brown adipose tissue (BAT) increases in association with the increased expression of some specific proteins and tissue hyperplasia, which are totally dependent on sympathetic innervation to this tissue. To clarify roles of the beta-adrenergic mechanism in BAT hyperplasia, the effects of chronic administration of various beta-adrenergic agonists on BAT were examined in rats, especially focusing on some agonists to the beta 3-adrenoceptor which is present specifically in adipocytes. Chronic administration of noradrenaline or isoproterenol for 7-10 days produced a marked increase in the tissue contents of DNA, total protein, mitochondrial uncoupling protein, and insulin-regulatable glucose transporter protein. The trophic effects of noradrenaline and isoproterenol were mimicked by chronic administration of beta 3-adrenergic agonists, such as CL316,243, BRL 26830A, and ICI D7114. These results suggest that the beta 3-adrenoceptor plays important roles for hyperplasia of BAT, and thereby increasing in the capacity of thermogenesis. PMID:7745877

  3. Role of nitric oxide/cyclic GMP and cyclic AMP in beta3 adrenoceptor-chronotropic response.

    PubMed

    Sterin-Borda, Leonor; Bernabeo, Gustavo; Ganzinelli, Sabrina; Joensen, Lilian; Borda, Enri

    2006-04-01

    In this study we determine different signaling pathways involved in beta(3) adrenoceptor (beta(3)-AR) dependent frequency stimulation in isolated rodent atria. Promiscuous coupling between different G-proteins and beta(3)-AR could explain the multiple functional effects of beta(3)-AR stimulation. We examine the mechanisms and functional consequences of dual adenylate cyclase and guanylate cyclase pathways coupling to beta(3)-AR in isolated rodent atria. The beta(3)-AR selective agonists ZD 7114 and ICI 215001 stimulated in a dose-dependent manner the contraction frequency that significantly correlated with cyclic AMP (cAMP) accumulation. Inhibition of adenylate cyclase shifted the chronotropic effect to the right. On the other hand, the ZD 7114 activity on frequency was enhanced by the inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase. This countervailing negative chronotropic nitric oxide-cyclic GMP (NO-cGMP) significantly correlated with the increase on NOS activity and cGMP accumulation. Current analysis showed a negative cross talk between cAMP chronotropic and NO-cGMP effects by inhibition of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), NOS isoforms and Gi-protein on the effects of beta(3)-AR stimulation. RT-PCR detected both eNOS and nNOS in isolated rat atria. NOS isoforms performed independently. Only nNOS participated in limiting the effect of beta(3)-AR stimulation. In eNOS-KO (eNOS-/-) mice the chronotropic effect of beta(3)-AR agonists did not differ from wild type (WT) mice atria, but it was increased by the inhibition of nNOS activity. Our results suggest that the increase in frequency by beta(3)-AR activation on isolated rodent atria is associated to a parallel increases in cAMP. The nNOS-cGMP pathway negatively modulates beta(3)-AR activation. Multiple signal transduction pathways between G-protein and beta(3)-AR may protect myocardium from catecholamine-induced cardiotoxic effects. PMID:16510153

  4. 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

  5. Minute dosages of alpha(nu)beta3-targeted fumagillin nanoparticles impair Vx-2 tumor angiogenesis and development in rabbits.

    PubMed

    Winter, Patrick M; Schmieder, Anne H; Caruthers, Shelton D; Keene, Jeffery L; Zhang, Huiying; Wickline, Samuel A; Lanza, Gregory M

    2008-08-01

    Fumagillin suppresses angiogenesis in cancer models and clinical trials, but it is associated with neurotoxicity at systemic doses. In this study, alpha(nu)beta(3)-targeted fumagillin nanoparticles were used to suppress the neovasculature and inhibit Vx-2 adenocarcinoma development using minute drug doses. Tumor-bearing rabbits were treated on days 6, 9, and 12 postimplantation with alpha(nu)beta(3)-targeted fumagillin nanoparticles (30 microg/kg), alpha(nu)beta(3)-targeted nanoparticles without drug, nontargeted fumagillin nanoparticles (30 microg/kg) or saline. On day 16, MRI was performed with alpha(nu)beta(3)-targeted paramagnetic nanoparticles to quantify tumor size and assess neovascularity. Tumor volume was reduced among rabbits receiving alpha(nu)beta(3)-targeted fumagillin nanoparticles (470+/-120 mm(3)) compared with the three control groups: nontargeted fumagillin nanoparticles (1370+/-300 mm(3), P<0.05), alpha(nu)beta(3)-targeted nanoparticles without drug (1080+/-180 mm(3), P<0.05) and saline (980+/-80 mm(3), P<0.05). MR molecular imaging of control rabbits (no fumagillin) revealed a predominant peripheral distribution of neovascularity representing 7.2% of the tumor rim volume, which decreased to 2.8% (P<0.05) with alpha(nu)beta(3)-targeted fumagillin nanoparticle treatment. Microscopically, the tumor parenchyma tended to show T-cell infiltration after targeted fumagillin treatment, which was not appreciated in control animals. These results suggest that alpha(nu)beta(3)-targeted fumagillin nanoparticles could provide a safe and effective means to deliver MetAP2 inhibitors alone or in combination with cytotoxic or immunotherapy.

  6. 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.

  7. Characteristics of cyanopindolol analogues active at the beta 3-adrenoceptor in rat ileum.

    PubMed Central

    Hoey, A. J.; Jackson, C. M.; Pegg, G. G.; Sillence, M. N.

    1996-01-01

    1. Cyanopindolol (CYP) is a potent antagonist at the beta 3-adrenoceptor in rat ileum. Several analogues of CYP and pindolol were synthesized that also produced antagonist effects at the beta 3-adrenoceptor. However, at high concentrations, these compounds appear to act as "partial agonists'. This study was conducted to determine the structural requirements of CYP analogues necessary for antagonist activity and to examine the possibility that the agonist effects of CYP and its analogues may occur through a mechanism independent of beta-adrenoceptor activation. 2. Analogues of CYP and pindolol were tested for antagonist activity in rat ileum in which the beta 1- and beta 2-adrenoceptors were blocked. Fourteen compounds were tested against (-)-isoprenaline, and four of the more potent analogues were then tested against BRL 37344. The two most potent antagonists were CYP and iodocyanopindolol. The pKb values (negative log of equilibrium dissociation constant) obtained against (-)-isoprenaline were significantly higher than those obtained against BRL 37344, but the cause of this difference is not known. 3. Several structural requirements were determined for antagonist activity. Modification at the carbon atom alpha to the secondary amine caused the antagonist potency to fall as the level of saturation was reduced. Thus, a quaternary carbon group, such as t-butyl, produced the most potent antagonist. Substitution with a large moiety such as a cyclohexyl or benzyl group reduced antagonist activity, probably due to steric hindrance. Inclusion of an electron-withdrawing group, such as a cyano or ethylester moiety, alpha to the indole nitrogen, also increased the potency. Iodination of CYP and ethylesterpindolol at the 3-position of the indole ring did not increase antagonist potency. In contrast, iodination of the almost inactive analogues produced a significant increase in potency, suggesting that a beneficial electronic effect on the indole ring imparted by the iodo

  8. Lateral paracapsular GABAergic synapses in the basolateral amygdala contribute to the anxiolytic effects of beta 3 adrenoceptor activation.

    PubMed

    Silberman, Yuval; Ariwodola, Olusegun J; Chappell, Ann M; Yorgason, Jordan T; Weiner, Jeff L

    2010-08-01

    Norepinephrine (NE) is known to play an integral role in the neurobiological response to stress. Exposure to stressful stimuli increases NE levels in brain regions that regulate stress and anxiety, like the basolateral amygdala (BLA). NE is thought to increase excitability in these areas through alpha- and beta-adrenoceptors (ARs), leading to increased anxiety. Surprisingly, recent studies have shown that systemic beta 3-AR agonist administration decreases anxiety-like behaviors, suggesting that beta 3-ARs may inhibit excitability in anxiety-related brain regions. Therefore, in this study we integrated electrophysiological and behavioral approaches to test the hypothesis that the anxiolytic effects of beta 3-AR agonists may be mediated by an increase in BLA GABAergic inhibition. We examined the effect of a selective beta 3-AR agonist, BRL37344 (BRL), on GABAergic synapses arising from local circuit interneurons and inhibitory synapses originating from a recently described population of cells called lateral paracapsular (LPCS) interneurons. Surprisingly, BRL selectively enhanced LPCS-evoked inhibitory postsynaptic currents (eIPSCs) with no effect on local GABAergic inhibition. BRL also had no effect on glutamatergic synaptic excitation within the BLA. BRL potentiation of LPCS eIPSCs was blocked by the selective beta 3-AR antagonist, SR59230A, or by intracellular dialysis of Rp-CAMPS (cAMP-dependent protein kinase inhibitor), and this enhancement was not associated with any changes in spontaneous IPSCs or LPCS paired-pulse ratio. BRL also increased the amplitude of unitary LPCS IPSCs (uIPSCs) with no effect on uIPSC failure rate. Finally, bilateral BLA microinjection of BRL reduced anxiety-like behaviors in an open-field assay and the elevated plus-maze. Collectively, these data suggest that beta 3-AR activation selectively enhances LPCS, but not local, BLA GABAergic synapses, and that increases in LPCS-mediated inhibition may contribute to the anxiolytic profile of

  9. Sequence specific protein binding to and activation of the TGF-beta 3 promoter through a repeated TCCC motif.

    PubMed Central

    Lafyatis, R; Denhez, F; Williams, T; Sporn, M; Roberts, A

    1991-01-01

    We have previously characterized the TGF-beta 3 promoter and shown that the activity of this promoter is highly variable in different cell types. Although the promoter contains a proximal cAMP responsive element, which is critical to basal and forskolin-induced promoter activity, this element is not responsible for the variable, cell-specific regulation of the promoter. In this paper, we identify a 25 base pair sequence in the proximal region of the TGF-beta 3 promoter that binds a novel DNA-binding protein. This region includes the sequence T-CCCTCCCTCCC, (3 x TCCC), and mutation of these T-CCC repeats inhibits protein binding. Further, we show that in the cell line A375, which we have previously shown expresses high levels of TGF-beta 3 mRNA, this region is responsible for mediating high level TGF-beta 3 promoter activity. Immediately 3' to the 3 x TCCC sequence is a consensus AP-2 binding site, however, we show that this region does not bind AP-2, and AP-2 does not transactivate the TGF-beta 3 promoter. Therefore, we provide strong evidence that high level expression of TGF-beta 3 in A375 cells results from transactivation of the TGF-beta 3 promoter by a protein that binds to a repeated TCCC motif in the promoter and suggest that this DNA-binding protein likely also regulates aspects of developmental and tissue-specific expression of this cytokine. Images PMID:1754378

  10. Zeneca ZD7114 acts as an antagonist at beta 3-adrenoceptors in rat isolated ileum.

    PubMed

    Growcott, J W; Holloway, B; Green, M; Wilson, C

    1993-12-01

    1. The relaxant effects of Zeneca ZD7114, BRL37344 (putative beta 3-adrenoceptor agonists) and various phenylethylamine-based agonists were studied in isolated ileum of the rat where tone was increased with carbachol (0.5 microM). Agonist-induced relaxation.was measured under equilibrium conditions with alpha-, beta 1- and beta 2-adrenoceptors inhibited. 2. Relaxant responses were obtained to isoprenaline, noradrenaline, and BRL37344, although, the efficacy of this latter agent was significantly.lower than that of isoprenaline. Salbutamol caused weak relaxation (< 20%) at high concentrations (10 microM) and ZD7114 was without significant relaxant effect even at high concentrations (10 microM). 3. Relaxant responses to isoprenaline and BRL37344 were weakly antagonized by high concentrations of (+/-)-propranolol (10 and 100 microM) yielding pKB values of 5.7 with isoprenaline as the agonist and 5.5 with BRL37344 as the agonist. 4. The non-selective beta-adrenoceptor antagonist, (+/-)-alprenolol (1-100 microM) caused competitive antagonism of the relaxant responses to isoprenaline (pA2 value = 6.5). A similar pKB value was obtained when BRL37344 was used as the agonist (6.4). 5. Relaxant effects of isoprenaline and BRL37344 were also antagonized by ZD7114 (1-100 microM) yielding pA2 and pKB values of 6.3 and 6.7 respectively. 6. The low potencies of (+/-)-propranolol and (+/-)-alprenolol as antagonists of the relaxant responses to isoprenaline and BRL37344 indicate that both the agonists and antagonists employed in the current study may interact with beta 3-adrenoceptors in the rat isolated ileum. Contrary to the previous findings in guinea-pig ileum, where BRL37344 and ZD7114 were full agonists, in the current study, BRL37344 was a partial agonist and ZD7114 an antagonist at the beta 3-adrenoceptor in rat ileum. PMID:7905770

  11. Two-Dimensional Infrared (2DIR) Spectroscopy of the Peptide Beta3s Folding

    PubMed Central

    Lai, Zaizhi; Preketes, Nicholas K; Jiang, Jun; Mukamel, Shaul; Wang, Jin

    2013-01-01

    Probing underlying free energy landscape, pathways, and mechanism is the key for understanding protein folding in theory and experiment. Recently time-resolved two-dimensional infrared (2DIR) with femtosecond laser pulses, has emerged as a promising tool for investigating the protein folding dynamics on faster timescales than possible by NMR. We have employed molecular dynamics simulations to compute 2DIR spectra of the folding process of a peptide, Beta3s. Simulated non-chiral and chiral 2DIR signals illustrate the variation of the spectra as the peptide conformation evolves along the free energy landscape. Chiral spectra show stronger changes than the non-chiral signals because cross peaks caused by the formation of the β-sheet are clearly resolved. Chirality-induced 2DIR may be used to detect the folding of β-sheet proteins with high spectral and temporal resolution. PMID:23956818

  12. 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.

  13. Zeneca ZD7114 acts as an antagonist at beta 3-adrenoceptors in rat isolated ileum.

    PubMed Central

    Growcott, J. W.; Holloway, B.; Green, M.; Wilson, C.

    1993-01-01

    1. The relaxant effects of Zeneca ZD7114, BRL37344 (putative beta 3-adrenoceptor agonists) and various phenylethylamine-based agonists were studied in isolated ileum of the rat where tone was increased with carbachol (0.5 microM). Agonist-induced relaxation.was measured under equilibrium conditions with alpha-, beta 1- and beta 2-adrenoceptors inhibited. 2. Relaxant responses were obtained to isoprenaline, noradrenaline, and BRL37344, although, the efficacy of this latter agent was significantly.lower than that of isoprenaline. Salbutamol caused weak relaxation (< 20%) at high concentrations (10 microM) and ZD7114 was without significant relaxant effect even at high concentrations (10 microM). 3. Relaxant responses to isoprenaline and BRL37344 were weakly antagonized by high concentrations of (+/-)-propranolol (10 and 100 microM) yielding pKB values of 5.7 with isoprenaline as the agonist and 5.5 with BRL37344 as the agonist. 4. The non-selective beta-adrenoceptor antagonist, (+/-)-alprenolol (1-100 microM) caused competitive antagonism of the relaxant responses to isoprenaline (pA2 value = 6.5). A similar pKB value was obtained when BRL37344 was used as the agonist (6.4). 5. Relaxant effects of isoprenaline and BRL37344 were also antagonized by ZD7114 (1-100 microM) yielding pA2 and pKB values of 6.3 and 6.7 respectively. 6. The low potencies of (+/-)-propranolol and (+/-)-alprenolol as antagonists of the relaxant responses to isoprenaline and BRL37344 indicate that both the agonists and antagonists employed in the current study may interact with beta 3-adrenoceptors in the rat isolated ileum.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7905770

  14. 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

  15. Alteration of medial-edge epithelium cell adhesion in two Tgf-beta3 null mouse strains.

    PubMed

    Martínez-Sanz, Elena; Del Río, Aurora; Barrio, Carmen; Murillo, Jorge; Maldonado, Estela; Garcillán, Beatriz; Amorós, María; Fuerte, Tamara; Fernández, Alvaro; Trinidad, Eva; Rabadán, María Angeles; López, Yamila; Martínez, María Luisa; Martínez-Alvarez, Concepción

    2008-04-01

    Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-beta3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-beta3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the alpha5- and beta1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-beta3 or neutralizing antibodies against fibronectin or the alpha5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-beta3 null mutants; the importance of TGF-beta3 to restore their normal pattern of expression; and the crucial role of fibronectin and the alpha5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-beta3 null mutant mice.

  16. beta3-adrenoceptor agonist prevents alterations of muscle diacylglycerol and adipose tissue phospholipids induced by a cafeteria diet.

    PubMed

    Darimont, Christian; Turini, Marco; Epitaux, Micheline; Zbinden, Irène; Richelle, Myriam; Montell, Eulàlia; Ferrer-Martinez, Andreu; Macé, Katherine

    2004-08-17

    BACKGROUND: Insulin resistance induced by a high fat diet has been associated with alterations in lipid content and composition in skeletal muscle and adipose tissue. Administration of beta3-adrenoceptor (beta3-AR) agonists was recently reported to prevent insulin resistance induced by a high fat diet, such as the cafeteria diet. The objective of the present study was to determine whether a selective beta3-AR agonist (ZD7114) could prevent alterations of the lipid profile of skeletal muscle and adipose tissue lipids induced by a cafeteria diet. METHODS: Male Sprague-Dawley rats fed a cafeteria diet were treated orally with either the beta3-AR agonist ZD7114 (1 mg/kg per day) or the vehicle for 60 days. Rats fed a chow diet were used as a reference group. In addition to the determination of body weight and insulin plasma level, lipid content and fatty acid composition in gastronemius and in epididymal adipose tissue were measured by gas-liquid chromatography, at the end of the study. RESULTS: In addition to higher body weights and plasma insulin concentrations, rats fed a cafeteria diet had greater triacylglycerol (TAG) and diacylglycerol (DAG) accumulation in skeletal muscle, contrary to animals fed a chow diet. As expected, ZD7114 treatment prevented the excessive weight gain and hyperinsulinemia induced by the cafeteria diet. Furthermore, in ZD7114 treated rats, intramyocellular DAG levels were lower and the proportion of polyunsaturated fatty acids, particularly arachidonic acid, in adipose tissue phospholipids was higher than in animals fed a cafeteria diet. CONCLUSIONS: These results show that activation of the beta3-AR was able to prevent lipid alterations in muscle and adipose tissue associated with insulin resistance induced by the cafeteria diet. These changes in intramyocellular DAG levels and adipose tissue PL composition may contribute to the improved insulin sensitivity associated with beta3-AR activation. PMID:15507149

  17. Interactions between TGF-beta1 and TGF-beta3 and their role in medial edge epithelium cell death and palatal fusion in vitro.

    PubMed

    Murillo, Jorge; Maldonado, Estela; Barrio, Maria Carmen; Del Río, Aurora; López, Yamila; Martínez-Sanz, Elena; González, Ignacio; Martín, Concepción; Casado, Inmaculada; Martínez-Alvarez, Concepción

    2009-02-01

    In recent decades, studies have shown that both TGF-beta(1) and TGF-beta(3) play an important role in the induction of medial edge epithelium (MEE) cell death and palatal fusion. Many of these experiments involved the addition or blockage of one of these growth factors in wild-type (WT) mouse palate cultures, where both TGF-beta(1) and TGF-beta(3) are present. Few studies have addressed the existence of interactions between TGF-beta(1) and TGF-beta(3), which could modify their individual roles in MEE cell death during palatal fusion. We carried out several experiments to test this possibility, and to investigate how this could influence TGF-beta(1) and TGF-beta(3) actions on MEE cell death and palatal shelf fusion. We double-immunolabelled developing mouse palates with anti-TGF-beta(1) or anti-TGF-beta(3) antibodies and TUNEL, added rhTGF-beta(1) or rhTGF-beta(3) or blocked the TGF-beta(1) and TGF-beta(3) action at different concentrations to WT or Tgf-beta(3) null mutant palate cultures, performed in situ hybridizations with Tgf-beta(1) or Tgf-beta(3) riboprobes, and measured the presence of TUNEL-positive midline epithelial seam (MES) cells and MES disappearance (palatal shelf fusion) in the different in vitro conditions. By combining all these experiments, we demonstrate great interaction between TGF-beta(1) and TGF-beta(3) in the developing palate and confirm that TGF-beta(3) has a more active role in MES cell death than TGF-beta(1), although both are major inductors of MES disappearance. Finally, the co-localization of TGF-beta(1), but not TGF-beta(3), with TUNEL in the MES allows us to suggest a possible role for TGF-beta(1) in MES apoptotic clearance.

  18. CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts.

    PubMed

    Chabadel, Anne; Bañon-Rodríguez, Inmaculada; Cluet, David; Rudkin, Brian B; Wehrle-Haller, Bernhard; Genot, Elisabeth; Jurdic, Pierre; Anton, Ines M; Saltel, Frédéric

    2007-12-01

    The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with beta3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)-/- OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP-/- OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP-/- OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.

  19. 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

  20. Effects of ZD7114, a selective beta3-adrenoceptor agonist, on neuroendocrine mechanisms controlling energy balance.

    PubMed

    Savontaus, E; Pesonen, U; Rouru, J; Huupponen, R; Koulu, M

    1998-04-24

    Selective beta3-adrenoceptor agonists increase energy expenditure by increasing non-shivering thermogenesis in brown adipose tissue. The aim of this study was to investigate how changes in energy balance affect energy intake and interaction of peripheral metabolic feedback signals with central neuroendocrine mechanisms participating in the control of body energy balance. Expression of preproneuropeptide Y (preproNPY) mRNA in the arcuate nucleus and preprocorticotropin-releasing factor (CRF) mRNA in the paraventricular nucleus were measured by in situ hybridisation technique after 1 day, 1 and 5 weeks of treatment with ZD7114 ((S)-4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]-N-(2-methoxyet hyl)phenoxyacetamide, 3 mg kg(-1) day(-1) in drinking water) in obese fa/fa Zucker rats. In addition, expression of leptin mRNA in epididymal fat and serum levels of leptin were analysed. Food intake, body weights, binding of GDP to brown adipose tissue mitochondria, plasma insulin and glucose were also measured. Treatment with ZD7114 significantly reduced weight gain and activated brown adipose tissue thermogenesis, but had no effect on food intake. Expressions of preproNPY or preproCRF mRNAs were similarly not changed by treatment with ZD7114. Furthermore, ZD7114 had no effect on plasma insulin or leptin and the expression of leptin mRNA in epididymal fat. However, statistically significant correlations were found between preproNPY and preproCRF mRNA expressions and brown fat thermogenic activity and plasma insulin levels in the ZD7114 treated rats, but not in the control rats. It is concluded that treatment with ZD7114 markedly activated brown fat thermogenesis, but did not affect neuropeptide Y (NPY) and CRF gene expression per se. However, the correlation analyses suggest that ZD7114 may modulate feedback connections of brown adipose tissue thermogenesis and plasma insulin with the hypothalamic neuroendocrine mechanisms integrating body energy balance. PMID:9653893

  1. Gastroprotective effect of beta3 adrenoreceptor agonists ZD 7114 and CGP 12177A in rats.

    PubMed

    Sevak, Rajkumar; Paul, Arindam; Goswami, Sunita; Santani, Devdas

    2002-10-01

    The effects of beta(3) adrenergic receptor agonists, (S)-4-[2-hydroxy-3-phenoxy-propylamino-ethoxy]-N-(2-methoxyethyl)-phenoxyacetamide (ZD 7114) and (+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one (CGP 12177A), were studied on aspirin plus pylorus ligation-induced gastric ulcers, gastric mucosal blood flow and gastric motility in rats. Pretreatment with ZD 7114 (3 mg kg(-1), p.o.) and CGP 12177A (3.5 mg kg(-1), p.o.) resulted in significant reduction in the incidences of gastric ulceration in aspirin plus pylorus ligated rats and results were comparable with the cimetidine treated group. Ulcer index was significantly reduced by ZD 7114 (0.71+/-0.05, P<0.05) and CGP 12177A (1.15+/-0.27, P<0.05) when compared with the control group (4.47+/-0.38). Further, significant increase in total carbohydrates to protein content ratio (mucin activity) was also observed. However, they did not alter the acid secretory parameters such as total acidity, total acid output and pepsin activity. Effects of ZD 7114 and CGP 12177A on gastric mucosal blood flow were studied using neutral red clearance method. Both the treatments showed significant increase in gastric mucosal blood flow (GV/Bt) as compared to control group. Effect on gastric motility was evaluated by estimation of phenol red concentration in rat stomach. Significantly higher concentrations of phenol red in the stomach were observed in ZD 7114 and CGP 12177A treated rats. Both ZD 7114 and CGP 12177A showed significant gastroprotective effect in the present study. The mechanism of this effect may be attributed to enhancement of gastric mucosal blood flow, reduction in gastric motility and strengthening of gastric mucosal barrier. PMID:12361698

  2. Involvement of PLC-beta3 in the effect of morphine on memory retrieval in passive avoidance task.

    PubMed

    Bianchi, E; Lehmann, D; Vivoli, E; Norcini, M; Ghelardini, C

    2010-06-01

    Phospholipase C (PLC) is one signalling effector enzyme whose activity is directly modulated by opioids. Several physiological studies have implicated PLC-linked pathways in in-vivo pain regulation and opioid tolerance. Co-administration of PLC-beta(2/3) activity blocker M119 with morphine resulted in a dramatic increase in morphine-induced amnesic effect in mice, proving a role for beta subunit of PLC enzyme in these processes. Administration of morphine to mice at amnesic dose increased PLC-beta(3) activity, with respect to basal value, in the membrane-soluble material from anterior cortex and hippocampal formation in brain areas. PLC-beta(3) appears to be simultaneously implicated in both analgesic and amnesic effects induced by administration of morphine to mice suggesting a commonality in the molecular mechanisms of morphine-induced analgesia and memory impairment.

  3. Urinary tract toxicity in rats following administration of beta 3-adrenoceptor agonists.

    PubMed

    Waghe, M; Westwood, R; Nunn, G; Kalinowski, A; Aldridge, A

    1999-01-01

    ZD7114, [(S)-4-[2-(2-hydroxy-3 phenoxypropylamine)ethoxy]-N-(2-methoxyethyl) phenoxyacetamide], and ZD2079, [(R)-N-(2-[4- (carboxymethyl)phenoxy]ethyl)-N-(beta-hydroxyphenethyl)ammonium chloride], are beta 3-adrenoceptor stimulants with selectivity for brown adipose tissue. ZD7144 is the hydrochloride salt of the S-enantiomer of the racemic amide ZD2079. They were developed as potential novel treatments for obesity and non-insulin-dependent diabetes mellitus. Male and female rats were dosed separately by gavage for a minimum of 28 days with 0, 10, 50, and 500 mg/kg/day of ZD7114 or with 0, 10, 30, and 150 mg/kg/day of ZD2079. Two further groups of male and female rats were dosed with 0 and 500 mg/kg/day of ZD7114 for 28 days and were then allowed a 6-wk, undosed withdrawal period. At high doses, both compounds caused urinary tract toxicity, which primarily affected the distal tubules and collecting ducts of the kidney via tubular necrosis. They also caused ureteric inflammation, cystitis, and accumulation of crystalline inclusions throughout the urinary tract. As a result of urinary tract toxicity, affected animals from one or both studies showed reduced red blood cell indices, lower platelet counts, and higher white cell counts. Blood chemistry revealed lower plasma concentrations of glucose (7.28 +/- 1.37 compared to 8.11 +/- 0.65 for the control) and total protein (63.42 +/- 3.65 compared to 69.17 +/- 3.24 for the control) and increased plasma urea (37.15 +/- 19.96 compared to 8.09 +/- 0.87 for the control). Urinalysis showed an increase in the number of crystals, blood, and protein. In the urinary tract, the severe crystalluria with accumulation of crystalline material indicated that this may have a role in the etiology of the target organ toxicity. Poor solubility of the compounds at normal urinary pH was considered a possible mechanism for the crystalluria. PMID:10207980

  4. 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

  5. Dissecting the role of integrin subunits alpha 2 and beta 3 in rotavirus cell entry by RNA silencing.

    PubMed

    Isa, Pavel; Sánchez-Alemán, Miguel Angel; López, Susana; Arias, Carlos F

    2009-11-01

    Several cell surface molecules have been implicated in rotavirus cell entry, however, their individual relevance during this process is unknown. In this work, the expression of integrins alpha2, beta2, and alpha v beta 3, the heat shock cognate protein 70, and of ganglioside GM1 in different cell lines of human and simian origin was correlated with the infectivity of four rotavirus strains. We observed that different combinations of receptor expression correlated with the infectivity of rotavirus strains, suggesting that the participation of several receptors is important for rotavirus infection. To characterize the relevance of integrins alpha2 and alpha v beta 3 in more detail, their expression was silenced using RNA interference. About 80% decrease in the cell content of integrins resulted in 15-30% decrease of infectivity of strains RRV and Wa when measured by a focus-forming assay, while there was no decrease of infectivity when measured by flow cytometry in integrin-deficient cells. Altogether these data suggest that integrins alpha2 and alpha v beta 3 do not play a major role in the rotavirus entry process.

  6. The acid metabolite of ZD7114 is a partial agonist of lipolysis mediated by the rat beta 3-adrenoceptor.

    PubMed

    Mayers, R M; Quayle, S P; Thompson, A J; Grant, T L; Holloway, B R

    1996-01-11

    Experiments were performed to characterise the lipolytic effects of the acid metabolite, ZM215001, ((S)-4-[2-hydroxy-3-phenoxy-propylamino-ethoxy] phenoxyacetic acid) of the putative beta 3-adrenoceptor agonist, ZD7114 ((S)-4-[2-hydroxy-3-phenoxy-propylamino-ethoxy]-N-(2-methoxyethyl) phenoxyacetamide) on isolated rat white adipocytes. ZM215001 was used for these studies since it is the predominant moiety after in vivo administration of ZD7114. The agonist properties of ZM215001 were assessed in comparison to the standard nonselective beta-adrenoceptor agonist (+/-)-isoprenaline and the beta 3-adrenoceptor-selective agonist BRL 37344. Isoprenaline, BRL 37344 and ZM215001 all stimulated the rate of free fatty acid release from isolated adipocytes with the order of potency being BRL > isoprenaline > ZM215001. The maximum effect of BRL 37344 was equivalent to that of isoprenaline, but ZM215001 achieved only 30% of the maximum isoprenaline response. ZM215001 competitively antagonised the lipolytic response to BRL 37344 (pA2 = 7.26), whereas the agonist effects of BRL 37344 were not antagonised competitively by the selective antagonists ICI 118551 and CGP 20712A, at concentrations which would be expected to block beta 1- and beta 2-adrenoceptors respectively. These results indicate that ZM215001 has low intrinsic activity at the rat adipocyte beta 3-adrenoceptor, and is a partial agonist of lipolysis in rat white adipocytes. PMID:8720584

  7. Effect of conventional (mixed beta 1/beta 2) and novel (beta 3) adrenergic agonists on thermoregulatory behavior.

    PubMed

    Carlisle, H J; Stock, M J

    1991-10-01

    The effects of submaximal and maximal thermogenic doses of isoproterenol (ISO) on operant thermoregulatory responses in a cold (-8 degrees C) environment were tested in lean (+/?) Zucker rats trained to barpress for radiant heat. Contrary to expectations, ISO rats pressed for twice as much exogenous heat as controls, but showed a smaller rise in colonic temperature. Conversely, a beta 3-selective adrenergic agonist (RO40-2148) decreased the requirement for exogenous heat and produced larger rises in colonic temperature. RO40-2148 and another beta 3-agonist (ICI D7114) produced similar responses in obese (fa/fa) Zucker rats, but tests with ISO were terminated because it caused profound, and lethal hypothermia. The hypothermic effects of ISO on colonic temperature were also observed in Sprague-Dawley rats at room temperature (22 degrees C), whereas RO40-2148 produced hyperthermia. These results provide behavioral evidence for the high thermogenic selectivity of these novel adrenergic agonists and support the existence of an atypical beta 3-adrenoceptor. The hypothermic effects of ISO are presumed to be due to actions on beta 1- and/or beta 2-adrenoceptors. PMID:1687163

  8. Mapping early conformational changes in alphaIIb and beta3 during biogenesis reveals a potential mechanism for alphaIIbbeta3 adopting its bent conformation.

    PubMed

    Mitchell, W Beau; Li, Jihong; Murcia, Marta; Valentin, Nathalie; Newman, Peter J; Coller, Barry S

    2007-05-01

    Current evidence supports a model in which the low-affinity state of the platelet integrin alphaIIbbeta3 results from alphaIIbbeta3 adopting a bent conformation. To assess alphaIIbbeta3 biogenesis and how alphaIIbbeta3 initially adopts the bent conformation, we mapped the conformational states occupied by alphaIIb and beta3 during biogenesis using conformation-specific monoclonal antibodies (mAbs). We found that alphaIIbbeta3 complex formation was not limited by the availability of either free pro-alphaIIb or free beta3, suggesting that other molecules, perhaps chaperones, control complex formation. Five beta3-specific, ligand-induced binding site (LIBS) mAbs reacted with much or all free beta3 but not with beta3 when in complex with mature alphaIIb, suggesting that beta3 adopts its mature conformation only after complex formation. Conversely, 2 alphaIIb-specific LIBS mAbs directed against the alphaIIb Calf-2 region adjacent to the membrane reacted with only minor fractions of free pro-alphaIIb, raising the possibility that pro-alphaIIb adopts a bent conformation early in biogenesis. Our data suggest a working model in which pro-alphaIIb adopts a bent conformation soon after synthesis, and then beta3 assumes its bent conformation by virtue of its interaction with the bent pro-alphaIIb.

  9. Evidence for beta3-adrenoceptor subtypes in relaxation of the human urinary bladder detrusor: analysis by molecular biological and pharmacological methods.

    PubMed

    Takeda, M; Obara, K; Mizusawa, T; Tomita, Y; Arai, K; Tsutsui, T; Hatano, A; Takahashi, K; Nomura, S

    1999-03-01

    The purpose of the present study was to confirm the presence of beta3-adrenoceptor subtype in the relaxation of human urinary bladder detrusor tissue by reverse transcription-polymerase chain reaction (PCR); direct sequencing of the PCR product, in situ hybridization; and isometric contraction. Using reverse transcription-PCR, the mRNAs of three receptor subtypes (beta1, beta2, and beta3) were expressed in the human urinary bladder detrusor tissue. Direct sequencing of the PCR product of the above beta3-adrenoceptor revealed no mutation in the amplified regions. In situ hybridization with digoxygenin-labeled oligonucleotide probe revealed the presence of the mRNA of beta3-adrenoceptor subtype in the smooth muscle of the urinary bladder. The relaxant effects of isoproterenol (a nonselective beta-adrenoceptor agonist); ZD7114, BRL37344, and CGP12177A (putative selective beta3-adrenoceptor agonists); and SR59230A (a putative selective beta3-adrenoceptor antagonist) were tested using an isometric contraction technique. Isoproterenol in either the presence or absence of both atenolol (a beta1-adrenoceptor-selective antagonist) and butoxamine (a beta2-adrenoceptor-selective antagonist) revealed a relaxant effect on the carbachol-induced contraction of the human urinary bladder detrusor. Both BRL37344 and CGP12177A also revealed relaxant effects on the human urinary bladder detrusor, but ZD7114 did not elicit any relaxation. These results suggest that beta3-adrenoceptor may have some role in urine storage in the human urinary bladder. PMID:10027879

  10. 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

  11. The crystal structure of TGF-beta 3 and comparison to TGF-beta 2: implications for receptor binding.

    PubMed Central

    Mittl, P. R.; Priestle, J. P.; Cox, D. A.; McMaster, G.; Cerletti, N.; Grütter, M. G.

    1996-01-01

    Transforming growth factors beta belong to a group of cytokines that control cellular proliferation and differentiation. Five isoforms are known that share approximately 75% sequence identity, but exert different biological activities. The structure of TGF-beta 3 was solved by X-ray crystallography and refined to a final R-factor of 17.5% at 2.0 A resolution. Comparison with the structure of TGF-beta 2 (Schlunegger MP, Grütter MG, 1992, Nature 358:430-434; Daopin S, Piez KA, Ogawa Y, Davies DR, 1992, Science 257:369-373) reveals a virtually identical central core. Differences exist in the conformations of the N-terminal alpha-helix and in the beta-sheet loops. In TGF-beta 3, the N-terminal alpha-helix has moved approximately 1 A away from the central core. This movement can be correlated with the mutation of Leu 17 to Val and Ala 47 to Pro in TGF-beta 3. The beta-sheet loops rotate as a rigid body 9 degrees around an axis that runs approximately parallel to the dimer axis. If these differences are recognized by the TGF-beta receptors, they might account for the individual cellular responses. A molecule of the precipitating agent dioxane is bound in a crystal contact, forming a hydrogen bond with Trp 32. This dioxane may occupy a carbohydrate-binding site, because dioxane possesses some structural similarity with a carbohydrate. The dioxane is in contact with two tryptophans, which are often involved in carbohydrate recognition. PMID:8819159

  12. Effect of beta 3-adrenergic agonist (Zeneca D7114) on thermoregulation in near-term lambs delivered by cesarean section.

    PubMed

    Clarke, L; Bird, J A; Lomax, M A; Symonds, M E

    1996-08-01

    We investigated the effect of a beta 3-adrenergic agonist, Zeneca D7114, on thermoregulation in near-term lambs delivered by cesarean section. Lambs were delivered into a cool ambient temperature of 15 degrees C, and between 60 and 80 min of life were given an oral dose of Zeneca D7114 (10 mg.kg body weight-1) dissolved in 20 mL of milk, or milk alone. During the first 0.5 h of life colonic temperature decreased in all lambs, and then increased to plateau levels (39.6-40.4 degrees C) after 120-150 min of life, in 19 out of 23 lambs studied. In the remaining lambs, colonic temperature failed to return to normothermic values, plateauing at 34.3 degrees C. All control lambs were observed to shiver throughout the study, but after Zeneca D7114 treatment 7 out of 10 normothermic lambs stopped shivering, and plateau colonic temperature was 0.8 degree C higher. Hypothermic beta 3-agonist-treated lambs had significantly lower rates of heat production, breathing frequency, and plasma triiodothyronine and cortisol concentrations than normothermic lambs. the level of GDP binding and norepinephrine content of brown adipose tissue (BAT) sampled from hypothermic beta 3-agonist-treated lambs was significantly lower than in normothermic lambs. There was no difference in GDP binding in BAT between control and Zeneca D7114-treated groups, but the Hb content was higher in the latter group. It is concluded that administration of Zeneca D7114 to euthyroid lambs enhances their ability to thermoregulate and restore colonic temperature without altering the thermogenic activity of BAT. This response may be mediated by increasing blood flow to BAT and/or an improvement in the animal's thermal efficiency (i.e. decreased heat loss) due to a reduced reliance on shivering thermogenesis. PMID:8827786

  13. Beta 1- and beta 2-adrenoceptor antagonist activities of ICI-215001, a putative beta 3-adrenoceptor agonist.

    PubMed Central

    Tesfamariam, B.; Allen, G. T.

    1994-01-01

    1. The present study was undertaken to characterize the beta 3-adrenoceptor agonist activity of ICI-215001 and to determine whether it exhibits additional activities on beta 1- and beta 2-adrenoceptors in isolated spontaneously beating atrium, trachea and ileum of guinea-pig. 2. In guinea-pig atrium, isoprenaline, a non-selective beta-adrenoceptor agonist, caused concentration-dependent, positive chronotropic effects that were inhibited by atenolol, a selective beta 1-antagonist. ICI-215001 also competitively antagonized the increase in heart rate caused by isoprenaline. 3. ICI-215001 exhibited low intrinsic activity at increasing the beating rate of atrium and no activity on resting or induced tone of tracheal strips. 4. In strips of guinea-pig trachea, contracted submaximally with carbachol, isoprenaline, caused concentration-dependent relaxations. Both ICI-118551, a selective beta 2-adrenoceptor antagonist, and ICI-215001 competitively inhibited the relaxations caused by isoprenaline. 5. In isolated strips of guinea-pig ileum longitudinal smooth muscle contracted with histamine, isoprenaline and ICI-215001 caused relaxations which were inhibited by alprenolol, a beta-adrenoceptor antagonist with modest affinity for beta 3-adrenoceptors, but were resistant to ICI-118551 and atenolol. 6. These results indicate that ICI-215001 exhibits beta 3-adrenoceptor agonist activity as demonstrated by relaxations mediated via atypical beta-adrenoceptors in the longitudinal smooth muscle of guinea-pig ileum. Further, the studies demonstrate that ICI-215001 can act as an antagonist at beta 1- and beta 2-adrenoceptors in situations where its intrinsic agonist activity is low. PMID:7913381

  14. Basic Fibroblast Growth Factor-2/beta3 Integrin Expression Profile: Signature of Local Progression After Chemoradiotherapy for Patients With Locally Advanced Non-Small-Cell Lung Cancer

    SciTech Connect

    Massabeau, Carole; Rouquette, Isabelle; Lauwers-Cances, Valerie; Mazieres, Julien; Bachaud, Jean-Marc; Armand, Jean-Pierre; Delisle, Marie-Bernadette; Favre, Gilles; Toulas, Christine; Cohen-Jonathan-Moyal, Elizabeth

    2009-11-01

    Purpose: No biologic signature of chemoradiotherapy sensitivity has been reported for patients with locally advanced non-small-cell lung cancer (NSCLC). We have previously demonstrated that basic fibroblast growth factor (FGF-2) and alphavbeta3 integrin pathways control tumor radioresistance. We investigated whether the expression of the proteins involved in these pathways might be associated with the response to treatment and, therefore, the clinical outcome. Methods and Materials: FGF-2, beta3 integrin, angiopoietin-2, and syndecan-1 expression was studied using immunohistochemistry performed on biopsies obtained, before any treatment, from 65 patients exclusively treated with chemoradiotherapy for locally advanced NSCLC. The response to treatment was evaluated according to the Response Evaluation Criteria in Solid Tumors criteria using computed tomography at least 6 weeks after the end of the chemoradiotherapy. Local progression-free survival, metastasis-free survival, and disease-free survival were studied using the log-rank test and Cox proportional hazard analysis. Results: Among this NSCLC biopsy population, 43.7% overexpressed beta3 integrin (beta3{sup +}), 43% FGF-2 (FGF-2{sup +}), 41.5% syndecan-1, and 59.4% angiopoietin-2. Our results showed a strong association between FGF-2 and beta3 integrin expression (p = .001). The adjusted hazard ratio of local recurrence for FGF-2{sup +}/beta3{sup +} tumors compared with FGF-2{sup -}/beta3{sup -} tumors was 6.1 (95% confidence interval, 2.6-14.6, p = .005). However, the risk of local recurrence was not increased when tumors overexpressed beta3 integrin or FGF-2 alone. Moreover, the co-expression of these two proteins was marginally associated with the response to chemoradiotherapy and metastasis-free survival. Conclusion: The results of this study have identified the combined profile FGF-2/beta3 integrin expression as a signature of local control in patients treated with chemoradiotherapy for locally advanced

  15. 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

  16. 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

  17. Beta 3-adrenergic receptor stimulation restores message and expression of brown-fat mitochondrial uncoupling protein in adult dogs.

    PubMed Central

    Champigny, O; Ricquier, D; Blondel, O; Mayers, R M; Briscoe, M G; Holloway, B R

    1991-01-01

    Brown adipose tissue (BAT) is present throughout life in rodents and plays an important role in energy balance. However, whereas BAT is clearly recognizable in the neonates of larger mammals (including dogs, cats, sheep, cattle, and humans), it is undetectable or present in only small quantities in adults of these species and is replaced by a tissue with the gross characteristics of white adipose tissue. Here we provide evidence that treatment of adult dogs with a beta 3-adrenergic receptor agonist (ICI D7114) that has thermogenic and antiobesity properties leads to the appearance of BAT at several anatomical sites. The presence of BAT was primarily demonstrated by monitoring the inner mitochondrial membrane uncoupling protein and its mRNA, which are unique to the tissue. Neither message nor protein was detected in adipose tissue samples from control dogs but both were detected in samples from dogs treated with ICI D7114. The data suggest that stimulation of beta 3-adrenergic receptors can reactivate nascent BAT (which has the appearance of white adipose tissue) by increasing expression of the gene coding for uncoupling protein or lead to the recruitment of fully differentiated BAT from preadipocyte precursor cells. Images PMID:1720550

  18. Beta 3-adrenergic receptor stimulation restores message and expression of brown-fat mitochondrial uncoupling protein in adult dogs.

    PubMed

    Champigny, O; Ricquier, D; Blondel, O; Mayers, R M; Briscoe, M G; Holloway, B R

    1991-12-01

    Brown adipose tissue (BAT) is present throughout life in rodents and plays an important role in energy balance. However, whereas BAT is clearly recognizable in the neonates of larger mammals (including dogs, cats, sheep, cattle, and humans), it is undetectable or present in only small quantities in adults of these species and is replaced by a tissue with the gross characteristics of white adipose tissue. Here we provide evidence that treatment of adult dogs with a beta 3-adrenergic receptor agonist (ICI D7114) that has thermogenic and antiobesity properties leads to the appearance of BAT at several anatomical sites. The presence of BAT was primarily demonstrated by monitoring the inner mitochondrial membrane uncoupling protein and its mRNA, which are unique to the tissue. Neither message nor protein was detected in adipose tissue samples from control dogs but both were detected in samples from dogs treated with ICI D7114. The data suggest that stimulation of beta 3-adrenergic receptors can reactivate nascent BAT (which has the appearance of white adipose tissue) by increasing expression of the gene coding for uncoupling protein or lead to the recruitment of fully differentiated BAT from preadipocyte precursor cells. PMID:1720550

  19. 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.

  20. Mass spectral study of alkali-cationized Boc-carbo-beta3-peptides by electrospray tandem mass spectrometry.

    PubMed

    Srikanth, R; Reddy, P Nagi; Srinivas, R; Sharma, G V M; Reddy, K Ravinder; Krishna, Palakodety Radha

    2004-01-01

    Electrospray tandem mass spectrometry was used to study the dissociation reactions of [M+Cat]+ (Cat = Na+ and Li+) of Boc-carbo-beta3-peptides. The collision-induced dissociation (CID) spectra of [M+Cat-Boc]+ of these peptides are found to be significantly different from those of [M+H-Boc]+ ions. The spectra are more informative and display both C- and N-terminus metallated ions in addition to characteristic fragment ions of the carbohydrate moiety. Based on the fragmentations observed in the CID spectra of the [M+Cat-Boc]+ ions, it is suggested that the dissociation involves complexes in which the metal ion is coordinated in a multidentate arrangement involving the carbonyl oxygen atoms. The CID spectra of [M+Cat-Boc]+ ions of the peptide acids show an abundant N-terminal rearrangement ion [b(n)+17+Cat]+ which is absent for esters. Further, two pairs of positionally isomeric Boc-carbo-beta3-peptide acids, Boc-NH-Caa(S)-beta-hGly-OH (11) and Boc-NH-beta-hGly-Caa(S)-OH (12), and [Boc-NH-Caa(S)-beta-hGly-Caa(S)-beta-hGly-OH] (13) and [Boc-NH-beta-hGly-Caa(S)-beta-hGly-Caa(S)-OH] (14), were differentiated by the CID of [M+Cat-Boc]+ ions. The CID spectra of compounds 11 and 13 are significantly different from those of 12 and 14, respectively. The abundance of [b(n)+17+Cat]+ ions is higher for peptide acids 12 and 14 with a sugar group at the C-terminus when compared to 11 and 13 which contain a sugar moiety at the N-terminus. The observed differences between the CID spectra of these isomeric peptides are attributed to the difference in the preferential site of metal ion binding and also on the structure of the cyclic intermediate involved in the formation of the rearrangement ion.

  1. Polymorphism at the ovine beta3-adrenergic receptor locus: associations with birth weight, growth rate, carcass composition and cold survival.

    PubMed

    Forrest, R H; Hickford, J G H; Hogan, A; Frampton, C

    2003-02-01

    The beta3-adrenergic receptors (ADRB3s) are predominantly found on the surface of adipocytes and are the major mediators of the lipolytic and thermogenic effects of high catecholamine concentrations. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of part of the ovine beta3-adrenergic receptor gene (ADRB3) intron was used to screen 12 large Merino half-sib families for sequence variation. Six different alleles that segregated in a Mendelian fashion were observed. The genetic basis for the allelic differences were identified by sequencing the ADRB3 (coding and non-coding regions) from animals that were homozygous for each of the alleles. Five sire lines (two Merino x Merino, two Merino x Coopworth, one Dorset Down x Coopworth) provided phenotypic and genotypic data used to ascertain the effects of allelic variation at the ADRB3 locus on birth weight, weaning weight, growth rate (up until weaning), carcass composition at 63 days post-weaning and cold survival. Statistical analyses within each half-sib family showed that in some sire lines (S13, S15, and S17) the inheritance of a particular allele was associated with increased birth weights and/or increased growth rates up until weaning. The inheritance of a particular sire allele was associated with fatter carcasses in sire line S16. Chi-squared analysis revealed the association of the E allele with cold survival and the D allele with cold-related mortality in sire line S14. Such associations support the hypothesis that ADRB3s are involved in energy homeostasis. With more research, the variation detected at the ADRB3 locus may assist in the genetic selection for desirable animal production traits.

  2. Inhibitory effects of SR 58611A on canine colonic motility: evidence for a role of beta 3-adrenoceptors.

    PubMed Central

    De Ponti, F; Cosentino, M; Costa, A; Girani, M; Gibelli, G; D'Angelo, L; Frigo, G; Crema, A

    1995-01-01

    1. In order to clarify whether atypical or beta 3-adrenoceptors can modulate canine colonic motility in vivo, we studied the effects of SR 58611A (a selective agonist for atypical beta-adrenoceptors) alone and after pretreatment with beta-adrenoceptor antagonists on colonic motility in the conscious dog. The gastrocolonic response (postprandial increase in motility) was monitored by means of electrodes and strain-gauge force transducers chronically implanted along the distal colon. In some experiments, heart rate was also measured. The possible role of beta 3-adrenoceptors in mediating the effects of SR 58611A was also tested in vitro in circular muscle strips taken from the canine distal colon. 2. Intravenous infusion of SR 58611A, ritodrine or isoprenaline at doses inducing the same degree of tachycardia inhibited the gastrocolonic response to a different extent, with SR 58611A and ritodrine being more effective than isoprenaline. 3. In a dose-response study, SR 58611A was more potent in inhibiting colonic motility than in inducing tachycardia: the ED35 values for inhibition of colonic motility and induction of tachycardia were 23 and 156 micrograms kg-1, i.v., respectively. 4. The inhibitory effect of SR 58611A 100 micrograms kg-1, i.v., on the gastrocolonic response was reversed by alprenolol (non-selective beta-adrenoceptor antagonist), but resistant to CGP 20712A (beta 1-adrenoceptor antagonist) or ICI 118551 (beta 2-adrenoceptor antagonist). 5. In vitro, SR 58611A concentration-dependently relaxed circular muscle strips, an effect that was competitively antagonized by alprenolol with a pA2 value of 7.1, but resistant to CGP 20712A (100 nM), ICI 118551 (100 nM) or tetrodotoxin (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7606348

  3. Ethanolic extracts of Brassica campestris spp. rapa roots prevent high-fat diet-induced obesity via beta(3)-adrenergic regulation of white adipocyte lipolytic activity.

    PubMed

    An, Sojin; Han, Jang-Il; Kim, Min-Jung; Park, Ji-Seon; Han, Jong-Min; Baek, Nam-In; Chung, Hae-Gon; Choi, Myung-Sook; Lee, Kyung-Tae; Jeong, Tae-Sook

    2010-04-01

    The influence of ethanolic extracts of Brassica campestris spp. rapa roots (EBR) on obesity was examined in imprinting control region (ICR) mice fed a high-fat diet (HFD) and in 3T3-L1 adipocytes. The ICR mice used were divided into regular diet, HFD, EBR (50 mg/kg/day EBR administered orally), and orlistat (10 mg/kg/day orlistat administered orally) groups. The molecular mechanism of the anti-obesity effect of EBR was investigated in 3T3-L1 adipocytes as well as in HFD-fed ICR mice. In the obese mouse model, both weight gain and epididymal fat accumulation were highly suppressed by the daily oral administration of 50 mg/kg EBR for 8 weeks, whereas the overall amount of food intake was not affected. EBR treatment induced the expression in white adipocytes of lipolysis-related genes, including beta(3)-adrenergic receptor (beta(3)-AR), hormone-sensitive lipase (HSL), adipose triglyceride lipase, and uncoupling protein 2. Furthermore, the activation of cyclic AMP-dependent protein kinase, HSL, and extracellular signal-regulated kinase was induced in EBR-treated 3T3-L1 cells. The lipolytic effect of EBR involved beta(3)-AR modulation, as inferred from the inhibition by the beta(3)-AR antagonist propranolol. These results suggest that EBR may have potential as a safe and effective anti-obesity agent via the inhibition of adipocyte lipid accumulation and the stimulation of beta(3)-AR-dependent lipolysis.

  4. 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.

  5. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro.

    PubMed Central

    Liaw, L; Skinner, M P; Raines, E W; Ross, R; Cheresh, D A; Schwartz, S M; Giachelli, C M

    1995-01-01

    Osteopontin is an arginine-glycine-aspartate containing acidic glycoprotein postulated to mediate adhesion, migration, and biomineralization in diverse tissues. The mechanisms explaining this multifunctionality are not well understood, although it is known that one osteopontin receptor is the alpha v beta 3 integrin. In this work, we studied human smooth muscle cells varying in alpha v beta 3 levels to identify additional osteopontin receptors. We report that, in addition to alpha v beta 3, both alpha v beta 5 and alpha v beta 1 are osteopontin receptors. Moreover, the presence or absence of alpha v beta 3 on the cell surface altered the adhesive and migratory responses of smooth muscle cells to osteopontin. Adhesion of alpha v beta 3-deficient cell populations to osteopontin was only half that of cells containing alpha v beta 3, and migration toward an osteopontin gradient in the Boyden chamber was dependent on cell surface alpha v beta 3. Although alpha v beta 3-deficient smooth muscle cells were unable to migrate to osteopontin, they did migrate significantly in response to vitronectin and fibronectin. These findings represent the first description of alpha v beta 5 and alpha v beta 1 as osteopontin receptors and suggest that, while adhesion to osteopontin is supported by integrins containing beta 1, beta 3, and beta 5, migration in response to osteopontin appears to depend on alpha v beta 3. Thus, interaction with distinct receptors is one mechanism by which osteopontin may initiate multiple functions. Images PMID:7532190

  6. Effect of postnatal age and a beta(3)-adrenergic agonist (Zeneca D7114) administration on uncoupling protein-1 abundance in the lamb.

    PubMed

    Bird, J A; Mostyn, A; Clarke, L; Juniper, D T; Budge, H; Stephenson, T; Symonds, M E

    2001-01-01

    We examined the effect of time after birth and beta(3)-adrenergic agonist (Zeneca D7114) administration on uncoupling protein-1 (UCP1) abundance and thermoregulation in the lamb. Forty twin lambs, all born normally at term, were maintained at a cold ambient temperature of between 3 and 8 degrees C. At 0.5, 1.75, 5.25, 11.25 and 23.25 h after birth eight sets of twins were fed 20 ml of formula milk +/- 10 mg kg(-1) of beta(3)-adrenergic agonist, and 45 min after feeding brown adipose tissue (BAT) was sampled. Colonic temperature was measured and BAT analysed for UCP1 abundance, GDP-binding to mitochondrial protein (i.e. thermogenic activity) and catecholamine content. Colonic temperature declined between 1.25 and 6 h from 40.2 degrees C to 39.2 degrees C and then increased to 39.8 degrees C at 12 h, but increased after feeding at all ages. UCP1 abundance increased from 1.25 h after birth, to peak at 2 h after birth in controls, compared with 6 h after birth in beta(3)-adrenergic agonist-treated lambs. The level of GDP-binding to mitochondrial protein did not change significantly with age but was increased by beta(3)-adrenergic agonist treatment. The noradrenaline (norepinephrine) content of BAT increased between 1.25 and 12 h after birth, irrespective of beta(3)-adrenergic agonist administration. The total weight of perirenal BAT plus its lipid, protein and mitochondrial protein content declined over the first 6 h of life. UCP1 development continues over the first 24 h of neonatal life, and can be manipulated by beta(3)-adrenergic agonist administration. This may represent one method of improving thermoregulation in newborn lambs. Experimental Physiology (2001) 86.1, 65-70. PMID:11429621

  7. 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

  8. 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

  9. 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.

  10. Impact of aging and beta3-adrenergic-receptor polymorphism on thermic and sympathetic responses to a high-fat meal.

    PubMed

    Nagai, Narumi; Sakane, Naoki; Moritani, Toshio

    2006-10-01

    The present study was designed to investigate the effect of aging and beta3-adrenergic-receptor (beta3-AR) polymorphism on the thermic effect of meal (TEM) and sympathetic nervous system (SNS) response to a high-fat meal in 13 boys, 12 young men, and 11 middle-aged men. SNS activity was assessed via power spectral analysis of heart rate variability. Significantly higher very-low-frequency (VLF) components associated with thermogenic SNS activity and energy expenditure per lean body mass (EE(LBM)) were observed in boys during the pre- and postprandial periods. There were no significant differences in VLF and EE(LBM) in the preprandial period between the young and middle-aged men. After feeding, however, the middle-aged men showed a significantly lower TEM (% test-meal energy) and VLF compared to the young men. A multiple regression analysis revealed that age was the only significant variable contributing to both TEM and VLF, but beta3-AR polymorphism and percentage of body fat were not statistically significant. In conclusion, age likely has a greater influence on TEM and SNS thermoregulation than genetic factors such as beta3-AR polymorphism, suggesting that this age-related decrease in thermogenic response may be involved in the development of obesity among middle-aged men.

  11. 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.

  12. Control of O-glycan synthesis: specificity and inhibition of O-glycan core 1 UDP-galactose:N-acetylgalactosamine-alpha-R beta 3-galactosyltransferase from rat liver.

    PubMed

    Brockhausen, I; Möller, G; Pollex-Krüger, A; Rutz, V; Paulsen, H; Matta, K L

    1992-02-01

    The specificity of glycosyltransferases is a major control factor in the biosynthesis of O-glycans. The enzyme that synthesizes O-glycan core 1, i.e., UDP-galactose:N-acetylgalactosamine-alpha-R beta 3-galactosyltransferase (beta 3-Gal-T; EC 2.4.1.122), was partially purified from rat liver. The enzyme preparation, free of pyrophosphatases, beta 4-galactosyltransferase, beta-galactosidase, and N-acetylglucosaminyltransferase I, was used to study the specificity and inhibition of the beta 3-Gal-T. beta 3-Gal-T activity is sensitive to changes in the R-group of the GalNAc alpha-R acceptor substrate and is stimulated when the R-group is a peptide or an aromatic group. Derivatives of GalNAc alpha-benzyl were synthesized and tested as potential substrates and inhibitors. Removal or substitution of the 3-hydroxyl or removal of the 4-hydroxyl of GalNAc abolished beta 3-Gal-T activity. Compounds with modifications of the 3- or 4-hydroxyl of GalNAc alpha-benzyl did not show significant inhibition. Removal or substitution of the 6-hydroxyl of GalNAc reduced activity slightly and these derivatives acted as competitive substrates. derivatives with epoxide groups attached to the 6-position of GalNAc acted as substrates and not as inhibitors, with the exception of the photosensitive 6-O-(4,4-azo)pentyl-GalNAc alpha-benzyl, which inhibited Gal incorporation into GalNAc alpha-benzyl. The results indicate that the enzyme does not require the 6-hydroxyl of GalNAc, but needs the 3- and the axial 4-hydroxyl as essential requirements for binding and activity. In the usual biochemical O-glycan pathway, core 2 (GlcNAc beta 6[Gal beta 3] GalNAc alpha-) is formed from core 1 (Gal beta 3GalNAc-R). We have now demonstrated an alternate pathway that may be of importance in human tissues.

  13. CP beta3, a novel isoform of an actin-binding protein, is a component of the cytoskeletal calyx of the mammalian sperm head.

    PubMed

    von Bülow, M; Rackwitz, H R; Zimbelmann, R; Franke, W W

    1997-05-25

    In the mammalian sperm head, the nucleus is tightly associated with the calyx, a cell type-specific cytoskeletal structure. Previously, we have identified and characterized some basic proteins such as calicin and cylicins I and II as major calyx components of bovine and human spermatids and spermatozoa. Surprisingly we have now discovered another calyx constituent which by amino acid sequencing and cDNA cloning was recognized as a novel isoform of the widespread beta subunit of the heterodimeric actin-binding "capping protein" (CP). This polypeptide, CP beta3, of sperm calices, is identical with the beta2 subunit present in diverse somatic cell types, except that it shows an amino-terminal extension of 29 amino acids and its mRNA is detected only in testis and, albeit in trace amounts, brain. This CP beta3 mRNA contains the additional sequence, encoded by exon 1 of the gene, which is missing in beta2 mRNAs. Antibodies specific for the beta3 amino-terminal addition have been used to identify the protein by immunoblotting and to localize it to the calyx structure by immunofluorescence microscopy. We conclude that in spermiogenesis the transcription of the gene encoding the beta1, beta2, and beta3 CP subunits is regulated specifically to include exon 1 and to give rise to the testis isoform CP beta3, which is integrated into the calyx structure of the forming sperm head. This surprising finding of an actin-binding protein isoform in an insoluble cytoskeletal structure is discussed in relation to the demonstrated roles of actin and certain actin-binding proteins, such as Limulus alpha-scruin, in spermiogenesis and spermatozoa.

  14. 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

  15. Probing conformational changes in the I-like domain and the cysteine-rich repeat of human beta 3 integrins following disulfide bond disruption by cysteine mutations: identification of cysteine 598 involved in alphaIIbbeta3 activation.

    PubMed

    Chen, P; Melchior, C; Brons, N H; Schlegel, N; Caen, J; Kieffer, N

    2001-10-19

    We have investigated receptor function and epitope expression of recombinant alpha(IIb)beta(3) mutated at Cys(177) or Cys(273) in the I-like domain as well as Cys(598), located in the fourth repeat of the membrane-proximal cysteine-rich region and mutated in a Glanzmann's thrombasthenia type II patient. The beta(3) mutants beta(3)C177A, beta(3)C273A, and beta(3)C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel electrophoresis under nonreducing conditions, confirming the disruption of the respective disulfide loops. Despite reduced surface expression, the alpha(IIb)beta(3)C177A, alpha(IIb)beta(3)C273A, and alpha(IIb)beta(3)C598Y receptors mediated cell adhesion to immobilized fibrinogen and translocated into focal adhesion plaques. The beta(3)C598Y mutation, but not the beta(3)C177A or beta(3)C273A mutations, induced spontaneous binding of the ligand mimetic monoclonal antibody PAC-1, while the beta(3)C177A and beta(3)C273A mutants exhibited reduced complex stability in the absence of Ca(2+). Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, pl2-46, 10E5, and P256 did not interact with alpha(IIb)beta(3)C273A and bound only weakly to alpha(IIb)beta(3)C177A, while mAbs AP2, LM609 and 7E3 bound normally to mutant alpha(IIb)beta(3)C273A, but interacted only weakly with mutant alpha(IIb)beta(3)C177A. Furthermore, a cryptic epitope recognized by mAb 4D10G3 and not exposed on wild type alpha(IIb)beta(3) became accessible only on mutant alpha(IIb)beta(3)C177A and was mapped to the 60-kDa chymotrypsin fragment of beta(3). Finally, the ligand-induced binding site (LIBS) epitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provide evidence that disruption of a single cysteine disulfide bond in the cysteine-rich repeat domain, but not in the I-like domain, activates integrin

  16. [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.

  17. Ligand binding properties of putative beta 3-adrenoceptors compared in brown adipose tissue and in skeletal muscle membranes.

    PubMed Central

    Sillence, M. N.; Moore, N. G.; Pegg, G. G.; Lindsay, D. B.

    1993-01-01

    1. The beta-adrenoceptor population was characterized in membrane preparations from rat brown adipose tissue (BAT) and from soleus muscle by use of the radioligand [125I]-iodocyanopindolol ([125I]-ICYP). In addition, atypical binding sites for [125I]-ICYP found in both tissues were examined, and the relationship between these sites and the putative rat beta 3-adrenoceptor is discussed. 2. It was established that BAT membranes host a mixed population of beta 1- and beta 2-adrenoceptors. Of these two sites, 55% showed a high affinity for the beta 1-selective ligand CGP 20712A (pK 8.5), and 45% showed a high affinity for the beta 2-selective antagonist ICI 118551 (pK 8.6). Soleus muscle membranes were found to host a population of beta 2-adrenoceptors, characterized by a high affinity for ICI 118551 (pK 9.1), but beta 1-adrenoceptors could not be detected in this preparation. 5-Hydroxytryptamine receptors were not detected in either preparation. 3. In addition to beta 1- and beta 2-adrenoceptors, atypical binding sites were identified in both tissues using high concentrations of radioligand (0.5-0.6 nM) and in the presence of 1 microM (-)-propranolol. The atypical sites were abundant, representing 80 and 81% of the total [125I]-ICYP binding sites in BAT and soleus muscle respectively. When the pK values for 11 ligands were compared, the correlation coefficient for atypical sites in BAT and soleus muscle was 0.94.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8104645

  18. Molecular imaging of angiogenesis in nascent Vx-2 rabbit tumors using a novel alpha(nu)beta3-targeted nanoparticle and 1.5 tesla magnetic resonance imaging.

    PubMed

    Winter, Patrick M; Caruthers, Shelton D; Kassner, Andrea; Harris, Thomas D; Chinen, Lori K; Allen, John S; Lacy, Elizabeth K; Zhang, Huiying; Robertson, J David; Wickline, Samuel A; Lanza, Gregory M

    2003-09-15

    Early noninvasive detection and characterization of solid tumors and their supporting neovasculature is a fundamental prerequisite for effective therapeutic intervention, particularly antiangiogenic treatment regimens. Emerging molecular imaging techniques now allow recognition of early biochemical, physiological, and anatomical changes before manifestation of gross pathological changes. Although new tumor, vascular, extracellular matrix, and lymphatic biomarkers continue to be discovered, the alpha(nu)beta(3)-integrin remains an attractive biochemical epitope that is highly expressed on activated neovascular endothelial cells and essentially absent on mature quiescent cells. In this study, we report the first in vivo use of a magnetic resonance (MR) molecular imaging nanoparticle to sensitively detect and spatially characterize neovascularity induced by implantation of the rabbit Vx-2 tumor using a common clinical field strength (1.5T). New Zealand White rabbits (2 kg) 12 days after implantation of fresh Vx-2 tumors (2 x 2 x 2 mm(3)) were randomized into one of three treatment groups: (a) alpha(nu)beta(3)-targeted, paramagnetic formulation; (b) nontargeted, paramagnetic formulation; and (c) alpha(nu)beta(3)-targeted nonparamagnetic nanoparticles followed by (2 h) the alpha(nu)beta(3)-targeted, paramagnetic formulation to competitively block magnetic resonance imaging (MRI) signal enhancement. After i.v. systemic injection (0.5 ml of nanoparticles/kg), dynamic T(1)-weighted MRI was used to spatially and temporally determine nanoparticle deposition in the tumor and adjacent tissues, including skeletal muscle. At 2-h postinjection, alpha(nu)beta(3)-targeted paramagnetic nanoparticles increased MRI signal by 126% in asymmetrically distributed regions primarily in the periphery of the tumor. Similar increases in MR contrast were also observed within the walls of some vessels proximate to the tumor. Despite their relatively large size, nanoparticles penetrated into the

  19. Regulatory elements in the first intron contribute to transcriptional regulation of the beta 3 tubulin gene by 20-hydroxyecdysone in Drosophila Kc cells.

    PubMed Central

    Bruhat, A; Tourmente, S; Chapel, S; Sobrier, M L; Couderc, J L; Dastugue, B

    1990-01-01

    We have studied the transcriptional regulation of the beta 3 tubulin gene by the steroid hormone 20-hydroxyecdysone (20-OH-E) in Drosophila Kc cells. A series of hybrid genes with varying tubulin gene lengths driving the bacterial chloramphenicol acetyl transferase (CAT) gene were constructed. The promoter activity was assayed after transient expression in Kc cells, in the presence or absence of 20-OH-E. We find that 0.91Kb upstream from the transcription start site contain one or several hormone independent positive cis-acting elements, responsible for the constitutive expression of the beta 3 tubulin gene. In the large (4.5 Kb) first intron of this gene, we identified additional hormone dependent negative and positive regulatory elements, which can act in both directions and in a position-independence manner. Then, the negative intron element(s), which repress the transcription in the absence of 20-OH-E has characteristics of silencer. Images PMID:2349088

  20. Genomic organization and complete cDNA sequence of the human phosphoinositide-specific phospholipase C {beta}3 gene (PLCB3)

    SciTech Connect

    Lagercrantz, J.; Carson, E.; Phelan, C.

    1995-04-10

    We have characterized the complete cDNA sequence, genomic structure, and expression of the human phosphoinositide-specific phospholipase C {beta}3 (PLC {beta}3) gene (gene symbol PLCB3). PLC {beta}3 plays an important role in initiating receptor-mediated signal transduction. Activation of PLC takes place in many cells as a response to stimulation by hormones, growth factors, neurotransmitters, and other ligands. The partial cDNA sequence of PLC {beta}3, previously published, was extended with 876 bp in the 5{prime} direction, giving a transcript of 4400 bp and a total open reading frame of 1234 amino acids. This was in accordance with expression analysis by Northern blotting that revealed a single 4.4-kb transcript in all tissues tested. Genomic data were obtained by sequencing plasmid subclones of a cosmid that contained the whole gene. The size of the complete transcription unit was estimated to be on the order of 15 kb. The gene contains 31 exons, with all splice donor and acceptor sites conforming to the GT/AG rule. No exon exceeds 571 bp in length, and the shortest exon spans only 36 bp. More than half of the introns are smaller than 200 bp, with the smallest being only 79 bp long. The transcription initiation site was determined to be within an 8-bp cluster 328-321 bp upstream of the translation initiation site. The 5{prime} flanking region is highly GC rich, with multiple CpG doublets, and contains multiple binding sites for Sp1. Lacking typical transcriptional regulatory sequences such as TATA and CAAT boxes, the putative promoter region conforms to the group of housekeeping promoters. 28 refs., 4 figs., 1 tab.

  1. Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta3 integrin family.

    PubMed

    Ahmad, Ishtiaq; Hoessli, Daniel C; Walker-Nasir, Evelyne; Choudhary, M Iqbal; Rafik, Saleem M; Shakoori, Abdul Rauf

    2006-10-15

    Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.

  2. 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

  3. The effects of uncoupling protein 1 and beta3-adrenergic receptor gene polymorphisms on weight loss and lipid profiles in obese women.

    PubMed

    Kim, Jung Yun; Lee, Sang Sun

    2010-04-01

    The purpose of this study was to investigate whether the genetic polymorphisms of the uncoupling protein 1 (UCP1) and beta 3 adrenergic receptor (beta3-AR) were associated with differences in weight loss and lipid profiles in obese premenopausal women exposed to low-calorie meal replacements over a period of six weeks. Forty women between the ages of 20 and 35 were randomly divided into two groups, each of which consumed one of two low-calorie meal replacements containing either white rice or mixed rice. Although body weight, body mass index (BMI), blood glucose concentration, triglycerides, total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were not significantly different by the UCP1 genotype in the white rice group, there were significant differences in body weight (p = 0.041), BMI (p = 0.027), and blood glucose concentration (p = 0.047) between carriers and non-carriers of the G allele in the mixed rice group after the six-week meal replacement intervention. The beta3-AR polymorphism showed no apparent affect on these parameters. Dietary fiber affects weight gain since it is closely related with absorption of nutrients. As a result, the AA type UCP1 genotype produced significant weight loss in the mixed rice group, but not in the white rice group.

  4. Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: immunolocalization according to the odontogenesis phases.

    PubMed

    Sassá Benedete, Ana Paula; Sobral, Ana Paula Veras; Lima, Dirce Mary Correia; Kamibeppu, Leonardo; Soares, Fernando Augusto; Lourenço, Silvia Vanessa

    2008-01-01

    Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo, including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunohistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3-were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

  5. 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.

  6. 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

  7. Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

    PubMed

    Dormond, Olivier; Ponsonnet, Lionel; Hasmim, Meriem; Foletti, Alessandro; Rüegg, Curzio

    2004-07-01

    Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

  8. Potentiation of the anti-obesity effect of the selective beta 3-adrenoceptor agonist BRL 35135 in obese Zucker rats by exercise.

    PubMed Central

    Santti, E; Huupponen, R; Rouru, J; Hänninen, V; Pesonen, U; Jhanwar-Uniyal, M; Koulu, M

    1994-01-01

    1. The effects of chronic treatments with a selective beta 3-adrenoceptor agonist and a selective alpha 2-adrenoceptor antagonist and their interactions with physical exercise training were studied in experimental obesity. 2. BRL 35135 (beta 3-agonist, 0.5 mg kg-1 day-1 p.o.), atipamezole (alpha 2-antagonist, 4.0 mg kg-1 day-1 p.o.) and placebo were given to genetically obese male Zucker rats. Half of the rats were kept sedentary whereas the other half were subjected to moderate treadmill exercise training. Body weight gain, cumulative food intake, the neuropeptide Y content of the hypothalamic paraventricular nucleus, brown adipose tissue thermogenic activity (measured as GDP binding), plasma insulin and glucose levels were measured after 3 weeks' treatment and exercise. 3. Treatment with BRL 35135 reduced weight gain by 19%, increased brown adipose tissue thermogenic activity 45-fold and reduced plasma insulin by 50%. Atipamezole slightly increased food intake and neuropeptide Y content in the paraventricular hypothalamic nucleus but had no effect on the other measured parameters. Exercise alone had no effect on weight gain, food intake or thermogenic activity, whereas it reduced plasma insulin and glucose levels. 4. The effect of BRL 35135 on weight gain and thermogenic activity was significantly potentiated by exercise; the reduction in weight gain was 56% in comparison with 19% in sedentary animals. Food intake was significantly reduced in the BRL 35135-treated-exercise-trained animals, although neither beta 3-agonist nor exercise alone affected it.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7889277

  9. Insulin-like growth factor-I signaling in smooth muscle cells is regulated by ligand binding to the 177CYDMKTTC184 sequence of the beta3-subunit of alphaVbeta3.

    PubMed

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

    2006-02-01

    The response of smooth muscle cells to IGF-I requires ligand occupancy of the alphaVbeta3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-alphaVbeta3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177-184 ((177)CYDMKTTC(184)) within the extracellular domain of beta3 have been proposed to confer the ligand specificity of alphaVbeta3; therefore, we hypothesized that ligand binding to the 177-184 cysteine loop of beta3 may be an important regulator of the cross talk between alphaVbeta3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the beta3 subunit of alphaVbeta3 (i.e. amino acids 177-184) blocked Vn binding to the beta3 subunit of alphaVbeta3 and correspondingly beta3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of beta3 in which three critical residues within the 177-184 sequence were altered beta3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177-184 sequence of beta3 is necessary for Vn binding to alphaVbeta3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I.

  10. Prolonged treatment with the beta3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 1) fat store depletion and desensitization of beta-adrenergic responses.

    PubMed

    Ferrand, C; Redonnet, A; Prévot, D; Carpéné, C; Atgié, C

    2006-06-01

    Beta3-adrenergic agonists have been considered as potent antiobesity and antidiabetic agents mainly on the basis of their beneficial actions discovered twenty years ago in obese and diabetic rodents. The aim of this work was to verify whether prolonged treatment with a beta3-adrenergic agonist known to stimulate lipid mobilisation, could promote desensitization of beta-adrenergic responses. Wistar rats and guinea pigs were treated during one week with CL 316243 (CL, 1 mg/kg/d) by implanted osmotic minipumps. In control animals, beta3-adrenergic agonists were lipolytic in rat but not in guinea pig adipocytes. CL-treatment did not alter body weight gain in both species, but reduced fat stores in rats. Lipolysis stimulation by forskolin was unmodified but responses to beta1-, beta2- and beta3-agonists were reduced in visceral or subcutaneous white adipose tissues of CL-treated rats. Similarly, the beta3-adrenergic-dependent impairment of insulin action on glucose transport and lipogenesis in rat adipocytes was diminished after CL-treatment. In rat adipocytes, [125I]ICYP binding and beta3-adrenoceptor mRNA levels were reduced after sustained CL administration. These findings show that CL 316243 exerts (beta3-adrenergic lipolytic and antilipogenic effects in rat adipocytes. These actions, which are likely involved in the fat depletion observed in rat, also lead to the desensitization of all beta-adrenergic responses. Therefore this desensitization, together with the lack of slimming action in guinea pig, seriously attenuates the usefulness of beta3-agonists as antiobesity agents, and may explain why such agonists have not been conducted to a widespread clinical use.

  11. Selective beta 3-adrenergic agonists of brown adipose tissue and thermogenesis. 2. [4-[2-[(2-Hydroxy-3-phenoxypropyl)amino]ethoxy]phenoxy]acetamides.

    PubMed

    Howe, R; Rao, B S; Holloway, B R; Stribling, D

    1992-05-15

    The ester methyl [4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]phenoxy]acetate (1) (R1 = OMe) had previously been identified as the most interesting member of a series of selective beta 3-adrenergic agonists of brown adipose tissue and thermogenesis in the rat. In vivo it acts mainly via the related acid 1 (R1 = OH). Amides have been examined to determine whether they have advantages over the ester. In particular, in the rat and dog the half-lives of amides of appropriate potency were no longer than those of the ester. The amide (S)-4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]-N-(2- methoxyethyl)phenoxyacetamide [S-27, ICI D7114] was selected as having properties consistent with a sustained-release formulation should that prove necessary. Unlike the ester it is resistant to hydrolysis in the gut lumen. Further testing of ICI D7114 has shown that in the rat, cat, and dog it stimulates the beta 3-adrenergic receptor in brown adipose tissue at doses lower than those at which it affects beta 1- and beta 2-adrenergic receptors in other tissues. Slimming effects were observed in the dog. ICI D7114 may be a selective thermogenic agent in man and may be useful in the treatment of obesity and diabetes. PMID:1350310

  12. In vitro and in vivo characterization of 64Cu-labeled Abegrin, a humanized monoclonal antibody against integrin alpha v beta 3.

    PubMed

    Cai, Weibo; Wu, Yun; Chen, Kai; Cao, Qizhen; Tice, David A; Chen, Xiaoyuan

    2006-10-01

    Abegrin (MEDI-522 or Vitaxin), a humanized monoclonal antibody against human integrin alpha(v)beta(3), is in clinical trials for cancer therapy. In vivo imaging using Abegrin-based probes is needed for better treatment monitoring and dose optimization. Here, we conjugated Abegrin with macrocyclic chelating agent 1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic (DOTA) at five different DOTA/Abegrin ratios. The conjugates were labeled with (64)Cu (half-life = 12.7 hours) and tested in three human (U87MG, MDA-MB-435, and PC-3) and one mouse (GL-26) tumor models. The in vitro and in vivo effects of these (64)Cu-DOTA-Abegrin conjugates were evaluated. The number of DOTA per Abegrin varied from 1.65 +/- 0.32 to 38.53 +/- 5.71 and the radiolabeling yield varied from 5.20 +/- 3.16% to 88.12 +/- 6.98% (based on 2 mCi (64)Cu per 50 microg DOTA-Abegrin conjugate). No significant difference in radioimmunoreactivity was found among these conjugates (between 59.78 +/- 1.33 % and 71.13 +/- 2.58 %). Micro-positron emission tomography studies revealed that (64)Cu-DOTA-Abegrin (1,000:1) had the highest tumor activity accumulation (49.41 +/- 4.54% injected dose/g at 71-hour postinjection for U87MG tumor). The receptor specificity of (64)Cu-DOTA-Abegrin was confirmed by effective blocking of MDA-MB-435 tumor uptake with coadministration of nonradioactive Abegrin. (64)Cu-DOTA-IgG exhibited background level tumor uptake at all time points examined. Integrin alpha(v)beta(3)-specific tumor imaging using (64)Cu-DOTA-Abegrin may be translated into the clinic to characterize the pharmacokinetics, tumor targeting efficacy, dose optimization, and dose interval of Abegrin and/or Abegrin conjugates. Chemotherapeutics or radiotherapeutics using Abegrin as the delivering vehicle may also be effective in treating integrin alpha(v)beta(3)-positive tumors.

  13. Complex role of the beta 2-beta 3 loop in the interaction of U1A with U1 hairpin II RNA.

    PubMed

    Katsamba, Phinikoula S; Bayramyan, Melina; Haworth, Ian S; Myszka, David G; Laird-Offringa, Ite A

    2002-09-01

    RNA recognition motifs (RRMs) are characterized by highly conserved regions located centrally on a beta-sheet, which forms the RNA binding surface. Variable flanking regions, such as the loop connecting beta-strands 2 and 3, are thought to be important in determining the RNA-binding specificities of individual RRMs. The N-terminal RRM of the spliceosomal U1A protein mediates binding to an RNA hairpin (U1hpII) in the U1 small nuclear RNA. In this complex, the beta(2)-beta(3) loop protrudes through the 10-nucleotide RNA loop. Shortening of the RNA loop strongly perturbs binding, suggesting that an optimal "fit" of the beta(2)-beta(3) loop into the RNA loop is an important factor in complexation. To understand this interaction further, we mutated or deleted loop residues Lys(50) and Met(51), which protrude centrally into the RNA loop but do not make any direct contacts to the bases. Using BIACORE, we analyzed the ability of these U1A mutants to bind to wild type RNAs, or RNAs with shortened loops. Alanine replacement mutations only modestly affected binding to wild type U1hpII. Interestingly, simultaneous replacement of Lys(50) and Met(51) with alanine appeared to alleviate the loss of binding caused by shortening of the RNA loop. Deletion of Lys(50) or Met(51) caused a dramatic loss in stability of the U1A.U1hpII complex. However, deletion of both residues simultaneously was much less deleterious. Simulated annealing molecular dynamics analyses suggest this is due to the ability of this mutant to rearrange flanking amino acids to substitute for the two deleted residues. The double deletion mutant also exhibited substantially reduced negative effects of RNA loop shortening, suggesting the rearranged loop is better able to accommodate a short RNA loop. Our results indicate that one of the roles of the beta(2)-beta(3) loop is to provide a steric fit into the RNA loop, thereby stabilizing the RNA.protein complex. PMID:12082087

  14. Identification of an interactive effect of beta3- and alpha2b-adrenoceptor gene polymorphisms on fat mass in Caucasian women.

    PubMed

    Dionne, I J; Turner, A N; Tchernof, A; Pollin, T I; Avrithi, D; Gray, D; Shuldiner, A R; Poehlman, E T

    2001-01-01

    Several adrenoceptor subtypes are expressed in adipocytes, which together exert their influence on adipocyte metabolism. Therefore, we specifically examined the interactive effect of Trp64Arg (beta3) and Glu12/Glu9 (alpha2b) adrenoceptor (AR) polymorphisms on energy metabolism and body composition in healthy women with a wide range of body habitus. We genotyped 909 unrelated women (age 55 +/- 12 [mean +/- SD] years, range 19-87; body weight 88 +/- 22 kg, range 40-167; and BMI 33 +/- 8 kg/m2, range 16-64) for Trp64Arg beta3AR and Glu12/Glu9 alpha2bAR variants. We examined the independent effect of the Glu12/Glu9 alpha2bAR variant on body composition and energy balance, in a large cohort of Caucasian women (n = 909). A second goal was to examine the interaction effect of Glu12/Glu9 alpha2bAR and Trp64Arg beta3AR on the same phenotypes. The obesity-related phenotypes studied were as follows: body weight, BMI, fat mass, visceral fat, fat-free mass, resting metabolic rate (RMR), VO2max, leisure time physical activity, and daily energy intake. Body composition and body fat distribution were measured by dual-energy X-ray absorptiometry and radiographic imagery, VO2max by a treadmill test to exhaustion, and RMR by indirect calorimetry. An analysis of covariance indicated that in the entire cohort, there was no significant difference between Glu12/Glu9 alpha2bAR carriers and control subjects for any of the obesity-related phenotypes that were examined. However, we observed a significant interaction effect of the Trp64Arg and Glu12/Glu9 variants on fat mass (P = 0.009) and percent fat (P = 0.016). Age, height, body weight, BMI, fat-free mass, visceral fat, energy expenditure, respiratory quotient, physical fitness, and energy intake were not different among groups. Collectively, these findings support an interaction effect of the two adrenoceptor variants on body fatness in Caucasian women, although the physiological mechanism by which they exert this effect remains to be

  15. Interactions among the alpha2-, beta2-, and beta3-adrenergic receptor genes and obesity-related phenotypes in the Quebec Family Study.

    PubMed

    Ukkola, O; Rankinen, T; Weisnagel, S J; Sun, G; Pérusse, L; Chagnon, Y C; Després, J P; Bouchard, C

    2000-08-01

    The gene-gene interactions between markers in the alpha2-, beta2-, and beta3-adrenergic receptor (ADR) genes and obesity-related phenotypes were studied in the Quebec Family Study (QFS) cohort. The prevalence of the Arg allele of the Arg16Gly polymorphism in the beta2-ADR gene was higher (49%) in males with a body mass index (BMI) of 35 kg/m2 or higher versus those with a BMI less than 35 kg/m2 (33%; P = .010). The beta2-ADR gene Arg16Gly and Gln27Glu polymorphisms were associated with plasma total and low-density lipoprotein (LDL) cholesterol concentrations. In addition, the homozygotes for the 6.3-kb allele of DraI polymorphism in the alpha2-ADR gene had the lowest mean abdominal subcutaneous fat area (P = .012) and total fat area (P = .003), as well as insulin area, under the curve during an oral glucose tolerance test ([OGTT] P = .004). Several ADR gene-gene interaction effects on abdominal fat distribution and plasma lipids were detected. First, significant interactions between alpha2- and beta3-ADR genes were observed on total (P = .015) and subcutaneous (P = .004) abdominal fat. Second, interaction effects between alpha2- and beta2-ADR gene variants influenced total, high-density lipoprotein (HDL), and LDL cholesterol concentrations. Finally, there were interactions between markers within the beta2-ADR gene affecting plasma triglyceride concentrations and subcutaneous abdominal fat. From these results, we conclude that polymorphisms in the ADR genes contribute to body fat and plasma lipid variability in men. Gene-gene interactions among the ADR genes contribute to the phenotypic variability in abdominal obesity and plasma lipid and lipoprotein, but not in visceral fat levels.

  16. The vitronectin receptor (alpha v beta 3) is implicated, in cooperation with P-selectin and platelet-activating factor, in the adhesion of monocytes to activated endothelial cells.

    PubMed Central

    Murphy, J F; Bordet, J C; Wyler, B; Rissoan, M C; Chomarat, P; Defrance, T; Miossec, P; McGregor, J L

    1994-01-01

    In this study we have investigated the presence on endothelial cells of potential glycoprotein receptors, other than P-selectin, which are involved in the adhesion of monocytes at the early stages of activation. We report that the majority of cells binding to thrombin-activated endothelial cells from a peripheral blood mononuclear cell (PBMC) preparation are monocytes. The adhesion of PBMC to thrombin-activated, but not resting, endothelial cells was inhibited (66%) by a monoclonal antibody (mAb) directed against alpha v beta 3. Elutriated monocytes or a monocytic cell line (U937) were also inhibited by this antibody, its F(ab)'2 fragments and three other anti-(alpha v beta 3) mAbs. alpha v beta 3 isolated from endothelial-cell lysates significantly inhibited the adhesion of monocytes and U937 cells to endothelial cells. A peptide motif (RGDF) known to interact with alpha v beta 3 inhibited U937 cell adhesion to activated endothelial cells by 53%. Finally, an anti-(P-selectin) mAb (LYP20) or a platelet-activating factor (PAF)-receptor antagonist (WEB 2086) inhibited monocyte adhesion to activated endothelial cells. This study shows for the first time that alpha v beta 3 is implicated, in addition to P-selectin and PAF, in the adhesion of monocytes to activated endothelial cells. PMID:7528011

  17. Expression of beta 1- and beta 3-adrenergic-receptor messages and adenylate cyclase beta-adrenergic response in bovine perirenal adipose tissue during its transformation from brown into white fat.

    PubMed Central

    Casteilla, L; Muzzin, P; Revelli, J P; Ricquier, D; Giacobino, J P

    1994-01-01

    Possible modifications of the beta-adrenergic effector system during the development of bovine perirenal brown adipose tissue (BAT) in utero and its transformation into white-like adipose tissue after birth were studied. The parameters assessed were the level of expression of beta 1-, beta 2- and beta 3-adrenergic receptor (AR) mRNAs and the response of the plasma-membrane adenylate cyclase to (-)-isoprenaline and to the beta 3-agonist BRL 37344. The beta 3-AR mRNA was found to be expressed very early in utero, i.e. before the third month of foetal life. Then it increased dramatically (9-fold) between month 6 of foetal life and birth. A high beta 3-AR mRNA level was maintained after birth up to an age of 3 months. After conversion of BAT into white-like adipose tissue, i.e. in the adult bovine, the beta 3-AR mRNA expression became small or not detectable, and the beta 1-AR mRNA, which was expressed much less than the beta 3-AR mRNA in foetal life, became predominant. A response of the adenylate cyclase to (-)-isoprenaline was observed in foetal life (3.1-fold stimulation). It decreased after birth (1.8-fold stimulation) and then remained constant until adulthood. A response to BRL 37344 was also observed in foetal life (1.8-fold stimulation). It was maintained after birth, but disappeared in the adult. A possible relationship between the beta-AR expression and the adenylate cyclase response to (-)-isoprenaline on the one hand and the uncoupling-protein expression on the other is discussed. The bovine might represent a good model to understand the transition from brown to white fat in the human. Images Figure 3 PMID:7904157

  18. Adrenergic stimulation of lipoprotein lipase gene expression in rat brown adipocytes differentiated in culture: mediation via beta3- and alpha1-adrenergic receptors.

    PubMed Central

    Kuusela, P; Rehnmark, S; Jacobsson, A; Cannon, B; Nedergaard, J

    1997-01-01

    In order to investigate whether the positive effect of adrenergic stimulation on lipoprotein lipase (LPL) gene expression in brown adipose tissue is a direct effect on the brown adipocytes themselves, the expression of the LPL gene was investigated by measuring LPL mRNA levels in brown adipocytes, isolated as precursors from the brown adipose tissue of rats and grown in culture in a fully defined medium before experimentation. Addition of noradrenaline led to an enhancement of LPL gene expression; the mRNA levels increased as a linear function of time for at least 5 h and were finally approx. 3 times higher than in control cells, an increase commensurate with that seen in vivo in both LPL mRNA levels and LPL activity during physiological stimulation. The increase was dependent on transcription. The effect of noradrenaline showed simple Michaelis-Menten kinetics with an EC50 of approx. 11 nM. beta3-Agonists (BRL-37344 and CGP-12177) could mimic the effect of noradrenaline; the beta1-agonist dobutamine and the beta2-agonist salbutamol could not; the alpha1-agonist cirazoline had only a weak effect. The effect of noradrenaline was fully inhibited by the beta-antagonist propranolol and was halved by the alpha1-antagonist prazosin; the alpha2-antagonist yohimbine was without effect. An increase in LPL mRNA level similar to (but not significantly exceeding) that caused by noradrenaline could also be induced by the cAMP-elevating agents forskolin and cholera toxin, and 8-Br-cAMP also increased LPL mRNA levels. The increase in LPL gene expression was not mediated via an increase in the level of an intermediary proteinaceous factor. It is concluded that the physiologically induced increase in LPL gene expression is a direct effect of noradrenaline on the brown adipocytes themselves, mediated via a dominant beta3-adrenergic pathway and an auxiliary alpha1-adrenergic pathway which converge at a regulatory point in transcriptional control. PMID:9032464

  19. 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

  20. Effect of chronic treatment with ICI D7114, a selective beta 3-adrenoceptor agonist, on macronutrient selection and brown adipose tissue thermogenesis in Sprague-Dawley rats.

    PubMed

    Santti, E; Rouvari, T; Rouru, J; Huupponen, R; Koulu, M

    1994-01-01

    ICI D7114 is a selective beta 3-agonist which stimulates brown adipose tissue thermogenesis. In the present study the effects of 18 days treatment with ICI D7114 (2 mg/kg/day orally) on macronutrient selection and brown adipose tissue thermogenesis were investigated in Sprague-Dawley rats. The rats were maintained on a free-feeding self-selection paradigm with three pure macronutrient diets of carbohydrate, fat and protein. Treatment with ICI D7114 did not change the macronutrient selection or total calories consumed by the rats. To monitor the thermogenic activation of brown adipose tissue the binding of [3H]GDP to brown adipose tissue mitochondria was measured. The treatment with ICI D7114 increased the binding of GDP both when expressed as total binding per lobe (P < 0.001) and per mg of protein (P < 0.01). It is concluded that ICI D7114, used in doses affecting brown adipose tissue thermogenesis, does not change the macronutrient selection or total energy intake in Sprague-Dawley rats. PMID:7800658

  1. Determination of a beta(3)-agonist in human plasma by LC/MS/MS with semi-automated 48-well diatomaceous earth plate.

    PubMed

    Wang, A Q; Fisher, A L; Hsieh, J; Cairns, A M; Rogers, J D; Musson, D G

    2001-10-01

    Methods for the determination of a beta(3)-agonist (A) in human plasma were developed and compared based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using a turbo ion spray (TIS) interface. Drug and internal standard were isolated from plasma by three sample preparation methods, liquid-liquid extraction, Chem Elut cartridges and 48-well diatomaceous earth plates, that successively improved sample throughput for LC/MS/MS. MS/MS detection was performed on a PE Sciex API 365 tandem mass spectrometer operated in positive ion mode and using multiple reaction monitoring (MRM). The precursor/product ion combinations of m/z 625/607 and 653/515 were used to quantify A and internal standard, respectively, after chromatographic separation of the analytes. Using liquid-liquid extraction and Chem Elut cartridges, the assay concentration range was 0.5-100 ng/ml. Using diatomaceous earth plates, the concentration range of the assay was extended to 0.5-200 ng/ml. For all three assays, the statistics for precision and accuracy is comparable. The assay accuracy ranged from 91-107% and intraday precision as measured by the coefficient of variation (CV) ranged 2-10%. The sample throughput was tripled when the diatomaceous earth plate method was compared with the original liquid-liquid extraction method. PMID:11489381

  2. 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

  3. Systematic determination of the peptide acceptor preferences for the human UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase).

    PubMed

    Perrine, Cynthia; Ju, Tongzhong; Cummings, Richard D; Gerken, Thomas A

    2009-03-01

    Mucin-type protein O-glycosylation is initiated by the addition of alpha-GalNAc to Ser/Thr residues of a polypeptide chain. The addition of beta-Gal to GalNAc by the UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase), forming the Core 1 structure (beta-Gal(1-3)-alpha-GalNAc-O-Ser/Thr), is a common and biologically significant subsequent step in O-glycan biosynthesis. What dictates the sites of Core 1 glycosylation is poorly understood; however, the peptide sequence and neighboring glycosylation effects have been implicated. To systematically address the role of the peptide sequence on the specificity of T-synthase, we used the oriented random glycopeptide: GAGAXXXX(T-O-GalNAc)XXXXAGAG (where X = G, A, P, V, I, F, Y, S, N, D, E, H, R, and K) as a substrate. The Core 1 glycosylated product was isolated on immobilized PNA (Arachis hypogaea) lectin and its composition determined by Edman amino acid sequencing for comparison with the initial substrate composition, from which transferase preferences were obtained. From these studies, elevated preferences for Gly at the +1 position with moderately high preferences for Phe and Tyr in the +3 position relative to the acceptor Thr-O-GalNAc were found. A number of smaller Pro enhancements were also observed. Basic residues, i.e., Lys, Arg, and His, in any position were disfavored, suggesting electrostatic interactions as an additional important component modulating transferase specificity. This work suggests that there are indeed subtle specific and nonspecific protein-targeting sequence motifs for this transferase.

  4. 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.

  5. Recognition of decay accelerating factor and alpha(v)beta(3) by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays.

    PubMed

    Buranda, Tione; Wu, Yang; Perez, Dominique; Jett, Stephen D; BonduHawkins, Virginie; Ye, Chunyan; Edwards, Bruce; Hall, Pamela; Larson, Richard S; Lopez, Gabriel P; Sklar, Larry A; Hjelle, Brian

    2010-07-15

    Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HFRS and HCPS and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. In this study, we developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then used flow cytometry and live cell confocal fluorescence microscopy imaging to show that ultraviolet (UV)-killed SNV particles bind to the cognate receptors of live virions, namely, decay accelerating factor (DAF/CD55) expressed on Tanoue B cells and alpha(v)beta(3) integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (K(d) approximately 26pM). Self-exchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of approximately 1:14,000. We configured the assay for measuring the binding of fluorescently labeled SNV to Tanoue B suspension cells using a high-throughput flow cytometer. In this way, we established a proof-of-principle high-throughput screening (HTS) assay for binding inhibition. This is a first step toward developing HTS format assays for small molecule inhibitors of viral-cell interactions as well as dissecting the mechanism of infection in a BSL-2 environment.

  6. 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 (

  7. Substitutions at Phe61 in the beta3-beta4 hairpin of HIV-1 reverse transcriptase reveal a role for the Fingers subdomain in strand displacement DNA synthesis.

    PubMed

    Fisher, Timothy S; Darden, Tom; Prasad, Vinayaka R

    2003-01-17

    Unlike most DNA polymerases, retroviral reverse transcriptases (RTs) are capable of strand displacement DNA synthesis in vitro, unassisted by other proteins. While human immunodeficiency virus type 1 (HIV-1) RT has been shown to possess this rare ability, the structural determinants responsible are unknown. X-Ray crystallographic and biochemical studies have indicated that the beta3-beta4 hairpin of the fingers subdomain of HIV-1 RT contains key contacts for the incoming template strand. In order to assess the possible role of the fingers subdomain in strand displacement synthesis, a set of substitutions was created at the highly conserved Phe61 residue, which is thought to contact the template strand immediately ahead of the dNTP-binding site. Purified heterodimeric RTs containing Phe61 substitutions displayed altered degrees of strand displacement synthesis on nicked and gapped duplex DNA templates with the relative order being: F61Y > or = F61L > wild-type = F61A > F61W. In order to verify that the effects on strand displacement synthesis were not an indirect effect of alterations in processivity, all Phe61 mutants were tested for processive polymerization. While the strand displacement activity of F61W RT variant was affected severely, it displayed a wild-type-like processivity. In contrast, both F61L and F61Y substitutions, despite showing enhanced strand displacement synthesis, displayed reduced processivity. In contrast, the processivity of F61A mutant, which had displayed nearly wild-type-like strand displacement synthesis, was affected most. These results showed that the effects of Phe61 substitutions on strand displacement are not due to global changes in polymerase processivity. Analysis of pause sites during DNA polymerization on double-stranded templates revealed that the wild-type and the Phe61 mutant RTs interact with the template quite differently. Modeling a 5 nt duplex DNA ahead of the dNTP-binding site of HIV-1 RT suggested a correlation between

  8. The beta 3-adrenoceptor agonist ICI-D7114 is not as efficient on reinduction of uncoupling protein mRNA in sheep as it is in dogs and smaller species.

    PubMed

    Nouguès, J; Reyne, Y; Champigny, O; Holloway, B; Casteilla, L; Ricquier, D

    1993-09-01

    Adipose tissue in newborn lambs is brown, but within a few days it is transformed into white adipose tissue. In the same way, preadipocytes cultured in serum-free chemically defined medium achieve full differentiation and express uncoupling protein (UCP), a marker of brown adipose tissue, when isolated from perirenal adipose tissue of the newborn, whereas they no longer express UCP when isolated from older lambs. The effects of a chronic stimulation of adipose tissue by novel beta 3-adrenoceptor agonist (ICI D7114) on the maintenance after birth and on the reinduction in older lambs of UCP mRNA in adipose tissue were studied. Treatment of newborn lambs with this agonist for 25 d maintained a slight level of UCP mRNA in perirenal and pericardiac, but not in omental and inguinal, adipose tissue depots. Preadipocytes isolated from perirenal adipose tissue of treated animals differentiated, in vitro, into adipocytes, but no UCP mRNA could be detected either in the absence or in the presence of the beta 3-adrenoceptor agonist in the culture medium. Treatment of 1-mo-old lambs with ICI D7114 for 12 d restored UCP mRNA in perirenal and pericardiac adipose tissues of two of four treated lambs, but at a much lower level than in the same tissues at birth. In both experiments, the final BW and the ADG of lambs treated with ICI D7114 were not statistically different from controls. These results are quite different from those obtained with the same beta 3-adrenoceptor agonist in dogs and rodents. PMID:8104921

  9. Expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated and nuclear transfer derived bovine pregnancies.

    PubMed

    Ravelich, S R; Shelling, A N; Wells, D N; Peterson, A J; Lee, R S F; Ramachandran, A; Keelan, J A

    2006-01-01

    Bovine nuclear transfer pregnancies are characterized by a high incidence of placental abnormalities, notably, increased placentome size and deficiencies in trophoblast cell function and establishment of placental vasculature. Alterations in gene expression during placental growth and development may contribute to the appearance of large placentomes in pregnancies derived from nuclear transfer. The placenta synthesizes a number of cytokines and growth factors, including the transforming growth factor-betas (TGF-betas) that are involved in the establishment, maintenance and/or regulation of pregnancy. All forms of TGF-beta and their receptors are present at the fetal-maternal interface of the bovine placentome, where they are thought to play an important role in regulating growth, differentiation, and function of the placenta. Using real-time RT-PCR, we have examined the expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated (AI) and nuclear transfer (NT)-derived bovine pregnancies at days 50, 100 and 150 of gestation. TGF-beta1, TGF-beta2 and TGF-beta3 mRNA expression increased by 2.0-2.8-fold, while TGF-betaRI and TGF-betaRII mRNA expression decreased by 1.7-2.0-fold in NT placentomes compared to AI controls at all gestational ages examined. These findings indicate that NT placentomes may be resistant to the growth suppressive effects of TGF-betas and could contribute to the placental proliferative abnormalities observed in NT-derived placentas. Alternatively, deficiencies in placentation may provide a mechanism whereby TGF-betas are dysregulated in NT pregnancies.

  10. 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

  11. The C-terminus of the {gamma}2 chain but not of the {beta}3 chain of laminin-332 is indirectly but indispensably necessary for integrin-mediated cell reactions

    SciTech Connect

    Navdaev, Alexei; Heitmann, Vanessa; Santana Evangelista, Karla de; Moergelin, Matthias; Wegener, Joachim; Eble, Johannes A.

    2008-02-01

    Using a recombinant mini-laminin-332, we showed that truncation of the three C-terminal amino acids of the {gamma}2 chain, but not of the C-terminal amino acid of the {beta}3 chain, completely abolished {alpha}3{beta}1 integrin binding and its cellular functions, such as attachment and spreading. However, a synthetic peptide mimicking the {gamma}2 chain C-terminus did not interfere with {alpha}3{beta}1 integrin binding or cell adhesion and spreading on laminin-332 as measured by protein interaction assays and electric cell-substrate impedance sensing. Nor was the soluble peptide able to restore the loss of integrin-mediated cell adhesiveness to mini-laminin-332 after deletion of the {gamma}2 chain C-terminus. These findings spoke against the hypothesis that the {gamma}2 chain C-terminus of laminin-332 is a part of the {alpha}3{beta}1 integrin interaction site. In addition, structural studies with electron microscopy showed that truncation of the {gamma}2 chain C-terminus opened up the compact supradomain structure of LG1-3 domains. Thus, by inducing or stabilizing an integrin binding-competent conformation or array of the LG1-3 domains, the {gamma}2 chain C-terminus plays an indirect but essential role in laminin-332 recognition by {alpha}3{beta}1 integrin and, hence, its cellular functions.

  12. Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines.

    PubMed Central

    Damstrup, L.; Rygaard, K.; Spang-Thomsen, M.; Skovgaard Poulsen, H.

    1993-01-01

    A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis of the binding data demonstrated that the cells bound between 4.5 and 27.5 fmol mg-1 protein with a KD ranging from 16 to 40 pM. TGF beta 1 binding to the receptors was confirmed by cross-linking TGF beta 1 to the TGF beta-r. Three classes of TGF beta-r were demonstrated, type I and type II receptors with M(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell lines express TGF beta-receptors and also produce TGF beta mRNAs. Images Figure 3 Figure 4 PMID:8388229

  13. GENETIC VARIATION IN THE BETA-3-ADRENORECEPTOR GENE (TRP64ARG POLYMORPHISM) AND THEIR INFLUENCE ON ANTHROPOMETRIC PARAMETERS AND INSULIN RESISTANCE AFTER A HIGH PROTEIN/LOW CARBOHYDRATE VERSUS A STANDARD HYPOCALORIC DIET.

    PubMed

    de Luis, Daniel Antonio; Aller, Rocío; Izaola, Olatz; de la Fuente, Beatriz; Romero, Enrique

    2015-08-01

    Introducción: la variante Trp64Arg del receptor Beta ha sido relacionada con un aumento del peso corporal y resistencia a la insulina. Objetivo: el objetivo de nuestro estudio fue investigar la influencia del polimorfismo (rs 4994) del gen del receptor adrenérgico-Beta-3 en la respuesta metabólica y la pérdida de peso en un estudio de intervención a medio plazo con una dieta con alto contenido en proteínas/baja en carbohidratos vs una dieta hipocalórica estándar (1.000 kcal / día). Material y métodos: se evaluó una muestra de 284 sujetos obesos con un diseño de ensayo aleatorio. Se realizó una evaluación nutricional al inicio y al final de un período de 9 meses en el que los sujetos recibieron una de las dos dietas (dieta HP: alta en proteínas/baja en carbohidratos vs dieta S: dieta estándar). Resultados: no hubo diferencias significativas entre los efectos positivos (sobre el peso, el índice de masa corporal, la circunferencia de la cintura, la masa grasa, la presión arterial sistólica y los niveles de leptina) en los dos genotipos con ambas dietas. Con ambas dietas y solo en el genotipo salvaje (dieta HP vs dieta S), colesterol total (-10,1 ± 3,9 mg / dl vs -10,1 ± 2,2 mg / dl; p> 0,05), colesterol LDL (-9,5 ± 2,1 mg / dl vs -8,5 ± 2,3 mg / dl; p> 0,05) y los triglicéridos (-19,1 ± 2,1 mg / dl vs -14,3 ± 2,1 mg / dl; p> 0,05) disminuyeron. La mejoría de estos parámetros fue similar en sujetos con dieta HP vs dieta HS. Con la dieta HP y solo en el genotipo salvaje, los niveles de insulina (-3,7 ± 1,9 UI / L; p.

  14. 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

  15. Beta-3 adrenoceptors as new therapeutic targets for cardiovascular pathologies.

    PubMed

    Gauthier, Chantal; Rozec, Bertrand; Manoury, Boris; Balligand, Jean-Luc

    2011-09-01

    Catecholamines play a key role in the regulation of cardiovascular function, classically through ß(1/2)-adrenoreceptors (AR) activation. After ß(3)-AR cloning in the late 1980s, convincing evidence for ß(3)-AR expression and function in cardiovascular tissues recently initiated a reexamination of their involvement in the pathophysiology of cardiovascular diseases. Their upregulation in diseased cardiovascular tissues and resistance to desensitization suggest they may be attractive therapeutic targets. They may substitute for inoperant ß(1/2)-AR to mediate vasodilation in diabetic or atherosclerotic vessels. In cardiac ventricle, their contractile effects are functionally antipathetic to those of ß(1/2)-AR; in normal heart, ß(3)-ARs may mediate a moderate negative inotropic effect, but in heart failure, it may protect against adverse effects of excessive catecholamine stimulation by action on excitation-contraction coupling, electrophysiology, or remodelling. Thus, prospective studies in animals and patients at different stages of heart failure should lead to identify the best therapeutic window to use ß(3)-AR agonists and/or antagonists. PMID:21633786

  16. 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.

  17. Beta 3-adrenoceptor in rat aorta: molecular and biochemical characterization and signalling pathway

    PubMed Central

    Rautureau, Yohann; Toumaniantz, Gilles; Serpillon, Sabrina; Jourdon, Philippe; Trochu, Jean-Noël; Gauthier, Chantal

    2002-01-01

    We have previously demonstrated that β3-adrenoceptor (β3-AR) stimulation induces endothelium-dependent vasorelaxation in rat aorta through the activation of an endothelial NO synthase associated with an increase in intracellular cGMP. The aim of the present study was to localise β3-AR to confirm our functional study and to complete the signalling pathway of β3-AR in rat aorta. By RT–PCR, we have detected β3-AR transcripts both in aorta and in freshly isolated endothelial cells. The absence of markers for adipsin or hormone-sensitive lipase in endothelial cells excluded the presence of β3-AR from adipocytes. The localization of β3-AR in aortic endothelial cells was confirmed by immunohistochemistry using a rat β3-AR antibody. To identify the G protein linked to β3-AR, experiments were performed in rat pre-treated with PTX (10 μg kg−1), a Gi/0 protein inhibitor. The blockage of Gi/0 protein by PTX was confirmed by the reduction of vasorelaxation induced by UK 14304, a selective α2-AR agonist. The cumulative concentration-response curve for SR 58611A, a β3-AR agonist, was not significantly modified on aorta rings from PTX pre-treated rats. At the same level of contraction, the relaxations induced by 10 μM SR 58611A were significantly reduced in 30 mM-KCl pre-constricted rings (Emax=16.7±8.4%, n=5), in comparison to phenylephrine (0.3 μM) pre-constricted rings (Emax=49.11±11.0%, n=5, P<0.05). In addition, iberotoxin (0.1 μM), glibenclamide (1 μM) and 4-aminopyridine (1 mM), selective potassium channels blockers of KCa, KATP, and Kv respectively, decreased the SR 58611A-mediated relaxation. We conclude that β3-AR is preferentially expressed in rat aortic endothelial cells. β3-AR-mediated aortic relaxation is independent of Gi/0 proteins stimulation, but results from the activation of several potassium channels, KCa, KATP, and Kv. PMID:12208771

  18. Transforming Growth Factor Beta 3 Is Required for Excisional Wound Repair In Vivo

    PubMed Central

    Le, Mark; Naridze, Rachelle; Morrison, Jasmine; Biggs, Leah C.; Rhea, Lindsey; Schutte, Brian C.; Kaartinen, Vesa; Dunnwald, Martine

    2012-01-01

    Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-ß isoforms–TGF- ß 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect. PMID:23110169

  19. 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

  20. 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.

  1. Tubulin Beta3 Serves as a Target of HDAC3 and Mediates Resistance to Microtubule-Targeting Drugs.

    PubMed

    Kim, Youngmi; Kim, Hyuna; Jeoung, Dooil

    2015-08-01

    We investigated the role of HDAC3 in anti-cancer drug-resistance. The expression of HDAC3 was decreased in cancer cell lines resistant to anti-cancer drugs such as celastrol and taxol. HDAC3 conferred sensitivity to these anti-cancer drugs. HDAC3 activity was necessary for conferring sensitivity to these anti-cancer drugs. The down-regulation of HDAC3 increased the expression of MDR1 and conferred resistance to anti-cancer drugs. The expression of tubulin β3 was increased in drug-resistant cancer cell lines. ChIP assays showed the binding of HDAC3 to the promoter sequences of tubulin β3 and HDAC6. HDAC6 showed an interaction with tubulin β3. HDAC3 had a negative regulatory role in the expression of tubulin β3 and HDAC6. The down-regulation of HDAC6 decreased the expression of MDR1 and tubulin β3, but did not affect HDAC3 expression. The down-regulation of HDAC6 conferred sensitivity to taxol. The down-regulation of tubulin β3 did not affect the expression of HDAC6 or MDR1. The down-regulation of tubulin β3 conferred sensitivity to anti-cancer drugs. Our results showed that tubulin β3 serves as a downstream target of HDAC3 and mediates resistance to microtubule-targeting drugs. Thus, the HDAC3-HDAC6-Tubulin β axis can be employed for the development of anti-cancer drugs.

  2. 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.

  3. Association between TGFBR2 gene polymorphisms and congenital heart defects in Han Chinese population.

    PubMed

    Huang, Fuhua; Li, Li; Shen, Chong; Wang, Hairu; Chen, Jinfeng; Chen, Wen; Chen, Xin

    2014-10-31

    Antecedentes: Factor de crecimiento transformante receptor II (TGFBR2) es un componente clave de la via de señalización de TGF - .TGFBR2 puede ser detectado en la generación de corazón. Los embriones de ratón de TGFBR2 gene knockout mostraron defectos congénitos del corazon. Métodos: Hemos realizado un estudio de casos y controles para investigar la asociación entre polimorfismos del gen TGFBR2 y defectos congénitos del corazón en la población china han. 125 pacientes con defectos congénitos del corazón y 615 unrelated controles fueron reclutados. Marcado de dos polimorfismos de nucleótido único (tagsnps) en 5 ‘aguas arriba del gen TGFBR2 (rs6785358, - 3779a / g; rs764522, - 1444c / g) fueron seleccionados y genotipados por reacción en cadena de la polimerasa (PCR) - polimorfismos de longitud de fragmentos de restricción (RFLP) de ensayo. Resultados: Se observó una diferencia significativa en la distribución de genotipos entre pacientes con defectos congénitos del corazón y controles para SNP rs6785358 (P = 0043). La SNP rs6785358 el porteador del alelo G (AG / GG genotipo) mostraron un importante crecimiento y mayor riesgo de defectos congénitos del corazón en comparación con AA homocigotos (OR = 1.545, IC del 95%: 1.013–2.356). Más análisis por sexo estratificación indicó que los individuos con alelo G (AG / GG genotipo) para SNP rs6785358 tienen una mayor susceptibilidad a defectos congénitos del corazón (OR = 2.088, IC del 95%: 1.123-3.883, p = 0.019) en machos, pero no en las mujeres (OR = 1.195, IC del 95%: 0.666-2.146, p = 0.55). No hay significación estadística fue detectado en la distribución de los genotipos y frecuencias de alelos de SNP rs764522 entre pacientes y controles. Conclusión: Nuestros resultados sugieren que el SNP rs6785358 de gen TGFBR2 se asoció con un mayor riesgo de defectos congénitos del corazón en los chinos han hombres y más investigación estaría justificada.

  4. Lyn- and PLC-beta3-dependent regulation of SHP-1 phosphorylation controls Stat5 activity and myelomonocytic leukemia-like disease.

    PubMed

    Xiao, Wenbin; Ando, Tomoaki; Wang, Huan-You; Kawakami, Yuko; Kawakami, Toshiaki

    2010-12-23

    Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)-β3-dependent manner. Thus, PLC-β3-deficient mice develop myeloproliferative neoplasm, like Lyn (Src family kinase)- deficient mice. Here we show that Lyn/PLC-β3 doubly deficient lyn(-/-);PLC-β3(-/-) mice develop a Stat5-dependent, fatal myelodysplastic/myeloproliferative neoplasm, similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn(-/-);PLC-β3(-/-) mice that cause the CMML-like disease, phosphorylation of SHP-1 at Tyr(536) and Tyr(564) is abrogated, resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore, SHP-1 phosphorylation at Tyr(564) by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand, Tyr(536) in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore, we identify a novel Lyn/PLC-β3-mediated regulatory mechanism of SHP-1 and Stat5 activities.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit.

    PubMed

    Wohlfarth, Kai M; Bianchi, Matt T; Macdonald, Robert L

    2002-03-01

    Attenuated behavioral sensitivity to neurosteroids has been reported for mice deficient in the GABA(A) receptor delta subunit. We therefore investigated potential subunit-specific neurosteroid pharmacology of the following GABA(A) receptor isoforms in a transient expression system: alpha1beta3gamma2L, alpha1beta3delta, alpha6beta3gamma2L, and alpha6beta3delta. Potentiation of submaximal GABA(A) receptor currents by the neurosteroid tetrahydrodeoxycorticosterone (THDOC) was greatest for the alpha1beta3delta isoform. Whole-cell GABA concentration--response curves performed with and without low concentrations (30 nm) of THDOC revealed enhanced peak GABA(A) receptor currents for isoforms tested without affecting the GABA EC50. Alpha1beta3delta currents were enhanced the most (>150%), whereas the other isoform currents were enhanced 15-50%. At a higher concentration (1 microm), THDOC decreased peak alpha1beta3gamma2L receptor current amplitude evoked by GABA (1 mm) concentration jumps and prolonged deactivation but had little effect on the rate or extent of apparent desensitization. Thus the polarity of THDOC modulation depended on GABA concentration for alpha1beta3gamma2L GABA(A) receptors. However, the same protocol applied to alpha1beta3delta receptors resulted in peak current enhancement by THDOC of >800% and prolonged deactivation. Interestingly, THDOC induced pronounced desensitization in the minimally desensitizing alpha1beta3delta receptors. Single channel recordings obtained from alpha1beta3delta receptors indicated that THDOC increased the channel opening duration, including the introduction of an additional longer duration open state. Our results suggest that the GABA(A) receptor delta subunit confers increased sensitivity to neurosteroid modulation and that the intrinsic gating and desensitization kinetics of alpha1beta3delta GABA(A) receptors are altered by THDOC.

  11. ...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…

  12. 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.

  13. 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

  14. Design, synthesis and biological evaluation of nonpeptide integrin antagonists.

    PubMed

    Nicolaou, K C; Trujillo, J I; Jandeleit, B; Chibale, K; Rosenfeld, M; Diefenbach, B; Cheresh, D A; Goodman, S L

    1998-08-01

    Recent studies demonstrated that peptide and antibody antagonists of integrin alpha v beta 3 block angiogenesis and tumor growth. In this article, the design, synthesis and biological evaluation of a series of nitroaryl ether-based, nonpeptide mimetics are described. The design of these compounds was based on Merck's arylether/alpha-aminoacid/guanidine framework and incorporates a novel nitroaryl system. The synthesized mimetics were tested against a variety of integrins (alpha v beta 3, alpha IIb beta 3, and alpha v beta 5) in order to determine their binding selectivity and ability to inhibit cell adhesion. Selected compounds were also tested for their ability to inhibit angiogenesis in vivo in the CAM (chick chorioallantoic membrane) assay. From the generated compound library, compounds 16 and 19 proved to be potent and selective inhibitors of alpha IIb beta 3 (IC50 = 14 nM) whereas compound 11 showed excellent in vivo inhibition of angiogenesis (at 30 micrograms/embryo).

  15. Auto-modulation of neuroactive steroids on GABA A receptors: a novel pharmacological effect.

    PubMed

    Wegner, Florian; Rassler, Cornelia; Allgaier, Clemens; Strecker, Karl; Wohlfarth, Kai

    2007-02-01

    GABA(A) receptor function is modulated by various important drugs including neuroactive steroids that act on allosteric modulatory sites and can directly activate GABA(A) receptor channels at high concentrations. We used whole cell patch-clamp recordings and rapid applications of the neuroactive steroid alphaxalone to investigate repetitive steroid effects. Alphaxalone potentiation of submaximal GABA-evoked currents was enhanced significantly by repetitive coapplications at all investigated recombinant isoforms (alpha1beta3delta, alpha1beta3gamma2L, alpha6beta3delta, alpha6beta3gamma2L) and at GABA(A) receptors of differentiated human NT2 neurons. A similar increase of current amplitudes was induced by repetitive applications of a high steroid concentration without GABA. We refer to these reversible effects as auto-modulation because repeated interactions of steroids enhanced their own pharmacological impact at the receptor sites in a time and concentration dependent manner without affecting GABA controls. Pronounced auto-modulatory actions were also measured using the neurosteroid 5alpha-THDOC in contrast to indiplon, THIP, and pentobarbital indicating a steroid specificity. Protein kinase A inhibition significantly reduced alphaxalone auto-modulation at alpha1beta3gamma2L, alpha6beta3gamma2L, and alpha6beta3delta subtypes while it enhanced potentiation at alpha1beta3delta isoforms suggesting a crucial influence of receptor subunit composition and phosphorylation for steroid actions. Especially at extrasynaptic GABA(A) receptor sites containing the delta subunit steroid auto-modulation may have a critical role in enhancing potentiation of GABA-induced currents.

  16. 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.

  17. [A very slow-growing exophytic hemisphere glioma: a case report].

    PubMed

    Velásquez, Carlos; Caballero, Hugo; Bucheli, Carlos; Berciano, José; Vázquez-Barquero, Alfonso; Martino, Juan

    2016-01-01

    Introduccion. Los gliomas de bajo grado presentan un patron de crecimiento caracteristico a traves de las fibras de la sustancia blanca. El crecimiento exofitico en gliomas de bajo grado hemisfericos no se ha descrito previamente. Se presenta un caso de glioma hemisferico de lenta progresion y con crecimiento exofitico. Caso clinico. Varon de 55 años, con crisis parciales motoras secundarias a un oligodendroglioma de grado II de la Organizacion Mundial de la Salud. El tumor infiltraba la circunvolucion frontal superior con extension exofitica que se extendia por encima de la circunvolucion precentral. Fue seguido con controles clinicorradiologicos durante 23 años. El analisis de la evolucion radiologica del tumor demostraba un crecimiento tumoral lento, con una velocidad de crecimiento de 0,5 mm al año. Durante la exeresis quirurgica se definio un plano subaracnoideo entre el componente exofitico y la circunvolucion precentral, que se encontraba desplazada inferiormente sin infiltracion tumoral. La estimulacion electrica intraoperatoria no evidencio funcion en el componente exofitico, pero si en la circunvolucion precentral. No se observaron deficits neurologicos postoperatorios. Conclusiones. La velocidad de crecimiento en gliomas de bajo grado se ha estimado en 4-6 mm al año. El tumor que se describe aqui tiene una velocidad de crecimiento de 0,5 mm al año, muy por debajo de esta media. La identificacion de la porcion exofitica es un paso importante en la planificacion preoperatoria. Este componente es mas facil de resecar debido al plano de clivaje subaracnoideo y a la ausencia de funcion.

  18. 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

  19. 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.

  20. [Efficacy of zinc on lineal growth on Latin American children younger than 5; systematic review].

    PubMed

    Jiménez-Morán, Elsa; Bacardí-Gascón, Montserrat; Jiménez-Cruz, Arturo

    2013-01-01

    Antecedentes: En la mayoría de los países latinoamericanos existe una deficiencia de micronutrientes, retraso en el crecimiento, y altas tasas de morbilidad y mortalidad infantil. En la actualidad se ha asociado la deficiencia de zinc con el retraso del crecimiento infantil. El propósito de esta revisión, fue analizar artículos sobre estudios aleatorizados que evaluaron el efecto de la suplementación con zinc y el crecimiento infantil. Metodología: Se realizó una búsqueda electrónica de la literatura en la base de datos de PubMed con las palabras claves “Micronutrient supplementation and growth”, publicados de enero de 2005 a abril de 2013. Se identificaron 34 estudios aleatorizados controlados (EAC) realizados en Latinoamérica, de los cuales cinco reunieron los criterios de inclusión. Se añadió un estudio referido por otros autores. Resultados: Los estudios se realizaron en Brasil, Cuba, Perú, Ecuador, y Guatemala. En ningún estudio se observó el efecto positivo del zinc sobre el crecimiento lineal. Conclusión: La revisión realizada sugiere que la suplementación con zinc no tiene un efecto significativo sobre el crecimiento lineal; sin embargo, podría tener beneficios sobre el crecimiento en niños con deficiencia de zinc si se añaden otros nutrientes en deficiencia.

  1. [Role of anaerobic blood culture in the simultaneous blood culture taking for the diagnosis of bacteremia].

    PubMed

    Guajardo-Lara, Claudia Elena; Saldaña-Ramírez, Martha Idalia; Ayala-Gaytán, Juan Jacobo; Valdovinos-Chávez, Salvador Bruno

    2016-01-01

    Introducción: la frecuencia de la septicemia va en aumento y su mortalidad es alta; por lo tanto, su detección, la identificación del microorganismo causal y su susceptibilidad son perentorias. Metodos: se revisaron los registros de 4110 botellas de cultivo de sangre obtenida de enero de 2013 a julio de 2014 de pacientes adultos en un hospital privado de tercer nivel. Resultados: se observó crecimiento de microorganismos en 559 cultivos (12.6 %). En 2648 hemocultivos (60 %) inoculados en pares de frascos uno con medio aeróbico y el otro anaeróbico (1324 sets), se detectó crecimiento en 182 frascos a los que les fueron inoculadas las muestras tomadas al mismo tiempo a 135 pacientes (13.7 %). En 86 pares de frascos con las muestras de 54 pacientes (40 %), el crecimiento solamente se dio en el frasco aeróbico (47.5 %); en 24 pares de frascos (13.19 %) tomados a 21 pacientes (15.5 %, p < 0.05), solamente hubo crecimiento en el frasco anaeróbico. En los hemocultivos de 32 de 60 pacientes con crecimiento en ambos frascos (53 %), el crecimiento se detectó primero en el frasco anaeróbico. Conclusiones: los hemocultivos anaeróbicos tienen una utilidad baja para la detección de bacteriemias por anaerobios estrictos; no obstante, en el 15.55 % de los pacientes estuvo presente el riesgo de pasar por alto la presencia de bacteriemia, y en 53 % de los pacientes con hemocultivos positivos, el diagnóstico de bacteriemia pudo establecerse de manera más temprana, lo que permitió anticipar con mejor precisión la toma de decisiones.

  2. Purification, characterization, and crystallization of single molecular species of beta-conglycinin from soybean seeds.

    PubMed

    Morita, S; Fukase, M; Yamaguchi, M; Fukuda, Y; Morita, Y

    1996-05-01

    Four major molecular species of beta-conglycinin, alpha 3, alpha 2 beta, alpha beta 2, and beta 3, were isolated and purified from seeds of an alpha' subunit-deficient strain of soybeans (Glycine max). All components were found to be homogeneous by high pressure liquid chromatography, SDS-polyacrylamide gel electrophoresis, and amino acid and amino terminal sequence analyses. The amino acid compositions of the alpha 3 and beta 3 components agreed fairly well with the compositions deduced from the cDNA sequences, and all of the components were highly glycosylated. The alpha 3 and beta 3 components were compared regarding their secondary structures. The secondary structure of the alpha 3 component deduced from CD measurements showed a higher alpha-helix content than that of the beta 3 component. The beta 3 component was crystallized by decreasing the ionic strength from 0.5 to 0.14 in phosphate buffer, pH 7.3, and the crystals grew to a size (1.0 mm x 0.2 mm x 0.2 mm) suitable for X-ray crystallographic analysis. A preliminary X-ray analysis showed that the crystal belonged to an orthorhombic crystal system having the space group P2(1)2(1)2(1) and unit cell dimensions of a = 185.1 A, b = 107.9 A, and c = 97.6 A.

  3. A novel beta1,3-N-acetylglucosaminyltransferase involved in invasion of cancer cells as assayed in vitro.

    PubMed

    Kataoka, Ken; Huh, Nam-ho

    2002-06-21

    Using a two-step screening system for genes involved in tissue invasion [Kataoka et al., Cancer Lett. 163(2) (2001) 213], we identified a cDNA whose expression level was higher in mouse placenta at later stages of gestation and in sublines of cancer cells with low degrees of invasiveness. The deduced amino acid sequence showed relatively high similarity with beta1,3-N-acetylglucosaminyltransferase2 approximately 5 (beta3GnT2 approximately 5), and the protein was therefore named beta3GnT7. A possible human ortholog was identified and its chromosomal locus was determined to be 2q37.1. In the mouse, beta3GnT7 was most strongly expressed in the placenta and colon. Moderate amounts of mRNA were detected in the lung, stomach, small intestine, and kidney. The expression of beta3GnT7 was very weak in the cerebrum, cerebellum, heart, and testis. Transfection of the antisense oligonucleotide significantly enhanced the motility of a lung cancer cell line (KLN205-MUC1) in a monolayer compared to the controls. Furthermore, the antisense oligonucleotide increased the number of cells that invaded the matrix-coated membrane in an in vitro invasion model. These results indicate that beta3GnT7 may play a role in preventing cells from migrating out of the original tissues and invading surrounding tissues.

  4. Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

    PubMed

    Paulhe, F; Racaud-Sultan, C; Ragab, A; Albiges-Rizo, C; Chap, H; Iberg, N; Morand, O; Perret, B

    2001-11-01

    Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

  5. Effects of chronic celiprolol treatment on brown fat, feeding, and drinking in fa/fa Zucker rats.

    PubMed

    Savontaus, E; Rouru, J; Malminiemi, K; Luukkaa, V; Pesonen, U; Koulu, M; Huupponen, R

    2000-04-01

    Celiprolol is a novel beta-adrenoceptor blocking drug that displays clinically favorable effects on glucose and lipid metabolism. Because some other atypical beta-adrenoceptor blocking drugs have been described to act as agonists on beta(3)-adrenoceptors, we aimed to investigate the effects of celiprolol on brown fat and beta(3)-adrenoceptors. Chronic treatment of obese fa/fa Zucker rats with celiprolol (50 mg/kg/day orally for 20 days) increased GDP binding to brown fat mitochondria by 1.5-fold, whereas beta(3)-adrenoceptor agonist ZD7114 ((S)-4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]-N-(2-methoxyet hyl )phenoxyacetamide, 3 mg/kg/day) increased the binding by 3.3-fold. Weight gain was reduced by 19% due to decreased water and food intakes in celiprolol-treated rats. Celiprolol did not activate lipolysis in rat adipocytes in vitro or stimulate human beta(3)-adrenoceptors expressed in Chinese hamster ovary cells as measured with Cytosensor microphysiometer. Therefore, celiprolol does not seem to activate brown fat via beta(3)-adrenoceptors. PMID:10764928

  6. Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors.

    PubMed

    Reynolds, Andrew R; Hart, Ian R; Watson, Alan R; Welti, Jonathan C; Silva, Rita G; Robinson, Stephen D; Da Violante, Georges; Gourlaouen, Morgane; Salih, Mishal; Jones, Matt C; Jones, Dylan T; Saunders, Garry; Kostourou, Vassiliki; Perron-Sierra, Françoise; Norman, Jim C; Tucker, Gordon C; Hodivala-Dilke, Kairbaan M

    2009-04-01

    Inhibitors of alpha(v)beta(3) and alpha(v)beta(5) integrin have entered clinical trials as antiangiogenic agents for cancer treatment but generally have been unsuccessful. Here we present in vivo evidence that low (nanomolar) concentrations of RGD-mimetic alpha(v)beta(3) and alpha(v)beta(5) inhibitors can paradoxically stimulate tumor growth and tumor angiogenesis. We show that low concentrations of these inhibitors promote VEGF-mediated angiogenesis by altering alpha(v)beta(3) integrin and vascular endothelial growth factor receptor-2 trafficking, thereby promoting endothelial cell migration to VEGF. The proangiogenic effects of low concentrations of RGD-mimetic integrin inhibitors could compromise their efficacy as anticancer agents and have major implications for the use of RGD-mimetic compounds in humans.

  7. Pathogenic hantaviruses direct the adherence of quiescent platelets to infected endothelial cells.

    PubMed

    Gavrilovskaya, Irina N; Gorbunova, Elena E; Mackow, Erich R

    2010-05-01

    Hantavirus infections are noted for their ability to infect endothelial cells, cause acute thrombocytopenia, and trigger 2 vascular-permeability-based diseases. However, hantavirus infections are not lytic, and the mechanisms by which hantaviruses cause capillary permeability and thrombocytopenia are only partially understood. The role of beta(3) integrins in hemostasis and the inactivation of beta(3) integrin receptors by pathogenic hantaviruses suggest the involvement of hantaviruses in altered platelet and endothelial cell functions that regulate permeability. Here, we determined that pathogenic hantaviruses bind to quiescent platelets via a beta(3) integrin-dependent mechanism. This suggests that platelets may contribute to hantavirus dissemination within infected patients and provides a means by which hantavirus binding to beta(3) integrin receptors prevents platelet activation. The ability of hantaviruses to bind platelets further suggested that cell-associated hantaviruses might recruit platelets to the endothelial cell surface. Our findings indicate that Andes virus (ANDV)- or Hantaan virus (HTNV)-infected endothelial cells specifically direct the adherence of calcein-labeled platelets. In contrast, cells comparably infected with nonpathogenic Tula virus (TULV) failed to recruit platelets to the endothelial cell surface. Platelet adherence was dependent on endothelial cell beta(3) integrins and neutralized by the addition of the anti-beta(3) Fab fragment, c7E3, or specific ANDV- or HTNV-neutralizing antibodies. These findings indicate that pathogenic hantaviruses displayed on the surface of infected endothelial cells bind platelets and that a platelet layer covers the surface of infected endothelial cells. This fundamentally changes the appearance of endothelial cells and has the potential to alter cellular immune responses, platelet activation, and endothelial cell functions that affect vascular permeability. Hantavirus-directed platelet quiescence and

  8. Hantaviruses direct endothelial cell permeability by sensitizing cells to the vascular permeability factor VEGF, while angiopoietin 1 and sphingosine 1-phosphate inhibit hantavirus-directed permeability.

    PubMed

    Gavrilovskaya, Irina N; Gorbunova, Elena E; Mackow, Natalie A; Mackow, Erich R

    2008-06-01

    Hantaviruses infect human endothelial cells and cause two vascular permeability-based diseases: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Hantavirus infection alone does not permeabilize endothelial cell monolayers. However, pathogenic hantaviruses inhibit the function of alphav beta3 integrins on endothelial cells, and hemorrhagic disease and vascular permeability deficits are consequences of dysfunctional beta3 integrins that normally regulate permeabilizing vascular endothelial growth factor (VEGF) responses. Here we show that pathogenic Hantaan, Andes, and New York-1 hantaviruses dramatically enhance the permeability of endothelial cells in response to VEGF, while the nonpathogenic hantaviruses Prospect Hill and Tula have no effect on endothelial cell permeability. Pathogenic hantaviruses directed endothelial cell permeability 2 to 3 days postinfection, coincident with pathogenic hantavirus inhibition of alphav beta3 integrin functions, and hantavirus-directed permeability was inhibited by antibodies to VEGF receptor 2 (VEGFR2). These studies demonstrate that pathogenic hantaviruses, similar to alphav beta3 integrin-deficient cells, specifically enhance VEGF-directed permeabilizing responses. Using the hantavirus permeability assay we further demonstrate that the endothelial-cell-specific growth factor angiopoietin 1 (Ang-1) and the platelet-derived lipid mediator sphingosine 1-phosphate (S1P) inhibit hantavirus directed endothelial cell permeability at physiologic concentrations. These results demonstrate the utility of a hantavirus permeability assay and rationalize the testing of Ang-1, S1P, and antibodies to VEGFR2 as potential hantavirus therapeutics. The central importance of beta3 integrins and VEGF responses in vascular leak and hemorrhagic disease further suggest that altering beta3 or VEGF responses may be a common feature of additional viral hemorrhagic diseases. As a result, our findings provide a potential mechanism

  9. Snail family members and cell survival in physiological and pathological cleft palates.

    PubMed

    Martínez-Alvarez, Concepción; Blanco, María J; Pérez, Raquel; Rabadán, M Angeles; Aparicio, Marta; Resel, Eva; Martínez, Tamara; Nieto, M Angela

    2004-01-01

    Palate fusion is a complex process that involves the coordination of a series of cellular changes including cell death and epithelial to mesenchymal transition (EMT). Since members of the Snail family of zinc-finger regulators are involved in both triggering of the EMT and cell survival, we decided to study their putative role in palatal fusion. Furthermore, Snail genes are induced by transforming growth factor beta gene (TGF-beta) superfamily members, and TGF-beta(3) null mutant mice (TGF-beta(3)-/-) show a cleft palate phenotype. Here we show that in the wild-type mouse at the time of fusion, Snail is expressed in a few cells of the midline epithelial seam (MES), compatible with a role in triggering of the EMT in a small subpopulation of the MES. We also find an intriguing relationship between the expression of Snail family members and cell survival associated to the cleft palate condition. Indeed, Snail is expressed in the medial edge epithelial (MEE) cells in TGF-beta(3)-/-mouse embryo palates, where it is activated by the aberrant expression of its inducer, TGF-beta(1), in the underlying mesenchyme. In contrast to Snail-deficient wild-type pre-adhesion MEE cells, Snail-expressing TGF-beta(3) mutant MEE cells survive as they do their counterparts in the chick embryo. Interestingly, Slug is the Snail family member expressed in the chick MEE, providing another example of interchange of Snail and Slug expression between avian and mammalian embryos. We propose that in the absence of TGF-beta(3), TGF-beta(1) is upregulated in the mesenchyme, and that in both physiological (avian) and pathological (TGF-beta(3)-/-mammalian) cleft palates, it induces the expression of Snail genes promoting the survival of the MEE cells and permitting their subsequent differentiation into keratinized stratified epithelium.

  10. A stretcher fiber for use in fs chirped pulse Yb amplifiers.

    PubMed

    Grüner-Nielsen, Lars; Jakobsen, Dan; Jespersen, Kim G; Pálsdóttir, Bera

    2010-02-15

    A newly developed fiber for use in pulse stretchers for chirped pulse amplifiers working in the 1 mum wavelength range of Yb fiber amplifiers is reported. The fiber has a record high numerical third order to second order dispersion beta(3)/beta(2) ratio of -7.7 fs. The fiber has very good dispersion match to a grating compressor for second, third, and fourth order dispersion. By combining the stretcher fiber with an anomalous dispersion fiber working in a higher order mode, even higher beta(3)/beta(2) ratio of -16.8 fs is demonstrated. The combined module shows very good dispersion match to a grating compressor.

  11. (18)F, (64)Cu, and (68)Ga labeled RGD-bombesin heterodimeric peptides for PET imaging of breast cancer.

    PubMed

    Liu, Zhaofei; Yan, Yongjun; Liu, Shuanglong; Wang, Fan; Chen, Xiaoyuan

    2009-05-20

    Radiolabeled RGD (Arg-Gly-Asp) and bombesin (BBN) radiotracers that specifically target integrin alpha(v)beta(3) and gastrin releasing peptide receptor (GRPR) are both promising radiopharmaceuticals for tumor imaging. We recently designed and synthesized a RGD-BBN heterodimeric peptide with both RGD and BBN motifs in one single molecule. The (18)F-labeled RGD-BBN heterodimer exhibited dual integrin alpha(v)beta(3) and GRPR targeting in a PC-3 prostate cancer model. In this study we investigated whether radiolabeled RGD-BBN tracers can be used to detect breast cancer by using microPET. Cell binding assay demonstrated that the high GRPR expressing breast cancer cells typically express low to moderate level of integrin alpha(v)beta(3), while high integrin alpha(v)beta(3) expressing breast cancer cells have negligible level of GRPR. We labeled RGD-BBN heterodimer with three positron emitting radionuclides (18)F, (64)Cu, and (68)Ga and investigated the corresponding PET radiotracers in both orthotopic T47D (GRPR(+)/low integrin alpha(v)beta(3)) and MDA-MB-435 (GRPR(-)/integrin alpha(v)beta(3)(+)) breast cancer models. The three radiotracers all possessed in vitro dual integrin alpha(v)beta(3) and GRPR binding affinity. The advantages of the RGD-BBN radiotracers over the corresponding BBN analogues are obvious for imaging MDA-MB-435 (GRPR(-)/integrin alpha(v)beta(3)(+)) tumor. (18)F-FB-PEG(3)-RGD-BBN showed lower tumor uptake than (64)Cu-NOTA-RGD-BBN and (68)Ga-NOTA-RGD-BBN but was able to visualize breast cancer tumors with high contrast. Synthesis of (64)Cu-NOTA-RGD-BBN and (68)Ga-NOTA-RGD-BBN is much faster and easier than (18)F-FB-PEG(3)-RGD-BBN. (64)Cu-NOTA-RGD-BBN showed prolonged tumor uptake but also higher liver retention and kidney uptake than (68)Ga-NOTA-RGD-BBN and (18)F-FB-PEG(3)-RGD-BBN. (68)Ga-NOTA-RGD-BBN possessed high tumor signals but also relatively high background uptake compared with the other two radiotracers. In summary, the prosthetic labeling

  12. 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.

  13. ADH2 gene polymorphisms are determinants of alcohol pharmacokinetics.

    PubMed

    Thomasson, H R; Beard, J D; Li, T K

    1995-12-01

    The class I hepatic alcohol dehydrogenases (ADHs) are primarily responsible for ethanol metabolism in humans. Genetic polymorphism at the ADH2 locus results in the inheritance of isozymes of strikingly different catalytic properties. The most common ADH2 allele, ADH2*1, encodes the low K(m) isozyme subunit beta 1. The ADH2*3 allele encodes a high-activity isozyme subunit of alcohol dehydrogenase, beta 3, identified in approximately 25% of African-Americans. The Vmax of beta 3 beta 3-ADH is 30 times greater than that of the beta 1 beta 1-ADH. Therefore, we hypothesized that the rate of ethanol metabolism, an important factor in the toxicity of ethanol, in persons with beta 3-containing ADH, either beta 3 beta 3- or beta 1 beta 3-ADH, would be faster than that of persons with only beta 1 beta 1-ADH. We tested this hypothesis with ethanol administered orally to healthy, young African-Americans. Three hundred and twenty-six African-American men and women were genotyped using polymerase chain reaction amplification of their leukocyte DNA followed by hybridization with allele-specific probes. One hundred twelve volunteers, selected by genotype, received an oral dose of ethanol designed to produce a blood ethanol concentration of 80 mg/dl (0.080 g/dl), when the blood alcohol concentration-time curve was extrapolated back to time 0. Ethanol metabolic rates (beta 60s) were determined in the 112 subjects from the slope of the pseudolinear portion of the blood ethanol concentration-time curves. The mean beta 60 of African-Americans having beta 3-containing ADH isozymes had significantly faster ethanol elimination rates than those with only beta 1 beta 1-ADH isozymes. There were no significant differences in body weight, ethanol intake in the week before testing, peak breath ethanol concentration, time to peak, or volume of distribution between the genotype groups. Within each of these groups, men had lower ethanol disappearance rates than women. These results demonstrate in

  14. 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…

  15. Finding new solutions in pediatric parenteral admixtures: how to improve quality and to deal with shortages.

    PubMed

    Watrobska-Swietlikowska, Dorota; Kwidzynska, Anna; Szlagatys-Sidorkiewicz, Agnieszka; Sznitowska, Malgorzata; Klek, Stanislaw

    2014-07-01

    INTRODUCCIÓN: La nutrición parenteral pediátrica permite un crecimiento normal incluso en lactantes pretérmino. Sin embargo, estos niños requieren una nutrición parenteral a medida y la formulación de tal nutrición puede suponer un reto por el riesgo de inestabilidad y el desabastecimiento.

  16. 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.

  17. [Reference curves for assessing the physical growth of male Wistar rats].

    PubMed

    Cossio-Bolaños, Marco; Gómez Campos, Rossana; Vargas Vitoria, Rodrigo; Hochmuller Fogaça, Rosalvo Tadeu; de Arruda, Miguel

    2013-11-01

    Introducción: Las ratas Wistar son una de las cepas más populares y utilizadas cotidianamente para la investigación en el laboratorio sirviendo como una importante herramienta de investigación, por lo que, exige el control estricto de variables como la edad, el sexo y el peso corporal, y de esta forma poder extrapolar los resultados al modelo humano. Objetivo: Desarrollar curvas de referencia para valorar el crecimiento físico de ratas machos Wistar en función de la edad cronológica y la maduración somática desde una perspectiva no-invasiva. Metodología: Fueron estudiadas 731 ratas machos Wistar de forma transversal. Se evaluó la edad, peso corporal y la superficie corporal. Se utilizó el método LMS para construir curvas de percentil en función del peso y la maduración somática. Resultados: Las curvas de crecimiento físico propuestas sirven para realizar el seguimiento del crecimiento físico y el diagnóstico del estado nutricional de ratas machos de cepa Wistar. Los puntos de corte prepuestos son: P3, P10, P25, P50, P75, P90 y P97. Conclusión: Los resultados sugieren que los científicos de diversas áreas puedan usar tales referencias, con el objetivo de extrapolar las fases del crecimiento somático de la rata de laboratorio al modelo humano y es una alternativa no-invasiva para valorar el crecimiento y el estado nutricional.

  18. 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 ...

  19. Expression of TGF-betas in the embryonic nervous system: analysis of interbalance between isoforms.

    PubMed

    Mecha, M; Rabadán, M A; Peña-Melián, A; Valencia, M; Mondéjar, T; Blanco, M J

    2008-06-01

    Transforming growth factor-beta (TGF-beta) is a family of growth factors with essential and multiple roles during embryonic development. In mammals, three isoforms (TGF-beta1, TGF-beta2, TGF-beta3) have been described. In the nervous system, the presence of TGF-beta1 has remained undetectable in other structures than meninges and choroids plexus, while TGF-beta2 and TGF-beta3 were considered as the neural members of the family. In the present study, we have analysed the expression pattern of the three isoforms in the neural tube, brain, and spinal cord during development in both mouse and chicken. The data reveal specific patterns for each isoform. This work also shows that both TGF-beta1 and TGF-beta3 are expressed in neural crest cells. In addition, we demonstrate the existence of interbalance between TGF-beta1 and TGF-beta3 with possible functional implications, which, together with the expression of TGF-beta1 in the CNS, represents one of the most important contributions of this work.

  20. GABA receptor subunit composition relative to insecticide potency and selectivity.

    PubMed

    Ratra, G S; Casida, J E

    2001-07-01

    Three observations on the 4-[(3)H]propyl-4'-ethynylbicycloorthobenzoate ([(3)H]EBOB) binding site in the gamma-aminobutyric acid (GABA) receptor indicate the specific target for insecticide action in human brain and a possible mechanism for selectivity. First, from published data, alpha-endosulfan, lindane and fipronil compete for the [(3)H]EBOB binding site with affinities of 0.3--7 nM in both human recombinant homooligomeric beta 3 receptors and housefly head membranes. Second, from structure-activity studies, including new data, GABAergic insecticide binding potency on the pentameric receptor formed from the beta 3 subunit correlates well with that on the housefly receptor (r=0.88, n=20). This conserved inhibitor specificity is consistent with known sequence homologies in the housefly GABA receptor and the human GABA(A) receptor beta 3 subunit. Third, as mostly new findings, various combinations of alpha 1, alpha 6, and gamma 2 subunits coexpressed with a beta 1 or beta 3 subunit confer differential insecticide binding sensitivity, particularly to fipronil, indicating that subunit composition is a major factor in insecticide selectivity.

  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. 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...

  3. Hyperglycosylation and reduced GABA currents of mutated GABRB3 polypeptide in remitting childhood absence epilepsy.

    PubMed

    Tanaka, Miyabi; Olsen, Richard W; Medina, Marco T; Schwartz, Emily; Alonso, Maria Elisa; Duron, Reyna M; Castro-Ortega, Ramon; Martinez-Juarez, Iris E; Pascual-Castroviejo, Ignacio; Machado-Salas, Jesus; Silva, Rene; Bailey, Julia N; Bai, Dongsheng; Ochoa, Adriana; Jara-Prado, Aurelio; Pineda, Gregorio; Macdonald, Robert L; Delgado-Escueta, Antonio V

    2008-06-01

    Childhood absence epilepsy (CAE) accounts for 10% to 12% of epilepsy in children under 16 years of age. We screened for mutations in the GABA(A) receptor (GABAR) beta 3 subunit gene (GABRB3) in 48 probands and families with remitting CAE. We found that four out of 48 families (8%) had mutations in GABRB3. One heterozygous missense mutation (P11S) in exon 1a segregated with four CAE-affected persons in one multiplex, two-generation Mexican family. P11S was also found in a singleton from Mexico. Another heterozygous missense mutation (S15F) was present in a singleton from Honduras. An exon 2 heterozygous missense mutation (G32R) was present in two CAE-affected persons and two persons affected with EEG-recorded spike and/or sharp wave in a two-generation Honduran family. All mutations were absent in 630 controls. We studied functions and possible pathogenicity by expressing mutations in HeLa cells with the use of Western blots and an in vitro translation and translocation system. Expression levels did not differ from those of controls, but all mutations showed hyperglycosylation in the in vitro translation and translocation system with canine microsomes. Functional analysis of human GABA(A) receptors (alpha 1 beta 3-v2 gamma 2S, alpha 1 beta 3-v2[P11S]gamma 2S, alpha 1 beta 3-v2[S15F]gamma 2S, and alpha 1 beta 3-v2[G32R]gamma 2S) transiently expressed in HEK293T cells with the use of rapid agonist application showed that each amino acid transversion in the beta 3-v2 subunit (P11S, S15F, and G32R) reduced GABA-evoked current density from whole cells. Mutated beta 3 subunit protein could thus cause absence seizures through a gain in glycosylation of mutated exon 1a and exon 2, affecting maturation and trafficking of GABAR from endoplasmic reticulum to cell surface and resulting in reduced GABA-evoked currents.

  4. Functional and molecular characterization of beta-adrenoceptors in the internal anal sphincter.

    PubMed

    Rathi, Sandeep; Kazerounian, Shiva; Banwait, Kuldip; Schulz, Stephanie; Waldman, Scott A; Rattan, Satish

    2003-05-01

    The purpose of the present study was to characterize different beta-adrenoceptors (beta-ARs) and determine their role in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The beta-AR subtypes in the opossum IAS were investigated by functional in vitro, radioligand binding, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR) studies. ZD 7114 [(S)-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-(2-methoxyethyl)phenoxyacetamide], a selective beta(3)-AR agonist, caused a potent and concentration-dependent relaxation of the IAS smooth muscle that was antagonized by the beta(3)-AR antagonist SR 59230A [1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride]. Conversely, the IAS smooth muscle relaxation caused by beta(1)- and beta(2)-AR agonists (xamoterol and procaterol, respectively) was selectively antagonized by their respective antagonists CGP 20712 [(+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt] and ICI 118551. Saturation binding of [(125)I]iodocyanopindolol to beta-AR subtypes revealed the presence of a high-affinity site (K(d1) = 96.4 +/- 8.7 pM; B(max1) = 12.5 +/- 0.6 fmol/mg protein) and a low-affinity site (K(d2) = 1.96 +/- 1.7 nM; B(max2) = 58.7 +/- 4.3 fmol/mg protein). Competition binding with selective beta-AR antagonists revealed that the high-affinity site correspond to beta(1)/beta(2)-AR and the low affinity site to beta(3)-AR. Receptor binding data suggest the predominant presence of beta(3)-AR over beta(1)/beta(2)-AR. Western blot studies identified beta(1)-, beta(2)-, and beta(3)-AR subtypes. The presence of beta(1)-, beta(2)-, and beta(3)-ARs was further demonstrated by mRNA analysis using RT-PCR. The studies demonstrate a comprehensive functional and molecular characterization of beta(1)-, beta(2)-, and beta(3)-ARs in IAS smooth muscle. These studies may have

  5. Atypical responses of rat ileum to pindolol, cyanopindolol and iodocyanopindolol.

    PubMed

    Hoey, A; Jackson, C; Pegg, G; Sillence, M

    1996-02-01

    1. Pindolol, cyanopindolol (CYP) and iodocyanopindolol (IodoCYP) have been reported to act either as antagonists, agonists or partial agonists at the beta 3-adrenoceptor in different preparations. A comprehensive investigation has not yet been described with these compounds tested in one tissue from one species. This study was conducted to delineate the pharmacological effects of pindolol, CYP and IodoCYP and to provide data on their affinities at the predominant beta-adrenoceptor in rat ileum. 2. The beta-adrenoceptors present in rat ileum were characterized in the presence of CGP 20712A and ICI 118 551, atropine and corticosterone, with (-)-isoprenaline used as an agonist. The role of the beta 1 and beta 2-adrenoceptors was determined by the omission of either CGP 20712A, ICI 118 551, or both, from the buffers. Conversely, the effectiveness of the beta 1- and beta 2-adrenoceptor blockade was examined by use of the beta 1-adrenoceptor-selective agonist, RO 363 and the beta 2-adrenoceptor-selective agonist, salbutamol. 3. There was no evidence for the presence of functional beta 1-adrenoceptors, and no strong evidence that beta 2-adrenoceptor stimulation contributed to the relaxant effects of (-)-isoprenaline. (-)-Phenylephrine did not produce relaxation of the tissue and 5-hydroxytryptamine produced contraction. 4. The beta 3-adrenoceptor-selective agonist, BRL 37344 and (-)-isoprenaline were potent full agonists (pD2 8.35 +/- 0.04 and 7.76 +/- 0.14 respectively), whereas ICI D7114 was less potent (pseudo pD2 6.92 +/- 0.15). These results indicate that the predominant functional beta-adrenoceptors in rat ileum are beta 3-adrenoceptors. 5. Partial agonist effects were produced by CYP (pD2 5.28 +/- 0.26) and IodoCYP (pD2 7.0 +/- 0.26), but not pindolol. All three compounds antagonized the effects of (-)-isoprenaline with pKb values of 6.68 +/- 0.10, 7.59 +/- 0.07 and 7.59 +/- 0.11 for pindolol, CYP and IodoCYP respectively. Likewise, CYP and IodoCYP antagonized the

  6. Atypical responses of rat ileum to pindolol, cyanopindolol and iodocyanopindolol.

    PubMed Central

    Hoey, A.; Jackson, C.; Pegg, G.; Sillence, M.

    1996-01-01

    1. Pindolol, cyanopindolol (CYP) and iodocyanopindolol (IodoCYP) have been reported to act either as antagonists, agonists or partial agonists at the beta 3-adrenoceptor in different preparations. A comprehensive investigation has not yet been described with these compounds tested in one tissue from one species. This study was conducted to delineate the pharmacological effects of pindolol, CYP and IodoCYP and to provide data on their affinities at the predominant beta-adrenoceptor in rat ileum. 2. The beta-adrenoceptors present in rat ileum were characterized in the presence of CGP 20712A and ICI 118 551, atropine and corticosterone, with (-)-isoprenaline used as an agonist. The role of the beta 1 and beta 2-adrenoceptors was determined by the omission of either CGP 20712A, ICI 118 551, or both, from the buffers. Conversely, the effectiveness of the beta 1- and beta 2-adrenoceptor blockade was examined by use of the beta 1-adrenoceptor-selective agonist, RO 363 and the beta 2-adrenoceptor-selective agonist, salbutamol. 3. There was no evidence for the presence of functional beta 1-adrenoceptors, and no strong evidence that beta 2-adrenoceptor stimulation contributed to the relaxant effects of (-)-isoprenaline. (-)-Phenylephrine did not produce relaxation of the tissue and 5-hydroxytryptamine produced contraction. 4. The beta 3-adrenoceptor-selective agonist, BRL 37344 and (-)-isoprenaline were potent full agonists (pD2 8.35 +/- 0.04 and 7.76 +/- 0.14 respectively), whereas ICI D7114 was less potent (pseudo pD2 6.92 +/- 0.15). These results indicate that the predominant functional beta-adrenoceptors in rat ileum are beta 3-adrenoceptors. 5. Partial agonist effects were produced by CYP (pD2 5.28 +/- 0.26) and IodoCYP (pD2 7.0 +/- 0.26), but not pindolol. All three compounds antagonized the effects of (-)-isoprenaline with pKb values of 6.68 +/- 0.10, 7.59 +/- 0.07 and 7.59 +/- 0.11 for pindolol, CYP and IodoCYP respectively. Likewise, CYP and IodoCYP antagonized the

  7. Aggressive parenteral nutrition and growth velocity in preterm infants.

    PubMed

    Ribed Sánchez, Almudena; Romero Jiménez, Rosa Ma; Sánchez Gómez de Orgaz, Ma Carmen; Sánchez Luna, Manuel; Sanjurjo Sáez, María

    2013-11-01

    Introducción: La administración parenteral de nutrientes para mantener el crecimiento en recién nacidos representa un importante reto terapéutico. Objetivo: Describir las prácticas de nutrición parenteral (NP) en un hospital de tercer nivel y evaluar el crecimiento postnatal en recién nacidos prematuros. Material y métodos: Estudio observacional retrospectivo de 3 meses de duración. Se incluyeron niños ingresados en el Servicio de Neonatología que iniciaron NP. Se recogieron datos demográficos, antropométricos, calorías diarias, ingesta de proteínas y componentes de la NP. La velocidad de crecimiento se midió mediante la media de la ganancia diaria de peso y se comparó con el crecimiento intrauterino. Resultados: 68 niños prematuros iniciaron NP durante el período de estudio. La mayoría de los niños (65%) nacieron por cesárea y la media de edad gestacional fue de 33 semanas. El 25% de los recién nacidos no recuperó el peso de nacimiento. El 75% restante recuperó el peso de nacimiento en el tercer día de NP y la media de la ganancia diaria de peso fue de 16 g/kg/d, con un rango de 12 a 22g/kg/d. A pesar de que la ganancia de peso se acercó a la tasa intrauterina, la mayoría de los niños nacidos con menos de 30 semanas de gestación no lograron la media de la población de referencia. La NP precoz e intensiva se administró con una media de 3, 11 y 3g/Kg/d de proteínas, hidratos de carbono y lípidos, respectivamente, alcanzando un máximo en el cuarto día de 4, 18, 4 g/kg/d, respectivamente. Discusión: La NP intensiva se utiliza en el ámbito hospitalario. Los recién nacidos prematuros alcanzaron antes el peso de nacimiento y presentaron una velocidad de crecimiento mayor que en otros estudios y similar al crecimiento intrauterino.

  8. 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

  9. [Effect of cryotherapy over the expression of vascular endothelial growth factor and pigment epithelium-derived factor].

    PubMed

    Toscano-Garibay, Julia Dolores; Quiroz-Mercado, Hugo; Espitia-Pinzón, Clara; Gil-Carrasco, Félix; Flores-Estrada, José Javier

    2014-01-01

    Antecedentes: la crioterapia es una técnica no invasiva que usa frio intenso para congelar y destruir los tejidos cancerosos. Sus efectos en la expresión del factor de crecimiento del endotelio vascular y el factor derivado del epitelio pigmentado no se han descrito. Material y métodos: estudio experimental en modelos experimentales de crioterapia. En la esclera del ojo derecho de 12 cerdos se aplicó un punto de congelamiento durante 10 segundos. Se usaron 3 cerdos como controles normales. Los animales se sacrificaron a los 7, 14 y 21 días y el tejido de coroides y retina se seccionó en áreas de aproximadamente 1 cm2 circundantes al punto de congelamiento. Los niveles de expresión del factor de crecimiento del endotelio vascular y factor derivado del epitelio pigmentado se determinaron y analizaron por reacción en cadena de la polimerasa acoplada a reverso-transcripción. Resultados: los niveles de factor de crecimiento del endotelio vascular disminuyeron significativamente (24%, p < 0.05) a los 7 días postratamiento, mientras que la expresión del factor derivado del epitelio pigmentado aumentó 44.8% (p< 0.05) en comparación con los niveles de las muestras normales. Los niveles de expresión se mantuvieron hasta el día 14 y regresaron a valores basales en el día 21. Conclusiones: este trabajo expone la relación entre la crioterapia y la expresión de dos factores angiogénicos. Los resultados muestran cambios significativos en la expresión del factor de crecimiento del endotelio vascular y factor derivado del epitelio pigmentado, y evidencian que ambas proteínas son reguladas en respuesta al tratamiento criogénico en periodos relativamente cortos (21 días).

  10. [Programme review of somatropin deficit in pediatrics at the Hospital Universitario Virgen del Rocío].

    PubMed

    Lavaredas, A; de la Puerta, R; Álvarez del Vayo, C

    2013-01-01

    Objetivo: Elaborar una revisión del programa de déficit de somatropina aplicado en pediatría en el Hospital Universitario Virgen del Rocío, utilizando dos grupos de pacientes, los diagnosticados con déficit de esta hormona y los nacidos pequeños para edad gestacional, con la intención de evaluar su efectividad en el primer año de tratamiento. Método: Realización de un estudio retrospectivo de la cohorte de pacientes en tratamiento con la hormona del crecimiento bajo los diagnósticos mencionados, con metodología observacional y transversal, a los cuales se aplicó un análisis estadístico con el programa Statistical Package for Social Sciences®. Resultados: Tras inicio del tratamiento la velocidad de crecimiento y la talla aumentaron y la edad ósea se aproximó a la edad cronológica. En los dos grupos tratados, en el primer año de tratamiento fueron los pacientes del sexo femenino con edad comprendida entre los 0 a 12 años con déficit de la hormona del crecimiento que respondieron mejor a la terapéutica establecida. Conclusiones: Pudimos observar que el tratamiento instituido se presentó altamente efectivo en ambos grupos de pacientes, permitiendo obtener un aumento favorable de estatura.

  11. [Influence of the composition of essential oils on their antioxidant and antiradical properties].

    PubMed

    Misharina, T A; Alinkina, E S; Fatkullina, L D; Vorob'eva, A K; Medvedeva, I B; Burlakova, E B

    2012-01-01

    The antiradical and antioxidant properties of essential oil mixtures (EOMs) with various compositions were studied and compared in model systems of hexanal autooxidation, thermal oxidation of methyl linoleate and beta3-carotene, and in a reaction with the stable diphenylpicrylhydrazyl radical. It was found that all EOMs under investigation exhibited antiradical activity. The highest antiradical activity was observed for an EOM containing monoterpene hydrocarbons as the main components (the phenol content was low). The antioxidant activity of all EOMs was from 60 to 98% and depended on the model system composition and the method of assay. The mixture with the highest phenol content exhibits the highest antioxidant activity level in the hexanal autooxidation system. EOMs with a high content of phenols and alpha- and y-terpinenes were the most efficient antioxidants in the beta3-carotene model system. The study confirmed the possibility to vary the antioxidant and antiradical properties of essential oils by preparing their mixtures with a specific composition.

  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. Lifelong ethanol consumption and brain regional GABAA receptor subunit mRNA expression in alcohol-preferring rats.

    PubMed

    Sarviharju, Maija; Hyytiä, Petri; Hervonen, Antti; Jaatinen, Pia; Kiianmaa, Kalervo; Korpi, Esa R

    2006-11-01

    Brain regional gamma-aminobutyric acid type A (GABAA) receptor subunit mRNA expression was studied in ethanol-preferring AA (Alko, Alcohol) rats after moderate ethanol drinking for up to 2 years of age. In situ hybridization with oligonucleotide probes specific for 13 different subunits was used with coronal cryostat sections of the brains. Selective alterations were observed by ethanol exposure and/or aging in signals for several subunits. Most interestingly, the putative highly ethanol-sensitive alpha4 and beta3 subunit mRNAs were significantly decreased in several brain regions. The age-related alterations in alpha4 subunit expression were parallel to those caused by lifelong ethanol drinking, whereas aging had no significant effect on beta3 subunit expression. The results suggest that prolonged ethanol consumption leading to blood concentrations of about 10 mM may downregulate the mRNA expression of selected GABAA receptor subunits and that aging might have partly similar effects.

  14. Preparation of high-specific-activity D-(3-/sup 3/H)pantothenic acid. [Escherichia coli strain DVI

    SciTech Connect

    Vallari, D.S.; Rock, C.O.

    1986-05-01

    High-specific-activity D-(3-/sup 3/H)pantothenic acid (5 Ci/mmol) was prepared from commercially available ..beta..-(3-/sup 3/H)alanine employing Escherichia coli strain DV1 (panD2 pan Fl). This strain is defective in ..beta..-alanine synthesis and pantothenate uptake, and under appropriate growth conditions converted 85 to 90% of the input ..beta..-(3-/sup 3/H)alanine to extracellular D-(3-/sup 3/h)pantothenate. The radiolabeled vitamin was purified from the medium by thin-layer chromatography followed by reverse phase high-performance liquid chromatography. The overall yield of D-(3-/sup 3/H)pantothenic acid was 30% and radiochemical purity was >99%.

  15. 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.

  16. 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

  17. Platelets prevent IFN-alpha/beta-induced lethal hemorrhage promoting CTL-dependent clearance of lymphocytic choriomeningitis virus.

    PubMed

    Iannacone, Matteo; Sitia, Giovanni; Isogawa, Masanori; Whitmire, Jason K; Marchese, Patrizia; Chisari, Francis V; Ruggeri, Zaverio M; Guidotti, Luca G

    2008-01-15

    We found that mice infected with different isolates of lymphocytic choriomeningitis virus (LCMV) develop a mild hemorrhagic anemia, which becomes severe and eventually lethal in animals depleted of platelets or lacking integrin beta3. Lethal hemorrhagic anemia is mediated by virus-induced IFN-alpha/beta that causes platelet dysfunction, mucocutaneous blood loss and suppression of erythropoiesis. In addition to the life-threatening hemorrhagic anemia, platelet-depleted mice fail to mount an efficient cytotoxic T lymphocyte (CTL) response and cannot clear LCMV. Transfusion of functional platelets into these animals reduces hemorrhage, prevents death and restores CTL-induced viral clearance in a manner partially dependent on CD40 ligand (CD40L). These results indicate that, upon activation, platelets expressing integrin beta3 and CD40L are required for protecting the host against the induction of an IFN-alpha/beta-dependent lethal hemorrhagic diathesis and for clearing LCMV infection through CTLs.

  18. Regulation of UCP gene expression in brown adipocytes differentiated in primary culture. Effects of a new beta-adrenoceptor agonist.

    PubMed

    Champigny, O; Holloway, B R; Ricquier, D

    1992-07-01

    Primary cultures of precursor cells from mouse and rat brown adipose tissue (BAT) were used to study the effect of a new beta-agonist (ICI D7114) on the uncoupling protein (UCP) gene expression. ICI 215001 (the active metabolite of D7114) increased the expression of UCP and its mRNA in brown adipocytes differentiating in vitro in a dose-dependent manner. This stimulating effect was not inhibited by propranolol, a non-specific beta-antagonist, but was partially reduced by bupranolol, a beta 3-antagonist. No expression of UCP mRNA was ever induced by ICI 215001 in white adipocytes differentiated in vitro. It was concluded that the drug could affect the brown adipose cells through a beta 3-pathway. It could clearly modulate the expression of UCP in brown adipocytes differentiated in vitro, but was not able by itself to turn on the gene. PMID:1355051

  19. The in vitro synthesis and secretion of alpha-ecdysone by the ring glands of the fly, Sarcophaga bullata.

    PubMed

    Bollenbacher, W E; Goodman, W; Vedeckis, W V; Gilbert, L I

    1976-03-01

    The in vitro secretory product of larval Sarcophage bullata ring glands has been identified as 2beta, 3beta, 14alpha, 22R, 25-pentahydroxy-5beta-cholest-7-en-6-one (alpha-ecdysone). Mid to late 3rd instar larval ecdysones were isolated and identified as 2beta, 3beta, 14alpha, 20R, 22R, 25-hexahydroxy-5beta-cholest-7-en-6-one (beta-ecdysone) and alpha-ecdysone at a ratio of 27:1. The low level of alpha-ecdysone in vivo, relative to its exclusive in vitro synthesis and secretion by the ring glands, is a function of the very active C20 hydroxylation mechanism in tissues peripheral to the ring gland. The role of alpha-ecdysone as a prohormone in dipteran metamorphosis is discussed.

  20. Effect of hospital nutrition support on growth velocity and nutritional status of low birth weight infants.

    PubMed

    Azzeh, Firas S; Alazzeh, Awfa Y; Dabbour, Ibrahim R; Jazar, Abdelelah S; Obeidat, Ahmed A

    2014-10-01

    Introducción: Los bebés con bajo peso al nacer reciben asistencia nutricional hospitalaria para reforzar su supervivencia y peso corporal. Sin embargo, cada hospital tiene su propia fórmula de asistencia nutricional. Por lo tanto, la efectividad de esta asistencia nutricional debería ser investigada. Objetivo: Evaluar el efecto de la asistencia nutricional hospitalaria sobre la velocidad del crecimiento velocidad del crecimiento y el estado nutricional de bebés con bajo peso al nacer en el hospital Al-Noor, Arabia Saudita. Métodos: Se llevó a cabo un estudio interseccional entre octubre de 2010 y diciembre de 2012. Se seleccionó a trescientos recién nacidos del hospital Al-Noor de la ciudad de Makkah, Arabia Saudita. Los bebés fueron seleccionados según su peso al nacer y fueron separados en tres grupos iguales: (i) Bajo peso al nacer (BPN) (peso de nacimiento 1501- 2500 g), (ii) Muy Bajo peso al nacer (MBPN) (peso de nacimiento 1001-1500 g) y (iii) Peso extremadamente bajo al nacer (PEBN) (peso de nacimiento < 1000 g). Los datos fueron recopilados en el momento del nacimiento y en el momento del alta. Los pesos fueron registrados y se calculó la velocidad del crecimiento. Se hizo la medición de algunos tests bioquímicos y niveles minerales. Resultados: los valores de índice de masa corporal del VBPN y EBPN fueron menores (p < 0.05) que los del grupo BPN. La velocidad de crecimiento de los niños de todos los grupos osciló entre 8.7 y 10.2 g/kg/d sin diferencias (p > 0.05) observadas entre grupos. Los niveles de suero, calcio, fósforo y potasio en el momento del alta fueron superiores (p < 0.05) que los del momento del nacimiento para los grupos de EBPN y VBPN, mientras que los niveles de sodio descendieron en el grupo de EBPN hasta un rango normal. El nivel de albúmina mejoró (p < 0.05) en el grupo de EBPN. Conclusión: La gestión de la atención sanitaria para bebés con bajo peso al nacer en el hospital Al-Noor no fue suficiente para alcanzar

  1. Two RGD-independent alpha vbeta 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties.

    PubMed

    Maeshima, Y; Colorado, P C; Kalluri, R

    2000-08-01

    Vascular basement membrane is an important regulator of angiogenesis and undergoes many alterations during angiogenesis and these changes are speculated to influence neovascularization. Recently, fragments of collagen molecules have been identified to possess anti-angiogenic activity. Tumstatin (alpha3(IV)NC1 domain) is one such novel molecule with distinct anti-tumor properties and possesses an N-terminal (amino acids 54-132) anti-angiogenic and a C-terminal (amino acids 185-203) anti-tumor cell activity (Maeshima, Y., et al. 2000) J. Biol. Chem. 275, 21340-21348). Previous studies have identified the 185-203 amino acid sequence as a ligand for alpha(v)beta(3) integrin (Shahan, T. A., et al. (1999) Cancer Res. 59, 4584-4590). In the present study, we found distinct additional RGD-independent alpha(v)beta(3) integrin binding site within 54-132 amino acids of tumstatin. This site is not essential for inhibition of tumor cell proliferation but necessary for the anti-angiogenic activity. A fragment of tumstatin containing 54-132 amino acid (tum-2) binds both endothelial cells and melanoma cells but only inhibited proliferation of endothelial cells, with no effect on tumor cell proliferation. A similar experiment with fragment of tumstatin containing the 185-203 amino acid (tum-4) demonstrates that it binds both endothelial cells and melanoma cells but only inhibits the proliferation of melanoma cells. The presence of cyclic RGD peptides did not affect the alpha(v)beta(3) integrin-mediated activity of tumstatin, although significant inhibition of endothelial cell binding to vitronectin was observed. The two distinct RGD-independent binding sites on tumstatin suggest unique alpha(v)beta(3) integrin-mediated mechanisms governing the two distinct anti-tumor properties of tumstatin. PMID:10837460

  2. Reciprocal regulation of transcription factors and PLC isozyme gene expression in adult cardiomyocytes.

    PubMed

    Singal, Tushi; Dhalla, Naranjan S; Tappia, Paramjit S

    2010-06-01

    By employing a pharmacological approach, we have shown that phospholipase C (PLC) activity is involved in the regulation of gene expression of transcription factors such as c-Fos and c-Jun in cardiomyocytes in response to norepinephrine (NE). However, there is no information available regarding the identity of specific PLC isozymes involved in the regulation of c-Fos and c-Jun or on the involvement of these transcription factors in PLC isozyme gene expression in adult cardiomyocytes. In this study, transfection of cardiomyocytes with PLC isozyme specific siRNA was found to prevent the NE-mediated increases in the corresponding PLC isozyme gene expression, protein content and activity. Unlike PLC gamma(1) gene, silencing of PLC beta(1), beta(3) and delta(1) genes with si RNA prevented the increases in c-Fos and c-Jun gene expression in response to NE. On the other hand, transfection with c-Jun si RNA suppressed the NE-induced increase in c-Jun as well as PLC beta(1), beta(3) and delta(1) gene expression, but had no effect on PLC gamma(1) gene expression. Although transfection of cardiomyocytes with c-Fos si RNA prevented NE-induced expression of c-Fos, PLC beta(1) and PLC beta(3) genes, it did not affect the increases in PLC delta(1) and PLC gamma(1) gene expression. Silencing of either c-Fos or c-Jun also depressed the NE-mediated increases in PLC beta(1), beta(3) and gamma(1) protein content and activity in an isozyme specific manner. Furthermore, silencing of all PLC isozymes as well as of c-Fos and c-Jun resulted in prevention of the NE-mediated increase in atrial natriuretic factor gene expression. These findings, by employing gene silencing techniques, demonstrate that there occurs a reciprocal regulation of transcription factors and specific PLC isozyme gene expression in cardiomyocytes.

  3. (-)-Norcocaine.

    PubMed

    Zhu, N; Reynolds, M; Klein, C L; Trudell, M

    1994-12-15

    The title compound, [2R,3S-(2 beta,3 beta)]-methyl 3-(benzoyloxy)-8-azabicyclo[3.2.1]octane-2-carboxylate, C16H19NO4, is a metabolite of the tropane alkaloid cocaine. The molecule crystallized as the free base with the piperidine ring in a chair conformation. The tropane ring system and its methoxycarbonyl and benzoyl groups are rigid; only rotational flexibility is allowed in the conformation of the substituents.

  4. 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

  5. Distinct activities of GABA agonists at synaptic- and extrasynaptic-type GABAA receptors.

    PubMed

    Mortensen, Martin; Ebert, Bjarke; Wafford, Keith; Smart, Trevor G

    2010-04-15

    The activation characteristics of synaptic and extrasynaptic GABA(A) receptors are important for shaping the profile of phasic and tonic inhibition in the central nervous system, which will critically impact on the activity of neuronal networks. Here, we study in isolation the activity of three agonists, GABA, muscimol and 4,5,6,7-tetrahydoisoxazolo[5,4-c]pyridin-3(2H)-one (THIP), to further understand the activation profiles of alpha 1 beta 3 gamma 2, alpha 4 beta 3 gamma 2 and alpha 4 beta 3 delta receptors that typify synaptic- and extrasynaptic-type receptors expressed in the hippocampus and thalamus. The agonists display an order of potency that is invariant between the three receptors, which is reliant mostly on the agonist dissociation constant. At delta subunit-containing extrasynaptic-type GABA(A) receptors, both THIP and muscimol additionally exhibited, to different degrees, superagonist behaviour. By comparing whole-cell and single channel currents induced by the agonists, we provide a molecular explanation for their different activation profiles. For THIP at high concentrations, the unusual superagonist behaviour on alpha 4 beta 3 delta receptors is a consequence of its ability to increase the duration of longer channel openings and their frequency, resulting in longer burst durations. By contrast, for muscimol, moderate superagonist behaviour was caused by reduced desensitisation of the extrasynaptic-type receptors. The ability to specifically increase the efficacy of receptor activation, by selected exogenous agonists over that obtained with the natural transmitter, may prove to be of therapeutic benefit under circumstances when synaptic inhibition is compromised or dysfunctional.

  6. The microbial oxidation of (-)-beta-pinene by Botrytis cinerea.

    PubMed

    Farooq, Afgan; Choudhary, M Iqbal; Tahara, Satoshi; Rahman, Atta-ur; Başer, K Hüsnü Can; Demirci, Fatih

    2002-01-01

    (-)-beta-pinene, a flavor and fragrance monoterpene is an important constituent of essential oils of many aromatic plants. It was oxidized by a plant-pathogenic fungus, Botrytis cinerea to afford four metabolites characterized as (-)-6a-hydroxy-beta-pinene, (-)-4beta,5beta-dihydroxy-beta-pinene, (-)-2beta,3beta-dihydroxypinane, and (-)-4beta-hydroxy-beta-pinene-6-one by detailed spectroscopic studies along with other known metabolites.

  7. 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.

  8. The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.

    PubMed

    Böttinger, E P; Factor, V M; Tsang, M L; Weatherbee, J A; Kopp, J B; Qian, S W; Wakefield, L M; Roberts, A B; Thorgeirsson, S S; Sporn, M B

    1996-06-11

    All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.

  9. The matricellular protein CYR61 inhibits osteoclastogenesis by a mechanism independent of alphavbeta3 and alphavbeta5.

    PubMed

    Crockett, Julie C; Schütze, Norbert; Tosh, Denise; Jatzke, Susanne; Duthie, Angela; Jakob, Franz; Rogers, Michael J

    2007-12-01

    Cysteine-rich protein 61 (CYR61/CCN1) belongs to the family of CCN matricellular proteins. Most of the known effects of CCN proteins appear to be due to binding to extracellular growth factors or integrins, including alpha(v)beta(3) and alpha(v)beta(5). Although CYR61 can stimulate osteoblast differentiation, until now the effect of CYR61 on osteoclasts was unknown. We demonstrate that recombinant human CYR61 inhibits the formation of multinucleated, alpha(v)beta(3)-positive, or tartrate-resistant acid phosphatase-positive human, mouse, and rabbit osteoclasts in vitro. CYR61 markedly reduced the expression of the osteoclast phenotypic markers tartrate-resistant acid phosphatase, matrix metalloproteinase-9, calcitonin receptor, and cathepsin K. However, CYR61 did not affect the formation of multinucleated osteoclasts when added to osteoclast precursors prior to fusion or affect the number or resorptive activity of osteoclasts cultured on dentine discs, indicating that CYR61 affects early osteoclast precursors but not mature osteoclasts. CYR61 did not affect receptor activator of nuclear factor-kappaB (RANK) ligand-induced phosphorylation of p38 or ERK1/2 in human macrophages and did not affect RANK ligand-induced activation of nuclear factor-kappaB, indicating that CYR61 does not appear to inhibit osteoclastogenesis by affecting RANK signaling. Furthermore, a mutant form of CYR61 defective in binding to alpha(v)beta(3) also inhibited osteoclastogenesis, and CYR61 inhibited osteoclastogenesis similarly in cultures of mouse wild-type or beta(5)(-/-) macrophages. Thus, CYR61 does not appear to inhibit osteoclast formation by interacting with alpha(v)beta(3) or alpha(v)beta(5). These observations demonstrate that CYR61 is a hitherto unrecognized inhibitor of osteoclast formation, although the exact mechanism of inhibition remains to be determined. Given that CYR61 also stimulates osteoblasts, CYR61 could represent an important bifunctional local regulator of bone

  10. A novel pentacyclic triterpene from Leontodon filii.

    PubMed

    Tostão, Zélia; Noronha, João P; Cabrita, Eurico J; Medeiros, Jorge; Justino, Jorge; Bermejo, Jaime; Rauter, Amélia P

    2005-03-01

    A novel oleanene triterpenetetrol was isolated from the chloroform extract of the aerial parts of Leontodon filii. Its structure was shown to be 2beta,3beta,15alpha,21beta-olean-12-ene-2,3,15,21-tetrol by chemical and spectroscopic methods. The fungicidal efficacy of the chloroform and methanol extracts of the plant was also evaluated, a protective effect being found against Plasmopara viticola, Botrytis cinerea, particularly powerful against Pyricularia oryzae. PMID:15752627

  11. Integrin-like proteins in the pollen tube: detection, localization and function.

    PubMed

    Sun, Y; Qian, H; Xu, X D; Han, Y; Yen, L F; Sun, D Y

    2000-10-01

    The distribution of integrin-like proteins in the pollen tube was examined by immunofluorescent labeling and western blotting techniques using antibodies against human placenta integrin vitronectin receptor (VnR), and alpha(v), beta3 and beta1 integrin subunits. Pseudocolor-coded confocal images showed intense immunostaining within 10 and 5 microm of the tip of the pollen tube in Lilium davidii and Nicotiana tabacum respectively. In both segments the site near the plasma membrane was labeled. Western blotting analyses revealed cross-reaction of anti-beta3, anti-alpha(v) and anti-VnR with the proteins in the plasma membrane preparation of L. davidii and Hemerocallis citrina pollen tube. These studies provide evidence for the first time that the integrin-like protein is present in pollen tubes, and it may be mainly composed of alpha(v) and beta3 subunits in lily pollen tubes. In a functional assay, neither anti-VnR antibody nor the Arg-Gly-Asp-Ser tetrapeptide inhibited pollen tube growth of N. tabacum in vitro, but both of them depressed tube growth on the stigma and in style under quasi in vivo culture conditions. The integrin-like proteins localized in the tip and periphery of the pollen tube appeared to play roles in growth of the pollen tube tip and interaction with the extracellular matrix of the style. PMID:11148272

  12. 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

  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. 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.

  15. 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.

  16. Chloroplast molecular chaperone-assisted refolding and reconstitution of an active multisubunit coupling factor CF1 core.

    PubMed Central

    Chen, G G; Jagendorf, A T

    1994-01-01

    The chloroplast coupling factor 1 (CF1) is composed of five kinds of subunits with a stoichiometry of alpha 3 beta 3 gamma delta epsilon. Reconstitution of a catalytically active alpha 3 beta 3 gamma core from urea-denatured subunits at a physiological pH is reported here. A restoration of approximately 90% of the CF1 ATPase activity has been observed. The reconstitution was achieved by using subunits overexpressed in Escherichia coli, purified, and combined in the presence of MgATP, K+, and a mixture of several chloroplast molecular chaperones at pH 7.5. The combination of chaperonin 60 and chaperonin 24 failed to reconstitute the active CF1 core, as did the GroEL/GroES pair (E. coli chaperonin 60/10 homologues). Characteristics of the reconstituted ATPase were very close to those of the native complex, including methanol-reversible inhibition by the purified epsilon subunit of CF1 and sensitivity to inhibition by azide and by tentoxin. In reconstitution with a mixture of tentoxin-resistant and -sensitive beta subunits, the extent of inhibition by tentoxin depended on the proportion of sensitive subunits in the reconstitution mixture. Finally, a model for the assembly of the CF1 core alpha 3 beta 3 gamma structure is proposed. Images PMID:7972091

  17. 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.

  18. Differential expression of transforming growth factor-beta in the interstitial tissue of testis during aging.

    PubMed

    Jung, Jae-Chang; Park, Geun-Tae; Kim, Kook-Hee; Woo, Ju Hyung; An, Jung-Min; Kim, Ki-Chul; Chung, Hae Young; Bae, Young-Seuk; Park, Jeen Woo; Kang, Shin-Sung; Lee, Young-Sup

    2004-05-01

    Transforming growth factor-betas (TGF-betas) have significant effects on testis development. The pattern of TGF-beta expression in aging testis has not been established to date. We examined age-related changes in the expression of TGF-beta and its receptors in the testis using Western blot analysis. TGF-beta1 expression increased continuously in aging rat testis, whereas no age-associated changes were observed for TGF-beta3. Strong expression of TGF-beta2, as well as type I and II receptors was observed in 12-month-old testis, but following this time, expression decreased dramatically. Interestingly, TGF-beta2 and -beta3 displayed strong and similar expression patterns in liver, regardless of age, suggesting that the down-regulation of TGF-beta2 is testis-specific. We observed significant induction of p53 and p21WAF1 in 18-month-old testis that appeared to correspond with aging. Moreover, caloric restriction (CR) prevented age-related decrease in TGF-beta2 expression. Using immunohistochemistry, we showed that all TGF-beta1, -beta2, and -beta3 proteins are expressed primarily in interstitial cells, which are located in the space between adjoining seminiferous tubules. Our data collectively indicate that aging in the testis is regulated by differential expression of TGF-beta proteins, and decreased levels of TGF-beta2 contribute to the aging process.

  19. 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.

  20. 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.

  1. 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

  2. Effect of different beta-adrenergic agonists on the intestinal absorption of galactose and phenylalanine.

    PubMed

    Díez-Sampedro, A; Pérez, M; Cobo, M T; Martínez, J A; Barber, A

    1998-08-01

    Nutrient transport across the mammalian small intestine is regulated by several factors, including intrinsic and extrinsic neural pathways, paracrine modulators, circulating hormones and luminal agents. Because beta-adrenoceptors seem to regulate gastrointestinal functions such as bicarbonate and acid secretion, intestinal motility and gastrointestinal mucosal blood flow, we have investigated the effects of different beta-adrenergic agonists on nutrient absorption by the rat jejunum in-vitro. When intestinal everted sacs were used the beta2-agonist salbutamol had no effect either on galactose uptake by the tissue or mucosal-to-serosal flux whereas mixed beta1- and beta2-agonists (isoproterenol and orciprenaline) and beta3-agonists (BRL 35135, Trecadrine, ICI 198157 and ZD 7114) inhibited galactose uptake and transfer of D-galactose from the mucosal-to-serosal media across the intestinal wall (although the inhibiting effects of isoproterenol and Trecadrine were not statistically significant). In intestinal everted rings both Trecadrine and BRL 35135 clearly reduced galactose uptake, the effect being a result of inhibition of the phlorizin-sensitive component. Total uptake of phenylalanine by the intestinal rings was also reduced by those beta3-adrenergic agonists. These results suggest that beta1- and beta3-adrenergic receptors could be involved in the regulation of intestinal active transport of sugars and amino acids. PMID:9751456

  3. 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.

  4. Pharmacological characterization of KUR-1246, a selective uterine relaxant.

    PubMed

    Kobayashi, M; Takeda, K; Murata, S; Kojima, M; Akahane, M; Inoue, Y; Kitamura, K; Kawarabayashi, T

    2001-05-01

    The aim of the present study was to evaluate the efficacy and beta 2-adrenoceptor (AR) selectivity of KUR-1246, a new uterine relaxant. Inhibition of spontaneous or drug-induced uterine contractions by KUR-1246 was evaluated in pregnant rats and rabbits by an organ bath method or by a balloon method. The selectivity of KUR-1246 was assessed simultaneously in organs isolated from late-pregnant rats. The affinity of KUR-1246 for human beta 1-, beta 2-, and beta 3-ARs was determined using two radioligands. KUR-1246 suppressed both spontaneous and drug-induced contractions in isolated uteri, the rank order of potency being isoproterenol > KUR-1246 > terbutaline > ritodrine. ICI-118551 (selective beta 2-AR antagonist) competitively antagonized the KUR-1246-induced inhibition of spontaneous uterine contractions, but CGP-20712A (selective beta 1-AR antagonist) and SR-58894A (selective beta 3-AR antagonist) did not. All beta-AR agonists tested produced significant inhibition of spontaneous uterine contractions in vivo: ED(30) value for KUR-1246 was 0.13 microg/kg/min, a potency about 6 times and 400 times greater than that of terbutaline and ritodrine, respectively. In contrast, the positive chronotropic effect was minimal in KUR-1246-treated rats. KUR-1246 displaced radioligand binding to beta 1-, beta 2-, and beta 3-ARs, the pK(i) values being 5.75 +/- 0.03, 7.59 +/- 0.08, and 4.75 +/- 0.03 for beta 1-, beta 2-, and beta 3-ARs, respectively. For the selectivity of KUR-1246 for human beta 2-AR, we obtained values of 39.2 ([IC(50) for beta 1-AR]/[IC(50) for beta 2-AR]) and 198.2 ([IC(50) for beta 3-AR]/[IC(50) for beta 2-AR]), indicating an apparently higher affinity for human beta 2-AR than for other beta-AR subtypes. The present study clearly demonstrated that KUR-1246 is a more selective beta 2-AR agonist than the drugs presently used for relaxing uterine muscle.

  5. Central roles of alpha5beta1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies.

    PubMed

    Francis, Sheila E; Goh, Keow Lin; Hodivala-Dilke, Kairbaan; Bader, Bernhard L; Stark, Margaret; Davidson, Duncan; Hynes, Richard O

    2002-06-01

    Vascular development and maturation are dependent on the interactions of endothelial cell integrins with surrounding extracellular matrix. Previous investigations of the primacy of certain integrins in vascular development have not addressed whether this could also be a secondary effect due to poor embryonic nutrition. Here, we show that the alpha5 integrin subunit and fibronectin have critical roles in blood vessel development in mouse embryos and in embryoid bodies (EBs) differentiated from embryonic stem cells (a situation in which there is no nutritional deficit caused by the mutations). In contrast, vascular development in vivo and in vitro is not strongly dependent on alpha(v) or beta3 integrin subunits. In mouse embryos lacking alpha5 integrin, greatly distended blood vessels are seen in the vitelline yolk sac and in the embryo itself. Additionally, overall blood vessel pattern complexity is reduced in alpha5-null tissues. This defective vascular phenotype is correlated with a decrease in the ligand for alpha5 integrin, fibronectin (FN), in the endothelial basement membranes. A striking and significant reduction in early capillary plexus formation and maturation was apparent in EBs formed from embryonic stem cells lacking alpha5 integrin or FN compared with wild-type EBs or EBs lacking alpha(v) or beta3 integrin subunits. Vessel phenotype could be partially restored to FN-null EBs by the addition of whole FN to the culture system. These findings confirm a clear role for alpha5 and FN in early blood vessel development not dependent on embryo nutrition or alpha(v) or beta3 integrin subunits. Thus, successful early vasculogenesis and angiogenesis require alpha5-FN interactions.

  6. Micro-CT molecular imaging of tumor angiogenesis using a magnetite nano-cluster probe.

    PubMed

    Liu, Ping; Li, Jing; Zhang, Chunfu; Xu, Lisa X

    2013-06-01

    Due to its high resolution, micro-CT is desirable for molecular imaging of tumor angiogenesis. However, the sensitivity of micro-CT to contrast agents is relatively low. Therefore, the purpose of this study is to develop high micro-CT sensitive molecular imaging probes for direct visualization and dynamic monitoring of tumor angiogenesis. To this end, Arg-Gly-Asp (RGD) peptides conjugated magnetite nano clusters (RGD-MNCs) were developed by assembling individual magnetite nano particles into clusters with amphiphilic (maleimide) methoxypoly(ethylene glycol)-b-poly(lactic acid) ((Mal)mPEG-PLA) copolymer and subsequently encoding RGD peptides onto the clusters for specific targeting alpha(v)beta3 integrin. The hydrodynamic size of RGD-MNCs was about 85 nm. To test its specificity, alpha(v)beta3 positive cells (H1299) were incubated with magnetite nano clusters (MNCs), RGD-MNCs or RGD-MNCs competition with free RGD peptides. Prussian Blue staining and inductively coupled plasma optical emission spectrometer (ICP-OES) measurements indicated that the cell uptake of RGD-MNCs was significantly more than that of MNCs, which could be inhibited by free RGD peptides. For detection of tumor angiogenesis, mice bearing H1299 tumors were injected intravenously with RGD-MNCs at the dose of 400 micro mol Fe/kg. Tumor angiogenic hot spots as well as individual angiogenic vessels could be clearly manifested by micro-CT imaging 12 h post injection, which was dynamically monitored with the extension of probe circulation time. Subsequent histological studies of tumor tissues verified that RGD-MNCs registered tumor angiogenic vessels. Our study demonstrated that RGD-MNC probes fabricated in this study could be used to effectively target alpha(v)beta3 integrin. Using high resolution micro-CT in combination with the probes, tumor angiogenesis could be studied dynamically.

  7. Enhanced behavioral sensitivity to the competitive GABA agonist, gaboxadol, in transgenic mice over-expressing hippocampal extrasynaptic alpha6beta GABA(A) receptors.

    PubMed

    Saarelainen, Kati S; Ranna, Martin; Rabe, Holger; Sinkkonen, Saku T; Möykkynen, Tommi; Uusi-Oukari, Mikko; Linden, Anni-Maija; Lüddens, Hartmut; Korpi, Esa R

    2008-04-01

    The behavioral and functional significance of the extrasynaptic inhibitory GABA(A) receptors in the brain is still poorly known. We used a transgenic mouse line expressing the GABA(A) receptor alpha6 subunit gene in the forebrain under the Thy-1.2 promoter (Thy1alpha6) mice ectopically expressing alpha6 subunits especially in the hippocampus to study how extrasynaptically enriched alphabeta(gamma2)-type receptors alter animal behavior and receptor responses. In these mice extrasynaptic alpha6beta receptors make up about 10% of the hippocampal GABA(A) receptors resulting in imbalance between synaptic and extrasynaptic inhibition. The synthetic GABA-site competitive agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; 3 mg/kg) induced remarkable anxiolytic-like response in the light : dark exploration and elevated plus-maze tests in Thy1alpha6 mice, while being almost inactive in wild-type mice. The transgenic mice also lost quicker and for longer time their righting reflex after 25 mg/kg gaboxadol than wild-type mice. In hippocampal sections of Thy1alpha6 mice, the alpha6beta receptors could be visualized autoradiographically by interactions between gaboxadol and GABA via [(35)S]TBPS binding to the GABA(A) receptor ionophore. Gaboxadol inhibition of the binding could be partially prevented by GABA. Electrophysiology of recombinant GABA(A) receptors revealed that GABA was a partial agonist at alpha6beta3 and alpha6beta3delta receptors, but a full agonist at alpha6beta3gamma2 receptors when compared with gaboxadol. The results suggest strong behavioral effects via selective pharmacological activation of enriched extrasynaptic alphabeta GABA(A) receptors, and the mouse model represents an example of the functional consequences of altered balance between extrasynaptic and synaptic inhibition.

  8. Interaction of alphaVbeta3 and alphaVbeta6 integrins with human parechovirus 1.

    PubMed

    Seitsonen, Jani; Susi, Petri; Heikkilä, Outi; Sinkovits, Robert S; Laurinmäki, Pasi; Hyypiä, Timo; Butcher, Sarah J

    2010-09-01

    Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to alpha(V) integrins. The interaction of HPEV1 with alpha(V) integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins alpha(V)beta(3) and alpha(V)beta(6) to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, alpha(V)beta(6) bound more efficiently than alpha(V)beta(3) to immobilized HPEV1. Moreover, soluble alpha(V)beta(6), but not alpha(V)beta(3), blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints.

  9. 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.

  10. Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone.

    PubMed

    Carron, C P; Meyer, D M; Engleman, V W; Rico, J G; Ruminski, P G; Ornberg, R L; Westlin, W F; Nickols, G A

    2000-06-01

    Osteoclasts are actively motile on bone surfaces and undergo alternating cycles of migration and resorption. Osteoclast interaction with the extracellular matrix plays a key role in the osteoclast resorptive process and a substantial body of evidence suggests that integrin receptors are important in osteoclast function. These integrin receptors bind to the Arg-Gly-Asp (RGD) sequence found in a variety of extracellular matrix proteins and it is well established that the interaction of osteoclast alpha v beta 3 integrin with the RGD motif within bone matrix proteins is important in osteoclast-mediated bone resorption. In this study, we characterized the effects of two synthetic peptidomimetic antagonists of alpha v beta 3, SC-56631 and SC-65811, on rabbit osteoclast adhesion to purified matrix proteins and bone, and on bone resorption in vitro. SC-56631 and SC-65811 are potent inhibitors of vitronectin binding to purified alpha v beta 3. Both SC-56631 and SC-65811 inhibited osteoclast adhesion to osteopontin- and vitronectin-coated surfaces and time-lapse video microscopy showed that osteoclasts rapidly retract from osteopontin-coated surfaces when exposed to SC-56631 and SC-65811. SC-56631 and SC-65811 blocked osteoclast-mediated bone resorption in a dose-responsive manner. Further analysis showed that SC-65811 and SC-56631 reduced the number of resorption pits produced per osteoclast and the average pit size. SC-65811 was a more potent inhibitor of bone resorption and the combination of reduced pit number and size led to a 90% inhibition of bone resorption. Surprisingly, however, osteoclasts treated with SC-65811, SC-56631 or the disintegrin echistatin, at concentrations that inhibit bone resorption did not inhibit osteoclast adhesion to bone. These results suggest that alphavbeta3 antagonists inhibited bone resorption by decreasing osteoclast bone resorptive activity or efficiency but not by inhibiting osteoclast adhesion to bone per se.

  11. 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

  12. Resting EEG in offspring of male alcoholics: beta frequencies.

    PubMed

    Rangaswamy, Madhavi; Porjesz, Bernice; Chorlian, David B; Wang, Kongming; Jones, Kevin A; Kuperman, Samuel; Rohrbaugh, John; O'Connor, Sean J; Bauer, Lance O; Reich, Theodore; Begleiter, Henri

    2004-02-01

    This study examines the differences in beta (12-28 Hz) band power in offspring of male alcoholics from densely affected alcoholic families. We have attempted to investigate if the increase in beta power is a 'state' or 'trait' marker for alcoholism. This study also explores the gender differences in the expression of this potential risk marker. Absolute beta power in three bands-beta 1(12-16 Hz), beta 2 (16-20 Hz), and beta 3 (20-28 Hz)-in the eyes closed EEG of 171 high risk (HR) subjects who were offspring of male alcoholics and 204 low risk (LR) subjects with no family history of alcoholism, were compared for each gender separately using a repeated measures analysis of variance design. Alcoholic and non-alcoholic subjects within the high risk group were compared using a repeated measures design as a follow-up analysis. The present study demonstrated increased beta power in the resting EEG of offspring of male alcoholics. Male HR subjects had higher beta 1 (12-16 Hz) power and female HR subjects had increased power in beta 2 (16-20 Hz) and beta 3 (20-28 Hz) as compared with low risk participants. Female HR subjects also showed significantly increased beta 2 and beta 3 power if they had two or more alcoholic first-degree relatives when compared with HR females having only an affected father. Risk characteristics are expressed differentially in males and females and may be an index of differential vulnerability to alcoholism. The results indicate that increased EEG beta power can be considered as a likely marker of risk for developing alcoholism and may be used as a predictive endophenotype.

  13. The GABA(A) receptor alpha1 subtype in the ventral pallidum regulates alcohol-seeking behaviors.

    PubMed

    Harvey, Scott C; Foster, Katrina L; McKay, Pete F; Carroll, Michelle R; Seyoum, Regat; Woods, James E; Grey, Collette; Jones, Cecily M; McCane, Shannan; Cummings, Rancia; Mason, Dynesha; Ma, Chunrong; Cook, James M; June, Harry L

    2002-05-01

    We investigated the potential role of the alpha1-containing GABA(A) receptor in regulating the reinforcing properties of alcohol. To accomplish this, we developed 3-propoxy-beta-carboline hydrochloride (3-PBC), a mixed agonist-antagonist benzodiazepine site ligand with binding selectivity at the alpha1 receptor. We then tested the capacity of 3-PBC to block alcohol-maintained responding in the ventral pallidum (VP), a novel alcohol reward substrate, which primarily expresses the alpha1-receptor isoform. Our results demonstrated that bilateral microinfusion of 3-PBC (0.5-40 microg) in the anterior and medial VP produced marked reductions in alcohol-maintained responding in a genetically selected rodent model of alcohol drinking. The VP infusions showed both neuroanatomical and reinforcer specificity because no effects were seen in sites dorsal to the VP (e.g., nucleus accumbens, caudate putamen). The saccharin-maintained responding was reduced only with the highest dose (40 microg). Parenteral injections of 3-PBC (1-20 mg/kg) also showed a similar selectivity on alcohol-maintained responding. Complementary in vitro studies revealed that 3-PBC exhibited a low partial agonist efficacy profile at recombinant diazepam-sensitive receptors (e.g., alpha1beta3gamma2, alpha2beta3gamma, and alpha3beta3gamma2). The selective suppression of 3-PBC on alcohol-maintained responding after central and parenteral administrations, together with its low-efficacy agonist profile, suggest that the reduction in alcohol-maintained behaviors was not attributable to a general suppression on consummatory behaviors. These results demonstrate that the alpha1-containing GABA(A) receptors in both the anterior and medial VP are important in regulating the reinforcing properties of alcohol. These receptors represent novel targets in the design and development of pharmacotherapies for alcohol-dependent subjects. PMID:11978852

  14. 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.

  15. Association between body composition and blood pressure in a contemporary cohort of 9-year-old children.

    PubMed

    Brion, M A; Ness, A R; Davey Smith, G; Leary, S D

    2007-04-01

    Elevated blood pressure (BP) in children is an early risk factor for cardiovascular disease and is positively associated with body mass index (BMI). However, BMI does not distinguish between fat and lean masses, and the relationship of BP in children to different elements of body composition is not well established. BP, BMI and body composition were measured in 6863 children enrolled in the Avon Longitudinal Study of Parents and Children. Fat mass, lean mass and trunk fat were assessed using dual-energy X-ray absorptiometry. After full adjustment for confounders, total body fat and BMI were positively associated with systolic blood pressure (SBP) (beta=3.29, 95% confidence interval CI 3.02, 3.57 mm Hg/standard deviation (s.d.) and beta=3.97, 95% CI 3.73, 4.21 mm Hg/s.d., respectively) and diastolic blood pressure (DBP) (beta=1.26, 95% CI 1.05, 1.46 mm Hg/s.d. and beta=1.37, 95% CI 1.19, 1.54 mm Hg/s.d., respectively). SBP was also positively associated with lean mass (beta=3.38, 95% CI 2.95, 3.81 mm Hg/s.d.), and weakly associated with trunk fat (beta=1.42, 95% CI -0.06, 2.90 mm Hg/s.d., independent of total fat mass), which was robust in girls only. The association between lean mass and SBP remained even after accounting for fat mass. SBP in 9-year-old children is independently associated with fat mass and lean mass and, to a lesser extent, trunk fat in girls. In this analysis, because both fat and lean masses are associated with BP, BMI predicts BP at least as well as these components of body composition.

  16. Analysis of expression of chorionic gonadotrophin transcripts in prostate cancer by quantitative Taqman and a modified molecular beacon RT-PCR.

    PubMed

    Span, P N; Thomas, C M G; Heuvel, J J; Bosch, R R; Schalken, J A; vd Locht, L; Mensink, E J B M; Sweep, C G J

    2002-03-01

    Expression of human chorionic gonadotrophin (hCG) is associated with trophoblastic, testicular and other malignancies such as bladder, pancreatic, cervical, breast and prostate cancer. In the prostate, however, hCG expression, associated with neuroendocrine cells, is also found in normal tissue. Of the six highly homologous genes that all encode the beta-subunit of hCG, the beta 7 gene is reportedly the only gene expressed in several non-transformed tissues. The beta 3, 5 and 8 genes would be variably expressed in malignant tissue and placenta, but not in normal tissue. To assess to what extent this expression difference can also be found in the prostate, we compared the levels of the different hCG beta transcripts in concurrent normal and cancerous prostate tissues obtained from 17 patients. To this end, we developed a Taqman real-time fluorescent RT-PCR assay for hCG beta, and a quantitative assay specific for the beta 3, 5 and 8 genes, modified from the molecular beacon principle. This latter assay proved highly specific and capable of reliably distinguishing between these hCG beta transcripts that differ in only one nucleotide. Surprisingly, median expression levels of hCG beta were lower in prostate cancer when compared with normal tissue from the same patient. In contrast, hCG beta 3, 5 and 8 transcripts were found in normal tissue and did not differ in prostate cancer, arguing against a specific role of these transcripts in the development of prostate cancer.

  17. Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same site.

    PubMed

    Chen, Ligong; Durkin, Kathleen A; Casida, John E

    2006-03-28

    Several major insecticides, including alpha-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human beta(3) homopentameric GABA(A) receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the beta(3) homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1'C, A2'C, T6'C, and L9'C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane and [(3)H] 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile was dramatically reduced with 8 of the 15 mutated positions, focusing attention on A2', T6', and L9' as proposed binding sites, consistent with earlier mutagenesis studies. The cytoplasmic half of the beta3 homopentamer pore was modeled as an alpha-helix. The six NCAs listed above plus t-butylbicyclophosphorothionate fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. Thus, widely diverse NCA structures fit the same GABA receptor beta subunit site with important implications for insecticide cross-resistance and selective toxicity between insects and mammals.

  18. Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8

    PubMed Central

    1990-01-01

    The involvement of integrins in mediating interaction of cells to well- characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody- sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules

  19. Structurally similar Drosophila alpha-tubulins are functionally distinct in vivo.

    PubMed Central

    Hutchens, J A; Hoyle, H D; Turner, F R; Raff, E C

    1997-01-01

    We used transgenic analysis in Drosophila to compare the ability of two structurally similar alpha-tubulin isoforms to support microtubule assembly in vivo. Our data revealed that even closely related alpha-tubulin isoforms have different functional capacities. Thus, in multicellular organisms, even small changes in tubulin structure may have important consequences for regulation of the microtubule cytoskeleton. In spermatogenesis, all microtubule functions in the postmitotic male germ cells are carried out by a single tubulin heterodimer composed of the major Drosophila alpha-84B tubulin isoform and the testis-specific beta 2-tubulin isoform. We tested the ability of the developmentally regulated alpha 85E-tubulin isoform to replace alpha 84B in spermatogenesis. Even though it is 98% similar in sequence, alpha 85E is not functionally equivalent to alpha 84B. alpha 85E can support some functional microtubules in the male germ cells, but alpha 85E causes dominant male sterility if it makes up more than one-half of the total alpha-tubulin pool in the spermatids. alpha 85E does not disrupt meiotic spindle or cytoplasmic microtubules but causes defects in morphogenesis of the two classes of singlet microtubules in the sperm tail axoneme, the central pair and the accessory microtubules. Axonemal defects caused by alpha 85E are precisely reciprocal to dominant defects in doublet microtubules we observed in a previous study of ectopic germ-line expression of the developmentally regulated beta 3-tubulin isoform. These data demonstrate that the doublet and singlet axoneme microtubules have different requirements for alpha- and beta-tubulin structure. In their normal sites of expression, alpha 85E and beta 3 are coexpressed during differentiation of several somatic cell types, suggesting that alpha 85E and beta 3 might form a specialized heterodimer. Our tests of different alpha-beta pairs in spermatogenesis did not support this model. We conclude that if alpha 85E and beta

  20. 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

  1. Analysis of cytokine production and V beta T-cell receptor subsets in irradiated recipients receiving portal or peripheral venous reconstitution with allogeneic bone marrow cells, with or without additional anti-cytokine monoclonal antibodies.

    PubMed Central

    Gorczynski, R M; Chen, Z; Zeng, H; Gorczynski, L; Terzioglu, E

    1998-01-01

    Irradiated (800 rads) AKR mice received intravenous (i.v.) reconstitution with a mixture of B10.BR T-depleted bone marrow cells and spleen cells. Only in groups of mice treated additionally with i.v. cyclophosphamide (Cy; 150 mg/kg), 24 hr before transplantation, was long-term (> 60% at 50 days) survival seen. In mice receiving only irradiation all animals died by 30 days post-transplantation. Histological changes consistent with graft-versus-host disease (GVHD) were seen in the liver of reconstituted mice at 30 days, along with an organ-specific increase in V beta 3 T-cell receptor-positive (TCR+) cells. No such increase in V beta 3 TCR+ cells was seen in the spleen from the same mice. These data are consistent with a tissue antigen-driven expansion of V beta 3 TCR+ cells associated with GVHD in the liver in this model. When we analysed cytokine production in vitro from CD3+ cells restimulated with 'host' (AKR) antigen-presenting cells (APC), we found a transition in cytokine production from preferential synthesis of type-1 cytokines [interleukin-2 (IL-2) and interferon-gamma (IFN-gamma)] at early times (day 15) post-reconstitution to increased production of type-2 cytokines [IL-4, transforming growth factor-beta (TGF-beta) and IL-10] at later times (day 30) post-reconstitution in Cy-treated recipients. Animals not receiving Cy did not show this 'switch' in cytokine production at later time points. We have observed a similar polarization in cytokine production, along with increased graft survival, in recipients of vascularized and non-vascularized allografts after portal venous (p.v.), but not i.v., pretransplant donor-specific immunization. We next studied AKR mice receiving 800 rads and subsequently reconstituted with B10.BR stem cells via the p.v. route. Again these mice showed prolonged survival (> 50% at 50 days), with polarization to IL-4, IL-10 and TGF-beta on restimulation of CD3+ cells in vitro at 30 days post-transplant and increased V beta 3 TCR+ cells

  2. 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

  3. Cytotoxic, antioxidant activities and structure activity relationship of some newly synthesized terpenoidal oxaliplatin analogs.

    PubMed

    Amr, Abd El-Galil E; Ali, Korany A; Abdalla, Mohamed M

    2009-02-01

    The terpenoidal oxaliplatin derivatives (6) and (12) were newly synthesized using 2beta,3alpha-dihydroxy-11-oxo-18beta-olean-12-ene-30-oic acid (1) and 2alpha,2beta-dihydroxy-18beta-ursan-12-ene-28-oic acid (7) as starting materials. The synthesized compounds were evaluated for their cytotoxicity and antioxidant activities and were compared to Oxaliplatin and vitamin C as positive controls. Some of the compounds exhibited better cytotoxicity and antioxidant activities than the reference controls. The detailed synthesis, spectroscopic data, toxicity (LD(50)) and pharmacological screening for the synthesized compounds were reported.

  4. [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.

  5. Morphological and biochemical changes in a hematopoietic cell line induced by jacalin, a lectin derived from Artocarpus integrifolia.

    PubMed

    Yagi, M; Campos-Neto, A; Gollahon, K

    1995-04-01

    Treatment of the human erythroleukemia cell line K562 with the galactose-binding lectin, jacalin, results in rapid and profound alterations in the morphology and biochemistry of the cells. Within minutes of lectin addition, the cells adhere to the plastic tissue culture surface, and within hours, the cells spread on the surface, acquiring a monocyte-like appearance. Jacalin treatment results in elevated expression of CD61 (integrin beta 3) and CD14, a monocyte-associated cell surface antigen. These results suggest that jacalin treatment of K562 cells triggers intracellular events that result in differentiation along the monocyte lineage.

  6. Inhibition of tyrosinase by flavonoids, stilbenes and related 4-substituted resorcinols: structure-activity investigations.

    PubMed

    Shimizu, K; Kondo, R; Sakai, K

    2000-02-01

    Several flavonoids, stilbenes and related 4-substituted resorcinols, obtained from Artocarpus incisus and other plants or synthesized, were tested for their inhibitory activity against tyrosinase. The structure-activity relationships suggested that specific natural or synthesized compounds having the 4-substituted resorcinol skeleton have potent tyrosinase inhibitory ability. Kinetic studies have indicated that specific compounds having the 4-substituted resorcinol skeleton exhibit competitive inhibition of the oxidation of DL-beta-(3,4-dihydroxyphenyl)alanine (DL-DOPA) by mushroom tyrosinase. These findings could lead to the design and discovery of new tyrosinase inhibitors.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. Phenylethanoids, iridoids and a spirostanol saponin from Veronica turrilliana.

    PubMed

    Kostadinova, Emanuela P; Alipieva, Kalina I; Kokubun, Tetsuo; Taskova, Rilka M; Handjieva, Nedjalka V

    2007-05-01

    From the aerial parts of Veronica turrilliana two phenylethanoid glycosides, turrilliosides A and B and a steroidal saponin, turrillianoside were isolated and their structures elucidated as beta-(3,4-dihydroxyphenyl)ethyl-4-O-E-caffeoyl-O-[beta-glucopyranosyl-(1-->4)-alpha-rhamnopyranosyl-(1-->6)]-beta-glucopyranoside, beta-(3,4-dihydroxyphenyl)ethyl-4-O-E-caffeoyl-[6-O-E-feruloyl-beta-glucopyranosyl-(1-->4)-alpha-rhamnopyranosyl-(1-->6)]-beta-glucopyranoside and (23S,25S)-12beta,23-dihydroxyspirost-5-en-3beta-yl O-alpha-rhamnopyranosyl-(1-->4)-beta-glucopyranoside, respectively. Furthermore, eight known glucosides are reported namely, catalpol, catalposide, verproside, amphicoside, isovanilloylcatalpol, aucubin, arbutin, and 6-O-E-caffeoylarbutin, the latter two for the first time in the genus Veronica. The two phenylethanoid glycosides were found to be potent DPPH radical scavengers. All of the tested compounds were inactive against the representative species of fungi and bacteria.

  13. 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

  14. Potential therapeutic radiotracers: preparation, biodistribution and metabolic characteristics of 177Lu-labeled cyclic RGDfK dimer.

    PubMed

    Shi, Jiyun; Liu, Zhaofei; Jia, Bing; Yu, Zilin; Zhao, Huiyun; Wang, Fan

    2010-06-01

    In this study, we reported the preparation and evaluation of (177)Lu-DOTA-RGD2, (177)Lu-DOTA-Bz-RGD2 and (177)Lu-DTPA-Bz-RGD2 (RGD2 = E[c(RGDfK)](2)) as a potential therapeutic radiotracers for the treatment of integrin alpha(v)beta(3)-positive tumors. The BALB/c nude mice bearing the U87MG human glioma xenografts were used to evaluate the biodistribution characteristics and excretion kinetics of (177)Lu-DOTA-RGD2, (177)Lu-DOTA-Bz-RGD2 and (177)Lu-DTPA-Bz-RGD2. It was found that there were no major differences in their lipophilicity and biodistribution characteristics, particularly at latter time points. A major advantage of using DTPA-Bz as the bifunctional chelator (BFC) was its high radiolabeling efficiency (fast and high yield radiolabeling) at room temperature. Using DOTA and DOTA-Bz as BFCs, the radiolabeling kinetics was slow, and heating at 100 degrees C and higher DOTA-conjugate concentration were needed for successful (177)Lu-labeling. Therefore, DTPA-Bz is an optimal BFC for routine preparation of (177)Lu-labeled cyclic RGDfK peptides, and (177)Lu-DTPA-Bz-RGD2 is worthy of further investigation for targeted radiotherapy of integrin alpha(v)beta(3)-positive tumors.

  15. A 70 kDa MHC class I associated protein (MAP-70) identified as a receptor molecule for Coxsackievirus A9 cell attachment.

    PubMed

    Triantafilou, M; Triantafilou, K; Wilson, K M

    2000-09-01

    One of the major categories of disease-causing micro-organisms are viruses. New studies on many different viruses have shown that virus attachment and cell entry is often a multistep process, requiring many interactions between the virus and cell surface molecules. In this study, we have attempted to identify the cell surface molecules involved in Coxsackievirus A9 (CAV-9), a common human pathogen and a member of the Picornavirus family, infectious process. GMK cells susceptible to virus infection were surfaced labeled with biotin and then solubilized in non-ionic and zwiterionic detergents. Free CAV-9 virions were used as an affinity surface, allowing the virus to bind to the solubilized receptors. The virus-receptor complexes were then immunoprecipitated by an anti CAV-9 serum and protein-A sepharose beads. SDS-PAGE and two-dimensional electrophoresis revealed the presence of integrin alpha v beta 3 molecules and a 70 kDa protein with apparent isoelectric point (pI) 5.5. The identity of the integrin alpha v beta 3 molecules was confirmed by immunoprecipitation and Western blotting; whereas the 70 kDa protein was also found to co-immunoprecipitate with MHC class I molecules in non-stringent conditions. Sequential immunoprecipitation experiments confirmed that the MHC class I associated protein (MAP-70) and the 70 kDa protein utilized by CAV-9 were identical. The role of MAP-70 in CAV-9 infectious process is discussed.

  16. Effect of green tea (Camellia sinensis L.) extract on morphological and functional changes in adult male gonads of albino rats.

    PubMed

    Chandra, Amar K; Choudhury, Shyamosree Roy; De, Neela; Sarkar, Mahitosh

    2011-09-01

    Green tea, prepared from the steamed and dried leaves of the shrub Camellia sinensis, is known for its antioxidant and anti-carcinogenic effects. However, its effects on male gonadal functions have not been explored adequately and the present investigation has been undertaken to evaluate the effect of green tea extract on gonads of adult male albino rats. Results of in vivo studies showed that green tea extract (GTE) at mild (1.25 g%, identical to 5 cups of tea/day), moderate (2.5 g%, identical to 10 cups of tea/day) and high (5.0 g%, identical to 20 cups of tea/day) doses, for a period of 26 days, altered morphology and histology of testis and accessory sex organs. A significant dose-dependent decrease in the sperm counts, inhibited activities of testicular delta(5)3beta-and 17beta-hydroxysteroid dehydrogenase (delta5-3beta3-HSD and 17beta3-HSD respectively) and decreased serum testosterone level were noticed. Significant increase in serum LH level was observed after moderate and high doses; serum FSH level also increased but not significantly. Histopathological examination showed inhibition of spermatogenesis evidenced by preferential loss of matured and elongated spermatids. Results of this study showed that GTE at relatively high dose may cause impairment of both the morphological and normal functional status of testis in rodents and thus its consumption at relatively high doses raises concern on male reproductive function in spite of its other beneficial effects.

  17. 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.

  18. Integrin-associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins

    PubMed Central

    1990-01-01

    Phagocytosis by monocytes or neutrophils can be enhanced by interaction with several proteins or synthetic peptides containing the Arg-Gly-Asp sequence. Recently we showed that an mAb, B6H12, specifically inhibited this enhancement of neutrophil phagocytosis by inhibiting Arg-Gly-Asp binding to the leukocyte response integrin (Gresham, H. D., J. L. Goodwin, P. M. Allen, D. C. Anderson, and E. J. Brown. 1989. J. Cell Biol. 108:1935-1943). Now, we have purified the antigen recognized by B6H12 to homogeneity. Surprisingly, it is a 50-kD molecule that is expressed on the plasma membranes of all hematopoietic cells, including erythrocytes, which express no known integrins. On platelets and placenta, but not on erythrocytes, this protein is associated with an integrin that can be recognized by an anti-beta 3 antibody. In addition, both the anti-beta 3 and several mAbs recognizing the 50-kD protein inhibit Arg-Gly-Asp stimulation of phagocytosis. These data demonstrate an association between integrins and the 50-kD protein on several cell types. For this reason, we call it Integrin-associated Protein (IAP). We hypothesize that IAP may play a role in signal transduction for enhanced phagocytosis by Arg-Gly-Asp ligands. PMID:2277087

  19. Role of the alpha subunit in the modulation of GABA(A) receptors by anabolic androgenic steroids.

    PubMed

    Yang, Paul; Jones, Brian L; Henderson, Leslie P

    2005-09-01

    Neural transmission mediated by circuits expressing alpha2 subunit-containing gamma-aminobutyric acid type A (GABA(A)) receptors is critical for the expression of behaviors known to be altered by anabolic androgenic steroids (AAS). Here we show that micromolar concentrations of AAS, which reflect levels found in steroid abusers, induce positive modulation of currents from alpha2beta3 gamma2L recombinant receptors elicited by pulses of GABA that mimic synaptic conditions in a manner that is mechanistically distinct from modulation induced at alpha1beta3 gamma2L receptors. Specifically, at alpha2-containing receptors, the AAS, 17alpha-methyltestosterone (17alpha-MeT) enhanced peak current, slowed deactivation, diminished desensitization, and promoted entry of receptors into more distal states along the activation pathway. Analysis of GABA(A) receptor-mediated synaptic currents in primary cortical neurons followed by single cell real-time RT-PCR demonstrated that 17alpha-MeT enhancement of synaptic currents is proportional to the ratio of alpha2 to alpha1 subunit mRNA. Finally, we show that the modulation elicited by AAS is not comparable to that produced by micromolar concentrations of other positive allosteric modulators at alpha2-containing receptors. In sum, these data indicate that AAS elicit effects on GABA(A) receptor function that depend significantly on alpha subunit composition and that the mechanism of AAS modulation of GABA(A) receptors is distinct from that of other positive allosteric modulators.

  20. Conserved site for neurosteroid modulation of GABA A receptors.

    PubMed

    Hosie, Alastair M; Clarke, Laura; da Silva, Helena; Smart, Trevor G

    2009-01-01

    This study addresses whether the potentiation site for neurosteroids on GABA(A) receptors is conserved amongst different GABA(A) receptor isoforms. The neurosteroid potentiation site was previously identified in the alpha1beta2gamma2S receptor by mutation of Q241 to methionine or leucine, which reduced the potentiation of GABA currents by the naturally occurring neurosteroids, allopregnanolone or tetrahydrodeoxycorticosterone (THDOC). By using heterologous expression of GABA(A) receptors in HEK cells, in combination with whole-cell patch clamp recording methods, a relatively consistent potentiation by allopregnanolone of GABA-activated currents was evident for receptors composed of one alpha subunit isoform (alpha2-5) assembled with beta3 and gamma2S subunits. Using mutant alphabetagamma receptors, the neurosteroid potentiation was universally dependent on the conserved glutamine residue in M1 of the respective alpha subunit. Studying wild-type and mutant receptors composed of alpha4beta3delta subunits revealed that the delta subunit is unlikely to contribute to the neurosteroid potentiation binding site and probably affects the efficacy of potentiation. Thus, in keeping with the ability of neurosteroids to potentiate GABA currents via a broad variety of GABA(A) receptor isoforms in neurons, the potentiation site is structurally highly conserved on this important neurotransmitter receptor family.

  1. In vivo characterization of endothelial cell activation in a transgenic mouse model of Alzheimer's disease.

    PubMed

    Schultheiss, Caroline; Blechert, Birgit; Gaertner, Florian C; Drecoll, Enken; Mueller, Jan; Weber, Georg F; Drzezga, Alexander; Essler, Markus

    2006-01-01

    Alzheimer's disease (AD) is the most common cause of dementia worldwide. AD is characterized by an excessive cerebral amyloid deposition leading to degeneration of neurons and eventually to dementia. It has been shown by epidemiological studies that cardiovascular drugs with an anti-angiogenic effect can influence the outcome of AD patients. Therefore, it has been speculated that in AD angiogenesis in the brain vasculature may play an important role. Here we report that in the brain of APP23 mice--a transgenic model of AD--after deposition of amyloid in blood vessels endothelial cell activation occurs in an age-dependent manner. Amyloid deposition is followed by the expression of beta3-integrin, a specific marker molecule of activated endothelium. The beta3-integrin expression is restricted to amyloid-positive vessels. Moreover, homogenates of the brains of APP23 mice induced the formation of new vessels in an in vivo angiogenesis assay. Vessel formation could be blocked by the VEGF antagonist SU 4312 as well as by statins, suggesting that these drugs may interfere with endothelial cell activation in AD. In conclusion our results indicate that amyloid deposition in the vasculature leads to endothelial cell apoptosis and endothelial cell activation, which can be modulated by anti-angiogenic drugs.

  2. Linkage arrangement of Na,K-ATPase genes in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss).

    PubMed

    Gharbi, K; Semple, J W; Ferguson, M M; Schulte, P M; Danzmann, R G

    2004-08-01

    As part of our efforts to characterize Na,K-ATPase isoforms in salmonid fish, we investigated the linkage arrangement of genes coding for the alpha and beta-subunits of the enzyme complex in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss). Genetic markers were developed from four of five previously characterized alpha-subunit isoforms (alpha1b, alpha1c, alpha2 and alpha3) and four expressed sequence tags derived from yet undescribed beta-subunit isoforms (beta1a, beta1b, beta3a and beta3b). Sex-specific linkage analysis of polymorphic loci in a reference meiotic panel revealed that Na,K-ATPase genes are generally dispersed throughout the rainbow trout genome. A notable exception was the colocalization of two alpha-subunit genes and one beta-subunit gene on linkage group RT-12, which may thus share a conserved orthologous segment with linkage group 1 in zebrafish (Danio rerio). Consistent with previously reported homeologous relationships among the chromosomes of the rainbow trout, primers designed from the alpha3-isoform detected a pair of duplicated genes on linkage groups RT-27 and RT-31. Similarly, the evolutionary conservation of homeologous regions on linkage groups RT-12 and RT-16 was further supported by the map localization of gene duplicates for the beta1b isoform. The detection of homeologs within each gene family also raises the possibility that novel isoforms may be discovered as functional duplicates. PMID:15265073

  3. Genetic maps of polymorphic DNA loci on rat chromosome 1

    SciTech Connect

    Ding, Yan-Ping; Remmers, E.F.; Longman, R.E.

    1996-09-01

    Genetic linkage maps of loci defined by polymorphic DNA markers on rat chromosome 1 were constructed by genotyping F2 progeny of F344/N x LEW/N, BN/SsN x LEW/N, and DA/Bkl x F344/Hsd inbred rat strains. In total, 43 markers were mapped, of which 3 were restriction fragment length polymorphisms and the others were simple sequence length polymorphisms. Nineteen of these markers were associated with genes. Six markers for five genes, {gamma}-aminobutyric acid receptor {beta}3 (Gabrb3), syntaxin 2 (Stx2), adrenergic receptor {beta}3 (Gabrb3), syntaxin 2 (Stx2), adrenergic receptor {beta}1 (Adrb1), carcinoembryonic antigen gene family member 1 (Cgm1), and lipogenic protein S14 (Lpgp), and 20 anonymous loci were not previously reported. Thirteen gene loci (Myl2, Aldoa, Tnt, Igf2, Prkcg, Cgm4, Calm3, Cgm3, Psbp1, Sa, Hbb, Ins1, and Tcp1) were previously mapped. Comparative mapping analysis indicated that the large portion of rat chromosome 1 is homologous to mouse chromosome 7, although the homologous to mouse chromosome 7, although the homologs of two rat genes are located on mouse chromosomes 17 and 19. Homologs of the rat chromosome 1 genes that we mapped are located on human chromosomes 6, 10, 11, 12, 15, 16, and 19. 38 refs., 1 fig., 3 tabs.

  4. 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.

  5. Pressure-induced dissociation and denaturation of allophycocyanin at subzero temperatures.

    PubMed

    Foguel, D; Weber, G

    1995-12-01

    The thermodynamics of assembly of the allophycocyanin hexamer was examined employing hydrostatic pressures in the range of 1 bar to 2.4 kbar and temperatures of 20 to -12 degrees C, the latter made possible by the decrease of the freezing point of water under pressure. The existence of two processes, dissociation of the hexamer into dimers, (alpha beta)3-->3 (alpha beta), and dissociation of the alpha beta dimers into monomers, (alpha beta)-->alpha + beta have been recognized previously by changes in the absorbance and fluorescence of the tetrapyrrolic chromophores owing to added ligands. The same changes are observed in the absence of ligands at pressures of under 2.4 kbar and temperatures down to -12 degrees C. On decompression from 2.4 kbar at 0 degrees C, appreciable hysteresis and a persistent loss of 50% in the absorbance at 653 nm is observed. It results from the conformational drift of the isolated subunits and is reduced to 10% when the highest pressure is limited to 1.6 kbar. The thermodynamic parameters of the reaction alpha + beta-->alpha beta can be determined from pressure effects on perchlorate solutions of allophycocyanin, which consist of dimers alone. Their previous knowledge permits estimation, under suitable hypotheses, of the thermodynamic parameters of the reaction 3(alpha beta)-->(alpha beta)3 from the overall pressure effects on the hexamers. Both association reactions have positive enthalpy changes, and the whole hexamer assembly is made possible by the excess entropy.

  6. 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

  7. 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

  8. The interaction of the transforming growth factor-betas with heparin/heparan sulfate is isoform-specific.

    PubMed

    Lyon, M; Rushton, G; Gallagher, J T

    1997-07-18

    We have undertaken a comparative study of the interaction of the three mammalian transforming growth factor-betas (TGF-beta) with heparin and heparan sulfate. TGF-beta1 and -beta2, but not -beta3, bind to heparin and the highly sulfated liver heparan sulfate. These polysaccharides potentiate the biological activity of TGF-beta1 (but not the other isoforms), whereas a low sulfated mucosal heparan sulfate fails to do so. Potentiation is due to antagonism of the binding and inactivation of TGF-beta1 by alpha2-macroglobulin, rather than by modulation of growth factor-receptor interactions. TGF-beta2.alpha2-macroglobulin complexes are more refractory to heparin/heparan sulfate, and those involving TGF-beta3 cannot be affected. Comparison of the amino acid sequences of the TGF-beta isoforms strongly implicates the basic amino acid residue at position 26 of each monomer as being a vital binding determinant. A model is proposed in which polysaccharide binding occurs at two distinct sites on the TGF-beta dimer. Interaction with heparin and liver heparan sulfate may be most effective because of the ability of the dimer to co-operatively engage two specific sulfated binding sequences, separated by a distance of approximately seven disaccharides, within the same chain.

  9. Subunit composition of G(o) proteins functionally coupling galanin receptors to voltage-gated calcium channels.

    PubMed Central

    Kalkbrenner, F; Degtiar, V E; Schenker, M; Brendel, S; Zobel, A; Heschler, J; Wittig, B; Schultz, G

    1995-01-01

    The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin-induced inhibition of voltage-gated Ca2+ channels in the rat pancreatic B-cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels. Images PMID:7588602

  10. 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

  11. 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

  12. Neurological morphofunctional differentiation induced by REAC technology in PC12. A neuro protective model for Parkinson's disease.

    PubMed

    Maioli, Margherita; Rinaldi, Salvatore; Migheli, Rossana; Pigliaru, Gianfranco; Rocchitta, Gaia; Santaniello, Sara; Basoli, Valentina; Castagna, Alessandro; Fontani, Vania; Ventura, Carlo; Serra, Pier Andrea

    2015-01-01

    Research for the use of physical means, in order to induce cell differentiation for new therapeutic strategies, is one of the most interesting challenges in the field of regenerative medicine, and then in the treatment of neurodegenerative diseases, Parkinson's disease (PD) included. The aim of this work is to verify the effect of the radio electric asymmetric conveyer (REAC) technology on the PC12 rat adrenal pheochromocytoma cell line, as they display metabolic features of PD. PC12 cells were cultured with a REAC regenerative tissue optimization treatment (TO-RGN) for a period ranging between 24 and 192 hours. Gene expression analysis of specific neurogenic genes, as neurogenin-1, beta3-tubulin and Nerve growth factor, together with the immunostaining analysis of the specific neuronal protein beta3-tubulin and tyrosine hydroxylase, shows that the number of cells committed toward the neurogenic phenotype was significantly higher in REAC treated cultures, as compared to control untreated cells. Moreover, MTT and Trypan blue proliferation assays highlighted that cell proliferation was significantly reduced in REAC TO-RGN treated cells. These results open new perspectives in neurodegenerative diseases treatment, particularly in PD. Further studies will be needed to better address the therapeutic potential of the REAC technology. PMID:25976344

  13. Neurological morphofunctional differentiation induced by REAC technology in PC12. A neuro protective model for Parkinson’s disease

    PubMed Central

    Maioli, Margherita; Rinaldi, Salvatore; Migheli, Rossana; Pigliaru, Gianfranco; Rocchitta, Gaia; Santaniello, Sara; Basoli, Valentina; Castagna, Alessandro; Fontani, Vania; Ventura, Carlo; Serra, Pier Andrea

    2015-01-01

    Research for the use of physical means, in order to induce cell differentiation for new therapeutic strategies, is one of the most interesting challenges in the field of regenerative medicine, and then in the treatment of neurodegenerative diseases, Parkinson’s disease (PD) included. The aim of this work is to verify the effect of the radio electric asymmetric conveyer (REAC) technology on the PC12 rat adrenal pheochromocytoma cell line, as they display metabolic features of PD. PC12 cells were cultured with a REAC regenerative tissue optimization treatment (TO-RGN) for a period ranging between 24 and 192 hours. Gene expression analysis of specific neurogenic genes, as neurogenin-1, beta3-tubulin and Nerve growth factor, together with the immunostaining analysis of the specific neuronal protein beta3-tubulin and tyrosine hydroxylase, shows that the number of cells committed toward the neurogenic phenotype was significantly higher in REAC treated cultures, as compared to control untreated cells. Moreover, MTT and Trypan blue proliferation assays highlighted that cell proliferation was significantly reduced in REAC TO-RGN treated cells. These results open new perspectives in neurodegenerative diseases treatment, particularly in PD. Further studies will be needed to better address the therapeutic potential of the REAC technology. PMID:25976344

  14. Mechanisms involved in the blood-testis barrier increased permeability induced by EMP.

    PubMed

    Wang, Xiao-Wu; Ding, Gui-Rong; Shi, Chang-Hong; Zeng, Li-Hua; Liu, Jun-Ye; Li, Jing; Zhao, Tao; Chen, Yong-Bin; Guo, Guo-Zhen

    2010-09-30

    The blood-testis barrier (BTB) plays an important role in male reproductive system. Lots of environmental stimulations can increase the permeability of BTB and then result in antisperm antibody (AsAb) generation, which is a key step in male immune infertility. Here we reported the results of male mice exposed to electromagnetic pulse (EMP) by measuring the expression of tight-junction-associated proteins (ZO-1 and Occludin), vimentin microfilaments, and transforming growth factor-beta (TGF-beta3) as well as AsAb level in serum. Male BALB/c mice were sham exposed or exposed to EMP at two different intensities (200kV/m and 400kV/m) for 200 pulses. The testes were collected at different time points after EMP exposure. Immunofluorescence histocytochemistry, western blotting, laser confocal microscopy and RT-PCR were used in this study. Compared with sham group, the expression of ZO-1 and TGF-beta3 significantly decreased accompanied with unevenly stained vimentin microfilaments and increased serum AsAb levels in EMP-exposed mice. These results suggest a potential BTB injury and immune infertility in male mice exposed to a certain intensity of EMP.

  15. alpha- and beta-adrenergic receptor mechanisms in spontaneous contractile activity of rat ileal longitudinal smooth muscle.

    PubMed

    Seiler, Roland; Rickenbacher, Andreas; Shaw, Sidney; Balsiger, Bruno M

    2005-02-01

    Gastrointestinal motility is influenced by adrenergic modulation. Our aim was to identify specific subtypes of adrenergic receptors involved in inhibitory mechanisms that modulate gut smooth muscle contractile activity. Muscle strips of rat ileal longitudinal muscle were evaluated for spontaneous contractile activity and for equimolar dose-responses (10(-7) to 3 x 10(-5) M) to the adrenergic agents norepinephrine (nonselective agonist), phenylephrine (alpha(1)-agonist), clonidine (alpha(2)-agonist), prenalterol (beta(1)-agonist), ritodrine (beta(2)-agonist), and ZD7114 (beta(3)-agonist) in the presence and absence of tetrodotoxin (nonselective nerve blocker). Norepinephrine (3 x 10(-5) M) inhibited 65 +/- 6% (mean +/- SEM) of spontaneous contractile activity. The same molar dose of ritodrine, phenylephrine, or ZD7114 resulted in less inhibition (46 +/- 7%, 31 +/- 5%, and 39 +/- 3%, respectively; P < 0.05). The calculated molar concentration of ZD7114 needed to induce 50% inhibition was similar to that of norepinephrine, whereas higher concentrations of phenylephrine or ritodrine were required. Clonidine and prenalterol had no effect on contractile activity. Blockade of intramural neural transmission by tetrodotoxin affected the responses to ritodrine and phenylephrine (but not to norepinephrine or ZD7114), suggesting that these agents exert part of their effects via neurally mediated enteric pathways. Our results suggest that adrenergic modulation of contractile activity in the rat ileum is mediated primarily by muscular beta(3)-, beta(2)-, and alpha(1)-receptor mechanisms; the latter two also involve neural pathways. PMID:15694819

  16. Relationship between food intake and metabolic rate in rats treated with beta-adrenoceptor agonists.

    PubMed

    Yamashita, J; Onai, T; York, D A; Bray, G A

    1994-06-01

    The purpose of the present experiment was to clarify the interaction between food intake and activity of the thermogenic component of the sympathetic nervous system by studying the dose response relationships of typical beta 1-, beta 2-, and beta 3-adrenoceptor agonists on oxygen consumption and food intake. The data showed that the ED50 for the effects of Dobutamine (beta 1), Clenbuterol (beta 2) and ICI D7114 (beta 3) agonists on food intake were 8.9, 0.041 and > 1.1 mumol/kg respectively whereas the ED50 values for stimulation of metabolic rate were 7.1, 1.3 and 0.11 mumole/kg respectively. The marked differences in ED50 values for suppression of food intake and stimulation of metabolic rate for both clenbuterol and ICI D7114 suggest that the regulation of feeding and of metabolic rate in response to these agonists are independent of each other. The experiments also identified that Clenbuterol activated a very sensitive beta 2-adrenergic system for the regulation of feeding behavior. PMID:7915938

  17. Stimulation of nonshivering thermogenesis in the Syrian hamster by norepinephrine and beta-selective adrenergic agents: a phenomenon of refractoriness.

    PubMed

    Dicker, A; Cannon, B; Nedergaard, J

    1996-01-01

    The ability of different adrenergic agents to stimulate nonshivering thermogenesis in Syrian hamsters was investigated. The hamsters were cold-acclimated to 6 degrees C and their thermogenic response was investigated in an open-circuit system at 24 degrees C. Both norepinephrine and the beta 3-specific adrenergic agonist CGP-12177 induced a high rate of nonshivering thermogenesis. However, neither CGP-12177 nor other beta 3-selective agonists (BRL-37344, ICI-D7114) could induce nonshivering thermogenesis fully to the extent induced by norepinephrine. It was further observed that an apparent "thermogenic refractoriness" was induced by certain adrenergic agents (isoprenaline, CGP-12177) but not by others (norepinephrine, BRL-37344, ICI-D7114). It is discussed whether the refractoriness could be secondary to effects of these agents on the vascular system. It is pointed out that the thermogenic response to adrenergic stimulation observed in the intact animal does not always fully correspond to what would be predicted from corresponding studies with isolated brown-fat cells. PMID:8665400

  18. THE ASSOCIATION BETWEEN PRE-PREGNANCY OBESITY AND WEIGHT GAIN IN PREGNANCY, WITH GROWTH DEVIATIONS IN NEWBORNS.

    PubMed

    Camacho-Buenrostro, Dennis; Pérez-Molina, J Jesús; Vásquez-Garibay, Edgar M; Panduro-Barón, J Guadalupe

    2015-07-01

    Introducción: la obesidad en el embarazo se ha relacionado con mayor morbilidad para la madre y el feto. Objetivo: cuantificar la asociación entre obesidad en el embarazo con desviaciones del crecimiento de sus recién nacidos. Métodos: se realizó un estudio de casos y controles, no pareado, basado en el Nuevo Hospital Civil de Guadalajara “Dr. Juan I Menchaca” de 2012 a 2013. Las variables dependientes fueron recién nacido grande (GEG) y pequeño para edad gestacional (PEG), y la independiente obesidad pre-gestacional. Se recabaron datos socioeconómicos y ginecoobstétricos. La asociación entre las variables dependientes con las independientes, se evaluó con regresión logística. Resultados: se estudiaron 143 díadas madre-hijo con desviaciones en el crecimiento de sus RN, y 137 díadas madre-hijo sin desviaciones. La edad de las embarazadas fue 24.7 ± 6.3 vs 24.0 ± 6.0 años, y la edad gestacional 38±1.2 vs 38±1.5. Los factores asociados con desviaciones en el crecimiento fueron: obesidad pre-gestacional (RM 2.65, IC95% 1.29-5.44), ganancia de peso durante el embarazo elevada (RM 1.98, IC95% 1.04-3.76) y enfermedades durante el embarazo (RM 2.62, IC95% 1.05-6.76). Un modelo multivariado, con la variable dependiente GEG, y las covariables asociadas, demostró que la obesidad pregestacional y ganancia de peso gestacional elevada fueron predictores de GEG (RM 2.43, IC95% 1.10- 5.40) y (RM 3.31, IC95% 1.83-5.96). Conclusiones: en una población de mujeres jóvenes de escasos recursos económicos, la obesidad pregestacional y la ganancia de peso durante el embarazo alta, fueron predictores de productos GEG.

  19. [Effect of nutrition on growth and neurodevelopment in the preterm infant: a systematic review].

    PubMed

    Aguilar Cordero, María José; Sánchez López, A M; Mur Villar, N; Hermoso Rodríguez, E; Latorre García, J

    2014-10-31

    Introducción: Las necesidades energéticas de los bebés pretérmino son elevadas y se incrementan en la medida en que el peso corporal es menor; para ello, es importante el aporte calórico óptimo, como garantía del desarrollo posterior. Objetivo: Analizar los principales artículos relacionados con la nutrición del recién nacido pretérmino y los efectos en su crecimiento y desarrollo. Método: Revisión sistemática, con etapas dirigidas a la selección de los estudios y estrategia de búsqueda y entre los meses de marzo y septiembre del año 2014. Para la búsqueda bibliográfica se siguieron los métodos: Análisis de documentos y síntesis de la información para hacer posible la ordenación y la combinación de la información extractada, así como una evaluación comparativa. La validez de los artículos seleccionados estuvo dada por el grado de evidencias demostrado, por las recomendaciones del artículo y por la aplicabilidad a nuestro contexto. Resultados: El proceso de búsqueda permitió seleccionar 79 estudios que cumplieron con los criterios de selección. Se trata de un tema ampliamente tratado por la literatura y se constata su frecuencia en los estudios referidos a la nutrición del prematuro a corto plazo. Se evidencia la relación directa entre la ingesta nutricional y el crecimiento de la longitud de los recién nacidos prematuros. Conclusiones: Una nutrición adecuada del recién nacido pretérmino tiene efectos positivos en su crecimiento y neurodesarrollo. Se constata que, a mayor ingesta de proteínas y lípidos, la altura de los bebés prematuros resulta favorecida, pero no el peso corporal. Los estudios denotan el efecto beneficioso de la leche materna sobre el cerebro, la retina y los vasos arteriales, pero una correlación negativa entre la adiposidad y el volumen cerebral.

  20. [Prevalence of overweight and obesity in children from Monterrey, Nuevo León].

    PubMed

    Hernández-Herrera, Ricardo Jorge; Mathiew-Quirós, Álvaro; Díaz-Sánchez, Oscar; Reyes-Treviño, Norma Orelia; Álvarez-Álvarez, Criselda; Villanueva-Montemayor, Daniel; de la Garza-Salinas, Laura Hermila; González-Guajardo, Eduardo

    2014-01-01

    INTRODUCCIÓN: la obesidad infantil es un problema de salud pública mundial y se ha incrementado en forma alarmante en las últimas décadas hasta en 30 % en el 2010. El objetivo de este estudio fue establecer la prevalencia de sobrepeso y obesidad en niños de cero a 14 años de tres unidades de medicina familiar (UMF). MÉTODOS: estudio prospectivo, de prevalencia, que incluyó a niños de ambos sexos que acudieron a tres UMF y una escuela primaria. Se evaluó la somatometría y se comparó de acuerdo con estándares y referencias de crecimiento de la Organización Mundial de la Salud (OMS) 2006-2007.

  1. [Increased height in obese schoolchildren versus healthy weight schoolchildren].

    PubMed

    Larrosa-Haro, Alfredo; González-Pérez, Guillermo Julián; Vásquez-Garibay, Edgar Manuel; Romero-Velarde, Enrique; Chávez-Palencia, Clío; Salazar-Preciado, Laura Leticia; Lizárraga-Corona, Elizabeth

    2014-01-01

    IBTRODUCCIÓN: la asociación entre crecimiento y obesidad ha sido poco estudiada en la población de escolares obesos mexicanos. El objetivo de este estudio fue comparar la talla para la edad entre escolares obesos y con peso saludable. MÉTODOS: estudio transversal realizado en 369 escolares con peso saludable y 162 obesos en una escuela primaria; 49.4 % era de sexo femenino. Se utilizó la clasificación percentilar del Índice de masa corporal: peso saludable: del percentil 5 al 84, obesidad: valor > percentil 95; se agruparon por sexo e intervalos de clase de un año. El análisis estadístico se hizo con U de Mann-Whitney.

  2. [SOCIODEMOGRAPHIC AND HEALTH CHARACTERISTICS ASSOCIATED WITH LOW BIRTH WEIGHT IN THE CANARY ISLANDS].

    PubMed

    Bello Lujan, Luis M; Saavedra Santana, Pedro; Gutiérrez García, Luisa Esther; García Hernández, José Angel; Serra Majem, Lluis

    2015-10-01

    Introducción y objetivo: desde 1976, el término bajo peso al nacer (BPN) se aplica a todos los recién nacidos con un peso inferior a 2.500 g. y constituye el factor más importante que afecta a la mortalidad neonatal y a la morbilidad en la infancia. El objetivo de este trabajo es identificar las asociaciones entre los factores biológicos, socioeconómicos y sanitarios y el bajo peso en el recién nacido en las islas Canarias. Material y método: se realizó un estudio epidemiológico transversal, con los ficheros de las gestantes del 2011 y 2012 (n = 11,768) del Complejo Hospitalario Universitario Insular Materno de Gran Canaria, que representó el 66,3% de todos los partos de la provincia para esos años, excluyéndose del análisis los datos correspondientes a los embarazos múltiples (393) (3,3%). Se analizaron la distribución y la frecuencia del peso según características sociodemográficas; los porcentajes se compararon con el test de la 2, las medias con el t-test y las medianas con el test de Wilcoxon para datos independientes. Aquellas variables que mostraron asociación con el bajo peso en el análisis univariado fueron introducidas en un análisis logístico multidimensional. Resultados: en la distribución del peso al nacer se observó un exceso de niños con bajo peso (9,3%), de los cuales el 62,1% fueron pretérmino. Las madres de estos niños son más delgadas, de menor talla y tienen un IMC promedio de < 18,5 kg/m2, así como un promedio de edad de 31,5 años. Se observó igualmente que existe un incremento entre la edad de la madre y el bajo peso, aunque el efecto no es lineal; de hecho, los aumentos del riesgo de BPN se aceleran a medida que aumenta la edad. Cuando el IMC de la madre decrece, aproximadamente por debajo de 25, aumenta linealmente el riesgo de bajo peso. Las OR más elevadas corresponden al retraso fetal en el crecimiento intrauterino (CIR) (OR = 6,3; IC-95% = 5,3; 7,4), la hipertensión/eclampsia (OR = 3,2; IC-95% = 2

  3. 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 :

  4. [The height target prediction by the Tanner method infra evaluates the final height in youths from the rural area of South East Spain].

    PubMed

    Ríos, Rafael; Bosch, Vicente; Santonja, Fernando; López, José Manuel; Garaulet, Marta

    2014-10-16

    Introducción: Conocer la talla final de un individuo antes de finalizar el crecimiento presenta utilidad clínica para el seguimiento de la salud infantil. Objetivo: Calcular la talla diana de una población rural del sudeste de España y comparar con la talla final alcanzada. Métodos: Fueron incluidos 50 jóvenes de 18 a 22 años (44% hombres) y 100 progenitores. La selección de los jóvenes se realizó en 2 fases: 1. Estudio retrospectivo a partir de historias clínicas. 2. Estudio prospectivo: reclutamiento y determinaciones antropométricas. Se calculó talla diana y el desvío de talla. Resultados: La talla final de los chicos fue de 4,44 cm superior a la talla diana (p.

  5. 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

  6. Porous tricalcium phosphate and transforming growth factor used for anterior spine surgery.

    PubMed

    Steffen, T; Stoll, T; Arvinte, T; Schenk, R K

    2001-10-01

    Harvesting autologous bone graft from the iliac crest is associated with considerable secondary morbidity. Bone graft substitutes such as porous ceramics are increasingly used for spinal surgery. This paper presents the results of an animal study in which beta-tricalcium phosphate (beta-TCP) bone substitutes were used for anterior spinal surgery in sheep and baboons. The presented baboon study also investigated the effect of impregnating the ceramic material with transforming growth factor (TGF). In the first study, using the sheep model, a stand-alone instrumented anterior fusion was performed. The animals were randomized into three treatment groups: autologous bone, beta-TCP granules, and sham group. The results were analyzed biomechanically and histologically at three survival intervals: 8, 16 and 32 weeks. An additional animal group was added later, with ceramic pre-filled implants. In the second study, a baboon model was used to assess the osteointegration of a 15-mm-diameter porous beta-TCP block into the vertebral body. The experiment was partially motivated by a new surgical procedure proposed for local bone graft harvest. Three treatment groups were used: beta-TCP plug, beta-TCP plug impregnated with TGF-beta3, and a sham group with empty defect. The evaluation for all animals included computer tomograms at 3 and 6 months, as well as histology at 6 months. In the sheep model, the mechanical evaluation failed to demonstrate differences between treatment groups. This was because massive anterior bone bridges formed in almost all the animals, masking the effects of individual treatments. Histologically, beta-TCP was shown to be a good osteoconductor. While multiple signs of implant micromotion were documented, pre-filling the cages markedly improved the histological fusion outcomes. In the baboon study, the beta-TCP plugs were completely osteointegrated at 6 months. For the group that used ceramic plugs impregnated with TGF-beta3, no incremental advantage was

  7. Simultaneous quantification of GABAergic 3alpha,5alpha/3alpha,5beta neuroactive steroids in human and rat serum.

    PubMed

    Porcu, Patrizia; O'Buckley, Todd K; Alward, Sarah E; Marx, Christine E; Shampine, Lawrence J; Girdler, Susan S; Morrow, A Leslie

    2009-01-01

    The 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography-mass spectrometry to simultaneously identify serum levels of the eight 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3alpha,5alpha- and 3alpha,5beta-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3alpha,5alpha-THP (+1488%, p<0.001), (3alpha,5alpha)-3,21-dihydroxypregnan-20-one (3alpha,5alpha-THDOC, +205%, p<0.01), (3alpha,5alpha)-3-hydroxyandrostan-17-one (3alpha,5alpha-A, +216%, p<0.001), (3alpha,5alpha,17beta)-androstane-3,17-diol (3alpha,5alpha-A-diol, +190%, p<0.01). (3alpha,5beta)-3-hydroxypregnan-20-one (3alpha,5beta-THP) and (3alpha,5beta)-3-hydroxyandrostan-17-one (3alpha,5beta-A) were not altered, while (3alpha,5beta)-3,21-dihydroxypregnan-20-one (3alpha,5beta-THDOC) and (3alpha,5beta,17beta)-androstane-3,17-diol (3alpha,5beta-A-diol) were increased from undetectable levels to 271+/-100 and 2.4+/-0.9 pg+/-SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3alpha,5alpha-THP (+1806%, p<0.0001), 3alpha,5beta-THP (+575%, p<0.001), 3alpha,5alpha

  8. 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

  9. Pharmacological study of atypical beta-adrenoceptors in rat esophageal smooth muscle.

    PubMed

    Lezama, E J; Konkar, A A; Salazar-Bookaman, M M; Miller, D D; Feller, D R

    1996-07-11

    The chemical specificity for the beta-adrenoceptor mediated relaxation of rat esophageal smooth muscle was evaluated using selective and non-selective beta-adrenoceptor agonists and antagonists. Pindolol, ICI 89,406, ICI 118551 [erythro-1-(7-methylindan-4-yloxy)-3-(isopropylamine)-but an-2-ol] and the beta-adrenoceptor alkylating agent, pindobind, produced only small rightward shifts in the concentration-response curves of (-)-isoprenaline and (-)-trimetoquinol in this preparation. Rank order potency (pD2 values) of agonists was: (+/-)-trimetoquinol [1-(3',4',5'-trimethoxybenzyl)-6,7-dihydroxy-1,2,3, 4-tetrahydroisoquinoline] (8.34) = (-)-trimetoquinol (8.26) = BRL 37344 [(R* R*)-(+/-)-4-[2'-2-hydroxy 2-(3-chlorophenyl)ethylamino]propyl] phenoxyacetic acid] (8.16) = ICID7114 [(S)-4-(2-hydroxy- 3-phenoxy-propylamino-ethoxy)-N-(2-methoxyethyl) phenoxyacetamide] (8.03) > or = (-)-isoprenaline (7.82) > 3',5'-diiodotrimetoquinol [1-(3',5'-diiodo-4'-methoxybenzyl)-6, 7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] (7.28) > 3'-iodotrimetoquinol [1-(3'-iodo-4',5'-dimethoxybenzyl)-6, 7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] (7.04) > ractopamine (6.84) = 5,8-difluorotrimetoquinol [5,8-difluoro-6,7-dihydroxy-1- (3',4',5'-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline] (6.82) > 8-fluorotrimetoquinol [6,7-dihydroxy-8-fluoro-1-(3',4',5'- trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline] (6.56) > or = (-)-noradrenaline (6.46) > or = (-)-adrenaline (6.36) > (+/-)-noradrenaline (6.24) > (+/-)-adrenaline (6.00) > clenbuterol (5.83) > (-)-1-benzyl-6, 7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (5.75). Isomeric activity ratios of trimetoquinol isomers [(-)-(S)- > > (+)-(R)-] in esophageal smooth muscle in the presence and absence of 1 microM pindolol were 1995- and 2951-fold, respectively; and were much greater than those in rat atria (282-fold) and rat trachea (107-fold). The atypical beta/beta 3-adrenoceptor partial agonist, ICI D7114, produced concentration-dependent rightward

  10. 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

  11. [Exposition to drugs of abuse in pregnancy and breastfed babies growth in CONIN Valparaíso, Chile].

    PubMed

    Piñuñuri, Raúl; Mardones, Constanza; Valenzuela, Carina; Estay, Pamela; Llanos, Miguel

    2015-05-01

    Introducción: Las consecuencias de la exposición fetal a drogas de abuso en niños han sido estudiadas extensamente. El presente estudio pretende describir la realidad chilena en esta materia, caracterizando el crecimiento de niños expuestos a drogas durante la vida fetal. Objetivo: Comparar la antropometría entre neonatos expuestos a drogas por consumo materno durante el embarazo y controles no expuestos, de los 0 a 6 meses de vida. Materiales y métodos: Se obtuvieron los datos antropométricos desde el nacimiento y hasta los 6 meses de vida de 74 individuos sanos atendidos en Centros de Salud de la comuna de Valparaíso, y de 61 individuos con antecedentes de consumo de drogas de la madre durante el embarazo, atendidos en la Corporación para la Nutrición Infantil (CONIN). Posteriormente se compararon las diferencias entre ambos grupos mediante (T-Student por grupos). Resultados: Según la edad gestacional hay diferencias en la proporción de individuos de pretérmino entre grupos de estudio, observándose una prevalencia de menos del 11% en el grupo no expuesto y superior al 25% en el grupo expuesto. En base a las curvas de los Dres. Pittaluga y Alarcón para crecimiento intrauterino, el grupo expuesto tuvo mayor cantidad de sujetos pequeños para la edad gestacional en ambos sexos, con 37% en CONIN y menos del 6% en el grupo no expuesto. El análisis estadístico muestra que existen diferencias significativas (p.

  12. Prevalence and determinants of the dual burden of malnutrition at the household level in Puna and Quebrada of Humahuaca, Jujuy, Argentina.

    PubMed

    Bassete, M N; Romaguera, D; Giménez, M A; Lobo, M O; Samman, N C

    2014-02-01

    El objetivo del presente estudio fue identificar los hogares en los que coexisten el sobrepeso y bajo peso (doble carga de malnutrición) y explorar los factores que podrían contribuir a la doble carga de malnutrición en los hogares de esta población. Después de aplicar los criterios de exclusión, se incluyeron 136 hogares. Las madres fueron clasificadas como de peso normal o con sobrepeso/ obesidad basado en puntos de corte del índice de masa corporal (IMC), los niños y adolescentes fueron clasificados según z–score de la talla para la edad, si tenían o no retraso en el crecimiento (Stunted). Los hogares con una madre obesa y niños o adolescentes con retraso en el crecimiento fueron clasificados como hogares de doble carga. La prevalencia de hogares con doble carga fue del 12 %. En comparación con los otros hogares, en los hogares con doble carga de malnutrición habitan un mayor número de personas en la casa, y el nivel de educación del jefe de familia fue menor. Las personas que viven en hogares de doble carga mostraron ingestas de energía global más bajas y eran más propensos a tener una ingesta insuficiente de calcio y hierro. Una de las principales causas de la doble carga de malnutrición observada en esta comunidad podría ser la transición nutricional. Los resultados presentados aquí indican la necesidad de considerar a los programas que se centran en un solo tipo de problema nutricional y que en realidad podrían exacerbar el otro.

  13. [Infraorbital schwannoma. Case report].

    PubMed

    Mora-Ríos, Laura Evelyn; Ríos Y Valles-Valles, Dolores; Flores-Estrada, José Javier; Rodríguez-Reyes, Abelardo Antonio

    2014-01-01

    Antecedentes: el schwannoma infraorbitario es un tumor benigno de la vaina nerviosa periférica compuesto por células de Schwann. Suele aparecer entre los 20 y 70 años de edad, asintomático y producir proptosis progresiva e indolora durante su crecimiento. Caso clínico: paciente masculino de 32 años de edad que ingresó al hospital debido a un tumor no doloroso de crecimiento lentamente progresivo sobre el saco lagrimal izquierdo. A la exploración oftalmológica el tumor era de consistencia ahulada y estaba firmemente adherido a las estructuras vecinas. La transiluminación resultó negativa. El ultrasonido modo B mostró un ojo fáquico y un tumor infraorbitario homogéneo, bien circunstrito, con diámetro mayor de 19.7 mm, sin afectación de la vía lagrimal. El ultrasonido modo A mostró una reflectividad media-alta, con escasa vascularidad interna. La tomografía computada mostró un tumor de densidad homogénea, bien circunscrito a la región anterior y por debajo del globo ocular, sin erosión ósea. El tumor se extirpó mediante una incisión subdérmica. El diagnóstico histopatológico fue: schwannoma infraorbitario. Conclusiones: el schwannoma es un tumor benigno, poco frecuente en la órbita. Su diagnóstico definitivo se establece con base en los hallazgos histopatológicos, como: cápsula verdadera, áreas hiper e hipocelulares, engrosamiento y hialinización de las paredes vasculares. Sin esos hallazgos puede confundirse con tumores fusocelulares benignos. Se informa un nuevo caso de schwannoma infraorbitario y se compara con los casos previamente reportados.

  14. [Wolf-Hirschhorn syndrome. A series of 27 patients: their epidemiological and clinical characteristics. The current situation of the patients and the opinions of their caregivers regarding the diagnostic process].

    PubMed

    Blanco-Lago, Raquel; Málaga, Ignacio; García-Peñas, Juan José; García-Ron, Adrián

    2013-07-16

    Introduccion. El sindrome de Wolf-Hirschhorn (SWH) es una cromosomopatia producida por una delecion en la region distal del brazo corto del cromosoma 4. Se caracteriza por la presencia de un fenotipo peculiar, retraso en el crecimiento, retraso del desarrollo psicomotor y epilepsia. Objetivos. Describir las caracteristicas de una serie de niños con SWH, incluido el tiempo medio empleado para el diagnostico, y valorar la opinion de las familias sobre el proceso diagnostico. Pacientes y metodos. Se contacto con la Asociacion Nacional de SWH y, a traves de ella, con 29 familias afectadas. Se recogio informacion sobre la clinica del niño y la opinion sobre el proceso diagnostico, y se solicitaron informes medicos que confirmaran la informacion facilitada. Constituida una base de datos de pacientes, se procedio a su analisis estadistico. Resultados. Se obtuvo informacion de 27 familias. Los pacientes presentan una edad media actual de 6,94 ± 6,37 años. La edad media de diagnostico fue de 14,34 meses. Existe retraso del crecimiento intrauterino en el 92,6% de los embarazos. Un 92,6% de los pacientes presenta epilepsia, el 44,4% de ellos en monoterapia. Existe retraso del desarrollo psicomotor/cognitivo en todos los pacientes. Camina sin ayuda el 33%. Los padres califican con una nota media de 7,25 ± 2,17 el trato ofrecido por los facultativos y de 6,29 ± 2,11 la informacion recibida. Conclusiones. No se han encontrado referencias a la edad media de diagnostico para el SWH. En nuestra muestra, existen variaciones importantes en este aspecto, posiblemente condicionadas por el fenotipo del caso y la experiencia del medico. Las caracteristicas clinicas son similares a las esperadas. El grado de dependencia estimado es alto y la calidad de la informacion recibida por la familia, baja.

  15. 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

  16. 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

  17. 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

  18. 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

  19. Virus binding to a plasma membrane receptor triggers interleukin-1 alpha-mediated proinflammatory macrophage response in vivo.

    PubMed

    Di Paolo, Nelson C; Miao, Edward A; Iwakura, Yoichiro; Murali-Krishna, Kaja; Aderem, Alan; Flavell, Richard A; Papayannopoulou, Thalia; Shayakhmetov, Dmitry M

    2009-07-17

    The recognition of viral components by host pattern-recognition receptors triggers the induction of the antiviral innate immune response. Toll-like receptor 9 (TLR9) and NLRP3 inflammasome were shown to be the principal specific sensors of viral double-stranded DNA. Here we present evidence that macrophages in vivo activated an innate immune response to a double-stranded DNA virus, adenovirus (Ad), independently of TLR9 or NLRP3 inflammasome. In response to Ad, macrophage-derived IL-1 alpha triggered IL-1RI-dependent production of a defined set of proinflammatory cytokines and chemokines. The IL-1 alpha-mediated response required a selective interaction of virus arginine-glycine-aspartic acid (RGD) motifs with macrophage beta(3) integrins. Thus, these data identify IL-1 alpha-IL-1RI as a key pathway allowing for the activation of proinflammatory responses to the virus, independently of its genomic nucleic acid recognition.

  20. A stereochemical examination of the equine metabolism of 17alpha-methyltestosterone.

    PubMed

    McKinney, Andrew R; Suann, Craig J; Stenhouse, Allen M

    2007-01-01

    An investigation was conducted into the stereochemistry of the equine urinary metabolites of 17alpha-methyltestosterone observed after oral administration. Standards of the complete range of C3/C5/C16 stereoisomeric 17alpha-methylandrostane-3,17beta-diols, 17alpha-methylandrostane-3,16,17beta-triols and 17alpha-hydroxymethylandrostane-3,17beta-diols were purchased or synthesised, and were used to unequivocally identify the absolute structures of the metabolites. Phase I metabolism was found to involve combinations of Delta(4)-3-ketone reduction with both 5alpha,3beta- and 5beta,3alpha-stereochemistry, hydroxylation at C16 with both 16alpha- and 16beta-stereochemistry and hydroxylation of the 17alpha-methyl substituent. Phase II metabolism involved mainly sulfation with a lesser degree of beta-glucuronidation.

  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. Characterization of digital waveforms using thermodynamic analogs: detection of contrast-targeted tissue in vivo.

    PubMed

    Hughes, Michael S; Marsh, Jon N; Zhang, Hyuing; Woodson, Adam K; Allen, John S; Lacy, Elizabeth K; Carradine, Cordelia; Lanza, Gregory M; Wickline, Samuel A

    2006-09-01

    We describe characterization of backscatter from tumor tissue targeted with a nanoparticle-based ultrasound contrast agent in vivo using analogs of thermodynamic quantities. We apply these waveform characteristics to detection of tumor neovasculature in tumors implanted in athymic nude mice, which were imaged using a research ultrasound scanner over a 2-hour period after injection of alpha upsilon beta3-targeted perfluorocarbon nanoparticles. Images were constructed from backscattered ultrasound using two different approaches: fundamental B-mode imaging and a signal receiver based on a thermodynamic analog (H(C)). The study shows that the thermodynamic analog is capable of detecting differences in backscattered signals that are not apparent with the B-mode approach.

  3. Emerging nanomedicine opportunities with perfluorocarbon nanoparticles.

    PubMed

    Winter, Patrick M; Cai, Kejia; Caruthers, Shelton D; Wickline, Samuel A; Lanza, Gregory M

    2007-03-01

    Perfluorocarbon (PFC) nanoparticles can serve as a platform technology for molecular imaging and targeted drug-delivery applications. These nanoparticles are approximately 250 nm in diameter and are encapsulated in a phospholipid shell, which provides an ideal surface for the incorporation of targeting ligands, imaging agents and drugs. For molecular imaging, PFC nanoparticles can carry very large payloads of gadolinium to detect pathological biomarkers with magnetic resonance imaging. A variety of different epitopes, including alpha(v)beta(3)-integrin, tissue factor and fibrin, have been imaged using nanoparticles formulated with appropriate antibodies or peptidomimentics as targeting ligands. Lipophilic drugs can also be incorporated into the outer lipid shell of nanoparticles for targeted delivery. Upon binding to the target cell, the drug is exchanged from the particle surfactant monolayer to the cell membrane through a novel process called 'contact facilitated drug delivery'. By combining targeted molecular imaging and localized drug delivery, PFC nanoparticles provide diagnosis and therapy with a single agent.

  4. An ellagitannin, n-butyl gallate, two aryltetralin lignans, and an unprecedented diterpene ester from Pelargonium reniforme.

    PubMed

    Latté, Klaus Peter; Kaloga, Maki; Schäfer, Andreas; Kolodziej, Herbert

    2008-02-01

    The structural diversity of the metabolic pool of Pelargonium reniforme was extended by the characterization of the 1C4-glucose based ellagitannin pelargoniin E, gallic acid n-butyl ester, (-)-4,4',9'-trihydroxy-3',5'-dimethoxy-2,7'-cyclolignan 9-O-beta-glucopyranoside and reniformin, a diterpene ester comprised of a diterpene acid with an uncommon -(CH2)(2)- bridging element linked to 2-(4-hydroxyphenyl)ethansulfonic acid. These metabolites were associated with the known (alpha,beta)-3,4-di-O-galloyl-glucopyranoside, 4,6-dihydroxy-2beta-glucopyranosyloxyacetophenone, 1-O-galloylglycerol, 6'-O-galloylsalidroside and (+)-isolariciresinol-9'-O-beta-glucopyranoside. All structures were established on the basis of spectroscopic methods.

  5. [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.

  6. 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}.

  7. Recognition of multiple antibody epitopes throughout Borrelia burgdorferi p66, a candidate adhesin, in patients with early or late manifestations of Lyme disease.

    PubMed

    Ntchobo, H; Rothermel, H; Chege, W; Steere, A C; Coburn, J

    2001-03-01

    Antibody responses to p66, a candidate integrin ligand of Borrelia burgdorferi, were studied in 79 patients with early or late manifestations of Lyme disease. The central portion of p66 was previously shown to contain all of the information required for specific recognition of beta3-chain integrins, but work by others had suggested that the C-terminal portion of the protein contains a single surface-exposed, immunodominant loop. In examining antibody responses to full-length p66 and to three overlapping fragments of the protein, we found that the majority of Lyme disease patients had immunoglobulin M (IgM) and/or IgG responses to p66 and that, particularly early in the disease, epitopes throughout p66 were recognized. Among patients with later manifestations of the illness, antibody responses to the C-terminal portion of the protein were more prominent. These results demonstrate that Lyme disease patient sera recognize epitopes throughout p66.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. Binding properties of HABA-type azo derivatives to avidin and avidin-related protein 4.

    PubMed

    Repo, Susanna; Paldanius, Tiina A; Hytönen, Vesa P; Nyholm, Thomas K M; Halling, Katrin K; Huuskonen, Juhani; Pentikäinen, Olli T; Rissanen, Kari; Slotte, J Peter; Airenne, Tomi T; Salminen, Tiina A; Kulomaa, Markku S; Johnson, Mark S

    2006-10-01

    The chicken genome encodes several biotin-binding proteins, including avidin and avidin-related protein 4 (AVR4). In addition to D-biotin, avidin binds an azo dye compound, 4-hydroxyazobenzene-2-carboxylic acid (HABA), but the HABA-binding properties of AVR4 are not yet known. Differential scanning calorimetry, UV/visible spectroscopy, and molecular modeling were used to analyze the binding of 15 azo molecules to avidin and AVR4. Significant differences are seen in azo compound preferences for the two proteins, emphasizing the importance of the loop between strands beta3 and beta4 for azo ligand recognition; information on these loops is provided by the high-resolution (1.5 A) X-ray structure for avidin reported here. These results may be valuable in designing improved tools for avidin-based life science and nanobiotechnology applications.

  13. High-beta extended MHD simulations of stellarators with Spitzer resistivity

    NASA Astrophysics Data System (ADS)

    Bechtel, Torrin

    2015-11-01

    The nonlinear, extended MHD code NIMROD is used to study high-beta, 3D magnetic topology evolution of a toroidal stellarator. The configurations under investigation derive from the geometry of the Compact Toroidal Hybrid (CTH) experiment. However, the vacuum rotational transform profile is artificially raised by modifying applied magnetic fields in an effort to examine the sensitivity of low order rational surfaces and/or magnetic islands. Finite beta plasmas are created using a volumetric heating source and temperature dependent anisotropic heat conduction and resistivity. Flux surface dependent temperature and density profiles are used for the initial condition so that Spitzer resistivity can be applied. The onset of MHD instabilities and nonlinear consequences are monitored as a function of beta as well as the fragility of the magnetic surfaces. Research supported by US DOE under grant no. DE-FG02-99ER54546.

  14. 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

  15. 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.

  16. Triterpenoids and flavonoids from celery (Apium graveolens).

    PubMed

    Zhou, Kailan; Zhao, Feng; Liu, Zhihui; Zhuang, Yulei; Chen, Lixia; Qiu, Feng

    2009-09-01

    Three new triterpenoids, 11,21-dioxo-2beta,3beta,15alpha-trihydroxyurs-12-ene-2-O-beta-D-glucopyranoside (1), 11,21-dioxo-3beta,15alpha,24-trihydroxyurs-12-ene-24-O-beta-D-glucopyranoside (2), and 11,21-dioxo-3beta,15alpha,24-trihydroxyolean-12-ene-24-O-beta-D-glucopyranoside (3), and two new flavonoids, apigenin-7-O-[2''-O-(5'''-O-feruloyl)-beta-D-apiofuranosyl]-beta-D-glucopyranoside (4) and chrysoeriol-7-O-[2''-O-(5'''-O-feruloyl)-beta-D-apiofuranosyl]-beta-d-glucopyranoside (5), were isolated from the whole plant of fresh celery (Apium graveolens), together with 10 known flavonoids. The structures of the new compounds were elucidated by analysis of spectroscopic data. The inhibitory effects of the compounds isolated on nitric oxide production in lipopolysaccaride-activated macrophages were evaluated.

  17. [Is there a correlation between S100 beta and post-concussion symptoms after mild traumatic brain injury?].

    PubMed

    Kleinert, K; Schleich, F; Biasca, N; Simmen, H P

    2010-06-01

    In the past a lot of patients suffered from post-concussive symptoms (PCS) after mild traumatic brain injury (mTBI). The present prospective study (n = 73) was intended to help predict the outcome after mTBI with blood asservation for analysis of S100 beta 3 hours after trauma. There was no statistically significant correlation be-tween PCS or even of single symptoms and elevated marker levels. Serum S100 beta appears to be a poor predictor of the outcome following mild TBI. Establishing a diagnosis of "PCS" will still be hard in future, since no objective diagnostic -method exists. The most important facts are a precise examination and a history of the patient with a negative CT scan of the head. PMID:19842078

  18. The epicardium as modulator of the cardiac autonomic response during early development.

    PubMed

    Kelder, Tim P; Duim, Sjoerd N; Vicente-Steijn, Rebecca; Végh, Anna M D; Kruithof, Boudewijn P T; Smits, Anke M; van Bavel, Thomas C; Bax, Noortje A M; Schalij, Martin J; Gittenberger-de Groot, Adriana C; DeRuiter, Marco C; Goumans, Marie-José; Jongbloed, Monique R M

    2015-12-01

    The cardiac autonomic nervous system (cANS) modulates heart rate, contraction force and conduction velocity. The embryonic chicken heart already responds to epinephrine prior to establishment of the cANS. The aim of this study was to define the regions of the heart that might participate in modulating the early autonomic response to epinephrine. Immunofluorescence analysis reveals expression of neural markers tubulin beta-3 chain and neural cell adhesion molecule in the epicardium during early development. In addition, expression of the β2 adrenergic receptor, the receptor for epinephrine, was found in the epicardium. Ex-ovo micro-electrode recordings in hearts with inhibition of epicardial outgrowth showed a significantly reduced response of the heart rate to epinephrine compared to control hearts. This study suggests a role for the epicardium as autonomic modulator during early cardiac development. PMID:26527381

  19. An acidic polysaccharide with immunological activities from the root of Paeonia lactiflora.

    PubMed

    Tomoda, M; Matsumoto, K; Shimizu, N; Gonda, R; Ohara, N; Hirabayashi, K

    1994-09-01

    An acidic polysaccharide, called peonan PA, was isolated from the root of Paeonia lactiflora PALLAS. It was homogeneous on electrophoresis and gel chromatography, and its molecular mass was estimated to be 6.0 x 10(4). Peonan PA is composed of L-arabinose: D-galactose: D-galacturonic acid in the molar ratio of 2:1:10, in addition to small amounts of O-acetyl groups and peptide moieties. About forty percent of the hexuronic acid residues in peonan PA exist as methyl esters. Reduction of carboxyl groups, methylation analysis, lithium degradation and nuclear magnetic resonance studies indicated that its main structural features involve both alpha-1,5-linked L-arabino-beta-3,6-branched D-galactan type and alpha-1,4-linked D-galacturonan type structural units. The polysaccharide exhibited remarkable reticuloendothelial system-potentiating activity in a carbon clearance test and considerable anti-complementary activity. PMID:7841934

  20. Ballistic thermal conductance of graphene ribbons.

    PubMed

    Muñoz, Enrique; Lu, Jianxin; Yakobson, Boris I

    2010-05-12

    An elastic-shell-based theory for calculating the thermal conductance of graphene ribbons of arbitrary width w is presented. The analysis of vibrational modes of a continuum thin plate leads to a general equation for ballistic conductance sigma. At low temperature, it yields a power law sigma approximately T(beta), where the exponent beta varies with the ribbon width w from beta = 1 for a narrow ribbon (sigma approximately T, as a four-channel quantum wire) to beta = (3)/(2) (sigma approximately wT(3/2)) in the limit of wider graphene sheets. The ballistic results can be augmented by the phenomenological value of a phonon mean free path to account for scattering and agree well with the reported experimental observations. PMID:20402531

  1. Phenylpyrazole insecticide photochemistry, metabolism, and GABAergic action: ethiprole compared with fipronil.

    PubMed

    Caboni, Pierluigi; Sammelson, Robert E; Casida, John E

    2003-11-19

    Ethiprole differs from fipronil, the major phenylpyrazole insecticide, only in an ethylsulfinyl substituent replacing the trifluoromethylsulfinyl moiety. This study compares their photochemistry, metabolism, action at the gamma-aminobutyric acid (GABA) receptor, and insecticidal potency. On exposure to sunlight as a thin film, ethiprole undergoes oxidation (major), reduction, and desethylsulfinylation but not desulfinylation whereas the major photoreaction for fipronil is desulfinylation. Metabolic sulfone formation is more rapid with ethiprole than fipronil in human expressed CYP3A4 in vitro and mouse brain and liver in vivo. High biological activity is observed for the sulfide, sulfoxide, sulfone, and desulfinyl derivatives in both the ethiprole and the fipronil series in GABA receptor assays (human recombinant beta3 homomer and house fly head membranes) with [(3)H]EBOB and in topical toxicity to house flies with and without the P450-inhibiting synergist piperonyl butoxide. On an overall basis, the ethiprole series is very similar in potency to the fipronil series.

  2. A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae.

    PubMed

    Pineda-Lucena, Antonio; Liao, Jack C C; Cort, John R; Yee, Adelinda; Kennedy, Michael A; Edwards, Aled M; Arrowsmith, Cheryl H

    2003-05-01

    As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by ORF YML108W from Saccharomyces cerevisiae. YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown. Moreover, this protein does not have sequence similarity to any other protein. The NMR structure of YML108W consists of a four-stranded beta-sheet with strand order 2143 and two alpha-helices, with an overall topology of betabetaalphabetabetaalpha. Strand beta1 runs parallel to beta4, and beta2:beta1 and beta4:beta3 pairs are arranged in an antiparallel fashion. Although this fold belongs to the split betaalphabeta family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds.

  3. ICI D7114: a novel selective adrenoceptor agonist of brown fat and thermogenesis.

    PubMed

    Holloway, B R; Howe, R; Rao, B S; Stribling, D

    1992-01-01

    Increasing energy expenditure by treatment with thermogenic drugs is not new, but available drugs have suffered from the problem of lack of selectivity. In the last decade two key findings have allowed the development of selective thermogenic drugs that have promise in the treatment of obesity. 1) The recognition that brown adipose tissue (BAT) plays a role in compensatory increases in energy expenditure has allowed an approach directed at a target organ. 2) The demonstration showing that increases in the activity of BAT may be modulated by an atypical (beta 3) adrenoceptor has led to the development of a new peripherally acting beta-adrenoceptor agonist ICI D7114, which stimulates thermogenesis at doses that have little effect on beta 1 or beta 2 adrenoceptors. Treatment with the compound activates BAT and thermogenesis even in species and situations where the intrinsic capacity is low. 3) The compound has beneficial effects in animal models of obesity and disturbed glucose and lipid homeostasis. PMID:1345891

  4. The effects of thermogenic agents on hindlimb oxygen consumption in the dog: ICI D7114 and noradrenaline.

    PubMed

    Gainer, J L; Briscoe, M G; Holloway, B R

    1993-01-15

    The thermogenic action of beta-adrenoceptor agonists may be due, in part, to increased metabolism in skeletal muscle. Previous results suggest that vasoconstriction is also necessary, and that the effect can be blocked by vasodilators. Both noradrenaline and the beta-3 agonist, ICI D7114, were studied using two dog hindlimb protocols. During constant perfusion conditions, ICI D7114 caused a significant increase in hindlimb oxygen consumption although it is a vasodilator. Noradrenaline resulted in a smaller rise in oxygen consumption, and produced a marked vasoconstriction. Both noradrenaline and ICI D7114 resulted in decreased oxygen consumption when the blood flow was allowed to vary in response to the drug treatment. The results suggest that changes in tissue oxygen consumption caused by beta-agonists are not related to vasomotion. PMID:8094057

  5. 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

  6. 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

  7. 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

  8. 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

  9. N-containing sugars from Morus alba and their glycosidase inhibitory activities.

    PubMed

    Asano, N; Oseki, K; Tomioka, E; Kizu, H; Matsui, K

    1994-06-17

    The reexamination of N-containing sugars from the roots of Morus alba by improved purification procedures led to the isolation of eighteen N-containing sugars, including seven that were isolated from the leaves of Morus bombycis. These N-containing sugars are 1-deoxynojirimycin (1), N-methyl-1-deoxynojirimycin (2), fagomine (3), 3-epi-fagomine (4), 1,4-dideoxy-1,4-imino-D-arabinitol (5), 1,4-dideoxy-1,4-imino-D-ribitol (6), calystegin B2 (1 alpha,2 beta,3 alpha,4 beta-tetrahydroxy-nor-tropane, 7), calystegin C1 (1 alpha,2 beta,3 alpha,4 beta,6 alpha-pentahydroxy-nor-tropane, 8), 1,4-dideoxy-1,4-imino-(2-O-beta-D-glucopyranosyl)-D-arabinitol (9), and nine glycosides of 1. These glycosides consist of 2-O- and 6-O-alpha-D-galactopyranosyl-1-deoxynojirimycins (10 and 11, respectively), 2-O-, 3-O- and 4-O-alpha-D-glucopyranosyl-1-deoxynojirimycins (12, 13, and 14, respectively), and 2-O-, 3-O-, 4-O- and 6-O-beta-D-glucopyranosyl-1-deoxynojirimycins (15, 16, 17, and 18, respectively). Compound 4 is a new member of polyhydroxylated piperidine alkaloids, and the isolation of 6 is the first report of its natural occurrence. It has recently been found that the polyhydroxy-nor-tropane alkaloids possess potent glycosidase inhibitory activities. Calystegin A3 is the trihydroxy-nor-tropane, and calystegins B1 and B2 are the tetrahydroxy-nor-tropane. Calystegin C1, a new member of calystegins, is the first naturally occurring pentahydroxy-nor-tropane alkaloid. The inhibitory activities of these compounds were investigated against rat digestive glycosidases and various commercially available glycosidases.

  10. TGF-beta/extracellular matrix interactions in dentin matrix: a role in regulating sequestration and protection of bioactivity.

    PubMed

    Baker, S M; Sugars, R V; Wendel, M; Smith, A J; Waddington, R J; Cooper, P R; Sloan, A J

    2009-07-01

    TGF-beta isoforms sequestrated in dentin matrix potentially provide a reservoir of bioactive molecules that may influence cell behavior in the dentin-pulp complex following tissue injury. The association of these growth factors with dentin matrix and the influence of such associations on the bioactivity of growth factors are still unclear. We used surface plasmon resonance technology in the BIAcore 3000 system to investigate the binding of TGF-beta isoforms 1 and 3 to purified decorin, biglycan, and EDTA soluble dentin matrix components. TGF-beta isoforms 1 and 3 were immobilized on sensorchips CM4 through amine coupling. For kinetic studies of protein binding, purified decorin and biglycan, isolated EDTA soluble dentin matrix, and dentin matrix immunodepleted of decorin and/or biglycan were injected over TGF-beta isoforms and allowed to interact. Programmed kinetic analysis software provided sensorgrams for each concentration of proteoglycan or dentin matrix extract injected. Purified decorin and biglycan and dentin matrix extract bound to the TGF-beta isoforms. However, the association with TGF-beta3 was much weaker than that with TGF-beta1. After immunoaffinity depletion of the dentin matrix extract, the level of interaction between the dentin matrix extract and TGF-beta was significantly reduced. These results suggest isoform-specific interactions between decorin/biglycan and TGF-beta isoforms 1 and 3, which may explain why TGF-beta3 is not detected in the dentin matrix despite being expressed at higher levels than TGF-beta1 in odontoblasts. These proteoglycans appear to play a significant role in TGF-beta/extracellular matrix interactions and may be important in the sequestration of these growth factors in the dentin matrix.

  11. 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}.

  12. Recombinant albumins containing additional peptide sequences smaller than barbourin retain the ability of barbourin-albumin to inhibit platelet aggregation.

    PubMed

    Sheffield, William P; Wilson, Brianna; Eltringham-Smith, Louise J; Gataiance, Sharon; Bhakta, Varsha

    2005-05-01

    The previously described fusion protein BLAH(6) (Marques JA et al.,Thromb Haemost 2001; 86: 902-8) is a recombinant protein that combines the small disintegrin barbourin with hexahistidine-tagged rabbit serumalbumin (RSA) produced in Pichia pastoris yeast. We sought to determine: (1) if BLAH(6) was immunogenic; and (2) if its barbourin domain could be productively replaced with smaller peptides. Purified BLAH(6) was injected into rabbits, and anti-barbourin antibodies were universally detected in plasma 28 days later; BLAH(6) was, however, equally effective in reducing platelet aggregation in both naive and pre-treated rabbits. Thrombocytopenia was not observed, and complexing BLAH(6) to alpha(IIb)beta(3) had no effect on antibody detection. The barbourin moiety of BLAH(6) was replaced with each of four sequences: Pep I (VCKGDWPC); PepII (VCRGDWPC); PepIII (bar-bourin 41-54); and PepIV (LPSPGDWR). The corresponding fusion proteins were tested for their ability to inhibit ADP-induced platelet aggregation. PepIII-LAH(6) inhibited neither rabbit nor human platelets. PepI-LAH(6) and PepIV-LAH(6) inhibited rabbit platelet aggregation as effectively as BLAH(6), but PepIV-LAH(6) did not inhibit human platelet aggregation. PepI-LAH(6) and PepIILAH(6) inhibited human platelet aggregation with IC(50)s 10- and 20-fold higher than BLAH(6). Cross-immunoprecipitation assays with human platelet lysates confirmed that all proteins and peptides interacted with the platelet integrin alpha(IIb)beta(3), but with greatly varying affinities. Our results suggest that the antiplatelet activity of BLAH(6) can be retained in albumin fusion proteins in which smaller peptides replace the barbourin domain; these proteins may be less immunogenic than BLAH(6).

  13. 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.

  14. Modulation of integrin antagonist signaling by ligand binding of the heparin-binding domain of vitronectin to the alphaVbeta3 integrin.

    PubMed

    Maile, Laura A; Aday, Ariel W; Busby, Walker H; Sanghani, Ravi; Veluvolu, Umadevi; Clemmons, David R

    2008-10-01

    The interaction between the arginine glycine and aspartic acid motif (RGD) of integrin ligands such as vitronectin and the integrin receptor alphaVbeta3 in mediating cell attachment has been well described. Similarly, the ability of disintegrins, small RGD containing peptides, to inhibit cell attachment and other cellular processes has also been studied extensively. Recently, we characterized a second site of interaction between vitronectin and its integrin partner. We determined that amino acids within the heparin-binding domain of vitronectin bind to a cysteine loop (C-loop) region of beta3 and that this interaction is required for the positive effects of alphaVbeta3 ligand occupancy on IGF-I signaling in smooth muscle cells. In this study we examine the signaling events activated following ligand binding of disintegrins to the alphaVbeta3 and the ability of these signals to be regulated by binding of the heparin-binding domain of vitronectin. We demonstrate that disintegrin ligand binding activates a series of events including the sequential activation of the tyrosine kinases c-Src and Syk. This leads to the activation of calpain and the cleavage of the beta3 cytoplasmic tail. Addition of vitronectin or a peptide homologous to the heparin-binding domain inhibited activation of this pathway. Our results suggest that the signaling events that occur following ligand binding to the alphaVbeta3 integrin reflects a balance between the effects mediated through the RGD binding site interaction and the effects mediated by the heparin binding site interaction and that for intact vitronectin the effect of the heparin-binding domain predominates.

  15. RIII S/J (H-2r). An inbred mouse strain with a massive deletion of T cell receptor V beta genes

    PubMed Central

    1989-01-01

    We have identified an inbred strain of mouse, RIII S/J (H-2r), that has the largest known deletion of the TCR V beta genes by screening with mAb and TCR V beta specific probes. Upon screening of PBL with mAb F23.1, which is specific for V beta 8 TCR, RIII S/J was found to be negative. On further screening with mAb KJ 23a, which is specific for V beta 17a TCR, RIII S/J was completely negative. We next tested RIII S/J with mAb 44-22-1, which is specific for V beta 6 TCR, and found it also to be negative. The (B10 X RIII)F1 mice showed a 50% expression of V beta 6 gene, indicating a genomic rather than a clonal deletion. mAb KJ25, detecting V beta 3, was positive in RIII S/J, denoting the downstream boundary for the deletion. Southern blot analysis of liver DNA using TCR V beta-specific probes confirmed the deletion of V beta 8 gene subfamily and V beta 5 gene subfamily, along with V beta 9, V beta 11, V beta 12, and V beta 13 genes similar to the known TCR V beta deletion mutants (SWR, SJL, C57L, and C57Br). In addition, RIII S/J is missing V beta 6, V beta 15, and V beta 17 genes. Our mapping of the deletion indicates that RIII S/J has lost approximately 130 kb of V beta chromosome and with it 13 V beta genes out of the known 21 V beta genes of the TCR. The deletion is marked by the presence of V beta 10 gene upstream and V beta 3 gene downstream. PMID:2525171

  16. Angiotensin II increases mRNA levels of all TGF-beta isoforms in quiescent and activated rat hepatic stellate cells.

    PubMed

    Moreno-Alvarez, Paola; Sosa-Garrocho, Marcela; Briones-Orta, Marco A; González-Espinosa, Claudia; Medina-Tamayo, Jaciel; Molina-Jijón, Eduardo; Pedraza-Chaverri, José; Macías-Silva, Marina

    2010-10-01

    AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF-beta (transforming growth factor-beta). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF-beta actions. Here, we studied the effect of AII on the mRNA levels of TGF-beta isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF-beta1, TGF-beta2 and TGF-beta3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF-beta isoforms, particularly TGF-beta2and TGF-beta3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF-beta protein after AII treatment. The mRNA expression of TGF-beta isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF-beta expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.

  17. 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

  18. Asymmetry and structural changes in ECF1 examined by cryoelectronmicroscopy.

    PubMed

    Wilkens, S; Capaldi, R A

    1994-01-01

    The Escherichia coli ATPase (ECF1) has been studied by cryoelectronmicroscopy and an intrinsic asymmetry of the molecule in the hexagonal projection identified. The three beta subunits could be distinguished. One, which we have called beta 1, has a greater density in projection than the other two; the second, beta 2, is of intermediate density in projection, while the third, beta 3, is smeared out in density. These different features of the beta subunits were used to orient images, and the positions of the gamma and epsilon subunits then established. The location of the gamma subunit, as monitored by the central mass, was not fixed. This subunit could be found in positions that followed an arc from close to beta 2 to close to beta 3, a shift of around 10A, with respect to the center of the mass. The location of the epsilon subunit was monitored after reconstituting a complex of epsilon subunit-depleted ECF1 with a mutant epsilon subunit in which His at residue 38 had been replaced by Cys, and this Cys labeled with an approximately 14A gold particle. The epsilon subunit was found in positions described by an arc between an alpha subunit (alpha 1) and the neighboring beta subunit (beta 1), a shift of around 20A, with respect to the center of the gold particle. A nucleotide dependence of the position of the gamma subunit has been established by Gogol, E.P., Johnston, E., Aggeler, R. and Capaldi, R.A. (1990) Proc. Natl. Acad. Sci. USA 87, 9585-9589. A nucleotide dependence of the position of the epsilon subunit is shown here.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8003256

  19. Long-term hypoxia modulates expression of key genes regulating adipose function in the late-gestation ovine fetus.

    PubMed

    Myers, Dean A; Hanson, Krista; Mlynarczyk, Malgorzata; Kaushal, Kanchan M; Ducsay, Charles A

    2008-04-01

    A major function of abdominal adipose in the newborn is nonshivering thermogenesis. Uncoupling protein (UCP) UCP1 and UCP2 play major roles in thermogenesis. The present study tested the hypothesis that long-term hypoxia (LTH) modulates expression of UCP1 and UCP2, and key genes regulating expression of these genes in the late-gestation ovine fetus. Ewes were maintained at high altitude (3,820 m) from 30 to 138 days gestation (dG); perirenal adipose tissue was collected from LTH and age-matched, normoxic control fetuses at 139-141 dG. Quantitative real-time PCR was used to analyze mRNA for UCP1, UCP2, 11beta hydroxysteroid dehydrogenase type 1 (HSD11B1) and 2 (HSD11B2), glucocorticoid receptor (GR), beta3 adrenergic receptor (beta3AR), deiodinase type 1 (DIO1) and DIO2, peroxisome proliferator activated receptor (PPAR) alpha and gamma and PPARgamma coactivator 1 (PGC1alpha). Concentrations of mRNA for UCP1, HSD11B1, PPARgamma, PGC1, DIO1, and DIO2 were significantly higher in perirenal adipose of LTH compared with control fetuses, while mRNA for HSD11B2, GR, or PPARalpha in perirenal adipose did not differ between control and LTH fetuses. The increased expression of UCP1 is likely an adaptive response to LTH, assuring adequate thermogenesis in the event of birth under oxygen-limiting conditions. Because both glucocorticoids and thyroid hormone regulate UCP1 expression, the increase in HSD11B1, DIO1, and DIO2 implicate increased adipose capacity for local synthesis of these hormones. PPARgamma and its coactivator may provide an underlying mechanism via which LTH alters development of the fetal adipocyte. These findings have important implications regarding fetal/neonatal adipose tissue function in response to LTH.

  20. Microstructural development to toughen SiC

    SciTech Connect

    Moberlychan, W.J.; Cannon, R.M.; Chan, L.H.; Cao, J.J.; Gilbert, C.J.; Ritchie, R.O.; De Jonghe, L.C.

    1996-12-31

    SiC offers a promise for high strength applications at high temperature; however, poor fracture resistance has inhibited its utility. Recent developments to control microstructure during hot pressing have improved fracture toughness > 3 fold, while also improving strength 50% above that of a commercial SiC, Hexoloy. This ABC-SiC (designated for the Al, B, and C additives) utilizes liquid phase sintering to obtain full densification at 1,650 C, and to induce the {beta}-3C to {alpha}-4H phase transformation below 1,900 C. Interlocking, plate-like, {alpha} grains, coupled with a thin ({approximately}1 nm) amorphous layer, provide for tortuous intergranular fracture and high toughness. This study focuses on the developing microstructure; how the {alpha}-4H polytype grow as a stacking modification of the {beta}-3C grains, and how amorphous grain boundaries and crystalline triple point phases develop and interact with the crack geometry. HR-TEM and Image-Filtered EELS characterize the amorphous grain boundaries. Field Emission-SEM, EDS and Auger Electron Spectroscopy characterize the fracture morphology and the chemistry of grain boundaries and triple points. Electron Diffraction and HR-TEM depict an epitaxial relationship between triple point phases (Al{sub 8}B{sub 4}C{sub 7} and Al{sub 4}O{sub 4}C) and matrix {alpha}-SiC grains, the development of which affects the mechanical toughening. The transformation to toughen SiC is compared to the well-studied transformation processing in Si{sub 3}N{sub 4}. A distinct advantage is the interlocked nature of the plate-like grains, which causes strong elastic bridging behind the crack tip.

  1. 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

  2. Implementation of indicators through balanced scorecards in a nutritional therapy company.

    PubMed

    de Matos Nasser, Emanuele; Reis da Costa, Stella Regina

    2013-01-01

    Introducción: La Balanced Scorecard (BSC) es una herramienta que ayuda en la gestión estratégica, bajo las siguientes cuatro perspectivas: la financiera, la del cliente, los procesos internos de la empresa, y los procesos de crecimiento y aprendizaje. Con el fin de medir el rendimiento de las entidades, la BSC emplea como plataforma indicadores financieros y no financieros. Objetivos: Implantar la BSC en una empresa de terapias nutricionales. Material y métodos: Esta investigación comprende el estudio de un caso que tuvo lugar en una empresa de terapia nutricional, entre enero y noviembre de 2010. Para el análisis de la perspectiva de aprendizaje y crecimiento, se consideraron los 45 colaboradores de la compañía y para el análisis de los clientes, 124 clientes de atención domiciliaria. La muestra del estudio consistió de 39 colaboradores y 44 clientes que participaron en la investigación. Se elaboró material para la recogida de los datos y verificación de las tendencias de las perspectivas mediante el análisis de los principales procesos de la compañía, encuestas de satisfacción del colaborador y hojas de cálculo para la verificación del beneficio neto y el porcentaje de anulaciones. Los datos se introdujeron en una hoja de cálculo de la aplicación informática Excel. Resultados y discusión: Se escogieron los indicadores en función de los objetivos estratégicos y los perfiles organizativos. Perspectivas de aprendizaje y crecimiento personal: eficacia en la capacitación 94%, participación 77%, fidelidad/ retención 93%, satisfacción 75%, ambiente organizativo 88%, bienestar 100%, perspectiva del proceso: análisis microbiológico 100%, auditoría interna 100%, productividad 100%, evaluación nutricional 81%, soporte nutricional 100%, indicación de atención domiciliaria 94%, visitas s domicilio 98%, perspectiva del cliente: percepción de la compañía 97%, priorización 94%, retención 59%, insatisfacción 24%, logística 94%, servicio

  3. [In Process Citation].

    PubMed

    Sánchez-Ramírez, Carmen A; Larrosa-Haro, Alfredo; M Vásquez Garibay, Edgar; Rodríguez-Anguiano, Ana K; Cámara-López, María E

    2016-02-16

    Introducción: la ingestión de cáusticos (IC) en niños y adolescentes puede ocasionar esofagitis erosiva, estenosis esofágica y disfagia, entidades que pueden alterar el proceso de alimentación y originar desnutrición y retraso en el crecimiento.Objetivos: evaluar el estado nutricio de niños con IC y su asociación con disfagia y estenosis esofágica.Métodos: estudio transversal analítico en el que se incluyó a 62 niños atendidos en un hospital pediátrico de referencia que sufrieron IC. Lasvariables independientes fueron la presencia/ausencia de disfagia y/o estenosis esofágica; las dependientes fueron el crecimiento y el estado nutricio evaluados mediante antropometría. Análisis estadístico: c2, OR, IC 95%, kappa y t de Student.Resultados: la edad promedio fue 39,7 meses, el 39,7% eran niñas. Cuarenta y dos (77,8%) presentaron esofagitis erosiva (II-b, III-a, and III-b) en la endoscopia. En 24 (38,7%) ocurrió disfagia y en 40 (64,5%) estenosis esofágica. El puntaje z de la talla para la edad fue <-2 DE en cinco niños (8,1%) y el puntaje z del IMC < -2 DE en tres (4,8%). En 24.2% la z-IMC fue > +1 DE. El puntaje z de los indicadores del brazo relacionados a reservas grasa y masa muscular tanto en el grupo de estenosis como de disfagia se localizó en el lado negativo de la curva y ambos fueron significativamente menores a los del grupo sin disfagia o estenosis.Conclusiones: la proporción de esofagitis erosiva, estenosis o disfagia fue elevada. En los niños con disfagia o estenosis esofágica se identificaron reservas de grasa y masa muscular menores a las de los niños sin estas complicaciones.

  4. [Zinc deficiency and associated factors in colombian children; results from the 2010 national nutrition survey; a cross sectional study].

    PubMed

    Martínez-Torres, Javier; Ramírez-Vélez, Robinson

    2014-04-01

    Introducción: El zinc (Zn) es un micronutriente esencial en el crecimiento celular, la síntesis proteica y la diferenciación celular. La deficiencia de Zn afecta el crecimiento y desarrollo del niño, el metabolismo energético y la respuesta inmune. Objetivo: Examinar los factores asociados a la deficiencia de Zn en una muestra representativa de colombianos niños. Pacientes y métodos: Estudio descriptivo transversal, secundario de la información obtenida en la Encuesta Nacional de la Situación Nutricional 2010 (ENSIN 2010), en 4.279 niños entre 12 y 59 meses. Los niveles plasmáticos de Zn se determinaron por espectrofotometría de absorción atómica, y los factores asociados (sexo, edad, etnia, puntaje de SISBEN, región y área geográfica) se recogieron por encuesta estructurada. Se establecieron asociaciones mediante la construcción de modelos de regresión y factores asociados. Resultados: Se encontró un valor promedio de Zinc de 78,5 μg/dl (IC 95% = 76,7-80,4 μg/dl). El 43,3% (IC 95% 42,2-44,3%) de los niños presentaron niveles de Zn menores a 65 μg/dl. Los niños pertenecientes a grupos étnicos (indígena) o que residen en áreas rurales; presentaron mayor déficit de Zn (56,3% y 47,8%) respectivamente. Los modelos de regresión muestran que; ser indígena (OR 1,76 IC 95% 1,29-2,41); y residir en zonas rurales (OR 1,39 IC 95% 1,16-1,67), se asociaron al déficit de Zn. Conclusiones: La población estudiada presenta una alta prevalencia de déficit de Zn, por lo que se recomienda intervenciones integrales donde estén involucrados el componente nutricional y educativo.

  5. [Placental weight percentiles and its relationship with fetal weight according to gestational age in an urban area of Buenos Aires].

    PubMed

    Grandi, Carlos; Roman, Estela; Dipierri, Jose

    2015-01-01

    Antecedentes: El peso placentario (PP) y los índices de su relación con el peso al nacer (PN) (PN/PP, PP/PN) predicen morbi-mortalidad perinatal y resultados alejados de la salud. Objetivos: Calcular percentilos del PP e índices por sexo y edad gestacional correspondientes a 867 RNV de la Maternidad Sardá de Buenos Aires, Argentina y compararlos con referencias internacionales. Material y métodos: Se excluyeron feto muerto, embarazo múltiple, edad gestacional <22 y >42 semanas y PP<100g y >2500g. Características maternas y fetales: edad, educación, tabaco, paridad, diabetes, preeclampsia, corioamnionitis, restricción del crecimiento, malformación congénita y prematurez. Se calcularon estadísticos de resumen y percentilos con el método LMS. Las comparaciones se realizaron con test t-Student, ANOVA y referencias internacionales. Resultados: Edad materna media 24 años, educación 10.1 años, 24.5% primíparas, 12.6% fumadoras, 4.9% presentaron diabetes, 8.7% preeclampsia, 7.9% corioamnionitis y 13.0% restricción del crecimiento fetal. El 55.3% de los RN fueron varones, 51.6% prematuros, 18.9% PEG y 7.1% malformados. El PN y EG promedio fue de 2581g y 35.6 semanas respectivamente. Elevada correlación positiva de la EG con PP y PN/PP y negativa con PP/PN (p%lt;0.001); el peso de la placenta e índices fueron mayores en varones. Se presentan los percentiles de PP, PN/PP y PP/PN. Las diferencias con las referencias oscilaron de 0.46% -13%, 4.91% -12.1% y 5.81% -14% para el PP, PN/PP y PP/PN respectivamente. Conclusiones: los percentilos generados son aplicables en investigaciones sobre la relación de la placenta con resultados perinatales y la salud durante el ciclo vital.

  6. [Not Available].

    PubMed

    Moreno Villares, José Manuel

    2016-01-01

    El desarrollo durante el periodo fetal y los primeros años de vida se caracteriza por su crecimiento rápido y la maduración de órganos y sistemas. Los cambios tanto en la calidad como en la cantidad de los nutrientes que consumen la mujer embarazada y el niño en ese periodo pueden influir de forma permanente sobre esos órganos en desarrollo. Esos efectos se conocen como "programación" y constituyen un factor importante en la aparición de enfermedades no transmisibles a lo largo de toda la vida, fundamentalmente en la edad adulta; en especial, la enfermedad cardiovascular, el síndrome metabólico y la intolerancia a los hidratos de carbono. Las carencias nutricionales durante el embarazo que ocasionan un crecimiento intrauterino retardado se asocian a un riesgo aumentado de diabetes tipo 2 y de enfermedad coronaria en la descendencia. Esa influencia en la aparición de enfermedades no transmisibles en la vida adulta también está marcada por el tipo y duración de la lactancia y por la alimentación complementaria. La explicación más plausible es que estos factores nutricionales -junto a otros no nutricionales- modifican la expresividad de determinados genes, modificando la respuesta de determinados órganos y tejidos, remodelando su estructura y su función. Estos cambios epigenéticos pueden transmitirse de una generación a la siguiente, por lo que se pone aún más en evidencia la importancia del fenómeno de la programación metabólica temprana. Pero al mismo tiempo abren una ventana de oportunidad a que modificando las pautas de alimentación de la mujer embarazada y del niño pequeño podamos disminuir la carga asociada a este tipo de enfermedades. PMID:27571856

  7. [Not Available].

    PubMed

    Moreno Villares, José Manuel

    2016-07-12

    El desarrollo durante el periodo fetal y los primeros años de vida se caracteriza por su crecimiento rápido y la maduración de órganos y sistemas. Los cambios tanto en la calidad como en la cantidad de los nutrientes que consumen la mujer embarazada y el niño en ese periodo pueden influir de forma permanente sobre esos órganos en desarrollo. Esos efectos se conocen como "programación" y constituyen un factor importante en la aparición de enfermedades no transmisibles a lo largo de toda la vida, fundamentalmente en la edad adulta; en especial, la enfermedad cardiovascular, el síndrome metabólico y la intolerancia a los hidratos de carbono. Las carencias nutricionales durante el embarazo que ocasionan un crecimiento intrauterino retardado se asocian a un riesgo aumentado de diabetes tipo 2 y de enfermedad coronaria en la descendencia. Esa influencia en la aparición de enfermedades no transmisibles en la vida adulta también está marcada por el tipo y duración de la lactancia y por la alimentación complementaria. La explicación más plausible es que estos factores nutricionales -junto a otros no nutricionales- modifican la expresividad de determinados genes, modificando la respuesta de determinados órganos y tejidos, remodelando su estructura y su función. Estos cambios epigenéticos pueden transmitirse de una generación a la siguiente, por lo que se pone aún más en evidencia la importancia del fenómeno de la programación metabólica temprana. Pero al mismo tiempo abren una ventana de oportunidad a que modificando las pautas de alimentación de la mujer embarazada y del niño pequeño podamos disminuir la carga asociada a este tipo de enfermedades.

  8. [Effect of high altitude on birth weight and adverse perinatal outcomes in two Argentine populations].

    PubMed

    Grandi, Carlos; Dipierri, José; Luchtenberg, Guillermo; Moresco, Angélica; Alfaro, Emma

    2013-01-01

    Introducción: existe poca información sobre la relación entre tamaño al nacer y altura geográfica ajustada para factores maternos y obstétricos potencialmente confusores. Objetivo: analizar la variación, en función de la altitud geográfica, del Peso al Nacimiento (PN) y resultados perinatales adversos, en dos poblaciones argentinas. Material y Métodos: 4000 registros de recién nacidos (RN) de Jujuy y 4000 de Buenos Aires (Maternidad Sardá) (1996-2000), seleccionados y aleatorizados del Sistema Informático Perinatal. Los datos provenían de la Maternidad Sarda (20 msnm) y las regiones jujeñas: Ramal (500 msnm), Valle (1200 msnm), Quebrada (2500 msnm) y Puna (3500 msnm). Variables resultado: PN >3000 g, PN <2500 g, Índice Ponderal (IP), prematurez, pequeño para edad gestacional (PEG) y restricción del crecimiento intrauterino (FGR). Variables confusoras: edad, tipo de pareja, educación, sobrepeso-obesidad, tabaquismo, hipertensión arterial, preeclampsia, infección urinaria, restricción del crecimiento y terminación cesárea. Resultados: Se observó un gradiente altitudinal creciente para madres adolescentes y decreciente para las variables obstétrico-maternas. El PN, PN >3000 g, PN <2500 g e Índice Ponderal se asociaron negativamente con altitud (p<0.001). La prevalencia de prematurez, PEG y FGR mostraron un comportamiento opuesto (p<0.001). Ajustados para variables confusoras el PN<3000 g, PEG, FGR < 0.90 e IP<2.53 mostraron mayor riesgo con la altitud geográfica (p<0.05). Conclusiones: La altitud se asoció independientemente con restricción del PN y resultados perinatales adversos. Dado el impacto de la reducción del PN en el riesgo de enfermedades crónicas no transmisibles, se debería evaluar esta relación en otras poblaciones, independientemente de su localización altitudinal.

  9. [In Process Citation].

    PubMed

    Sánchez-Ramírez, Carmen A; Larrosa-Haro, Alfredo; M Vásquez Garibay, Edgar; Rodríguez-Anguiano, Ana K; Cámara-López, María E

    2016-01-01

    Introducción: la ingestión de cáusticos (IC) en niños y adolescentes puede ocasionar esofagitis erosiva, estenosis esofágica y disfagia, entidades que pueden alterar el proceso de alimentación y originar desnutrición y retraso en el crecimiento.Objetivos: evaluar el estado nutricio de niños con IC y su asociación con disfagia y estenosis esofágica.Métodos: estudio transversal analítico en el que se incluyó a 62 niños atendidos en un hospital pediátrico de referencia que sufrieron IC. Lasvariables independientes fueron la presencia/ausencia de disfagia y/o estenosis esofágica; las dependientes fueron el crecimiento y el estado nutricio evaluados mediante antropometría. Análisis estadístico: c2, OR, IC 95%, kappa y t de Student.Resultados: la edad promedio fue 39,7 meses, el 39,7% eran niñas. Cuarenta y dos (77,8%) presentaron esofagitis erosiva (II-b, III-a, and III-b) en la endoscopia. En 24 (38,7%) ocurrió disfagia y en 40 (64,5%) estenosis esofágica. El puntaje z de la talla para la edad fue <-2 DE en cinco niños (8,1%) y el puntaje z del IMC < -2 DE en tres (4,8%). En 24.2% la z-IMC fue > +1 DE. El puntaje z de los indicadores del brazo relacionados a reservas grasa y masa muscular tanto en el grupo de estenosis como de disfagia se localizó en el lado negativo de la curva y ambos fueron significativamente menores a los del grupo sin disfagia o estenosis.Conclusiones: la proporción de esofagitis erosiva, estenosis o disfagia fue elevada. En los niños con disfagia o estenosis esofágica se identificaron reservas de grasa y masa muscular menores a las de los niños sin estas complicaciones. PMID:27019238

  10. Self-similar collapse and accretion of radiative gas

    NASA Astrophysics Data System (ADS)

    Boily, C. M.; Lynden-Bell, D.

    1995-09-01

    The hydrodynamics of spherically symmetric radiative gas is discussed. Self-similar solutions are obtained for situations where the total emissivity epsilon of the gas has a power-law dependence on the density and temperature, epsilon~rho^alphaT^4+beta, in an optically thin medium. A perfect gas equation of state is used throughout. The flows have uniform density and contract uniformly initially. The region of uniform density becomes ever smaller with time, and gives way to power-law radial profiles. For each pair alpha and beta, there may exist three types of flows. Flows of the first type are far from dynamical equilibrium, and have gas converging steadily to the origin. These solutions develop strong velocity fields and supersonic motion. These are found for alpha, beta such that 1/2beta<~-3, where omega=(3/2-alpha)/(alpha+beta+3/2). All have a density profile that maximizes at the centre and dynamical properties reminiscent of the Penston-Larson solution for isothermal gas. The second branch of solutions proceed from near hydrostatic equilibrium, and the motion remains subsonic everywhere during infall. These occur only for values of the adiabatic exponent gamma<6/5. For each pair alpha, beta such that 0<=omega<1/4, an infinite, but discrete, number of solutions exist. They have the property that trajectories of shells of constant internal mass are non-monotonic functions of time (`breathing modes'). The third type of collapse flows develop supersonic motion, but have a maximum density on a sphere away from the origin. These occur for alpha<3/2. The overdense shell is unstable to fragmentation and the onset of convection. The collapse solution ends when the region of uniform density has shrunk to zero volume. At that time, provided gravity overcomes pressure gradients, a central point-mass forms. The solutions can be continued in time with accretion solutions in which the central mass grows at the expense of the surrounding envelope. Near the

  11. [Molecular characteristics of leiomyoma uteri based on selected compounds of the extracellular matrix].

    PubMed

    Auguściak-Duma, Aleksandra; Sieroń, Aleksander L

    2008-01-14

    Leiomyoma is a monoclonal benign tumor. It is often located in the muscle layer of the uterus in women of reproductive age. Its growth is accelerated by pregnancy and hormonal therapy. Its growth also depends on the concentration of sex hormones. Growth factors and cytokines may also participate in the formation of leiomyomas. The modulation of mitotic activity and abnormal extracellular matrix production are key elements of tumor growth. Elements of the TGFbeta superfamily are crucial factors in the proliferation of neoplasmic cells. TGF-beta1 and -beta3 stimulate the synthesis of various components of the extracellular matrix, but they also down-regulate the synthesis of proteinases which degrade the matrix, often leading to excessive overdeposition of connective tissue. Collagen types 1 and 3 are the main structural components of the extracellular matrix. The biosynthesis of collagens requires, among others, the action of procollagen C-endopeptidase, a protein of the BMP-1/mTLD subfamily. BMP-1/mTLD-like proteinases remove the carboxyl propeptides of procollagens 1, 2, and 3. Removal of the C-propeptides decreases the solubility of procollagens about 1000-fold to a concentration critical for their spontaneous self-assembly to collagen fibrils. Different substrates of BMP-1/mTLD are prolysyl oxidase, gamma2 chain of prolaminin, procollagen type VII, miostatin, dentin matrix protein 1, and perlekan. Due to the activation of various substrates by BMP-1/mTLDs, they are important regulators of the production of the extracellular matrix and its quality as well as of antiangiogenic responses by producing a factor from the basal membrane compound called perlekan. The BMP-1/mTLDs influence the formation of dorsal ventral patterning in embryos by releasing BMP-2/4 from the inhibitory protein chordin. Another aspect is induction of the development of muscle and neural tissue by activation of GDF8 and GDF11 as well as the regulation of growth and cell proliferation by

  12. Functional and phenotypic effects of AhR activation in inflammatory dendritic cells

    SciTech Connect

    Bankoti, Jaishree; Rase, Ben; Simones, Tom; Shepherd, David M.

    2010-07-15

    Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immune suppression. Dendritic cells (DCs) are key antigen presenting cells governing T cell activation and differentiation. However, the consequences of AhR activation in DCs are not fully defined. We hypothesized that AhR activation alters DC differentiation and generates dysfunctional DCs. To test this hypothesis, inflammatory bone marrow-derived DCs (BMDCs) from C57Bl/6 mice were generated in the presence of vehicle or TCDD. TCDD decreased CD11c expression but increased MHC class II, CD86 and CD25 expression on the BMDCs. The effects of TCDD were strictly AhR-dependent but not exclusively DRE-mediated. Similar effects were observed with two natural AhR ligands, 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid (ITE). TCDD increased LPS- and CpG-induced IL-6 and TNF-{alpha} production by BMDCs but decreased their NO production. TCDD decreased CpG-induced IL-12p70 production by BMDCs but did not affect their secretion of IL-10. TCDD downregulated LPS- and CpG-induced NF-kB p65 levels and induced a trend towards upregulation of RelB levels in the BMDCs. AhR activation by TCDD modulated BMDC uptake of both soluble and particulate antigens. Induction of indoleamine-2,3-dioxygenase (IDO) and TGF-{beta}3 has been implicated in the generation of regulatory T cells following AhR activation. TCDD increased IDO1, IDO2 and TGF-{beta}3 mRNA levels in BMDCs as compared to vehicle. Despite the induction of regulatory mediators, TCDD-treated BMDCs failed to suppress antigen-specific T cell activation. Thus, AhR activation can directly alter the differentiation and innate functions of inflammatory DCs without affecting their ability to successfully interact with T cells.

  13. Induction of a deficiency of steroid delta 4-5 alpha-reductase activity in liver by a porphyrinogenic drug.

    PubMed Central

    Kappas, A; Bradlow, H L; Bickers, D R; Alvares, A P

    1977-01-01

    The hepatic enzymes that catalyze drug oxidations and the reductive metabolism of steroid hormones to 5alpha-derivatives are localized in membranes of the endoplasmic reticulum. Phenobarbital, which exacerbates acute intermittent porphyria in man, induces drug-oxidizing enzymes in liver. Additionally, patients in whome the primary gene defect (uroporphyrinogen-I-synthetase deficiency) of acute intermittent porphyria has become clinically expressed have low levels of hepatic steroid delta4-5alpha-reductase activity. This 5alpha-reductase deficiency in acute intermittent porphyria leads to the disproportionate generation of 5beta-steroid metabolites from precursor hormones; such steroid metabolites have significant porphyria-inducing action experimentally. In this study the effects of phenobarbital on drug oxidation and steroid 5alpha-reduction in man were examined to determine if this drug could produce changes in steroid 5alpha-reductase activity which mimicked those seen in patients with acute intermittent porphyria. Metabolic studies with [14C]-testosterone and 11beta-[3H]hydroxyandrostenedione were carried out in five normal volunteers. In all five subjects phenobarbital administration (2 mg/kg/per day for 21 days) enhanced plasma removal of the test drugs antipyrine and phenylbutazone as expected; but in four subjects phenobarbital also substantially depressed 5alpha-metabolite formation from [14C]testosterone and resulted in a pattern of hormone biotransformation characterized by a high ratio of 5beta/5alpha-metabolite formation. Studies with 11beta-[3H]hydroxy-androstenedione in three subjects confirmed that phenobarbital produced this high 5beta/5alpha ratio of steroid metabolism by depressing 5alpha-reductase activity for steroid hormones in liver. The high ratio of 5beta/5alpha-metabolites formed in normals after drug treatment mimicks the high 5beta/5alpha-steroid metabolite ratio formed from endogenous hormones in acute intermittent porphyria. The

  14. An essential role for gp39, the ligand for CD40, in thymic selection.

    PubMed

    Foy, T M; Page, D M; Waldschmidt, T J; Schoneveld, A; Laman, J D; Masters, S R; Tygrett, L; Ledbetter, J A; Aruffo, A; Claassen, E; Xu, J C; Flavell, R A; Oehen, S; Hedrick, S M; Noelle, R J

    1995-11-01

    The interactions between CD40 on B cells and its ligand gp39 on activated T helper cells are known to be essential for the development of thymus-dependent humoral immunity. However, CD40 is also functionally expressed on thymic epithelial cells and dendritic cells, suggesting that gp39-CD40 interactions may also play a role in thymic education, the process by which self-reactive cells are deleted from the T cell repertoire. Six systems of negative selection were studied for their reliance on gp39-CD40 interactions to mediate negative selection. In all cases, when the antigen/superantigen was endogenously expressed (in contrast to exogenously administered), negative selection was blocked by loss of gp39 function. Specifically, blockade of gp39-CD40 interactions prevented the deletion of thymocytes expressing V beta 3, V beta 11, and V beta 12, specificities normally deleted in BALB/c mice because of the endogenous expression of minor lymphocyte-stimulating determinants. Independent verification of a role of gp39 in negative selection was provided by studies in gp39-deficient mice where alterations in T cell receptor (TCR) V beta expression were also observed. Studies were also performed in the AND TCR transgenic (Tg) mice, which bear the V alpha 11, V beta 3 TCR and recognize both pigeon cytochrome c (PCC)/IEk and H-2As. Neonatal administration of anti-gp39 to AND TCR Tg mice that endogenously express H-2As or endogenously produce PCC prevented the deletion of TCR Tg T cells. In contrast, deletion mediated by high-dose PCC peptide antigen (administered exogenously) in AND TCR mice was unaltered by administration of anti-gp39. In addition, deletion by Staphylococcus enterotoxin B in conventional mice was also unaffected by anti-gp39 administration. gp39 expression was induced on thymocytes by mitogens or by antigen on TCR Tg thymocytes. Immunohistochemical analysis of B7-2 expression in the thymus indicated that, in the absence of gp39, B7-2 expression was

  15. GABAA receptor pharmacology of fluorinated derivatives of the novel sedative-hypnotic pyrazolopyrimidine indiplon.

    PubMed

    Wegner, Florian; Deuther-Conrad, Winnie; Scheunemann, Matthias; Brust, Peter; Fischer, Steffen; Hiller, Achim; Diekers, Michael; Strecker, Karl; Wohlfarth, Kai; Allgaier, Clemens; Steinbach, Jörg; Hoepping, Alexander

    2008-02-01

    The function of gamma-aminobutyric acid type A receptors (GABA(A) receptors) is enhanced by various clinically important drugs including benzodiazepines that act on an allosteric site formed at the interface between the alpha and gamma subunits. In contrast to classical benzodiazepines, the novel pyrazolopyrimidine indiplon (N-methyl-N-{3-[7-(thiophene-2-carbonyl)-1,5,9-triazabicyclo[4.3.0]nona-2,4,6,8-tetraen-2-yl]phenyl}acetamide; N-methyl-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide) demonstrates relative binding selectivity for the alpha1 subunit containing receptor subtypes, which are the most frequently expressed in the mammalian central nervous system. To investigate the pharmacological properties at GABA(A) receptors and to promote the development of alpha1 subunit selective radiotracers for positron emission tomography imaging, we have started with the evaluation of various fluorinated indiplon derivatives. Binding affinities were determined in homogenates from newborn and adult rats suggesting an alpha1 preference of the reference compounds indiplon, zaleplon as well as for all newly synthesized indiplon derivatives. In homogenated cerebellar tissue obtained from adult rat brain, known to primarily express alpha1 containing GABA(A) receptors, the high affinity of the basic indiplon structure was only slightly affected by an elongation of the alkyl substituent of the amide N from methyl (indiplon; K(i) 3.1 nM) via ethyl (2a, N-(2-fluoro-ethyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide; K(i) 5.4 nM) to propyl (2b, N-(3-fluoro-propyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide; K(i) 2.4 nM). Whole cell patch-clamp recordings at neuronal and recombinant GABA(A) receptors indicated that the fluorinated derivatives 2a and 2b have a high potency at alpha1beta3gamma2L isoforms comparable to indiplon (EC(50): 105, 158, and 81 nM, respectively), with 2b

  16. Calystegins of Physalis alkekengi var. francheti (Solanaceae). Structure determination and their glycosidase inhibitory activities.

    PubMed

    Asano, N; Kato, A; Oseki, K; Kizu, H; Matsui, K

    1995-04-15

    Five calystegins were extracted from the roots of Physalis alkekengi var. francheti (Solanaceae) with hot water and purified to homogeneity by the combination of a variety of ion-exchange column chromatographies. Their structures have been determined from the 1H- and 13C-NMR spectral data, and two of the compounds were identified as calystegins A3 and B2, which have been isolated from the roots of Calystegia sepium (Convolvulaceae). Two of the remaining three were found to be 1 alpha, 3 alpha, 4 beta-trihydroxy-nor-tropane and 1 alpha, 2 alpha, 3 alpha, 4 beta-tetrahydroxy-nor-tropane and given the trivial name calystegins A5 and B3, respectively. The last calystegin was assigned as 1 alpha, 2 beta, 3 alpha, 6 alpha-tetrahydroxy-nor-tropane, which was the same as the relative configuration proposed in the literature for calystegin B1 isolated from C. sepium. However, the 13C-NMR spectral data for the compound from C. sepium differed substantially from our results. From a personal communication with the authors of the original paper on calystegins, it was clarified that the 13C-NMR chemical shifts of calystegin B1 in the original paper had been erroneous. Since their corrected 13C-NMR data of calystegin B1 and its 1H-NMR chemical shifts in the original paper are very close to our present data, we concluded that both compounds from C. sepium and P. alkekengi are identical. Calystegin B2 has been known to be a potent competitive inhibitor of almond beta-glucosidase (Ki = 1.2 microM) and coffee bean alpha-galactosidase (Ki = 0.86 microM). In this study calystegin B1 (1 alpha, 2 beta, 3 alpha, 6 alpha-tetrahydroxy-nor-tropane) proved to be a potent competitive inhibitor of almond beta-glucosidase (Ki = 1.9 microM) and bovine liver beta-galactosidase (Ki = 1.6 microM), but not an inhibitor of alpha-galactosidases. Calystegin A3 was found to be a weaker inhibitor compared to calystegin B2 but with the same inhibitory spectrum. Calystegin A5, a 2-deoxy derivative of

  17. Magnetic structure of light nuclei from lattice QCD

    SciTech Connect

    Chang, Emmanuel; Detmold, William; Orginos, Kostas; Parreño, Assumpta; Savage, Martin J.; Tiburzi, Brian C.; Beane, Silas R.

    2015-12-09

    Lattice QCD with background magnetic fields is used to calculate the magnetic moments and magnetic polarizabilities of the nucleons and of light nuclei with $A\\le4$, along with the cross-section for the $M1$ transition $np\\rightarrow d\\gamma$, at the flavor SU(3)-symmetric point where the pion mass is $m_\\pi\\sim 806$ MeV. These magnetic properties are extracted from nucleon and nuclear energies in six uniform magnetic fields of varying strengths. The magnetic moments are presented in a recent Letter. For the charged states, the extraction of the polarizability requires careful treatment of Landau levels, which enter non-trivially in the method that is employed. The nucleon polarizabilities are found to be of similar magnitude to their physical values, with $\\beta_p=5.22(+0.66/-0.45)(0.23) \\times 10^{-4}$ fm$^3$ and $\\beta_n=1.253(+0.056/-0.067)(0.055) \\times 10^{-4}$ fm$^3$, exhibiting a significant isovector component. The dineutron is bound at these heavy quark masses and its magnetic polarizability, $\\beta_{nn}=1.872(+0.121/-0.113)(0.082) \\times 10^{-4}$ fm$^3$ differs significantly from twice that of the neutron. A linear combination of deuteron scalar and tensor polarizabilities is determined by the energies of the $j_z=\\pm 1$ deuteron states, and is found to be $\\beta_{d,\\pm 1}=4.4(+1.6/-1.5)(0.2) \\times 10^{-4}$ fm$^3$. The magnetic polarizabilities of the three-nucleon and four-nucleon systems are found to be positive and similar in size to those of the proton, $\\beta_{^{3}\\rm He}=5.4(+2.2/-2.1)(0.2) \\times 10^{-4}$ fm$^3$, $\\beta_{^{3}\\rm H}=2.6(1.7)(0.1) \\times 10^{-4}$ fm$^3$, $\\beta_{^{4}\\rm He}=3.4(+2.0/-1.9)(0.2) \\times 10^{-4}$ fm$^3$. Mixing between the $j_z=0$ deuteron state and the spin-singlet $np$ state induced by the background magnetic field is used to extract the short-distance two-nucleon counterterm, ${\\bar L}_1$, of the pionless effective theory for $NN$ systems (equivalent to the meson-exchange current

  18. Compounds exhibiting selective efficacy for different beta subunits of human recombinant gamma-aminobutyric acid A receptors.

    PubMed

    Smith, Alison J; Oxley, Beth; Malpas, Sallie; Pillai, Gopalan V; Simpson, Peter B

    2004-11-01

    Inhibitory GABA(A) receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk(-) cells stably expressing human recombinant GABA(A) subunits (alpha1beta1-3gamma2s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl(-) buffer using a voltage/ion probe reader. The influence of different betasubunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at alpha1beta1gamma2, alpha1beta2gamma2, and alpha1beta3gamma2 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC(50) values of 2.4 +/- 0.2, 2.5 +/- 0.2, and 1.3 +/- 0.1 microM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC(50) values of 8.7 +/- 0.9, 9.2 +/- 0.5, and 11.7 +/- 1.2, and 43.7 +/- 6.4, 24.8 +/- 1.6, and 26.1 +/- 2.4 microM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, pentobarbital, and steroids, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one, were unaffected by the beta isoform present in the receptor complex. However, several compounds displayed beta2/3 subunit selectivity, notably loreclezole, R(-)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at beta2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at alpha1beta1gamma2. Diflunisal was the most efficacious compound, eliciting greater

  19. Capsaicin stimulates the non-store-operated Ca{sup 2+} entry but inhibits the store-operated Ca{sup 2+} entry in neutrophils

    SciTech Connect

    Wang, J.-P. . E-mail: w1994@vghtc.gov.tw; Tseng, C.-S.; Sun, S.-P.; Chen, Y.-S.; Tsai, C.-R.; Hsu, M.-F.

    2005-12-01

    Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) V1. However, capsaicin-stimulated [Ca{sup 2+}]{sub i} elevation occurred only at high concentrations ({>=}100 {mu}M). This response was substantially decreased in a Ca{sup 2+}-free medium. Vanilloids displayed similar patterns of Ca{sup 2+} response with the rank order of potency as follows: scutigeral>resiniferatoxin>capsazepine>capsaicin=olvanil>isovelleral. Arachidonyl dopamine (AAD), an endogenous ligand for TRPV1, failed to desensitize the subsequent capsaicin challenge. Capsaicin-induced Ca{sup 2+} response was not affected by 8-bromo-cyclic ADP-ribose (8-Br-cADPR), the ryanodine receptor blocker, but was slightly attenuated by 1-[6-[17{beta}-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,= 5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, 1-[{beta}-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), the blocker of receptor-gated and store-operated Ca{sup 2+} (SOC) channels, 2-aminoethyldiphenyl borate (2-APB), the blocker of D-myo-inositol 1,4,5-trisphospahte (IP{sub 3}) receptor and Ca{sup 2+} influx, and by ruthenium red, a blocker of TRPV channels, and enhanced by the Ca{sup 2+} channels blocker, cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12330A) and Na{sup +}-deprivation. In addition, capsaicin had no effect on the plasma membrane Ca{sup 2+}-ATPase activity or the production of nitric oxide (NO) and reactive oxygen intermediates (ROI) or on the total thiols content. Capsaicin ({>=}100 {mu}M) inhibited the cyclopiazonic acid (CPA)-induced store-operated Ca{sup 2+} entry (SOCE). In the absence of external Ca{sup 2+}, the robust Ca{sup 2+} entry after subsequent addition of Ca{sup 2+} was decreased by capsaicin in CPA-activated cells. Capsaicin alone increased the actin cytoskeleton, and also increased the actin filament content in cell activation with CPA. These results indicate that capsaicin

  20. Risk assessment of hand washing efficacy using literature and experimental data.

    PubMed

    Montville, Rebecca; Chen, Yuhuan; Schaffner, Donald W

    2002-03-01

    This study simulated factors that influence the levels of bacteria on foodservice workers' hands. Relevant data were collected from the scientific literature and from laboratory experiments. Literature information collected included: initial bacterial counts on hands and water faucet spigots, bacterial population changes during hand washing as effected by soap type, sanitizing agent, drying method, and the presence of rings. Experimental data were also collected using Enterobacter aerogenes as a surrogate for transient bacteria. Both literature and experimental data were translated into appropriate discrete or probability distribution functions. The appropriate statistical distribution for each phase of the hand washing process was determined. These distributions were: initial count on hands, beta (2.82, 2.32, 7.5); washing reduction using regular soap, beta (3.01, 1.91, -3.00, 0.60); washing reduction using antimicrobial soap, beta (4.19, 2.99, -4.50, 1.50); washing reduction using chlorhexidine gluconate (CHG), triangular (-4.75, -1.00, 0); reductions from hot air drying, beta (3.52, 1.92, -0.20, 1.00); reduction from paper towel drying, triangular (-2.25, -0.75, 0); reduction due to alcohol sanitizer, gamma (-1.23, 4.42) -5.8; reduction due to alcohol-free sanitizer, gamma (2.22, 5.38) -5.00; and the effect of rings, beta (8.55, 23.35, 0.10, 0.45). Experimental data were fit to normal distributions (expressed as log percentage transfer rate): hand-to-spigot transfer, normal (-0.80, 1.09); spigot to hand, normal (0.36, 0.90). Soap with an antimicrobial agent (in particular, CHG) was observed to be more effective than regular soap. Hot air drying had the capacity to increase the amount of bacterial contamination on hands, while paper towel drying caused a slight decrease in contamination. There was little difference in the efficacy of alcohol and alcohol-free sanitizers. Ring wearing caused a slight decrease in the efficacy of hand washing. The experimental data

  1. Visceral adiposity syndrome.

    PubMed

    Lopes, Heno F; Corrêa-Giannella, Maria Lúcia; Consolim-Colombo, Fernanda M; Egan, Brent M

    2016-01-01

    The association of anthropometric (waist circumference) and hemodynamic (blood pressure) changes with abnormalities in glucose and lipid metabolism has been motivation for a lot of discussions in the last 30 years. Nowadays, blood pressure, body mass index/abdominal circumference, glycemia, triglyceridemia, and HDL-cholesterol concentrations are considered in the definition of Metabolic syndrome, referred as Visceral adiposity syndrome (VAS) in the present review. However, more than 250 years ago an association between visceral and mediastinal obesity with hypertension, gout, and obstructive apnea had already been recognized. Expansion of visceral adipose tissue secondary to chronic over-consumption of calories stimulates the recruitment of macrophages, which assume an inflammatory phenotype and produce cytokines that directly interfere with insulin signaling, resulting in insulin resistance. In turn, insulin resistance (IR) manifests itself in various tissues, contributing to the overall phenotype of VAS. For example, in white adipose tissue, IR results in lipolysis, increased free fatty acids release and worsening of inflammation, since fatty acids can bind to Toll-like receptors. In the liver, IR results in increased hepatic glucose production, contributing to hyperglycemia; in the vascular endothelium and kidney, IR results in vasoconstriction, sodium retention and, consequently, arterial hypertension. Other players have been recognized in the development of VAS, such as genetic predisposition, epigenetic factors associated with exposure to an unfavourable intrauterine environment and the gut microbiota. More recently, experimental and clinical studies have shown the autonomic nervous system participates in modulating visceral adipose tissue. The sympathetic nervous system is related to adipose tissue function and differentiation through beta1, beta2, beta3, alpha1, and alpha2 adrenergic receptors. The relation is bidirectional: sympathetic denervation of

  2. Differentiation of Boc-protected alpha,delta-/delta,alpha- and beta,delta-/delta,beta-hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry.

    PubMed

    Raju, G; Ramesh, V; Srinivas, R; Sharma, G V M; Shoban Babu, B

    2010-06-01

    Two new series of Boc-N-alpha,delta-/delta,alpha- and beta,delta-/delta,beta-hybrid peptides containing repeats of L-Ala-delta(5)-Caa/delta(5)-Caa-L-Ala and beta(3)-Caa-delta(5)-Caa/delta(5)-Caa-beta(3)-Caa (L-Ala = L-alanine, Caa = C-linked carbo amino acid derived from D-xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MS(n) spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc-group, and the side chain. The dipeptide positional isomers are differentiated by the collision-induced dissociation (CID) of the protonated peptides. The loss of 2-methylprop-1-ene is more pronounced for Boc-NH-L-Ala-delta-Caa-OCH(3) (1), whereas it is totally absent for its positional isomer Boc-NH-delta-Caa-L-Ala-OCH(3) (7), instead it shows significant loss of t-butanol. On the other hand, second isomeric pair shows significant loss of t-butanol and loss of acetone for Boc-NH-delta-Caa-beta-Caa-OCH(3) (18), whereas these are insignificant for its positional isomer Boc-NH-beta-Caa-delta-Caa-OCH(3) (13). The tetra- and hexapeptide positional isomers also show significant differences in MS(2) and MS(3) CID spectra. It is observed that 'b' ions are abundant when oxazolone structures are formed through five-membered cyclic transition state and cyclization process for larger 'b' ions led to its insignificant abundance. However, b(1)(+) ion is formed in case of delta,alpha-dipeptide that may have a six-membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di-, tetra-, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers.

  3. Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

    SciTech Connect

    Zawawi, M.S.F.; Dharmapatni, A.A.S.S.K.; Cantley, M.D.; McHugh, K.P.; Haynes, D.R.; Crotti, T.N.

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. Black-Right-Pointing-Pointer Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. Black-Right-Pointing-Pointer FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcR{gamma}) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin ({beta}3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real

  4. Magnetic structure of light nuclei from lattice QCD

    DOE PAGES

    Chang, Emmanuel; Detmold, William; Orginos, Kostas; Parreño, Assumpta; Savage, Martin J.; Tiburzi, Brian C.; Beane, Silas R.

    2015-12-09

    Lattice QCD with background magnetic fields is used to calculate the magnetic moments and magnetic polarizabilities of the nucleons and of light nuclei withmore » $$A\\le4$$, along with the cross-section for the $M1$ transition $$np\\rightarrow d\\gamma$$, at the flavor SU(3)-symmetric point where the pion mass is $$m_\\pi\\sim 806$$ MeV. These magnetic properties are extracted from nucleon and nuclear energies in six uniform magnetic fields of varying strengths. The magnetic moments are presented in a recent Letter. For the charged states, the extraction of the polarizability requires careful treatment of Landau levels, which enter non-trivially in the method that is employed. The nucleon polarizabilities are found to be of similar magnitude to their physical values, with $$\\beta_p=5.22(+0.66/-0.45)(0.23) \\times 10^{-4}$$ fm$^3$ and $$\\beta_n=1.253(+0.056/-0.067)(0.055) \\times 10^{-4}$$ fm$^3$, exhibiting a significant isovector component. The dineutron is bound at these heavy quark masses and its magnetic polarizability, $$\\beta_{nn}=1.872(+0.121/-0.113)(0.082) \\times 10^{-4}$$ fm$^3$ differs significantly from twice that of the neutron. A linear combination of deuteron scalar and tensor polarizabilities is determined by the energies of the $$j_z=\\pm 1$$ deuteron states, and is found to be $$\\beta_{d,\\pm 1}=4.4(+1.6/-1.5)(0.2) \\times 10^{-4}$$ fm$^3$. The magnetic polarizabilities of the three-nucleon and four-nucleon systems are found to be positive and similar in size to those of the proton, $$\\beta_{^{3}\\rm He}=5.4(+2.2/-2.1)(0.2) \\times 10^{-4}$$ fm$^3$, $$\\beta_{^{3}\\rm H}=2.6(1.7)(0.1) \\times 10^{-4}$$ fm$^3$, $$\\beta_{^{4}\\rm He}=3.4(+2.0/-1.9)(0.2) \\times 10^{-4}$$ fm$^3$. Mixing between the $j_z=0$ deuteron state and the spin-singlet $np$ state induced by the background magnetic field is used to extract the short-distance two-nucleon counterterm, $${\\bar L}_1$$, of the pionless effective theory for $NN$ systems (equivalent to the

  5. Competitive product inhibition of aromatase by natural estrogens.

    PubMed

    Shimizu, Y; Yarborough, C; Osawa, Y

    1993-03-01

    In order to better understand the function of aromatase, we carried out kinetic analyses to assess the ability of natural estrogens, estrone (E1), estradiol (E2), 16 alpha-OHE1, and estriol (E3), to inhibit aromatization. Human placental microsomes (50 micrograms protein) were incubated for 5 min at 37 degrees C with [1 beta-3H]testosterone (1.24 x 10(3) dpm 3H/ng, 35-150 nM) or [1 beta-3H,4-14C]androstenedione (3.05 x 10(3) dpm 3H/ng, 3H/14C = 19.3, 7-65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1 beta-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The Ki of E1, E2, 16 alpha-OHE1, and E3 for testosterone aromatization was 1.5, 2.2, 95, and 162 microM, respectively, where the Km of aromatase was 61.8 +/- 2.0 nM (n = 5) for testosterone. The Ki of E1, E2, 16 alpha-OHE1, and E3 for androstenedione aromatization was 10.6, 5.5, 252, and 1182 microM, respectively, where the Km of aromatase was 35.4 +/- 4.1 nM (n = 4) for androstenedione. These results show that estrogen inhibit the process of androgen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogen binds to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.

  6. [Anthropometric measures in urban child population from 6 to 12 years from the northwest of México].

    PubMed

    Brito-Zurita, Olga Rosa; López-Leal, Josefa; Exiga-González, Emma Beatriz; Armenta-Llanes, Oscar; Jorge-Plascencia, Blanca; Domínguez-Banda, Alberto; López-Morales, Mónica; Ornelas-Aguirre, José Manuel; Sabag-Ruiz, Enrique

    2014-01-01

    INTRODUCCIÓN: el grado de sobrepeso-obesidad tendrá variaciones de acuerdo con las condiciones de cada población, según el ámbito geográfico, la raza o etnia, el status socioeconómico y la susceptibilidad de cada individuo. El objetivo de este estudio fue determinar medidas antropométricas en la población infantil urbana de 6 a 12 años de Ciudad Obregón, Sonora. MÉTODOS: se estudiaron 684 escolares de 6 a 12 años de edad, sanos y de ambos géneros de la zona urbana de Ciudad Obregón, Sonora, en quienes se midieron peso, talla, perímetro braquial (PB), cintura e índice de masa corporal (IMC). Se realizó estadística descriptiva mediante frecuencias, porcentajes, e inferencial mediante t de Student para comparar las tablas de crecimiento de Ramos Galván y las del Centro para el Control y Prevención de Enfermedades (CDC).

  7. [Low grade fibromixoid sarcoma: a purpose of 3 cases and review of the bibliography].

    PubMed

    Citores-Pascual, Miguel Angel; Tinoco-Carrasco, Claudia; Arenal-Vera, Juan José; Benito-Fernández, César; Torres-Nieto, María de Los Ángeles; Zamora-Martínez, Tomás

    2013-01-01

    Antecedentes: el sarcoma fibromixoide de bajo grado o tumor de Evans es una variedad de sarcoma de partes blandas que representa el 1% de todos los tumores malignos. Más frecuente en extremidades y tronco (50%), presentamos tres nuevos casos de localización retroperitoneal. Se revisan sus características y se realiza actualización bibliográfica. Casos clínicos: en su localización retroperitoneal destaca lo inespecífico de su sintomatología clínica, se manifiesta en función de su crecimiento y tamaño, como una tumoración abdominal, habitualmente indolora, o por la compresión y/o invasión de estructuras próximas. Los casos presentados se manifestaron, por la presencia de tumoración abdominal, sin otra sintomatología; a pesar de infiltrar a otros órganos. Conclusiones: el diagnóstico preoperatorio es difícil, y debe tenerse la presunción clínica para solicitar el estudio histológico, con tipificación inmuno-histo-química para su dentificación precisa.

  8. Hysteresis loop behaviors of ferroelectric thin films: A Monte Carlo simulation study

    NASA Astrophysics Data System (ADS)

    M. Bedoya-Hincapié, C.; H. Ortiz-Álvarez, H.; Restrepo-Parra, E.; J. Olaya-Flórez, J.; E. Alfonso, J.

    2015-11-01

    The ferroelectric response of bismuth titanate Bi4Ti3O12 (BIT) thin film is studied through a Monte Carlo simulation of hysteresis loops. The ferroelectric system is described by using a Diffour Hamiltonian with three terms: the electric field applied in the z direction, the nearest dipole-dipole interaction in the transversal (x-y) direction, and the nearest dipole-dipole interaction in the direction perpendicular to the thin film (the z axis). In the sample construction, we take into consideration the dipole orientations of the monoclinic and orthorhombic structures that can appear in BIT at low temperature in the ferroelectric state. The effects of temperature, stress, and the concentration of pinned dipole defects are assessed by using the hysteresis loops. The results indicate the changes in the hysteresis area with temperature and stress, and the asymmetric hysteresis loops exhibit evidence of the imprint failure mechanism with the emergence of pinned dipolar defects. The simulated shift in the hysteresis loops conforms to the experimental ferroelectric response. Project sponsored by the research departments of the Universidad Nacional de Colombia DIMA and DIB under Project 201010018227-“Crecimiento y caracterización eléctrica y estructural de películas delgadas de BixTiyOz producidas mediante Magnetrón Sputtering” and Project 12920-“Desarrollo teóricoexperimental de nanoestructuras basadas en Bismuto y materiales similares” and “Bisnano Project.”

  9. [Pathogenesis of spinal cord injuries and mechanisms of repair induced by olfactory ensheathing cells].

    PubMed

    Botero, Lucía; Gomez, Rosa Margarita; Chaparro, Orlando

    2013-05-16

    Introduccion. La lesion medular es un evento catastrofico, cuyas consecuencias persisten durante toda la vida del paciente. La investigacion en tratamiento se ha basado principalmente en el desarrollo de terapias que reduzcan la discapacidad, pero desde los anos noventa hay un avance significativo y se han probado varios trasplantes celulares en modelos animales de lesion medular, celulas de Schwann, astrocitos y celulas de la glia envolvente olfatoria (CGEO). Objetivo. Hacer un recuento detallado de la patogenia de la lesion medular primaria y secundaria y de los mecanismos por los cuales las CGEO inducirian sus posibles efectos regenerativos descritos en la bibliografia. Desarrollo. Despues del traumatismo, la lesion se desarrolla en dos fases, la primaria se caracteriza por las lesiones de compresion y la secundaria se produce por una serie de factores que se dan en paralelo y que incluyen factores vasculares, celulares, moleculares y formacion de cicatriz glial. La mayoria de los modelos de lesion medular y trasplante con CGEO han comunicado recuperacion funcional, remielinizacion y regeneracion axonal. Estas celulas ejercen su accion de manera indirecta a traves de la produccion de factores de crecimiento y de manera directa induciendo regeneracion neuronal, axonal y remielinizacion. Conclusiones. Las CGEO son una opcion terapeutica en pacientes con lesion medular debido a que inducen de modo directo o indirecto regeneracion neuronal, axonal, remielinizacion de axones, disminucion de cicatriz glial y otros efectos que conducen a la recuperacion funcional.

  10. [Nutrition and child development II; a comparison of physical development in two school children groups with different income level (Rev Clin Esp 1944; 12:155-164) ].

    PubMed

    Grande Covián, F; Rof Carballo, J; Jiménez García, F

    2014-09-01

    El estudio comparativo de dos grupos de niños en edad escolar y de nivel económico diferente demuestra que los que pertenecen a un suburbio madrileño económicamente pobre sufren un retraso en el crecimiento que se caracteriza por una talla y un peso inferiores en relación con la edad. El retraso del peso corporal es proporcionalmente mayor al de la talla. Cuando se calcula según WETZEL la edad que teóricamente corresponde al desarrollo corporal de estos niños se observa un retraso de 2,94 años para una edad término medio de 10,14 años. El retraso global del desarrollo somático representa por tanto el 29 por 100 de la edad cronológica media. Este retraso en el desarrollo debe atribuirse principalmente a causas dietéticas y no solo a carencias específicas, sino sobre todo a insuficiencia cuantitativa en la dieta consumida. Desde el punto de vista cualitativo es probablemente la falta de leche en la dieta uno de los más importantes factores en la génesis de este trastorno del desarrollo.

  11. [Neuronal communication and synaptic metabolism in childhood epilepsy].

    PubMed

    García-Cazorla, Àngels; Cortès-Saladelafont, Elisenda; Duarte, Sofia

    2015-03-01

    Introduccion. Los conocimientos que la neurociencia basica y el neurometabolismo estan aportando en epilepsia pediatrica, y en concreto en mecanismos de comunicacion sinaptica, crecen rapidamente. Existe, no obstante, una desconexion entre estos avances y una vision que los integre de manera global y en la practica clinica y terapeutica. Objetivos. Ofrecer una vision integradora de los diferentes mecanismos moleculares y metabolicos que se conocen y postulan en epilepsia pediatrica, y sugerir conceptos como el de 'metabolismo sinaptico' y 'fenotipos sinapticos' como herramientas utiles para desarrollar este enfoque. Desarrollo. Se revisan los estudios mas destacados que intentan explicar las caracteristicas esenciales de la comunicacion sinaptica en el cerebro en desarrollo, a traves de diferentes moleculas, basicamente proteinas sinapticas, canales ionicos (cotransportadores de cloro, sodio y potasio), la compartimentalizacion pre y postsinaptica, y los principales actores metabolicos (neurotransmisores, metabolismo energetico, factores de crecimiento y lipidos). A partir de esta combinacion de mecanismos biologicos se sugieren ejemplos de 'fenotipos sinapticos' en dos casos concretos de epilepsia genetica (SCN1A) y metabolica (epilepsia con respuesta a la piridoxina). Conclusiones. Una perspectiva holistica, entendiendo la diversidad de elementos relacionados y que suceden en determinados momentos del neurodesarrollo, puede ayudar a delinear fenotipos, vias de metabolismo sinaptico y conectividad cerebral, que faciliten no solo la comprension de la fisiopatologia, sino nuevas aproximaciones terapeuticas en epilepsia pediatrica.

  12. [Recurring post-traumatic growing skull fracture].

    PubMed

    San Martín-García, Isabel; Aguilera-Albesa, Sergio; Zazpe-Cenoz, Idoya; Yoldi-Petri, M Eugenia

    2015-04-16

    Introduccion. La fractura craneal evolutiva, tambien llamada absorcion osea postraumatica o quiste leptomeningeo, es una rara complicacion de los traumatismos craneoencefalicos y ocurre de forma casi exclusiva en ninos menores de 3 anos. Caso clinico. Nino de 6 meses que presentaba, dos meses despues de un traumatismo craneal aparentemente banal, persistencia de cefalohematoma temporooccipital izquierdo sin otros signos. El estudio de ecografia transfontanelar revelo un defecto oseo con herniacion cerebral, y la tomografia computarizada y la resonancia magnetica confirmaron, ademas, una fractura evolutiva. Se realizo reseccion del quiste encefalomeningeo, cierre dural y reparacion del defecto oseo con placas y material de lactato. Tres meses despues de la intervencion, presentaba persistencia de coleccion liquida y se confirmo recidiva de la fractura evolutiva. Tras la reintervencion, se coloco casco ortesico para evitar nuevas recidivas. Un ano despues del traumatismo, el paciente continua asintomatico. Conclusiones. Todo nino menor de 3 anos con cefalohematoma postraumatico deberia ser revisado de forma periodica hasta comprobar la resolucion de la coleccion, en particular si presenta fractura craneal. La presencia de un cefalohematoma persistente mas de dos semanas despues de un traumatismo craneoencefalico debe hacernos sospechar un proceso de fractura creciente, y son necesarias la reparacion de la duramadre y una craneoplastia para su tratamiento. La colocacion de material reabsorbible permite su remodelacion con el crecimiento craneal del paciente, pero su fragilidad conlleva riesgo de recidiva. La colocacion de un casco ortesico tras la intervencion podria prevenir complicaciones.

  13. Cabergoline or bromocriptine for prolactinoma?

    PubMed

    Triantafilo, Nicolás; Castro-Gutiérrez, Victoria; Rada, Gabriel

    2016-09-15

    Cabergolina y bromocriptina son los fármacos más utilizados en el manejo del prolactinoma. Si bien cabergolina, un agonista dopaminérgico de vida media larga, tendría algunas ventajas desde el punto de vista fisiopatológico, no está claro si esto se traduce en un real beneficio. Utilizando la base de datos Epistemonikos, la cual es mantenida mediante búsquedas en 30 bases de datos, identificamos dos revisiones sistemáticas que en conjunto comprenden 12 estudios relevantes para la pregunta, incluyendo cinco estudios controlados aleatorizados. Realizamos un metanálisis y tablas de resumen de los resultados utilizando el método GRADE. Concluimos que la cabergolina es superior a la bromocriptina en la mejoría de la oligomenorrea/amenorrea y la galactorrea, probablemente aumenta la probabilidad de embarazo, y se asocia a menos efectos adversos. No está claro si cabergolina también es más efectiva sobre el crecimiento del tumor porque la certeza de la evidencia es muy baja.

  14. [Applicability of BMI in adolescent students living at moderate altitude of Perú].

    PubMed

    Cossio Bolaños, Marco Antonio; Viveros Flores, Antonio; Eduardo Hespanhol, Jefferson; Camargo, Cristiane; Gomez Campos, Rossana

    2014-11-01

    Introducción y objetivos: El uso del IMC es muy cuestionado, sobretodo, en poblaciones en fase de crecimiento que se caracterizan por presentar baja estatura para su edad. El objetivo es verificar si el IMC es aplicable a una muestra de escolares adolescentes que viven en una región de moderada altitud del Perú. Material y Métodos: Estudio descriptivo transversal realizado en 319 adolescentes escolares (181 hombres y 138 mujeres) de 12,0 a 17,9 años de edad. Se evaluó las variables antropométricas del peso y la estatura. Se calculó el Índice de Masa Corporal (IMC). Se utilizó la referencia del CDC-2000 para comparar el peso y estatura a partir del Z-score y el IMC por medio de diferencia de medias. Resultados: El Z-score para el peso corporal mostró pequeñas variaciones (entre -0,3 a 0,3 kg). En la estatura se observa valores negativos para ambos sexos (hombres entre -0,3 a -1,3 cm y mujeres entre -0,5 a 1,3 cm). Respecto al IMC, hubo diferencias significativas en todas la edades y en ambos sexos (p.

  15. [Hemorrhagic colloid cyst of the third ventricle: fulminant deterioration].

    PubMed

    Martínez-Gómez, Debora; Joanes, Vicente; Herrera, Juan Manuel; Rivera-Paz, Marlon; Vanaclocha, Vicente

    2015-03-16

    Introduccion. Los quistes coloides son tumores benignos del tercer ventriculo. La mayoria permanecen asintomaticos. Sin embargo, algunos pacientes pueden desarrollar desde cefaleas intermitentes hasta un deterioro agudo e incluso muerte subita. Existen varias teorias por las que se produciria una muerte subita en estos pacientes, entre las que se incluyen el rapido crecimiento del quiste, su rotura, la alteracion del control cardiovascular reflejo mediado por el hipotalamo y, rara vez, el sangrado del quiste, con solo 15 casos descritos en la bibliografia. Caso clinico. Varon de 45 años, con hipertension arterial como unico antecedente de interes, que sufre un deterioro brusco del nivel de consciencia a causa de una hidrocefalia aguda debida a un quiste coloide hemorragico del tercer ventriculo. Se implanto un drenaje ventricular externo en cada lado e ingreso en la unidad de cuidados intensivos, donde se certifico la muerte encefalica. Tras la extirpacion del quiste mediante abordaje transcortical frontal, se confirmo el diagnostico de quiste coloide con restos de material hemolizado subagudo. Conclusion. La hemorragia en los quistes coloides es excepcional y puede producirse en pacientes tanto sintomaticos como asintomaticos, lo que dificulta, de forma extraordinaria, el reconocimiento de esta complicacion.

  16. La dinámica demográfica y la desigualdad educativa en Méxicoa

    PubMed Central

    Giorguli Saucedo, Silvia E.; Vargas Valle, Eunice D.; Ulloa, Viviana Salinas; Hubert, Celia; Potter, Joseph E.

    2015-01-01

    En este trabajo se presenta una investigación sobre el vínculo entre los procesos educativos y la dinámica demográfica en México. La tradición del pensamiento sobre población y desarrollo ha partido de la hipótesis de que el ritmo de crecimiento poblacional, el tamaño de las familias y la migración influyen sobre la acumulación de capital humano entre la población en edades escolares. Se explora aquí la relación del desempeño educativo de los jóvenes entre 14 y 23 años y la razón de dependencia juvenil, la fecundidad adolescente y la migración interna e internacional; para ello se utilizan datos agregados a escala municipal del año 2000. El análisis incluye indicadores de la oferta educativa municipal basados en estadísticas administrativas de la SEP. PMID:26146427

  17. Regulación del flujo sanguíneo uterino. II. Funciones de estrógeno y receptores estrogénicos α/β en acciones genómicas y no-genómicas del endotelio uterino *

    PubMed Central

    Mayra, Pastore R.; Rosalina, Villalón L.; López, Gladys; Iruretagoyena, Jesús; Magness, Ronald

    2015-01-01

    Resumen El embarazo está marcado por cambios y adaptaciones cardiovasculares que son importantes para el crecimiento y mantenimiento de la placenta y el feto. Durante este periodo, las adaptaciones vasculares uterinas manifiestan cambios clasificados como de corto o largo plazo los cuales están relacionados con adaptaciones vasodilatadoras, angiogénicas o de remodelación. El estrógeno y los receptores estrogénicos clásicos (REs), RE-α y RE-β, han demostrado ser parcialmente responsables por facilitar el incremento dramático en el fluido sanguíneo uterino necesario durante el embarazo. En ésta revisión bibliográfica se discuten la base estructural para la diversidad y selectividad funcional de los REs por el estrógeno, el papel de los REs sobre los efectos genómicos y no-genómicos en células endoteliales de arterias uterinas (CEAU). Estos temas integran el conocimiento científico sobre la regulación molecular de CEAU para mantener el incremento fisiológico en la perfusión útero-placentaria observada durante un embarazo normal. PMID:26113751

  18. [Anorexia-cachexia frequency and its gastrointestinal symptoms association in paliative patients at the Instituto Nacional de Cancerología, México].

    PubMed

    Pérez Camargo, Dana Aline; Allende Pérez, Silvia R; Meneses García, Abelardo; De Nicola Delfin, Luigina; Copca Mendoza, Erika Thalía; Sánchez López, Miriam S; Flores García, Martha Karen; Verástegui Avilés, Emma

    2014-10-01

    Introducción: La anorexia-caquexia es un síndrome frecuente en pacientes oncológicos, particularmente en etapas avanzadas: la prevalencia global de anorexia-caquexia para-neoplásica oscila entre el 20-40% en los enfermos en la fase diagnóstica, y entre el 70–80% en la fase avanzada de la enfermedad. En los enfermos oncológicos es frecuente la coexistencia de alteraciones digestivas estructurales o funcionales, secundarias al crecimiento tumoral y a los fenómenos sistémicos de carácter metabólico, determinados por la interrelación tumor-huésped específicos de la anorexia-caquexia. Objetivo: El objetivo de este trabajo es conocer la frecuencia de anorexia-caquexia, y su asociación con síntomas gastrointestinales en pacientes en terreno paliativo del Instituto Nacional de Cancerología, Ciudad de México, atendidos en el servicio de Cuidados Paliativos. Material y métodos: Se realizó un estudio transversal analítico. Se incluyeron 100 pacientes con diagnóstico de cáncer avanzado, con un rango de edad de 18 y 80 años, y una puntuación de Karnofsky > 50, así como un ECOG.

  19. [Weight-for-age percentile curves for the population of Catamarca province (Argentina)].

    PubMed

    López Barbancho, Diego; Terán de Frutos, José Manuel; Candelas González, Nieves; Díaz de Luna, María Cristina; Marrodán Serrano, María Dolores; Lomaglio, Delia Beatriz

    2014-09-12

    Introducción: La evaluación del desarrollo fetal y del peso al nacimiento es un objetivo prioritario en el ámbito de la epidemiología nutricional y de la salud pública. En la actualidad, la Organización Mundial de la Salud (OMS) no dispone de curvas específicas de peso al nacimiento para las distintas semanas gestacionales al considerar que las características socioeconómicas, ecológicas y étnicas de los distintos grupos humanos influyen en sus propios patrones de crecimiento. Objetivo: La presente investigación desarrolla las curvas percentilares de peso por edad gestacional para las poblaciones residentes en la provincia de Catamarca. Métodos: El procesamiento de los datos se llevó a cabo con el programa SPSS y para la posterior realización de las curvas percentilares se utilizó el programa LMS Chart Maker pro, a partir de un total de 22.576 nacimientos ocurridos entre 1994 y 2003. Discusión y conclusiones: Dichas curvas posibilitarán en el futuro una mejor valoración del desarrollo intrauterino y del peso de los recién nacidos en esta región del Noroeste de Argentina.

  20. Expression of CD24 in Human Bone Marrow-Derived Mesenchymal Stromal Cells Is Regulated by TGFβ3 and Induces a Myofibroblast-Like Genotype

    PubMed Central

    Schäck, Luisa Marilena; Buettner, Manuela; Wirth, Alexander; Krettek, Christian; Hoffmann, Andrea; Noack, Sandra

    2016-01-01

    Human bone marrow-derived stromal cells (hBMSCs) derived from the adult organism hold great promise for diverse settings in regenerative medicine. Therefore a more complete understanding of hBMSC biology to fully exploit the cells' potential for clinical settings is important. The protein CD24 has been reported to be involved in a diverse range of processes such as cancer, adaptive immunity, inflammation, and autoimmune diseases in other cell types. Its expression in hBMSCs, which has not yet been analyzed, may add an important aspect in the understanding of hBMSC biology. The present study therefore analyzes the expression, regulation, and functional implication of the surface protein CD24 in hBMSCs. Methods used are stimulation studies with TGF beta as well as shRNA-mediated knockdown and overexpression of CD24 followed by microarray, immunocytochemistry, and flow cytometric analyses. To our knowledge, we demonstrate for the first time that the expression of CD24 is an inherent property of hBMSCs. Importantly, the data links the upregulation of CD24 to the adoption of a myofibroblast-like gene expression pattern in hBMSCs. We demonstrate that CD24 is an important modulator in transforming growth factor beta 3 (TGFβ3) signaling with a reciprocal regulatory relationship between these two proteins. PMID:26788063

  1. Chondrogenic differentiation of amniotic fluid-derived stem cells.

    PubMed

    Kolambkar, Yash M; Peister, Alexandra; Soker, Shay; Atala, Anthony; Guldberg, Robert E

    2007-10-01

    For regenerating damaged articular cartilage, it is necessary to identify an appropriate cell source that is easily accessible, can be expanded to large numbers, and has chondrogenic potential. Amniotic fluid-derived stem (AFS) cells have recently been isolated from human and rodent amniotic fluid and shown to be highly proliferative and broadly pluripotent. The purpose of this study was to investigate the chondrogenic potential of human AFS cells in pellet and alginate hydrogel cultures. Human AFS cells were expanded in various media conditions, and cultured for three weeks with growth factor supplementation. There was increased production of sulfated glycosaminoglycan (sGAG) and type II collagen in response to transforming growth factor-beta (TGF-beta) supplementation, with TGF-beta1 producing greater increases than TGF-beta3. Modification of expansion media supplements and addition of insulin-like growth factor-1 during pellet culture further increased sGAG/DNA over TGF-beta1 supplementation alone. Compared to bone marrow-derived mesenchymal stem cells, the AFS cells produced less cartilaginous matrix after three weeks of TGF-beta1 supplementation in pellet culture. Even so, this study demonstrates that AFS cells have the potential to differentiate along the chondrogenic lineage, thus establishing the feasibility of using these cells for cartilage repair applications. PMID:17668282

  2. Nanomechanics of Engineered Articular Cartilage: Synergistic Influences of Transforming Growth Factor-β3 and Oscillating Pressure.

    PubMed

    Nazempour, Arshan; Quisenberry, Chrystal R; Van Wie, Bernard J; Abu-Lail, Nehal I

    2016-03-01

    Articular cartilage (AC), tissue with the lowest volumetric cellular density, is not supplied with blood and nerve tissue resulting in limited ability for self-repair upon injury. Because there is no treatment capable of fully restoring damaged AC, tissue engineering is being investigated. The emphasis of this field is to engineer functional tissues in vitro in bioreactors capable of mimicking in vivo envi- ronments required for appropriate cellular growth and differentiation. In a step towards engineering AC, human adipose-derived stem cells were differentiated in a unique centrifugal bioreactor under oscillating hydrostatic pressure (OHP) and supply of transforming growth factor beta 3 (TGF-β3) that mimic in vivo environments. Static micromass and pellet cultures were used as controls. Since withstanding and absorbing loads are among the main functions of an AC, mechanical properties of the engineered AC tissues were assayed using atomic force microscopy (AFM) under a controlled indentation depth of 100 nm. Young's moduli of elasticity were quantified by modeling AFM force-indentation data using the Hertz model of contact mechanics. We found exposure to OHP causes cartilage constructs to have 45-fold higher Young's moduli compared to static cultures. Addition of TGF-β3 further increases Young's moduli in bioreactor samples by 1.9-fold bringing it within 70.6% of the values estimated for native cartilage. Our results imply that OHP and TGF-β3 act synergistically to improve the mechanics of engineered tissues. PMID:27455774

  3. Optimized Markov state models for metastable systems

    NASA Astrophysics Data System (ADS)

    Guarnera, Enrico; Vanden-Eijnden, Eric

    2016-07-01

    A method is proposed to identify target states that optimize a metastability index amongst a set of trial states and use these target states as milestones (or core sets) to build Markov State Models (MSMs). If the optimized metastability index is small, this automatically guarantees the accuracy of the MSM, in the sense that the transitions between the target milestones is indeed approximately Markovian. The method is simple to implement and use, it does not require that the dynamics on the trial milestones be Markovian, and it also offers the possibility to partition the system's state-space by assigning every trial milestone to the target milestones it is most likely to visit next and to identify transition state regions. Here the method is tested on the Gly-Ala-Gly peptide, where it is shown to correctly identify the expected metastable states in the dihedral angle space of the molecule without a priori information about these states. It is also applied to analyze the folding landscape of the Beta3s mini-protein, where it is shown to identify the folded basin as a connecting hub between an helix-rich region, which is entropically stabilized, and a beta-rich region, which is energetically stabilized and acts as a kinetic trap.

  4. Hyaluronan content in experimental carcinoma is not correlated to interstitial fluid pressure.

    PubMed

    Jacobson, Annica; Salnikov, Alexei; Lammerts, Ellen; Roswall, Pernilla; Sundberg, Christian; Heldin, Paraskevi; Rubin, Kristofer; Heldin, Nils-Erik

    2003-06-13

    Mechanism(s) for generation of the high tumor interstitial fluid pressure (TIFP) that is characteristic of carcinoma is not known. We investigated the role of hyaluronan, the major water-binding polysaccharide of the extracellular matrix, for the generation of a high TIFP. A human anaplastic thyroid carcinoma (KAT-4) xenografted to athymic mice and a syngeneic rat colon carcinoma (PROb) were used. Neither KAT-4 nor PROb cells produced hyaluronan (HA) in culture, however, both cell lines produced factors that stimulated HA-synthesis by cultured fibroblasts. Modulating hyaluronan levels by transfection of PROb carcinoma cells with hyaluronan synthase-2 revealed no correlation between hyaluronan content and TIFP. Furthermore, lowering of TIFP by treating KAT-4 tumors with a specific inhibitor of TGF-beta 1 and -beta 3 did not change the concentration of hyaluronan in the tumors. In summary, our results suggest that a modulation of hyaluronan content is not a major pathogenetic mechanism for the generation of the characteristically high TIFP in malignant carcinomas.

  5. Transforming growth factor-beta regulates stearoyl coenzyme A desaturase expression through a Smad signaling pathway.

    PubMed

    Samuel, William; Nagineni, Chandrasekharam N; Kutty, R Krishnan; Parks, W Tony; Gordon, Joel S; Prouty, Stephen M; Hooks, John J; Wiggert, Barbara

    2002-01-01

    The regulation of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is physiologically important because the ratio of saturated to unsaturated fatty acids is thought to control cellular functions by modulating the structural integrity and fluidity of cell membranes. Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, increased SCD mRNA expression in cultured human retinal pigment epithelial cells. This response was elicited by all three TGF-beta isoforms, beta1, beta2, and beta3. However, SCD mRNA expression was not increased either by other members of the TGF-beta family or by other growth factors or cytokines. TGF-beta also increased SCD mRNA expression in several other cell lines tested. The increase in SCD mRNA expression was preceded by a marked increase in Smad2 phosphorylation in TGF-beta-treated human retinal pigment epithelial cells. TGF-beta did not induce SCD mRNA expression in a Smad4-deficient cell line. However, Smad4 overexpression restored the TGF-beta effect in this cell line. Moreover, TGF-beta-induced SCD mRNA expression was effectively blocked by the overexpression of Smad7, an inhibitory Smad. Thus, a TGF-beta signal transduction pathway involving Smad proteins appears to regulate the cellular expression of the SCD gene, and this regulation may play an important role in lipid metabolism.

  6. Sulfhydryl groups of the F1 adenosine triphosphatase of Escherichia coli and the stoichiometry of the subunits.

    PubMed

    Stan-Lotter, H; Bragg, P D

    1984-02-15

    The distribution and total number of sulfhydryl groups present in the F1 adenosine triphosphatase of Escherichia coli were used to calculate the stoichiometry of the alpha-delta subunits. Titration with 5,5'-dithiobis (2-nitrobenzoate) gave 19.1 +/- 2.2 sulfhydryl groups/mol ATPase. Labeling with [14C]iodoacetamide and [14C]N-ethylmaleimide showed that 11.9, 3.1, 1.9, and 1.8 sulfhydryl groups per molecule of ATPase were associated with the alpha, beta, gamma, and delta subunits, respectively. The epsilon subunit was not labeled. Application of the method of Creighton [Nature (London) (1980) 284, 487-489] showed that 4, 1, and 2 sulfhydryl groups were present in the alpha, beta, and gamma subunits, respectively. This, together with published data for the delta subunit, allowed a subunit stoichiometry of alpha 3 beta 3 gamma delta to be calculated. The presence of four cysteinyl residues in the alpha subunit, as shown by several different methods, does not agree with the results of DNA sequencing of the ATPase genes [H. Kanazawa, T. Kayano, K. Mabuchi, and M. Futai (1981) Biochem. Biophys. Res. Commun. 103, 604-612; N. J. Gay and J. E. Walker (1981) Nucl. Acids Res. 9, 2187-2194] where three cysteinyl residues/alpha subunit have been found. It is suggested that post-translational modification of the alpha subunit to add a fourth cysteinyl residue might occur.

  7. Detection of novel LAMA3 mutation in Herlitz junctional epidermolysis bullosa in a Jordanian family.

    PubMed

    Badran, Eman F; Battah, Hamdi A; Akl, Kamal F; Khalil, Raida W; Al Amori, Iyad

    2013-08-01

    The Herlitz junctional epidermolysis bullosa (H-JEB) subtype usually presents as a severe lethal inherited variant of epidermolysis bullosa (EB) caused by a homozygous mutation in the genes LAMA3, LMAB3, or LAMAC3. Each gene encodes one of the three chains of heterotrimer laminin-332 proteins (including the alpha-3 chain, beta-3 chain and gamma-2-chain) responsible for the adherence of the epidermis to the underlying dermis. The aim of this report is to add to the existing knowledge about EB by describing a novel mutation in a gene responsible for genodermatosis. A case of a Jordanian male neonate, born to healthy, first cousin consanguineous parents, who developed nonhealing blistering skin and mucous membrane lesions, crusted erosions with significant granulation tissue and dystrophic nails immediately after birth is described. The patient was diagnosed as having a novel LAMA3 mutation causing (H-JEB) by immunofluorescence mapping and molecular analysis. Both parents and this baby's sibling were shown to be heterozygous carriers of the same mutation. Pre-implantation diagnosis using molecular analysis for subsequent pregnancies in this family is crucial for managing any new pregnancy. PMID:22963541

  8. Maternal uniparental disomy of chromosome 1 with reduction to homozygosity of the LAMB3 locus in a patient with Herlitz junctional epidermolysis bullosa.

    PubMed

    Pulkkinen, L; Bullrich, F; Czarnecki, P; Weiss, L; Uitto, J

    1997-09-01

    Junctional epidermolysis bullosa (JEB) is an autosomal recessive disorder characterized by blister formation at the level of the lamina lucida within the cutaneous basement-membrane zone. Classic lethal JEB (Herlitz type [H-JEB]; OMIM 226700) is frequently associated with premature-termination-codon mutations in both alleles of one of the three genes (LAMA3, LAMC2, or LAMB3) encoding the subunit polypeptides (alpha3, beta3, and gamma2) of laminin 5. In this study, we describe a unique patient with H-JEB, who was homozygous for a nonsense mutation, Q243X, in the LAMB3 gene on chromosome 1 and who had normal karyotype 46,XY. The mother was found to be a carrier of the Q243X mutation, whereas the father had two normal LAMB3 alleles. Nonpaternity was excluded by use of 11 microsatellite markers from six different chromosomes. The use of 17 partly or fully informative microsatellite markers spanning the entire chromosome 1 revealed that the patient had both maternal uniparental meroisodisomy of a 35-cM region on 1q containing the maternal LAMB3 mutation and maternal uniparental heterodisomy of other regions of chromosome 1. Thus, the results suggested that reduction to homozygosity of the 1q region containing the maternal LAMB3 mutation caused the H-JEB phenotype. The patient was normally developed at term and did not show overt dysmorphisms or malformations. This is the first description of uniparental disomy of human chromosome 1. PMID:9326326

  9. The epitheliogenesis imperfecta locus maps to equine chromosome 8 in American Saddlebred horses.

    PubMed

    Lieto, L D; Cothran, E G

    2003-01-01

    Epitheliogenesis imperfecta (EI) is a hereditary junctional mechanobullous disease that occurs in newborn American Saddlebred foals. The pathological signs of epitheliogenesis imperfecta closely match a similar disease in humans known as Herlitz junctional epidermolysis bullosa, which is caused by a mutation in one of the genes (LAMA3, LAMB3 and LAMC2) coding for the subunits of the laminin 5 protein (laminin alpha3, laminin beta3 and laminin gamma2). The LAMA3 gene has been assigned to equine chromosome 8 and LAMB3 and LAMC2 have been mapped to equine chromosome 5. Linkage disequilibrium between microsatellite markers that mapped to equine chromosome 5 and equine chromosome 8 and the EI disease locus was tested in American Saddlebred horses. The allele frequencies of microsatellite alleles at 11 loci were determined for both epitheliogenesis imperfecta affected and unaffected populations of American Saddlebred horses by genotyping and direct counting of alleles. These were used to determine fit to Hardy-Weinberg equilibrium for control and EI populations using Chi square analysis. Two microsatellite loci located on equine chromosome 8q, ASB14 and AHT3, were not in Hardy-Weinberg equilibrium in affected American Saddlebred horses. In comparison, all of the microsatellite markers located on equine chromosome 5 were in Hardy-Weinberg equilibrium in affected American Saddlebred horses. This suggested that the EI disease locus was located on equine chromosome 8q, where LAMA3 is also located. PMID:14970704

  10. A homozygous nonsense mutation in the alpha 3 chain gene of laminin 5 (LAMA3) in lethal (Herlitz) junctional epidermolysis bullosa.

    PubMed

    Kivirikko, S; McGrath, J A; Baudoin, C; Aberdam, D; Ciatti, S; Dunnill, M G; McMillan, J R; Eady, R A; Ortonne, J P; Meneguzzi, G

    1995-05-01

    The inherited mechanobullous disorder, junctional epidermolysis bullosa (JEB), is characterized by extensive blistering and erosions of the skin and mucous membranes. The diagnostic hallmarks of JEB include ultrastructural abnormalities in the hemidesmosomes of the cutaneous basement membrane zone, as well as an absence of staining with antibodies against the anchoring filament protein, laminin 5. Therefore, the three genes encoding alpha 3, beta 3 and gamma 2 chains of laminin 5, known as LAMA3, LAMB3 and LAMC2, are candidate genes for JEB. We have previously demonstrated mutations in the LAMB3 and LAMC2 genes in several families with JEB. We initiated mutation analysis from an affected child by PCR amplification of individual LAMA3 exons, followed by heteroduplex analysis. Nucleotide sequencing of heteroduplexes identified a homozygous nonsense mutation within domain I/II of the alpha 3 chain. These findings provide the first evidence that nonsense mutations within the LAMA3 gene are also involved in the pathogenesis of JEB, and indicate that mutations of all three genes of laminin 5 can result in the JEB phenotype. PMID:7633458

  11. Abnormal development of glomerular endothelial and mesangial cells in mice with targeted disruption of the lama3 gene.

    PubMed

    Abrass, C K; Berfield, A K; Ryan, M C; Carter, W G; Hansen, K M

    2006-09-01

    Mice with targeted disruption of the lama3 gene, which encodes the alpha3 chain of laminin-5 (alpha3beta3gamma2, 332), develop a blistering skin disease similar to junctional epidermolysis bullosa in humans. These animals also develop abnormalities in glomerulogenesis. In both wild-type and mutant animals (lama3(-/-)), podocytes secrete glomerular basement membrane and develop foot processes. Endothelial cells migrate into this scaffolding and secrete a layer of basement membrane that fuses with the one formed by the podocyte. In lama3(-/-) animals, glomerular maturation arrests at this stage. Endothelial cells do not attenuate, develop fenestrae, or form typical lumens, and mesangial cells (MCs) were not identified. LN alpha3 subunit (LAMA3) protein was identified in the basement membrane adjacent to glomerular endothelial cells (GEnCs) in normal rats and mice. In developing rat glomeruli, the LAMA3 subunit was first detectable in the early capillary loop stage, which corresponds to the stage at which maturation arrest was observed in the mutant mice. Lama3 mRNA and protein were identified in isolated rat and mouse glomeruli and cultured rat GEnCs, but not MC. These data document expression of LAMA3 in glomeruli and support a critical role for it in GEnC differentiation. Furthermore, LAMA3 chain expression and/or another product of endothelial cells are required for MC migration into the developing glomerulus. PMID:16850021

  12. Inhibition of laminin-5 production in breast epithelial cells by overexpression of p300.

    PubMed

    Miller, K A; Chung, J; Lo, D; Jones, J C; Thimmapaya, B; Weitzman, S A

    2000-03-17

    The transcriptional coactivator p300 is essential for normal embryonic development and cellular differentiation. We have been studying the role of p300 in the transcription of a variety of genes, and we became interested in the role of this coactivator in the transcription of genes important in breast epithelial cell biology. From MCF-10A cells (spontaneously immortalized, nontransformed human breast epithelial cells), we developed cell lines that stably overexpress p300. These p300-overexpressing cells displayed reduced adhesion to culture dishes and were found to secrete an extracellular matrix deficient in laminin-5. Laminin-5 is the major extracellular matrix component produced by breast epithelium. Immunofluorescence studies, as well as experiments using normal matrix, confirmed that the decreased adhesion of p300-overexpressing cells is due to laminin-5-deficient extracellular matrix and not due to loss of laminin-5 receptors. Northern blots revealed markedly decreased levels of expression of two of the genes (designated LAMA3 and LAMC2) encoding the alpha3 and gamma2 chains of the laminin-5 heterotrimer in the cells that overexpress p300, whereas LAMB3 mRNA, encoding the third or beta3 chain of laminin-5, was not markedly reduced. Transient transfection experiments with a vector containing a murine LAMA3 promoter demonstrate that overexpressing p300 down-regulates the LAMA3 promoter. In summary, overexpression of p300 leads to down-regulation of laminin-5 production in breast epithelial cells, resulting in decreased adhesion. PMID:10713141

  13. The actions of neurotensin in rat bladder detrusor contractility

    PubMed Central

    Dong, Xingyou; Bai, Xinyu; Zhao, Jiang; Wang, Liang; Wang, Qingqing; Li, Longkun

    2015-01-01

    This study assessed the expression, distribution and function of neurotensin (NTs) and two main neurotensin receptors (NTSR), NTSR1 and NTSR2 in normal rat urinary bladders. NTs is primarily located in the suburothelium and the interstitium of smooth muscle bundles. The NTSR1 and NTSR2 receptor subtypes are found to co-localize with smooth muscle cells (SMCs). NTs not only can directly act on bladder SMCs to induce intracellular calcium mobilization by activating the phospholipase C/inositol triphosphate (PLC/IP3) pathway, promoting extracellular calcium influx through a non-selective cation channels, but may be also involved in the modulation of the cholinergic system. Nowadays, the selective antimuscarinic drugs (solifenacin) and the selective beta 3-adrenergic agonist (mirabegron) are used as the first-line pharmacotherapy for overactive bladder (OAB), but without satisfactory treatment benefits in some patients. This study provided evidence suggesting that bladder NTs may play an important role in the regulation of micturition. Further research is needed to investigate the effects of NTs on bladder contractility and the underlying mechanism, which might reveal that the administration of NTSR antagonists can potentially relieve the symptoms of OAB by coordination with antimuscarinic pharmacotherapy. PMID:26053252

  14. Microbial transformations of the antimelanoma agent betulinic acid.

    PubMed

    Kouzi, S A; Chatterjee, P; Pezzuto, J M; Hamann, M T

    2000-12-01

    Microbial transformation studies of the antimelanoma agent betulinic acid (1) were conducted. Screening experiments showed a number of microorganisms capable of biotransforming 1. Three of these cultures, Bacillus megaterium ATCC 14581, Cunninghamella elegans ATCC 9244, and Mucor mucedo UI-4605, were selected for preparative scale transformation. Bioconversion of 1 with resting-cell suspensions of phenobarbital-induced B. megaterium ATCC 14581 resulted in the production of the known betulonic acid (2) and two new metabolites: 3beta,7beta-dihydroxy-lup-20(29)-en-28-oic acid (3) and 3beta,6alpha, 7beta-trihydroxy-lup-20(29)-en-28-oic acid (4). Biotransformation of 1 with growing cultures of C. elegans ATCC 9244 produced one new metabolite characterized as 1beta,3beta, 7beta-trihydroxy-lup-20(29)-en-28-oic acid (5). Incubation of 1 with growing cultures of M. mucedo UI-4605 afforded metabolite 3. Structure elucidation of all metabolites was based on NMR and HRMS analyses. In addition, the antimelanoma activity of metabolites 2-5 was evaluated against two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid).

  15. Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies.

    PubMed

    Nagai, Masaru; Kawata, Maki; Watanabe, Hisayuki; Ogawa, Machiko; Saito, Kumiko; Takesawa, Toshikazu; Kanda, Katsuhiro; Sato, Toshitsugu

    2003-09-01

    A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58.0 kDa. The enzyme had an isoelectric point of around pH 6.9. The optimum pH for enzyme activity was around 3.0 against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 degrees C and stable up to 50 degrees C. The enzyme contained 8.6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. Beta-(3,4-dihydroxyphenyl)alanine (L-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of L-dopa was identified as L-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain. PMID:12949171

  16. Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes, and decolorization of chemically different dyes.

    PubMed

    Nagai, M; Sato, T; Watanabe, H; Saito, K; Kawata, M; Enei, H

    2002-11-01

    A laccase (EC 1.10.3.2) was isolated from the culture filtrate of Lentinula edodes. The enzyme was purified to a homogeneous preparation using hydrophobic, anion-exchange, and size-exclusion chromatographies. SDS-PAGE analysis showed the purified laccase, Lcc 1, to be a monomeric protein of 72.2 kDa. The enzyme had an isoelectric point of around pH 3.0. The optimum pH for enzyme activity was around 4.0, and it was most active at 40 degrees C and stable up to 35 degrees C. The enzyme contained 23.8% carbohydrate and some copper atoms. The enzyme oxidized 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, p-phenylendiamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol, and ferulic acid, but not veratryl alcohol, tyrosine, and beta-(3,4-dihydroxyphenyl) alanine. The N-terminal amino acid sequence of Lcc 1 showed close homology to the N-terminal sequences determined for laccases from Phlebia radiata, Trametes villosa, and Trametes versicolor, but only low similarity was observed to a previously reported laccase from L. edodes. Lcc 1 was effective in the decolorization of chemically different dyes - Remazole Brilliant Blue R, Bromophenol Blue, methyl red, and Naphtol Blue Black - without any mediators, but the decolorization of two dyes - red poly(vinylamine)sulfonate-anthrapyridone dye and Reactive Orange 16 - did require some redox mediators. PMID:12436315

  17. The regulation of body fat distribution and the modulation of insulin action.

    PubMed

    Cases, J A; Barzilai, N

    2000-11-01

    Body fat distribution may determine insulin resistance and its metabolic syndrome in humans, independent of obesity. Surgical removal of visceral fat (VF) in obese rats was associated with decreased leptin plasma levels and its gene expression in subcutaneous fat (SC). Chronic leptin treatment to rats decreased VF specifically supporting the role of leptin in determining fat distribution. Surgical removal of selected VF provided direct evidence of improved in vivo insulin action on hepatic glucose production (HGP) by over 2-fold vs sham-operated control. The impact of decreased VF on improved in vivo insulin action was further supported by obtaining similar decreases in VF by treating rats with leptin (Lep), beta3-aderenoreceptor agonist, or by severe caloric restriction (CR). All these three interventions improved insulin action on the modulation of HGP and were mostly attributed to preservation of hepatic glycogen stores. Because free fatty acids (FFA) plasma levels were unchanged, this effect may not be mediated portally by substrates. Improved peripheral insulin sensitivity and glycogen synthesis was demonstrated only in Lep. These data suggest that VF is a major determinant of hepatic insulin action. In obese rats, the ability of leptin to prevent visceral adiposity and its own expression is attenuated. Thus, the failure of leptin to regulate fat distribution and its own secretion suggest that 'leptin resistance' may be a pathologic feature in obesity.

  18. Extracellular Matrix (ECM) Multilayer Membrane as a Sustained Releasing Growth Factor Delivery System for rhTGF-β3 in Articular Cartilage Repair

    PubMed Central

    Park, Sang-Hyug; Kim, Moon Suk; Kim, Young Jick; Choi, Byung Hyune; Lee, Chun Tek; Park, So Ra; Min, Byoung-Hyun

    2016-01-01

    Recombinant human transforming growth factor beta-3 (rhTGF-β3) is a key regulator of chondrogenesis in stem cells and cartilage formation. We have developed a novel drug delivery system that continuously releases rhTGF-β3 using a multilayered extracellular matrix (ECM) membrane. We hypothesize that the sustained release of rhTGF-β3 could activate stem cells and result in enhanced repair of cartilage defects. The properties and efficacy of the ECM multilayer-based delivery system (EMLDS) are investigated using rhTGF-β3 as a candidate drug. The bioactivity of the released rhTGF-ß3 was evaluated through chondrogenic differentiation of mesenchymal stem cells (MSCs) using western blot and circular dichroism (CD) analyses in vitro. The cartilage reparability was evaluated through implanting EMLDS with endogenous and exogenous MSC in both in vivo and ex vivo models, respectively. In the results, the sustained release of rhTGF-ß3 was clearly observed over a prolonged period of time in vitro and the released rhTGF-β3 maintained its structural stability and biological activity. Successful cartilage repair was also demonstrated when rabbit MSCs were treated with rhTGF-β3-loaded EMLDS ((+) rhTGF-β3 EMLDS) in an in vivo model and when rabbit chondrocytes and MSCs were treated in ex vivo models. Therefore, the multilayer ECM membrane could be a useful drug delivery system for cartilage repair. PMID:27258120

  19. [Microbial alpha-amylases: physicochemical properties, substrate specificity and domain structure].

    PubMed

    Avdiiuk, K V; Varbanets', L D

    2013-01-01

    The current literature data on producers, physico-chemical properties and substrate specificity of a-amylases produced by microbes from different taxonomic groups such as bacteria, fungi and yeasts are discussed in the survey. Synthesis of alpha-amylase majority is an inducible process which is stimulated in the presence of starch or products of its hydrolysis. It is possible to increase enzymes activity level by optimization of cultivation conditions of strains-producers. alpha-Amylases, isolated from different sources are distinguished in their physico-chemical properties, particularly in their molecular weights, pH- and thermooptimums, inhibitors and activators. The enzymes hydrolyse soluble starch, amylose, amylopectin, glycogen, maltodextrins, alpha- and beta3-cyclodextrins and other carbohydrate substrates. It is well known that alpha-amylases belong to GH-13 family of glycosyl-hydrolases, which contain the catalytic domain A as (beta/alpha)8-barrel. In addition to domain A, alpha-amylases contain two other domains: B and C, which are localized approximately on opposite sides of (beta/alpha)8-barrel. Most of the known alpha-amylases contain calcium ion, which is located on the surface between domains A and B and plays an important role in stability and activity of the enzyme.

  20. Land surface temperature measurements from EOS MODIS data

    NASA Technical Reports Server (NTRS)

    Wan, Zhengming

    1995-01-01

    A significant progress has been made in TIR instrumentation which is required to establish the spectral BRDF/emissivity knowledge base of land-surface materials and to validate the land-surface temperature (LST) algorithms. The SIBRE (spectral Infrared Bidirectional Reflectance and Emissivity) system and a TIR system for measuring spectral directional-hemispherical emissivity have been completed and tested successfully. Optical properties and performance features of key components (including spectrometer, and TIR source) of these systems have been characterized by integrated use of local standards (blackbody and reference plates). The stabilization of the spectrometer performance was improved by a custom designed and built liquid cooling system. Methods and procedures for measuring spectral TIR BRDF and directional-hemispheric emissivity with these two systems have been verified in sample measurements. These TIR instruments have been used in the laboratory and the field, giving very promising results. The measured spectral emissivities of water surface are very close to the calculated values based on well established water refractive index values in published papers. Preliminary results show that the TIR instruments can be used for validation of the MODIS LST algorithm in homogeneous test sites. The beta-3 version of the MODIS LST software is being prepared for its delivery scheduled in the early second half of this year.

  1. Developmental expression analysis of the mouse and chick orthologues of IRF6: the gene mutated in Van der Woude syndrome.

    PubMed

    Knight, Alexandra S; Schutte, Brian C; Jiang, Rulang; Dixon, Michael J

    2006-05-01

    Development of the lip and palate involves a complex series of events that are frequently disturbed resulting in the congenital anomalies cleft lip and cleft palate. Van der Woude syndrome (VWS) is an autosomal dominant disorder that is characterised by cleft lip, cleft palate, lower lip pits, and hypodontia. VWS arises as the result of mutations in the gene encoding interferon regulatory factor 6 (IRF6). To provide insights into the role of IRF6 during embryogenesis, we have analysed the expression of this molecule during mouse and chick facial development. Irf6 was expressed in the ectoderm covering the facial processes during their fusion to form the upper lip and primary palate in both mouse and chick. However, while Irf6 was expressed in the medial edge epithelia of the developing secondary palate of the mouse, which fuses as in man, Irf6 was not expressed in the medial edge epithelia of the naturally cleft chick secondary palate. Similarly, Irf6 was found to be down-regulated in the medial edge epithelia of transforming growth factor beta3-null mice, which also exhibit cleft palate. Together, these results support a role for IRF6 during the fusion events that occur during development of the lip and palate. PMID:16245336

  2. The glucagon-like peptide 2 receptor is expressed in enteric neurons and not in the epithelium of the intestine.

    PubMed

    Pedersen, Jens; Pedersen, Nis B; Brix, Sophie W; Grunddal, Kaare Villum; Rosenkilde, Mette M; Hartmann, Bolette; Ørskov, Cathrine; Poulsen, Steen S; Holst, Jens J

    2015-05-01

    Glucagon-like peptide 2 (GLP-2) is a potent intestinotrophic growth factor with therapeutic potential in the treatment of intestinal deficiencies. It has recently been approved for the treatment of short bowel syndrome. The effects of GLP-2 are mediated by specific binding of the hormone to the GLP-2 receptor (GLP-2R) which was cloned in 1999. However, consensus about the exact receptor localization in the intestine has never been established. By physical, chemical and enzymatic tissue fragmentation, we were able to divide rat jejunum into different compartments consisting of: (1) epithelium alone, (2) mucosa with lamina propria and epithelium, (3) the external muscle coat including myenteric plexus, (4) a compartment enriched for the myenteric plexus and (5) intestine without epithelium. Expression of Glp2r; chromogranin A; tubulin, beta 3; actin, gamma 2, smooth muscle, enteric and glial fibrillary acidic protein in these isolated tissue fractions was quantified with qRT-PCR. Expression of the Glp2r was confined to compartments containing enteric neurons and receptor expression was absent in the epithelium. Our findings provide evidence for the expression of the GLP-2R in intestinal compartments rich in enteric neurons and, importantly they exclude significant expression in the epithelium of rat jejunal mucosa.

  3. The gamma subunit in chloroplast F(1)-ATPase can rotate in a unidirectional and counter-clockwise manner.

    PubMed

    Hisabori, T; Kondoh, A; Yoshida, M

    1999-12-10

    Rotation of the gamma subunit in chloroplast F(1)-ATPase (CF(1)) was investigated by using a single molecule observation technique, which is developed by Noji et al. to observe the rotation of a central gamma subunit portion in the alpha(3)beta(3)gamma sub-complex of F(1)-ATPase from thermophilic Bacillus PS3 (TF(1)) during ATP hydrolysis [Noji, H. et al. (1997) Nature 386, 299-302]. We used two cysteines of the gamma subunit (Cys-199 and Cys-205) of CF(1)-ATPase, which are involved in the regulation of this enzyme, to fix the fluorochrome-labeled actin filament. Then we successfully observed a unidirectional, counter-clockwise rotation of the actin filament with the fluorescent microscope indicating the rotation of the gamma subunit in CF(1)-ATPase. We conclude that the rotation of the gamma subunit in the F(1)-motor is a ubiquitous phenomenon in all F(1)-ATPases in prokaryotes as well as in eukaryotes.

  4. Efficient targeting of adenoviral vectors to integrin positive vascular cells utilizing a CAR-cyclic RGD linker protein.

    PubMed

    Krom, Y D; Gras, J C E; Frants, R R; Havekes, L M; van Berkel, T J; Biessen, E A L; van Dijk, K Willems

    2005-12-16

    Vascular smooth muscle (VSMC) and endothelial cells (EC) are particularly resistant to infection by type 5 adenovirus (Ad) vectors. To overcome this limitation and target Ad vectors to ubiquitously expressed alpha(V)beta(3/5) integrins, we have generated a linker protein consisting of the extracellular domain of the coxsackie adenovirus receptor (CAR) connected via avidin to a biotinylated cyclic (c) RGD peptide. After optimization of CAR to cRGD and to Ad coupling, infection of mouse heart endothelial cells (H5V) could be augmented significantly, as demonstrated by 600-fold increased transgene expression levels. In EOMAs, a hemangioendothelioma-derived cell line, the fraction of infected cells was enhanced 4- to 6-fold. Furthermore, the fraction of infected primary mouse VSMC was increased from virtually 0% to 25%. Finally, in human umbilical vein endothelial cells, the number of GFP positive cells was enhanced from 2% to 75%. In conclusion, CAR-cRGD is a versatile and highly efficient construct to target Ad vectors to both transformed and primary VSMC and EC.

  5. Expression and function of the AMF receptor by human melanoma in experimental and clinical systems.

    PubMed

    Tímár, J; Rásó, E; Döme, B; Ladányi, A; Bánfalvi, T; Gilde, K; Raz, A

    2002-01-01

    Motility of tumor cells is the rate limiting potential of metastatic cells and is regulated by autocrine and paracrine factors. Autocrine motility factor/neuroleukin/phosphohexose isomerase (AMF) is one of the best characterized autocrine motogenic cytokines. Here we have studied its in vitro effects on several human melanoma cell lines and found that neither cell line exhibited mitogenic response to AMF at a concentration where motogenic response could be initiated. Similar to previous studies on murine melanoma, activation of the AMF receptor upregulated beta3 while it downregulated beta1 integrins at the cell surface, inducing an integrin phenotype characteristic for invasive/metastatic melanoma. The gp78/AMF receptor protein expression in human melanoma cell lines correlated to their in vivo spontaneous metastatic potential. Furthermore, in two out of three human melanoma lines the expression significantly increased in the primary tumor when spontaneous metastases developed (immunosuppressed newborn rat model versus SCID mice). In a prospective study we have also analyzed AMF receptor protein expression in primary tumors of 54 skin melanoma patients using IHC. These studies revealed three types of AMF receptor phenotype: weak, heterogenous and strong expression profile. While in thin tumors weak/heterogenous AMFR expression predominated, in thick tumors the strong expression profile was predominant. The connection between AMFR expression and the invasive/metastatic potential of melanoma was further supported by our observation that SSM melanoma in the vertical growth phase expressed this motility receptor more strongly than tumors in the radial growth phase.

  6. SY 06-1 OBESITY, DIABETES AND HYPERTENSION: ARE ADIPOKINES RESPONSIBLE?

    PubMed

    Heagerty, Anthony

    2016-09-01

    There is now a considerable body of evidence to suggest that the fat cells that surround blood vessels (perivascular adipose tissue, PVAT) can influence profoundly arterial tone by releasing vasodilator adipokines which can act locally in a paracrine fashion. In healthy lean individuals the primary vasodilator released appears to be adiponectin and there is a complex interation between autonomic nerve firing in PVAT and the release of nitric oxide from adipocytes and an increased bioavailability of adiponectin. However the vasodilators that are released appear to be agonist dependent and adiponectin is certainly increased as a result of sympathomimetic stimulation. Primarily our work has focused on the role of sympathetic nerves running through PVAT and innovating adipocytes and releasing adiponectin. Electrical field stimulation studies have demonstrated clearly the release of adiponectin and the induction of relaxation which is abolished using 6-hydroxydopamine or tetrodotoxin to destroy the autonomic nerve fibres. In obesity there is evidence of beta-3 receptors on adipocytes and the loss of vasodilator activity which is clearly observed. In some models of obesity there is also evidence of an increase in vasoconstrictor prostaglandin bioavailability and increased contractility. This loss of balance between vasodilatation and vasoconstriction will lead to an increase in peripheral resistance which will decrease glucose uptake in skeletal muscle and bring about a rise in blood pressure: key components of the metabolic syndrome. Caloric restriction or weight reducing surgery can restore completely normal PVAT structure and function and this is seen even when individuals remain morbidly obese. PMID:27642933

  7. Structure of Protein Having Inhibitory Disintegrin and Leukotriene Scavenging Functions Contained in Single Domain

    SciTech Connect

    Xu, Xueqing; Francischetti, Ivo M.B.; Lai, Ren; Ribeiro, José M.C.; Andersen, John F.

    2012-08-10

    The antihemostatic/antiangiogenic protein tablysin-15 is a member of the CAP (cysteine-rich secretory, antigen 5, and pathogenesis-related 1 protein) superfamily and has been shown to bind the integrins {alpha}{sub IIb}{beta}{sub 3} and {alpha}{sub V}{beta}{sub 3} by means of an Arg-Gly-Asp (RGD) tripeptide sequence. Here we describe the x-ray crystal structure of tablysin-15 and show that the RGD motif is located in a novel structural context. The motif itself is contained in a type II {beta}-turn structure that is similar in its conformation to the RGD sequence of the cyclic pentapeptide cilengitide when bound to integrin {alpha}V{beta}3. The CAP domain also contains a hydrophobic channel that appears to bind a fatty acid molecule in the crystal structure after purification from Escherichia coli. After delipidation of the protein, tablysin-15 was found to bind proinflammatory cysteinyl leukotrienes with submicromolar affinities. The structure of the leukotriene E{sub 4}-tablysin-15 complex shows that the ligand binds with the nonfunctionalized end of the fatty acid chain buried in the hydrophobic pocket, whereas the carboxylate end of the ligand binds forms hydrogen bond/salt bridge interactions with polar side chains at the channel entrance. Therefore, tablysin-15 functions as an inhibitor of integrin function and as an anti-inflammatory scavenger of eicosanoids.

  8. Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MS(n) (part 1: methodology).

    PubMed

    Pfenninger, Anja; Karas, Michael; Finke, Berndt; Stahl, Bernd

    2002-11-01

    Underivatized neutral oligosaccharides from human milk were analyzed by nano-electrospray ionization (ESI) using a quadrupole ion trap mass spectrometer (QIT-MS) in the negative-ion mode. Under these conditions neutral oligosaccharides are observed as deprotonated molecules [M-H]- with high intensity. CID-experiments of these species with the charge localized at the reducing end lead to C-type fragment ions forming a "new" reducing end. Fragmentations are accompanied by cross-ring cleavages that yield information about linkages of internal monosaccharides. Several isomeric compounds with distinct structural features, such as different glycosidic linkages, fucosylation and branching sites were investigated. The rules governing the fragmentation behavior of this class of oligosaccharides were elucidated and tested for a representative number of certain isomeric glycoforms using the MS/MS and MS(n) capabilities of the QIT. On the basis of the specific fragmentation behavior of deprotonated molecules, the position of fucoses and the linkage type (Gal beta-->3 GlcNAc or Gal beta1-->4 GlcNAc) could be determined and linear and branched could be differentiated. Rules could be established which can be applied in further investigations of these types of oligosaccharides even from heterogenous mixtures. PMID:12443024

  9. ND-309, a novel compound, ameliorates cerebral infarction in rats by antioxidant action.

    PubMed

    Tian, Jingwei; Li, Guisheng; Liu, Zhifeng; Zhang, Shumin; Qu, Guiwu; Jiang, Wanglin; Fu, Fenghua

    2008-09-19

    Extract of danshen (Salvia miltiorrhiza Bunge.) has been clinically prescribed in China to treat patients with stroke. The novel compound designated ND-309, namely isopropyl-beta-(3,4-dihydroxyphenyl)-alpha-hydroxypropanoate is a new metabolite of danshen in rat brain. The present study was conducted to investigate whether ND-309 has a protective effect on brain injury after focal cerebral ischemia, and to determine the possible mechanism. Adult male SD rats were subjected to middle cerebral artery occlusion (MCAO) by bipolar electro-coagulation. Behavioral tests were used to evaluate the damage to central nervous system. The cerebral infarct volume and edema were assessed to evaluate the brain patho-physiological changes. Spectrophotometric or spectrofluorometric assay methods were used to determine the generation of reactive oxygen species (ROS), activities of superoxide dismutase (SOD) and glutathione-peroxidase (GSH-Px), contents of malondialdehyde (MDA) and adenosine triphosphate (ATP), as well as respiratory control ratio of the brain mitochondria. The results showed that treatment with ND-309 significantly decreased neurological deficit scores, reduced infarct volume and the edema compared with the model group. Meanwhile, ND-309 significantly increased the brain ATP content, improved mitochondrial energy metabolism, attenuated the elevation of MDA content, the decrease in SOD, GSH-Px activity and the generation of ROS in brain mitochondria. All of these findings indicate that ND-309 has the protective potential against cerebral ischemia injury and its protective effects may be due to the amelioration of cerebral energy metabolism and its antioxidant property. PMID:18652875

  10. Linkage study of nonsyndromic cleft lip with or without cleft palate using candidate genes and mapped polymorphic markers

    SciTech Connect

    Stein, J.D.; Nelson, L.D.; Conner, B.J.

    1994-09-01

    Nonsyndromic cleft lip with or without cleft palate (CL(P)) involves fusion or growth failure of facial primordia during development. Complex segregation analysis of clefting populations suggest that an autosomal dominant gene may play a role in this common craniofacial disorder. We have ascertained 16 multigenerational families with CL(P) and tested linkage to 29 candidate genes and 139 mapped short tandem repeat markers. The candidate genes were selected based on their expression in craniofacial development or were identified through murine models. These include: TGF{alpha}, TGF{beta}1, TGF{beta}2, TGF{beta}3, EGF, EGFR, GRAS, cMyc, FGFR, Jun, JunB, PDFG{alpha}, PDGF{beta}, IGF2R, GCR Hox7, Hox8, Hox2B, twirler, 5 collagen and 3 extracellular matrix genes. Linkage was tested assuming an autosomal dominant model with sex-specific decreased penetrance. Linkage to all of the candidate loci was excluded in 11 families. RARA was tested and was not informative. However, haplotype analysis of markers flanking RARA on 17q allowed exclusion of this candidate locus. We have previously excluded linkage to 61 STR markers in 11 families. Seventy-eight mapped short tandem repeat markers have recently been tested in 16 families and 30 have been excluded. The remaining are being analyzed and an exclusion map is being developed based on the entire study results.

  11. Anti-platelet effect of cumanastatin 1, a disintegrin isolated from venom of South American Crotalus rattlesnake.

    PubMed

    Da Silva, Manuel; Lucena, Sara; Aguilar, Irma; Rodríguez-Acosta, Alexis; Salazar, Ana M; Sánchez, Elda E; Girón, Maria E; Carvajal, Zoila; Arocha-Piñango, Carmen L; Guerrero, Belsy

    2009-03-01

    Disintegrins have been previously described in the venom of several snake families inhibiting signal transduction, cell-cell interactions, and cell-matrix interactions and may have therapeutic potential in heart attacks, thrombotic diseases, and cancers. This investigation describes the first disintegrin isolated from South American Crotalus venom (Venezuelan rattlesnake Crotalus durissus cumanensis), which inhibits platelet adhesion to matrix proteins. C. d. cumanensis crude venom was first separated on a Sephadex G-100 column into 4 fractions (SI to SIV). Crude venom and SIII fraction significantly diminished platelet adhesion to fibrinogen (Fg) and to fibronectin (Fn). Anti-adhesive SIII fraction was further separated by DEAE-Sephacel followed by C-18 reverse phase high performance liquid chromatography (HPLC). The platelet anti-adhesive fraction obtained was designated as cumanastatin-1. This disintegrin has a mass of 7.442 kDa as determined by mass spectrometry (MALDI-TOF/TOF) and pI of 8.5. Cumanastatin-1 also inhibited ADP-induced platelet aggregation with an IC(50) of 158 nM. However, it did not significantly inhibit collagen and thrombin-induced platelet aggregation. Cumanastatin-1 considerably inhibited anti-alpha(IIb)beta(3) integrin binding to platelets in a dose-dependent manner; however, it did not present any effect on the alpha(5)beta(1) integrin or on P-selectin.

  12. Assessment of estrogen receptor-monoclonal antibody interaction by high performance liquid chromatography

    SciTech Connect

    Brandt, D.W.; Wittliff, J.L.

    1986-05-01

    To define the interrelationships between the various isoforms of the estrogen receptors (ER), a monoclonal antibody-horse radish peroxidase conjugate H222 was used as a probe in conjunction with HPIEC (Synchrom AX-1000) and HPSEC (TSK-3000 SW Toyo Soda) columns. ER from breast tumors was assessed using (16..cap alpha..-/sup 125/I)-iodoestradiol-17..beta.. (3nM) +/-200 fold excess estradiol-17..beta.. and excess H222. When ER was analyzed by HPSEC (size and shape), with 400 mM KCl which caused the dissociation of ER into 4S isoforms, a shift in retension time to higher molecular weight species was seen. The H222 appeared to interact with most isoforms of ER. However, when ER was analyzed by HPIEC (surface charge) with H222, a shift in virtually all of the high salt (150mM) isoform to the flow-through was observed with only 46% shift in elution of the low salt (60-70mM) isoforms. H222 did not alter total ER binding capacity. These data suggest that H222 recognized discrete forms of the ER. Therefore, modification in the receptor may have occurred which masks or removes the antigenic determinant limiting the specificity of H222. These results indicate that H222 may be employed as a tool to elucidate the interrelationships between these ER species.

  13. What to do with targeted IL-2.

    PubMed

    Lode, H N; Xiang, R; Perri, P; Pertl, U; Lode, A; Gillies, S D; Reisfeld, R A

    2000-05-01

    A common strategy in immunotherapy of cancer is the induction of an increased immunogenicity of syngeneic malignancies. A novel approach to achieve this goal is the targeting of cytokines into the tumor microenvironment with antibody-cytokine fusion proteins, called immunocytokines. This report summarizes therapeutic efficacy and immune mechanisms involved in targeting IL-2 to syngeneic tumors and describes their extended use as a synergistic treatment modality for cancer vaccines and antiangiogenesis. Treatment of established melanoma and colon carcinoma metastases with IL-2 immunocytokines resulted in eradication of disease, followed by a vaccination effect protecting mice from lethal challenges with wild-type tumor cells. In a syngeneic neuroblastoma model, targeted IL-2 elicited effective antitumor responses mediated by NK cells in the absence of a T-cell memory. Interestingly, targeted IL-2 was effective in amplification of memory immune responses previously induced by cancer vaccines. Furthermore, a synergistic effect achieved by combining targeted IL-2-immunotherapy with an antiangiogenic inhibitor of integrin alpha(v)beta(3) extends the potential of this immunotherapeutic strategy in combination with antiangiogenesis as demonstrated in three syngeneic tumor models. Based on these findings, targeted IL-2 may provide an effective tool for the adjuvant treatment of cancer either applied as a single strategy or in combination with cancer vaccines and antiangiogenic strategies.

  14. Non-linear dynamic complexity of the human EEG during meditation.

    PubMed

    Aftanas, L I; Golocheikine, S A

    2002-09-20

    We used non-linear analysis to investigate the dynamical properties underlying the EEG in the model of Sahaja Yoga meditation. Non-linear dimensional complexity (DCx) estimates, indicating complexity of neuronal computations, were analyzed in 20 experienced meditators during rest and meditation using 62-channel EEG. When compared to rest, the meditation was accompanied by a focused decrease of DCx estimates over midline frontal and central regions. By contrast, additionally computed linear measures exhibited the opposite direction of changes: power in the theta-1 (4-6 Hz), theta-2 (6-8 Hz) and alpha-1 (8-10 Hz) frequency bands was increased over these regions. The DCx estimates negatively correlated with theta-2 and alpha-1 and positively with beta-3 (22-30 Hz) band power. It is suggested that meditative experience, characterized by less complex dynamics of the EEG, involves 'switching off' irrelevant networks for the maintenance of focused internalized attention and inhibition of inappropriate information. Overall, the results point to the idea that dynamically changing inner experience during meditation is better indexed by a combination of non-linear and linear EEG variables.

  15. THE EFFECT OF MASS LOSS ON THE TIDAL EVOLUTION OF EXTRASOLAR PLANET

    SciTech Connect

    Guo, J. H.

    2010-04-01

    By combining mass loss and tidal evolution of close-in planets, we present a qualitative study on their tidal migrations. We incorporate mass loss in tidal evolution for planets with different masses and find that mass loss could interfere with tidal evolution. In an upper limit case (beta = 3), a significant portion of mass may be evaporated in a long evolution timescale. Evidence of greater modification of the planets with an initial separation of about 0.1 AU than those with a = 0.15 AU can be found in this model. With the assumption of a large initial eccentricity, the planets with initial mass <=1 M{sub J} and initial distance of about 0.1 AU could not survive. With the supposition of beta = 1.1, we find that the loss process has an effect on the planets with low mass at a {approx} 0.05 AU. In both cases, the effect of evaporation on massive planets can be neglected. Also, heating efficiency and initial eccentricity have significant influence on tidal evolution. We find that even low heating efficiency and initial eccentricity have a significant effect on tidal evolution. Our analysis shows that evaporation on planets with different initial masses can accelerate (decelerate) the tidal evolution due to the increase (decrease) in tide of the planet (star). Consequently, the effect of evaporation cannot be neglected in evolutionary calculations of close-in planets. The physical parameters of HD 209458b can be fitted by our model.

  16. IFN-gamma induces endothelial cells to proliferate and to invade the extracellular matrix in response to the HIV-1 Tat protein: implications for AIDS-Kaposi's sarcoma pathogenesis.

    PubMed

    Fiorelli, V; Barillari, G; Toschi, E; Sgadari, C; Monini, P; Stürzl, M; Ensoli, B

    1999-01-15

    Previous studies indicated that the Tat protein of HIV functions as a progression factor in Kaposi's sarcoma (KS), an angioproliferative disease common and aggressive in HIV-1-infected individuals (AIDS-KS). In particular, Tat that is released by infected cells stimulates the growth and invasion of spindle cells of endothelial origin derived from KS lesions (KS cells). Other work suggested that inflammatory cytokines may act as initiating factors in KS since they induce normal endothelial cells to acquire the same phenotype and functional features of KS cells, including the responsiveness to Tat. In this study, we show that among the inflammatory cytokines increased in AIDS-KS lesions, IFN-gamma alone is sufficient to induce endothelial cells to proliferate and to invade the extracellular matrix in response to Tat. This is because IFN-gamma up-regulates the expression and activity of the receptors for Tat identified as the integrins alpha5beta1 and alpha(v)beta3. These results suggest that, by triggering Tat effects, IFN-gamma plays a major role in AIDS-KS pathogenesis.

  17. Ephrin-B1 transduces signals to activate integrin-mediated migration, attachment and angiogenesis.

    PubMed

    Huynh-Do, Uyen; Vindis, Cécile; Liu, Hua; Cerretti, Douglas Pat; McGrew, Jeffrey T; Enriquez, Miriam; Chen, Jin; Daniel, Thomas O

    2002-08-01

    Ephrin-B/EphB family proteins are implicated in bidirectional signaling and were initially defined through the function of their ectodomain sequences in activating EphB receptor tyrosine kinases. Ephrin-B1-3 are transmembrane proteins sharing highly conserved C-terminal cytoplasmic sequences. Here we use a soluble EphB1 ectodomain fusion protein (EphB1/Fc) to demonstrate that ephrin-B1 transduces signals that regulate cell attachment and migration. EphB1/Fc induced endothelial ephrin-B1 tyrosine phosphorylation, migration and integrin-mediated (alpha(v)beta(3) and alpha(5)beta(1)) attachment and promoted neovascularization, in vivo, in a mouse corneal micropocket assay. Activation of ephrin-B1 by EphB1/Fc induced phosphorylation of p46 JNK but not ERK-1/2 or p38 MAPkinases. By contrast, mutant ephrin-B1s bearing either a cytoplasmic deletion (ephrin-B1DeltaCy) or a deletion of four C-terminal amino acids (ephrin-B1DeltaPDZbd) fail to activate p46 JNK. Transient expression of intact ephin-B1 conferred EphB1/Fc migration responses on CHO cells, whereas the ephrin-B1DeltaCy and ephrin-B1DeltaPDZbd mutants were inactive. Thus ephrin-B1 transduces 'outside-in' signals through C-terminal protein interactions that affect integrin-mediated attachment and migration. PMID:12118063

  18. 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

    SciTech Connect

    McGrath, J.A. |; Ciatti, S.; Christiano, A.M.

    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 {r_arrow} 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. 15 refs., 1 fig.

  19. Isolation and identification in bovine cerebral cortex of n-butyl beta-carboline-3-carboxylate, a potent benzodiazepine binding inhibitor.

    PubMed

    Peña, C; Medina, J H; Novas, M L; Paladini, A C; De Robertis, E

    1986-07-01

    A substance having benzodiazepine-binding inhibitory activity has been extracted from 18 kg of gray matter of bovine cerebral cortex and purified to homogeneity. This substance inhibits competitively [3H]flunitrazepam and ethyl beta-[3H]carboline-3-carboxylate binding with high affinity (Ki, 3 nM), but it is inactive upon 3H-labeled Ro 5-4864, [3H]quinuclidinyl benzylate, [3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol, and upon high-affinity [3H]muscimol binding. This inhibitor has been identified as n-butyl beta-carboline-3-carboxylate (beta-CCB) by fast atom bombardment mass spectroscopy (Mr, 268) and electron bombardment fragmentography, ultraviolet and fluorescence spectra, coelution in HPLC with standard beta-CCB, and by the exact correspondence in Ki with beta-CCB on the displacement of [3H]flunitrazepam binding. The possible artificial formation of beta-CCB has been discarded by a series of control experiments including addition of tryptophan to the starting homogenate, extraction from liver, isolation and purification by an alternative procedure avoiding organic solvents, and by the impossibility of making beta-CCB from beta-carboline-3-carboxylic acid or its methyl ester in the conditions of our extraction and purification procedures.

  20. Potential application of N-carbamoyl-beta-alanine amidohydrolase from Agrobacterium tumefaciens C58 for beta-amino acid production.

    PubMed

    Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Pozo-Dengra, Joaquín; Tessaro, Davide; Servi, Stefano; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier

    2009-01-01

    An N-carbamoyl-beta-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (beta car(At)) has been characterized. Beta car(At) is most active at 30 degrees C and pH 8.0 with N-carbamoyl-beta-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn(2+), Ni(2+), and Co(2+). The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-alpha-, -beta-, -gamma-, and -delta-amino acids, with the greatest catalytic efficiency for N-carbamoyl-beta-alanine. Beta car(At) also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the beta-amino acids taurine and ciliatine, respectively. Beta car(At) is able to produce monosubstituted beta(2)- and beta(3)-amino acids, showing better catalytic efficiency (k(cat)/K(m)) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-beta-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make beta car(At) an outstanding candidate for application in the biotechnology industry.

  1. Leptin signalling and leptin-mediated activation of human platelets: importance of JAK2 and the phospholipases Cgamma2 and A2.

    PubMed

    Dellas, Claudia; Schäfer, Katrin; Rohm, Ilonka K; Lankeit, Mareike; Leifheit, Maren; Loskutoff, David J; Hasenfuss, Gerd; Konstantinides, Stavros V

    2007-11-01

    Leptin enhances agonist-induced platelet aggregation, and human platelets have been reported to express the leptin receptor. However, the pathways and mediators lying downstream of leptin binding to platelets remain, with few exceptions, unknown. In the present study, we sought to gain further insight into the possible role of leptin as a platelet agonist. Stimulation of platelets with leptin promoted thromboxane generation and activation of alpha(IIb)beta(3), as demonstrated by PAC-1 binding. Furthermore, it increased the adhesion to immobilised fibrinogen (p<0.001) and induced cytoskeletal rearrangement of both platelets and Meg01 cells. Leptin time- and dose-dependently phosphorylated the intracellular signalling molecules JAK2 and STAT3, although the importance of STAT3 for leptin-induced platelet activation remains to be determined. Important intracellular mediators and pathways activated by leptin downstream of JAK2 were found to include phosphatidylinositol-3 kinase, phospholipase Cgamma2 and protein kinase C, as well as the p38 MAP kinase-phospholipase A(2) axis. Accordingly, incubation with the specific inhibitors AG490, Ly294002, U73122, and SB203580 prevented leptin-mediated platelet activation. These results help delineate biologically relevant leptin signalling pathways in platelets and may improve our understanding of the mechanisms linking hyperleptinaemia to the increased thrombosis risk in human obesity. PMID:18000612

  2. Increased flow precedes remote arteriolar dilations for some microapplied agonists.

    PubMed

    Frame, M D

    2000-04-01

    This study asks which occurs first in time for remote responses: a dilation or a remote change in flow. Arteriolar diameter (approximately 20 microm) and fluorescently labeled red blood cell (RBC) velocity were measured in the cremaster muscle of anesthetized (pentobarbital sodium, 70 mg/kg) hamsters (n = 51). Arterioles were locally stimulated for 60 s with micropipette-applied 10 microg/ml LM-609 (alpha(v)beta(3)-integrin agonist), 10(-3) M adenosine, or 10(-3) M 3-morpholinosydnonimine (SIN-1, nitric oxide donor) as remote response agonists or with 10(-3) M papaverine, which dilates only locally. Observations were made at a remote site 1,200 microm upstream. With LM-609 or adenosine, the RBC velocity increased first (within 5 s), and the remote dilation followed 5-7 s later. N-nitro-L-arginine (100 microM) blocked the LM-609 (100%) and adenosine (60%) remote dilations. SIN-1 induced a concurrent remote dilation and decrease in RBC velocity (approximately 10 s), suggesting the primary signal was to dilate. Papaverine had no remote effects. This study suggests that, although remote responses to some agonists are induced by primary signals to dilate, additionally, network changes in flow can stimulate extensive remote changes in diameter.

  3. Carnitine palmitoyltransferase 1B 531K allele carriers sustain a higher respiratory quotient after aerobic exercise, but β3-adrenoceptor 64R allele does not affect lipolysis: a human model.

    PubMed

    Gómez-Gómez, Eduardo; Ríos-Martínez, Martín Efrén; Castro-Rodríguez, Elena Margarita; Del-Toro-Equíhua, Mario; Ramírez-Flores, Mario; Delgado-Enciso, Ivan; Pérez-Huitimea, Ana Lilia; Baltazar-Rodríguez, Luz Margarita; Velasco-Pineda, Gilberto; Muñiz-Murguía, Jesús

    2014-01-01

    Carnitine palmitoyltransferase IB (CPT1B) and adrenoceptor beta-3 (ADRB3) are critical regulators of fat metabolism. CPT1B transports free acyl groups into mitochondria for oxidation, and ADRB3 triggers lipolysis in adipocytes, and their respective polymorphisms E531K and W64R have been identified as indicators of obesity in population studies. It is therefore important to understand the effects of these mutations on ADRB3 and CPT1B function in adipose and skeletal muscle tissue, respectively. This study aimed to analyze the rate of lipolysis of plasma indicators (glycerol, free fatty acids, and beta hydroxybutyrate) and fat oxidation (through the non-protein respiratory quotient). These parameters were measured in 37 participants during 30 min of aerobic exercise at approximately 62% of maximal oxygen uptake, followed by 30 min of recovery. During recovery, mean respiratory quotient values were higher in K allele carriers than in non-carriers, indicating low post-exercise fatty acid oxidation rates. No significant differences in lipolysis or lipid oxidation were observed between R and W allele carriers of ADRB3 at any time during the aerobic load. The substitution of glutamic acid at position 531 by lysine in the CPT1B protein decreases the mitochondrial beta-oxidation pathway, which increases the non-protein respiratory quotient value during recovery from exercise. This may contribute to weight gain or reduced weight-loss following exercise. PMID:24905907

  4. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors.

    PubMed

    Takayama, Naoya; Nishikii, Hidekazu; Usui, Joichi; Tsukui, Hiroko; Sawaguchi, Akira; Hiroyama, Takashi; Eto, Koji; Nakauchi, Hiromitsu

    2008-06-01

    Human embryonic stem cells (hESCs) could potentially represent an alternative source for blood transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. When we cultured hESCs on either C3H10T1/2 or OP-9 cells to facilitate hematopoiesis, we found that exogenous administration of vascular endothelial growth factor promoted the emergence of sac-like structures, which we named embryonic stem cell-derived sacs (ES-sacs). These ES-sacs consisted of multiple cysts demarcated by cellular monolayers that retained some of the properties of endothelial cells. The spherical cells inside ES-sacs expressed primarily CD34, along with VE-cadherin, CD31, CD41a, and CD45, and were able to form hematopoietic colonies in semisolid culture and to differentiate into mature megakaryocytes by day 24 in the presence of thrombopoietin. Apparently, ES-sacs provide a suitable environment for hematopoietic progenitors. Relatively large numbers of mature megakaryocytes could be induced from the hematopoietic progenitors within ES-sacs, which were then able to release platelets that displayed integrin alpha IIb beta 3 activation and spreading in response to ADP or thrombin. This novel protocol thus provides a means of generating platelets from hESCs, which could serve as the basis for efficient production of platelets for clinical transfusion and studies of thrombopoiesis.

  5. RNA editing of the GABA(A) receptor alpha3 subunit alters the functional properties of recombinant receptors.

    PubMed

    Nimmich, Mitchell L; Heidelberg, Laura S; Fisher, Janet L

    2009-04-01

    RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the alpha3 subunit of the GABAA receptors has been shown to undergo RNA editing. As a result, a highly conserved isoleucine residue in the third transmembrane domain is replaced with a methionine. To determine the effect of this structural change on receptor function, we compared the GABA sensitivity, pharmacological properties and macroscopic kinetics of recombinant receptors containing either the edited or unedited forms of the alpha3 subunit along with beta3 and gamma2L. Editing substantially altered the GABA sensitivity and deactivation rate of the receptors, with the unedited form showing a lower GABA EC50 and slower decay. Comparable effects were observed with a mutation at the homologous location in the alpha1 subunit, suggesting a common role for this site in regulation of channel gating. Except for the response to GABA, the pharmacological properties of the receptor were unaffected by editing, with similar enhancement by a variety of modulators. Since RNA editing of the alpha3 subunit increases through development, our findings suggest that GABAergic neurotransmission may be more effective early in development, with greater GABA sensitivity and slower decay rates conferred by the unedited alpha3 subunit.

  6. Thrombospondin type I domain containing 7A (THSD7A) mediates endothelial cell migration and tube formation.

    PubMed

    Wang, Chieh-Huei; Su, Pei-Tsu; Du, Xiao-Yan; Kuo, Meng-Wei; Lin, Chia-Yi; Yang, Chung-Chi; Chan, Hau-Shien; Chang, Shing-Jyh; Kuo, Calvin; Seo, Kyunga; Leung, Lawrence L; Chuang, Yung-Jen

    2010-03-01

    Angiogenesis is a highly organized process controlled by a series of molecular events. While much effort has been devoted to identifying angiogenic factors and their reciprocal receptors, far less information is available on the molecular mechanisms underlying directed endothelial cell migration. To search for novel proteins that participate in this process, we used the serial analysis of gene expression (SAGE) transcript profiling approach to identify genes that are selectively expressed in endothelial cells (ECs). Two EC SAGE libraries were constructed from human umbilical vein and artery ECs to enable data-mining against other non-ECs. A novel endothelial protein, Thrombospondin Type I Domain Containing 7A (THSD7A), with preferential expression in placenta vasculature and in human umbilical vein endothelial cells (HUVECs) was identified and targeted for further characterization. Overexpression of a THSD7A carboxyl-terminal fragment in HUVECs inhibited cell migration and disrupted tube formation, while suppression of THSD7A expression enhanced HUVEC migration and tube formation. Immunohistological analysis revealed that THSD7A was expressed at the leading edge of migrating HUVECs, and it co-localized with alpha(V)beta(3) integrin and paxillin. This distribution was dispersed from focal adhesions after disruption of the actin cytoskeleton, suggesting the involvement of THSD7A in cytoskeletal organization. Our results show that THSD7A is a novel placenta endothelial protein that mediates EC migration and tube formation, and they highlight its potential as a new target for anti-angiogenic therapy.

  7. Effect of the alpha subunit subtype on the macroscopic kinetic properties of recombinant GABA(A) receptors.

    PubMed

    Picton, Amber J; Fisher, Janet L

    2007-08-24

    The GABA(A) receptors (GABARs) are chloride-permeable ligand-gated ion channels responsible for fast inhibitory neurotransmission. These receptors are structurally heterogeneous, and in mammals can be formed from a combination of sixteen different subunit subtypes. Much of this variety comes from the six different alpha subunit subtypes. All neuronal GABARs contain an alpha subunit, and the identity of the alpha subtype affects the pharmacological properties of the receptors. The expression of each of the different alpha subtypes is regulated developmentally and regionally and changes with both normal physiological processes such development and synaptic plasticity, and pathological conditions such as epilepsy. In order to understand the functional significance of this structural heterogeneity, we examined the effect of the alpha subtype on the receptor's response to GABA. Each of the six alpha subtypes was transiently co-expressed with the beta3 and gamma2L subunits in mammalian cells. The sensitivity to GABA was measured with whole-cell recordings. We also determined the activation, deactivation, desensitization, and recovery kinetics for the six isoforms using rapid application recordings from excised macropatches. We found unique characteristics associated with each alpha subunit subtype. These properties would be expected to influence the post-synaptic response to GABA, creating functional diversity among neurons expressing different alpha subunits.

  8. DNA sequencing of the gene encoding a bacterial superantigen, Yersinia pseudotuberculosis-derived mitogen (YPM), and characterization of the gene product, cloned YPM

    SciTech Connect

    Miyoshi-Akiyama, Tohru; Kato, Hidehito; Uchiyama, Takehiko

    1995-05-15

    Previously, we found a novel bacterial superantigen from Yersinia pseudotuberculosis, designated Y. pseudotuberculosis-derived mitogen (YPM). In the present study, we analyzed the DNA sequence of the gene encoding YPM. The YPM gene was cloned into a plasmid vector pMW119 and expressed in Escherichia coli DH10B. Like the native YPM, the cloned YPM required the expression of MHC class II molecules on accessory cells in the induction of IL-2 production by human T cells. TCR-V{beta} repertoire of human T cells reactive with the cloned YPM was V{beta}3, V{beta}9, V{beta}13.1, and V{beta}13.2. This repertoire is the same as that of T cells reactive with the native YPM. These results indicate that the cloned YPM expressed in E. coli is identical to the native YPM. Sequencing of the YPM gene revealed that the gene contained an open reading frame of 456 base pairs encoding a precursor form of 151 amino acid residues with m.w. 16,679 that is processed into a mature form of 131 amino acid residues with m.w. 14,529. Homology analysis revealed that the homology of amino acid sequence is quite low among YPM and other well known bacterial superantigens. We designated the gene encoding YPM as ypm. 30 refs., 5 figs., 2 tabs.

  9. The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence.

    PubMed Central

    Barillari, G; Gendelman, R; Gallo, R C; Ensoli, B

    1993-01-01

    Spindle-shaped cells of vascular origin are the probable tumor cells of Kaposi sarcoma (KS). These cells, derived from patients with KS and AIDS, proliferate in response to extracellular Tat protein of human immunodeficiency virus type 1. Normal vascular cells, believed to be the progenitors of AIDS-KS cells, acquire spindle morphology and become responsive to the mitogenic effect of Tat after culture with inflammatory cytokines. Such cytokines are increased in human immunodeficiency virus type 1-infected people, suggesting that immune stimulation (rather than immune deficiency) is a component of AIDS-KS pathogenesis. Here we show that (i) Tat promotes adhesion of AIDS-KS and normal vascular cells; (ii) adhesion of normal vascular cells to Tat is induced by exposure of the cells to the same cytokines; (iii) adhesion is associated with the amino acid sequence RGD of Tat through a specific interaction with the integrin receptors alpha 5 beta 1 and alpha v beta 3, although it is augmented by the basic region; and (iv) the expression of both integrins is increased by the same cytokines that promote these cells to acquire spindle morphology and become responsive to the adhesion and growth effects of Tat. The results also suggest that RGD-recognizing integrins mediate the vascular cell-growth-promoting effect of Tat. Images Fig. 1 Fig. 5 PMID:7690138

  10. Passaged adult chondrocytes can form engineered cartilage with functional mechanical properties: a canine model.

    PubMed

    Ng, Kenneth W; Lima, Eric G; Bian, Liming; O'Conor, Christopher J; Jayabalan, Prakash S; Stoker, Aaron M; Kuroki, Keiichi; Cook, Cristi R; Ateshian, Gerard A; Cook, James L; Hung, Clark T

    2010-03-01

    It was hypothesized that previously optimized serum-free culture conditions for juvenile bovine chondrocytes could be adapted to generate engineered cartilage with physiologic mechanical properties in a preclinical, adult canine model. Primary or passaged (using growth factors) adult chondrocytes from three adult dogs were encapsulated in agarose, and cultured in serum-free media with transforming growth factor-beta3. After 28 days in culture, engineered cartilage formed by primary chondrocytes exhibited only small increases in glycosaminoglycan content. However, all passaged chondrocytes on day 28 elaborated a cartilage matrix with compressive properties and glycosaminoglycan content in the range of native adult canine cartilage values. A preliminary biocompatibility study utilizing chondral and osteochondral constructs showed no gross or histological signs of rejection, with all implanted constructs showing excellent integration with surrounding cartilage and subchondral bone. This study demonstrates that adult canine chondrocytes can form a mechanically functional, biocompatible engineered cartilage tissue under optimized culture conditions. The encouraging findings of this work highlight the potential for tissue engineering strategies using adult chondrocytes in the clinical treatment of cartilage defects.

  11. Modulation of cerebral microvascular permeability by endothelial nicotinic acetylcholine receptors.

    PubMed

    Hawkins, Brian T; Egleton, Richard D; Davis, Thomas P

    2005-07-01

    Nicotine increases the permeability of the blood-brain barrier in vivo. This implies a possible role for nicotinic acetylcholine receptors in the regulation of cerebral microvascular permeability. Expression of nicotinic acetylcholine receptor subunits in cerebral microvessels was investigated with immunofluorescence microscopy. Positive immunoreactivity was found for receptor subunits alpha3, alpha5, alpha7, and beta2, but not subunits alpha4, beta3, or beta4. Blood-brain barrier permeability was assessed via in situ brain perfusion with [14C]sucrose. Nicotine increased the rate of sucrose entry into the brain from 0.3 +/- 0.1 to 1.1 +/- 0.2 microl.g(-1).min(-1), as previously described. This nicotine-induced increase in blood-brain barrier permeability was significantly attenuated by both the blood-brain barrier-permeant nicotinic antagonist mecamylamine and the blood-brain barrier-impermeant nicotinic antagonist hexamethonium to 0.5 +/- 0.2 and 0.3 +/- 0.2 microl.g(-1).min(-1), respectively. These data suggest that nicotinic acetylcholine receptors expressed on the cerebral microvascular endothelium mediate nicotine-induced changes in blood-brain barrier permeability.

  12. The co-occurrence of two pyridine alkaloids, mimosine and trigonelline, in Leucaena leucocephala.

    PubMed

    Ogita, Shinjiro; Kato, Misako; Watanabe, Shin; Ashihara, Hiroshi

    2014-01-01

    Leucaena leucocephala is a nitrogen-fixing tropical leguminous tree that produces two pyridine alkaloids, i. e. mimosine [beta-(3-hydroxy-4-pyridon-1-yl)-L-alanine] and trigonelline (1-methylpyridinium-3-carboxylate). Mimosine has been detected in leaves, flowers, pods, seeds, and roots, and it is one of the principal non-protein amino acids that occurs in all organs. Asparagine was the most abundant amino acid in flowers. The mimosine content varied from 3.3 micromol/g fresh weight (FW) in developing flowers to 171 micromol/g FW in mature seeds. Trigonelline was also detected in leaves, flowers, pods, and seeds, but not roots. The trigonelline content was lower than that of mimosine in all organs. It varied from 0.12 micromol/g FW in developing seeds to 2.6 micromol/g FW in mature seeds. [2-14C]Nicotinic acid supplied to the developing seeds was incorporated into trigonelline but not mimosine. This indicates that the pyridine and dihydroxypyridine structures of these two alkaloids are derived from distinct precursors. The physiological functions of mimosine and trigonelline are discussed briefly. PMID:24873033

  13. Combination therapy with tamsulosin and solifenacin for male lower urinary tract symptoms with predominant filling symptoms: a new approach to an old problem.

    PubMed

    Angulo, Javier C

    2015-11-01

    Benign prostatic enlargement is the main cause of male lower urinary tract symptoms (LUTS). The distinction and quantification of voiding, storage and postmicturition symptoms help clinicians decide on a modern pharmacological therapeutic approach for male LUTS. Muscarinic receptors antagonists are now considered safe therapy in males and a fixed-dose combination of oral controlled absorbed system solifenacin and tamsulosin is advantageous compared to tamsulosin monotherapy in males with predominant storage symptoms when validated instruments such as International Prostatic Score Symptoms (IPSS) or Total Urgency Frequency Score (TUFS) are used. This combination therapy is well tolerated and maintains symptomatic improvement in the long term. Different options of managing male LUTS with predominant storage symptoms include a combination of tamsulosin and solifenacin, antimuscarinics alone - some with flexible dosing, and the beta-3 agonist mirabegron. Tailored pharmacological therapy for the particular patient is not a reality yet, but judicious use of the different alternatives could bring varied new therapeutic solutions for male LUTS including benign prostatic enlargement and benign prostatic obstruction.

  14. Biologic activity of the iodoestrogens and their use in breast cancer

    SciTech Connect

    Longcope, C.; Arunachalam, T.; Rafkind, I.; Caspi, E.

    1981-01-01

    Several iodinated estrogens, 6-iodoestra-1,3,5(10), 6-tetraene-3, 17 beta-diol (4) 16 beta-iodo-estradiol (3) were shown to displace /sup 3/H-estradiol (1b) from the uterine cytosol receptor by a competitive type of inhibition. The three compounds translocated the cytosol receptor to the nucleus in vitro and increased mouse uterine weight in vivo. In all tests the relative activities were 16 alpha-(2a) greater than 16 beta-(3a) greater than 6-(4a). When the compounds were made with (/sup 125/I) the 16 alpha-(/sup 125/I)-iodoestradiol (2b) bound with high affinity, Kd . 0.4 x 10(-10), to the 8S cytosol receptor. No high affinity binding could be demonstrated for the 6-(/sup 125/I)-iodoestratetraene (4b). In in vivo experiments following the administration of 16 alpha-(/sup 125/I)-iodoestradiol (2b) to rats, high levels of radioactivity were found in the uterus, liver and thyroid but only in the liver and thyroid following administration of 6-(/sup 125/I)-iodoestratetraene (4b). After administration of (2b) to rats with DMBA-induced mammary tumors, no tumor concentration of radioactivity could be detected by imaging. When (4b) was administered similarly, radioactivity could be detected in some of the tumors by imaging. The radioactivity was associated with non-specific 4S proteins.

  15. Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization

    PubMed Central

    Gumy, Laura F.; Yeo, Giles S.H.; Tung, Yi-Chun Loraine; Zivraj, Krishna H.; Willis, Dianna; Coppola, Giovanni; Lam, Brian Y.H.; Twiss, Jeffery L.; Holt, Christine E.; Fawcett, James W.

    2011-01-01

    mRNAs are transported, localized, and translated in axons of sensory neurons. However, little is known about the full repertoire of transcripts present in embryonic and adult sensory axons and how this pool of mRNAs dynamically changes during development. Here, we used a compartmentalized chamber to isolate mRNA from pure embryonic and adult sensory axons devoid of non-neuronal or cell body contamination. Genome-wide microarray analysis reveals that a previously unappreciated number of transcripts are localized in sensory axons and that this repertoire changes during development toward adulthood. Embryonic axons are enriched in transcripts encoding cytoskeletal-related proteins with a role in axonal outgrowth. Surprisingly, adult axons are enriched in mRNAs encoding immune molecules with a role in nociception. Additionally, we show Tubulin-beta3 (Tubb3) mRNA is present only in embryonic axons, with Tubb3 locally synthesized in axons of embryonic, but not adult neurons where it is transported, thus validating our experimental approach. In summary, we provide the first complete catalog of embryonic and adult sensory axonal mRNAs. In addition we show that this pool of axonal mRNAs dynamically changes during development. These data provide an important resource for studies on the role of local protein synthesis in axon regeneration and nociception during neuronal development. PMID:21098654

  16. Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds.

    PubMed

    Stoff, Alexander; Rivera, Angel A; Mathis, J Michael; Moore, Steven T; Banerjee, N S; Everts, Maaike; Espinosa-de-los-Monteros, Antonio; Novak, Zdenek; Vasconez, Luis O; Broker, Thomas R; Richter, Dirk F; Feldman, Dale; Siegal, Gene P; Stoff-Khalili, Mariam A; Curiel, David T

    2007-05-01

    Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-beta(1) and TGF-beta(2) precursor proteins, but an increase in expression of TGF-beta(3) precursor protein and TGF-beta type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

  17. Thrombin activation of human platelets dissociates a complex containing gelsolin and actin from phosphatidylinositide-specific phospholipase Cgamma1.

    PubMed Central

    Baldassare, J J; Henderson, P A; Tarver, A; Fisher, G J

    1997-01-01

    We have examined the association of two cytoskeleton proteins, gelsolin and actin, with phosphatidylinositide-specific phospholipase Cgamma1 (PLCgamma1) in resting and thrombin-stimulated human platelets. In unstimulated platelets, gelsolin, actin and PLCgamma1 were immunoprecipitated as a complex by a polyclonal antibody to PLCgamma1. The association of gelsolin and actin was specific for PLCgamma1 because immunoprecipitates of PLCs beta2, beta3, gamma2 and delta1, which are also expressed in human platelets, did not contain detectable gelsolin or actin. Activation with thrombin resulted in platelet aggregation and the dissociation of gelsolin and actin from PLCgamma1. Inhibition of thrombin-induced platelet aggregation blocked the dissociation of gelsolin and actin from PLCgamma1. After stimulation with thrombin, PLCgamma1 activity in immunoprecipitates was increased 2-3-fold. This elevation in PLCgamma1 activity in response to thrombin activation was not observed when platelet aggregation was blocked. Although PLCgamma1 is tyrosine phosphorylated in response to many agonists, we could not detect, by Western analysis with anti-phosphotyrosine antibodies, tyrosine phosphorylation of PLCgamma1 immunoprecipitated from thrombin-stimulated platelets. These results demonstrate that PLCgamma1 is associated with gelsolin and actin in resting platelets, and that thrombin-induced platelet aggregation results in the dissociation of PLCgamma1 from gelsolin and actin, and the stimulation of PLCgamma1 activity. PMID:9164868

  18. Maturation State-Dependent Alterations in Meniscus Integration: Implications for Scaffold Design and Tissue Engineering

    PubMed Central

    Ionescu, Lara C.; Lee, Gregory C.; Garcia, Grant H.; Zachry, Tiffany L.; Shah, Roshan P.; Sennett, Brian J.

    2011-01-01

    The knee meniscus is a crucial component of the knee that functions to stabilize the joint, distribute load, and maintain congruency. Meniscus tears and degeneration are common, and natural healing is limited. Notably, few children present with meniscus injuries and other related fibrocartilaginous tissues heal regeneratively in immature animals and in the fetus. In this work, we evaluated fetal, juvenile, and adult bovine meniscus properties and repair capacity in vitro. Although no changes in cell behavior (migration and proliferation) were noted with age, drastic alterations in the density and distribution of the major components of meniscus tissue (proteoglycan, collagen, and DNA) occurred with development. Coincident with these marked tissue changes, the in vitro healing capacity of the tissue decreased with age. Fetal and juvenile meniscus formed a robust repair over 8 weeks on both a histological and mechanical basis, despite a lack of vascular supply. In contrast, adult meniscus did not integrate over this period. However, integration was improved significantly with the addition of the growth factor transforming growth factor-beta 3. Finally, to evaluate engineered scaffold integration in the context of aging, we monitored cellular infiltration from native tissue into engineered nanofibrous constructs. Our findings suggest that maturation processes that enable load bearing in the adult limit endogenous healing potential and identify new metrics for the development of tissue-engineered meniscus implants. PMID:20712419

  19. Structure of the three beta-tubulin-encoding genes of the unicellular alga, Polytomella agilis.

    PubMed

    Conner, T W; Thompson, M D; Silflow, C D

    1989-12-14

    The quadriflagellate, unicellular, colorless alga, Polytomella agilis, contains several distinct microtubule arrays. To study the genetic basis of microtubule heterogeneity in P. agilis, we characterized its tubulin(Tub)-encoding genes (tub). The three beta tub genes detected in blots of P. agilis DNA were isolated from a genomic library. The structure and organization of the genes were examined by restriction mapping and nucleotide (nt) sequencing. S1 nuclease protection studies showed that all three genes are expressed. The predicted amino acid (aa) sequences are more than 98% conserved with the Chlamydomonas reinhardtii and Volvox carteri beta-Tubs, underscoring the close phylogenetic relationship of these species. Evolutionary divergence among the P. agilis genes is demonstrated by differences in intron number, nt sequences in noncoding regions, and silent nt substitutions in the coding regions. However, the proteins encoded by the beta 1 and beta 3 tub genes are identical; the beta 2 gene product differs by one conservative aa substitution. These results are in striking contrast to the C-terminal aa diversity reported within beta tub gene families in animal, higher plant and fungal systems. The data support the hypothesis that those tub genes whose products assemble into axonemal microtubules are subject PMID:2533130

  20. Design of the muon collider lattice: Present status

    SciTech Connect

    Garren, A.; Courant, E.; Gallardo, J.

    1996-05-01

    The last component of a muon collider facility, as presently envisioned, is a colliding-beam storage ring. Design studies on various problems for this ring have been in progress over the past year. In this paper we discuss the current status of the design. The projected muon currents require very low beta values at the IP, {beta}* = 3 mm, in order to achieve the design luminosity of L = 10{sup 35} cm{sup -2} s{sup -1}. The beta values in the final-focus quadrupoles are roughly 400 km. To cancel the corresponding chromaticities, sextupole schemes for local correction have been included in the optics of the experimental insertion. The hour-glass effect constraints the bunch length to be comparable too. To obtain such short bunches with reasonable rf voltage requires a very small value of the momentum compaction a, which can be obtained by using flexible momentum compaction (FMC) modules in the arcs. A preliminary design of a complete collider ring has now been made; it uses an experimental insertion and arc modules as well as a utility insertion. The layout of this ring is shown schematically, and its parameters are summarized. Though some engineering features are unrealistic, and the beam performance needs some improvement, we believe that this study can serve as the basis for a workable collider design. The remaining sections of the paper will describe the lattice, show beam behaviour, and discuss future design studies.

  1. A novel PPAR{gamma} agonist, KR62776, suppresses RANKL-induced osteoclast differentiation and activity by inhibiting MAP kinase pathways

    SciTech Connect

    Park, Ju-Young; Bae, Myung-Ae; Cheon, Hyae Gyeong; Kim, Sung Soo; Hong, Jung-Min; Kim, Tae-Ho; Choi, Je-Yong; Kim, Sang-Hyun; Lim, Jiwon; Choi, Chang-Hyuk; Shin, Hong-In; Kim, Shin-Yoon Park, Eui Kyun

    2009-01-16

    We investigated the effects of a novel peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, KR62776, on osteoclast differentiation and function, and on the underlying signaling pathways. KR62776 markedly suppressed differentiation into osteoclasts in various osteoclast model systems, including bone marrow mononuclear (BMM) cells and a co-culture of calvarial osteoblasts and BMM cells. KR62776 suppressed the activation of tartrate-resistant acid phosphatase (TRAP) and the expression of genes associated with osteoclast differentiation, such as TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), and osteoclast-associated receptor (OSCAR). Furthermore, KR62776 reduced resorption pit formation in osteoclasts, and down-regulated genes essential for osteoclast activity, such as Src and {alpha}v{beta}3 integrin. An analysis of a signaling pathway showed that KR62776 inhibited the receptor activator of nuclear factor-{kappa}B ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and nuclear factor-{kappa}B (NF-{kappa}B). Together, these results demonstrate that KR62776 negatively affects osteoclast differentiation and activity by inhibiting the RANKL-induced activation of MAP kinases and NF-{kappa}B.

  2. Rationale for active vitamin D and analogs in the treatment of osteoporosis.

    PubMed

    Nishii, Yasuho

    2003-02-01

    In 1981, Chugai Pharmaceutical succeeded in marketing alfacalcidol, a prodrug of calcitriol, as a therapeutic agent for renal osteodystrophy. In 1983, Chugai succeeded in extending the application of alfacalcidol to the treatment of osteoporosis as well. Clinicians in Japan have accepted alfacalcidol as a remedy for osteoporosis. However, the use of calcitriol and its analogs for the treatment of osteoporosis is still controversial. Some misunderstandings exist internationally about the efficacy of the active form of vitamin D for the treatment of osteoporosis. It is important to emphasize that patients with osteoporosis have intestinal calcium malabsorption and dysfunction in renal activation of vitamin D. When massive doses of parent vitamin D were administered to OVX rats, bone mass increased, but surprisingly, many porotic area were observed in the cortical bone. On the other hand, administration of alfacalcidol increased physiological bone without porotic observation. It is necessary to give the active form of vitamin D, D-hormone, with an RDA-equivalent supply of calcium. Alfacalcidol forms physiological strong bones that are hardly fractured by regulating calcium and bone metabolism. We proposed a new vitamin D analog, 2beta (3-hydroxypropoxy)calcitriol [ED-71] as a therapeutic drug for osteoporosis, which is more potent than calcitriol. ED-71 is now being investigated in phase 2 clinical studies in Japan. ED-71 will appear as more improved drugs for osteoporosis until 2010. PMID:12520540

  3. Mechanosensitivity of human osteosarcoma cells and phospholipase C {beta}2 expression

    SciTech Connect

    Hoberg, M. . E-mail: Maik.Hoberg@med.uni-tuebingen.de; Gratz, H.-H.; Noll, M.; Jones, D.B.

    2005-07-22

    Bone adapts to mechanical load by osteosynthesis, suggesting that osteoblasts might respond to mechanical stimuli. We therefore investigated cell proliferation and phospholipase C (PLC) expression in osteoblasts. One Hertz uniaxial stretching at 4000 {mu}strains significantly increased the proliferation rates of human osteoblast-like osteosarcoma cell line MG-63 and primary human osteoblasts. However, U-2/OS, SaOS-2, OST, and MNNG/HOS cells showed no significant changes in proliferation rate. We investigated the expression pattern of different isoforms of PLC in these cell lines. We were able to detect PLC {beta}1, {beta}3, {gamma}1, {gamma}2, and {delta}1 in all cells, but PLC {beta}2 was only detectable in the mechanosensitive cells. We therefore investigated the possible role of PLC {beta}2 in mechanotransduction. Inducible antisense expression for 24 h inhibited the translation of PLC {beta}1 in U-2/OS cells by 35% and PLC {beta}2 in MG-63 by 29%. Fluid shear flow experiments with MG-63 lacking PLC {beta}2 revealed a significantly higher level of cells losing attachment to coverslips and a significantly lower number of cells increasing intracellular free calcium.

  4. Sustained phenotypic reversion of junctional epidermolysis bullosa dog keratinocytes: Establishment of an immunocompetent animal model for cutaneous gene therapy

    SciTech Connect

    Spirito, Flavia; Capt, Annabelle; Rio, Marcela Del; Larcher, Fernando; Guaguere, Eric; Danos, Olivier; Meneguzzi, Guerrino . E-mail: meneguzz@unice.fr

    2006-01-20

    Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 ({alpha}3{beta}3{gamma}2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the {alpha}3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretion of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials.

  5. Direct stimulation of BAT thermogenesis does not affect hypothalamic neuropeptide Y.

    PubMed

    Bing, C; Hopkins, D; Wang, Q; Frankish, H; Buckingham, R; Williams, G

    1998-01-01

    Acute cold exposure which significantly stimulated thermogenesis in brown adipose tissue (BAT), also increased neuropeptide Y (NPY) levels in its hypothalamic sites of release without affecting NPY synthesis, suggesting that NPY release is acutely inhibited. To clarify whether these changes in NPY are the cause or consequence of BAT activation, we studied whether hypothalamic NPY and NPY mRNA levels in rats were affected by acute intraperitoneal injection of the beta 3-adrenoceptor agonist BRL 35135 (500 micrograms/kg), which directly activates BAT thermogenesis. BRL 35135 treatment doubled BAT uncoupling protein mRNA levels (p < 0.05), and increased core temperature by 0.4 degree C (p < 0.05), but neither hypothalamic regional NPY levels nor hypothalamic NPY mRNA levels were affected by BRL 35135. This suggests that the NPY changes induced by cold exposure are not the result of BAT activation, and is consistent with the hypothesis that decreased NPY release during cold exposure might disinhibit the sympathetic innervation that drives BAT thermogenesis.

  6. Tumor therapy with an antibody-targeted superantigen generates a dichotomy between local and systemic immune responses.

    PubMed Central

    Litton, M. J.; Dohlsten, M.; Hansson, J.; Rosendahl, A.; Ohlsson, L.; Kalland, T.; Andersson, J.; Andersson, U.

    1997-01-01

    Repeated injections of a fusion protein containing the superantigen staphylococcal enterotoxin A (SEA) combined with a Fab fragment of a tumor-specific antibody is a highly efficient immunotherapy for mice expressing lung melanoma micrometastasis. In the present study, the systemic and local immune responses generated by this therapy were analyzed at a cellular level. Two distinct but coupled immune reactions occurred after repeated therapy. Tumor necrosis factor and macrophage inflammatory protein-1 alpha and -1 beta were immediately synthesized, in the absence of T lymphocytes, at the local tumor site in the lung. This was followed by the induction of VCAM-1 adhesion molecule expression on pulmonary vascular endothelial cells. Concurrently, the early response in the spleen was characterized by the induction of selective T cells producing interleukin (IL)-2. The primed and expanded SEA-reactive V beta 3- and V beta 11-expressing T lymphocytes accumulated to the tumor area only after Fab-SEA therapy and were not present in the lung when SEA, Fab fragment, or recombinant IL-2 was injected. The tumor-infiltrating T cells produced large amounts of interferon-gamma, but no IL-2 or Th2 type of lymphokines were detected at the tumor site in the Fab-SEA-targeted antitumor immune response. These results emphasize the necessity to investigate several sites of antigen presentation to elucidate the effects of immunotherapy. Images Figure 1 Figure 7 PMID:9137087

  7. Bardoxolone Methyl Prevents Fat Deposition and Inflammation in Brown Adipose Tissue and Enhances Sympathetic Activity in Mice Fed a High-Fat Diet

    PubMed Central

    Dinh, Chi H. L.; Szabo, Alexander; Yu, Yinghua; Camer, Danielle; Zhang, Qingsheng; Wang, Hongqin; Huang, Xu-Feng

    2015-01-01

    Obesity results in changes in brown adipose tissue (BAT) morphology, leading to fat deposition, inflammation, and alterations in sympathetic nerve activity. Bardoxolone methyl (BARD) has been extensively studied for the treatment of chronic diseases. We present for the first time the effects of oral BARD treatment on BAT morphology and associated changes in the brainstem. Three groups (n = 7) of C57BL/6J mice were fed either a high-fat diet (HFD), a high-fat diet supplemented with BARD (HFD/BARD), or a low-fat diet (LFD) for 21 weeks. BARD was administered daily in drinking water. Interscapular BAT, and ventrolateral medulla (VLM) and dorsal vagal complex (DVC) in the brainstem, were collected for analysis by histology, immunohistochemistry and Western blot. BARD prevented fat deposition in BAT, demonstrated by the decreased accumulation of lipid droplets. When administered BARD, HFD mice had lower numbers of F4/80 and CD11c macrophages in the BAT with an increased proportion of CD206 macrophages, suggesting an anti-inflammatory effect. BARD increased phosphorylation of tyrosine hydroxylase in BAT and VLM. In the VLM, BARD increased energy expenditure proteins, including beta 3-adrenergic receptor (β3-AR) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Overall, oral BARD prevented fat deposition and inflammation in BAT, and stimulated sympathetic nerve activity. PMID:26066016

  8. Targeting a homogeneously glycosylated antibody Fc to bind cancer cells using a synthetic receptor ligand.

    PubMed

    Xiao, Junpeng; Chen, Rui; Pawlicki, Mark A; Tolbert, Thomas J

    2009-09-30

    The targeting of a glycosylated antibody Fc fragment to bind to cancer cells by site-selective incorporation of a synthetic ligand is described. Homogeneously glycosylated immunoglobulin G subclass 1 fragment crystallizable (IgG1 Fc) was produced by expression in a glycosylation-deficient yeast strain and subsequent treatment with mannosidase IA. A N-terminal cysteine was generated on the expressed IgG1 Fc by utilizing proteolytic processing enzymes in the yeast secretory pathway. A cyclic RGD peptide thioester 2 was synthesized and then site-selectively attached to the N-terminus of the IgG1 Fc glycoprotein using native chemical ligation. The resulting chemically modified antibody fragment, RGD-Man(5)-IgG1 Fc (5), retained biological activity similar to that of the free cyclic RGD peptide 1 when assayed for its ability to both promote and inhibit the adhesion of alpha(v)beta(3) integrin receptor-expressing WM-115 melanoma cells. In addition, fluorescent microscopy experiments were conducted using FITC-labeled 5 and confirmed binding of 5 to WM-115 melanoma cells. Site-selectively modified antibody fragments such as the one described here may be used to combine the beneficial properties of synthetic receptor ligands with antibody fragments to develop useful biochemical tools and improved therapeutics. The methods described here can also be used to produce glycoprotein fragments for the chemoenzymatic synthesis of homogeneous glycoproteins.

  9. Development of the dopamine transporter selective RTI-336 as a pharmacotherapy for cocaine abuse.

    PubMed

    Carroll, F Ivy; Howard, James L; Howell, Leonard L; Fox, Barbara S; Kuhar, Michael J

    2006-01-01

    The discovery and preclinical development of selective dopamine reuptake inhibitors as potential pharmacotherapies for treating cocaine addiction are presented. The studies are based on the hypothesis that a dopamine reuptake inhibitor is expected to partially substitute for cocaine, thus decreasing cocaine self-administration and minimizing the craving for cocaine. This type of indirect agonist therapy has been highly effective for treating smoking addiction (nicotine replacement therapy) and heroin addiction (methadone). To be an effective pharmacotherapy for cocaine addiction, the potential drug must be safe, long-acting, and have minimal abuse potential. We have developed several 3-phenyltropane analogs that are potent dopamine uptake inhibitors, and some are selective for the dopamine transporter relative to the serotonin and norepinephrine transporters. In animal studies, these compounds substitute for cocaine, reduce the intake of cocaine in rats and rhesus monkeys trained to self-administer cocaine, and have demonstrated a slow onset and long duration of action and lack of sensitization. The 3-phenyltropane analogs were also tested in a rhesus monkey self-administration model to define their abuse potential relative to cocaine. Based on these studies, 3beta-(4-chlorophenyl)-2beta-[3-(4'-methylphenyl)isoxazol-5-yl]tropane (RTI-336) has been selected for preclinical development. PMID:16584128

  10. Synthesis of 8-thiabicyclo[3.2.1]octanes and their binding affinity for the dopamine and serotonin transporters.

    PubMed

    Pham-Huu, Duy-Phong; Deschamps, Jeffrey R; Liu, Shanghao; Madras, Bertha K; Meltzer, Peter C

    2007-01-15

    Cocaine is a potent stimulant of the central nervous system. Its reinforcing and stimulant properties have been associated with inhibition of the dopamine transporter (DAT) on presynaptic neurons. In the search for medications for cocaine abuse, we have prepared 2-carbomethoxy-3-aryl-8-thiabicyclo[3.2.1]octane analogues of cocaine. We report that this class of compounds provides potent and selective inhibitors of the DAT and SERT. The selectivity resulted from reduced activity at the SERT. The 3beta-(3,4-dichlorophenyl) analogue inhibits the DAT and SERT with a potency of IC(50)=5.7 nM and 8.0 nM, respectively. The 3-(3,4-dichlorophenyl)-2,3-unsaturated analogue inhibits the DAT potently (IC(50)=4.5 nM) and selectively (>800-fold vs SERT). Biological enantioselectivity of DAT inhibition was limited for both the 3-aryl-2,3-unsaturated and the 3alpha-aryl analogues (2-fold), but more robust (>10-fold) for the 3beta-aryl analogues. The (1R)-configuration provided the eutomers. PMID:17070057

  11. Effect of high-fat diet on lypolisis in isolated adipocytes from visceral and subcutaneous WAT.

    PubMed

    Portillo, M P; Simón, E; García-Calonge, M A; Del Barrio, A S

    1999-08-01

    Variations in total energy intake and composition of daily food play an important role in the regulation of metabolic processes and so, in the control of body weight. This study was designed in order to investigate the effect of a high-fat diet on lipolysis in isolated adipocytes. For this purpose, fourteen Wistar rats were divided into two groups and fed either a standard-fat diet or a high-fat diet ad libitum for 7 weeks. Adipocytes were prepared from fat pads by collagenase digestion and incubated in vitro in the absence or presence of various lipolytic agents. Lipolysis was measured by the release of glycerol into the medium during 90 min of incubation. We observed that a high amount of fat in the diet induced an enlargement of adipose tissue, which was accompanied by a reduction of beta-adrenergic agonist-induced lipolysis, that could be due to a loss of beta(1) and beta(3)-adrenoceptor number or to alterations of their coupling to adenylate-cyclase through the guanine nucleotide regulatory protein. New data about regional differences were provided by comparing two adipose locations (subcutaneous and visceral). PMID:10502029

  12. Microbial transformations of the antimelanoma agent betulinic acid.

    PubMed

    Kouzi, S A; Chatterjee, P; Pezzuto, J M; Hamann, M T

    2000-12-01

    Microbial transformation studies of the antimelanoma agent betulinic acid (1) were conducted. Screening experiments showed a number of microorganisms capable of biotransforming 1. Three of these cultures, Bacillus megaterium ATCC 14581, Cunninghamella elegans ATCC 9244, and Mucor mucedo UI-4605, were selected for preparative scale transformation. Bioconversion of 1 with resting-cell suspensions of phenobarbital-induced B. megaterium ATCC 14581 resulted in the production of the known betulonic acid (2) and two new metabolites: 3beta,7beta-dihydroxy-lup-20(29)-en-28-oic acid (3) and 3beta,6alpha, 7beta-trihydroxy-lup-20(29)-en-28-oic acid (4). Biotransformation of 1 with growing cultures of C. elegans ATCC 9244 produced one new metabolite characterized as 1beta,3beta, 7beta-trihydroxy-lup-20(29)-en-28-oic acid (5). Incubation of 1 with growing cultures of M. mucedo UI-4605 afforded metabolite 3. Structure elucidation of all metabolites was based on NMR and HRMS analyses. In addition, the antimelanoma activity of metabolites 2-5 was evaluated against two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid). PMID:11141108

  13. Hydrogenotrophic denitrification in a microporous membrane bioreactor.

    PubMed

    Mansell, Bruce O; Schroeder, Edward D

    2002-11-01

    Hydrogenotrophic denitrification of nitrate contaminated groundwater in a bench-scale microporous membrane bioreactor has been investigated. To prevent microbial contamination of the effluent from the reactor the nitrate-laden water treated was separated from the denitrifying culture with a 0.02 microm pore diameter membrane. Equal pressure was maintained across the membrane and nitrate was removed by molecular diffusion through the membrane and into the denitrifying culture. The system was operated with a hydrogenotrophic denitrification culture to circumvent the addition of an organic substrate to the water. Removal efficiencies ranging from 96% to 92% were achieved at influent concentrations ranging from 20 to 40 mg/L NO3(-)-N. The flux values achieved in this study were 2.7-5.3 g NO3-N m 2d(-1). The microporous membrane served as an effective barrier for preventing microbial contamination of the product water as evidenced by the effluent heterotrophic plate count of 9 (+/- 3.5) CFU/mL. The hydrogenotrophic culture was analyzed using available 16S and 23S rRNA-targeted oligonucleotide probes. It was determined that the enrichment process selected for organisms belonging to the beta subclass of Proteobacteria. Further analysis of the hydrogenotrophic culture indicated that the organisms may belong to the beta-3 subgroup of Proteobacteria and have yet to be identified as hydrogenotrophic denitrifiers.

  14. Specific expression of GFP{sub uv}-{beta}1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide

    SciTech Connect

    Kato, Tatsuya; Park, Enoch Y. . E-mail: yspark@agr.shizuoka.ac.jp

    2007-08-03

    Bombyxin (bx) and prophenoloxidase-activating enzyme (ppae) signal peptides from Bombyx mori, their modified signal peptides, and synthetic signal peptides were investigated for the secretion of GFP{sub uv}-{beta}1,3-N-acetylglucosaminyltransferase 2 (GGT2) fusion protein in B. mori Bm5 cells and silkworm larvae using cysteine protease deficient B. mori multiple nucleopolyhedrovirus (BmMNPV-CP{sup -} ) and its bacmid. The secretion efficiencies of all signal peptides were 15-30% in Bm5 cells and 24-30% in silkworm larvae, while that of the +16 signal peptide was 0% in Bm5 cells and 1% in silkworm larvae. The fusion protein that contained the +16 signal peptide was expressed specifically in the endoplasmic reticulum (ER) and in the fractions of cell precipitations. Ninety-four percent of total intracellular {beta}1,3-N-acetylglucosaminyltransferase ({beta}3GnT) activity was detected in cell precipitations following the 600, 8000, and 114,000g centrifugations. In the case of the +38 signal peptide, 60% of total intracellular activity was detected in the supernatant from the 114,000g spin, and only 1% was found in the precipitate. Our results suggest that the +16 signal peptide might be situated in the transmembrane region and not cleaved by signal peptidase in silkworm or B. mori cells. Therefore, the fusion protein connected to the +16 signal peptide stayed in the fat body of silkworm larvae with biological function, and was not secreted extracellularly.

  15. Glucose regulation of thrombospondin and its role in the modulation of smooth muscle cell proliferation.

    PubMed

    Maile, Laura A; Allen, Lee B; Hanzaker, Christopher F; Gollahon, Katherine A; Dunbar, Paul; Clemmons, David R

    2010-01-01

    Smooth muscle cells (SMC) maintained in high glucose are more responsive to IGF-I than those in normal glucose. There is significantly more thrombospondin-1 (TSP-1) in extracellular matrix surrounding SMC grown in 25 mM glucose. In this study we investigated 1) the mechanism by which glucose regulates TSP-1 levels and 2) the mechanism by which TS-1 enhances IGF-I signaling. The addition of TSP-1 to primary SMC was sufficient to enhance IGF-I responsiveness in normal glucose. Reducing TSP-1 protein levels inhibited IGF-I signaling in SMC maintained in high glucose. We determined that TSP-1 protected IAP/CD47 from cleavage and thereby facilitated its association with SHP substrate-1 (SHPS-1). We have shown previously that the hyperglycemia induced protection of IAP from cleavage is an important component of the ability of hyperglycemia to enhance IGF-I signaling. Furthermore we determined that TSP-1 also enhanced phosphorylation of the beta3 subunit of the alphaVbeta3 integrin, another molecular event that we have shown are critical for SMC response to IGF-I in high glucose. Our studies also revealed that the difference in the amount of TSP-1 in the two different glucose conditions was due, at least in part, to a difference in the cellular uptake and degradation of TSP-1.

  16. Regulation of ob gene and overexpression in obesity.

    PubMed

    Guerre-Millo, M

    1997-01-01

    The ob gene product, called leptin, is a recently discovered hormone secreted by the adipose cells. By acting as a satiety factor and increasing energy expenditure, leptin plays a major role in body weight homeostasis in mice. Ob gene and leptin production by the adipose cells are under the control of various hormonal and metabolic factors. Ob mRNA levels are markedly reduced by fasting and restored to normal by refeeding. High-fat feeding increases ob gene and plasma leptin, and induces a state of resistance to leptin. Two hormones, insulin and corticosterone, increase leptin production in rodent and human adipose cells. In contrast, the activity of the sympathetic nervous system exerts an opposite effect, mainly through activation of the adipose beta 3-adrenergic receptors. Leptin synthesis is also decreased by thiazolidinediones, a new class of antidiabetic drugs. The obese Zucker fa/fa rats bear a mutation in the leptin receptor gene (OB-R) and are leptin resistant. In these rats, ob mRNA levels are increased early in life and are not reduced by fasting. This suggests that functional OB-Rs are required for the generation of the signal(s) that downregulates ob gene expression in the adipose cell. The extent to which this is relevant to human obesities, which are characterized by increased leptin levels, remains to be determined.

  17. Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression.

    PubMed

    Chellaiah, M A; Kizer, N; Biswas, R; Alvarez, U; Strauss-Schoenberger, J; Rifas, L; Rittling, S R; Denhardt, D T; Hruska, K A

    2003-01-01

    Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.

  18. Physical and biological characterization of a growth-inhibitory activity purified from the neuroepithelioma cell line A673.

    PubMed Central

    Stam, K; Stewart, A A; Qu, G Y; Iwata, K K; Fenyö, D; Chait, B T; Marshak, D R; Haley, J D

    1995-01-01

    Epithelial- and haematopoietic-cell growth-inhibitory activities have been identified in the conditioned medium of the human peripheral neuroepithelioma cell line A673. An A673-cell-derived growth-inhibitory activity was previously fractionated into two distinct components which inhibited the proliferation of human carcinoma and leukaemia cells in culture. One inhibitory activity was shown to comprise interleukin-1 alpha (IL-1 alpha). Here, we have purified to homogeneity a distinct activity which inhibited the growth of the epithelial cells in vitro. Using a combination of protein-sequence analysis and mass spectrometry, we demonstrated that biological activity can be assigned to a dimeric protein with a molecular mass of 25,576 (+/- 4) Da and an N-terminal sequence identical with that of transforming growth factor-beta 1 (TGF-beta 1). Further characterization of the growth inhibitor with TGF-beta-isoform-specific antibodies showed that > 90% of the bioactivity consists of TGF-beta 1 and not TGF-beta 2 or TGF-beta 3. Although A673 cells were growth-inhibited by exogenous TGF-beta 1, we showed that TGF-beta 1 in A673-cell-conditioned media was present in the latent, biologically inactive, form which did not act as an autocrine growth modulator of A673 cells in vitro. Images Figure 2 Figure 3 PMID:7826358

  19. Chemical composition and anti-inflammation activity of essential oils from Citrus unshiu flower.

    PubMed

    Kim, Min-Jin; Yang, Kyong-Wol; Kim, Sang Suk; Park, Suk Man; Park, Kyung Jin; Kim, Kwang Sik; Choi, Young Hun; Cho, Kwang Keun; Hyun, Chang-Gu

    2014-05-01

    Though many essential oils from citrus peels are claimed to have several medicinal functions, the chemical composition and biological activities of the essential oils of Citrus flowers have not been well described. Therefore, this study intended to investigate the chemical composition and anti-inflammatory potential of essential oils from C. unshiu flower (CEO) to support its purported beneficial health effects. The chemical constituents of the CEO, analyzed by gas chromatography-mass spectrometry (GC-MS), included y-terpinene (24.7%), 2-beta-pinene (16.6%), 1-methyl-2-isopropylbenzene (11.5%), L-limonene (5.7%), beta3-ocimene (5.6%), and alpha-pinene (4.7%). The effects of the CEO on nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages were also examined. The results indicate that the CEO is an effective inhibitor of LPS-induced NO and PGE2 production in RAW 264.7 cells. Additionally, CEO was shown to suppress the production of inflammatory cytokines including interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and IL-6. Based on these results, CEO may be considered a potential anti-inflammatory candidate with human health benefits.

  20. ATP synthase: a tentative structural model.

    PubMed

    Engelbrecht, S; Junge, W

    1997-09-15

    Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft. The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer. PMID:9323021

  1. Carnitine Palmitoyltransferase 1B 531K Allele Carriers Sustain a Higher Respiratory Quotient after Aerobic Exercise, but β3-Adrenoceptor 64R Allele Does Not Affect Lipolysis: A Human Model

    PubMed Central

    Gómez-Gómez, Eduardo; Ríos-Martínez, Martín Efrén; Castro-Rodríguez, Elena Margarita; Del-Toro-Equíhua, Mario; Ramírez-Flores, Mario; Delgado-Enciso, Ivan; Pérez-Huitimea, Ana Lilia; Baltazar-Rodríguez, Luz Margarita; Velasco-Pineda, Gilberto; Muñiz-Murguía, Jesús

    2014-01-01

    Carnitine palmitoyltransferase IB (CPT1B) and adrenoceptor beta-3 (ADRB3) are critical regulators of fat metabolism. CPT1B transports free acyl groups into mitochondria for oxidation, and ADRB3 triggers lipolysis in adipocytes, and their respective polymorphisms E531K and W64R have been identified as indicators of obesity in population studies. It is therefore important to understand the effects of these mutations on ADRB3 and CPT1B function in adipose and skeletal muscle tissue, respectively. This study aimed to analyze the rate of lipolysis of plasma indicators (glycerol, free fatty acids, and beta hydroxybutyrate) and fat oxidation (through the non-protein respiratory quotient). These parameters were measured in 37 participants during 30 min of aerobic exercise at approximately 62% of maximal oxygen uptake, followed by 30 min of recovery. During recovery, mean respiratory quotient values were higher in K allele carriers than in non-carriers, indicating low post-exercise fatty acid oxidation rates. No significant differences in lipolysis or lipid oxidation were observed between R and W allele carriers of ADRB3 at any time during the aerobic load. The substitution of glutamic acid at position 531 by lysine in the CPT1B protein decreases the mitochondrial beta-oxidation pathway, which increases the non-protein respiratory quotient value during recovery from exercise. This may contribute to weight gain or reduced weight-loss following exercise. PMID:24905907

  2. Carnitine palmitoyltransferase 1B 531K allele carriers sustain a higher respiratory quotient after aerobic exercise, but β3-adrenoceptor 64R allele does not affect lipolysis: a human model.

    PubMed

    Gómez-Gómez, Eduardo; Ríos-Martínez, Martín Efrén; Castro-Rodríguez, Elena Margarita; Del-Toro-Equíhua, Mario; Ramírez-Flores, Mario; Delgado-Enciso, Ivan; Pérez-Huitimea, Ana Lilia; Baltazar-Rodríguez, Luz Margarita; Velasco-Pineda, Gilberto; Muñiz-Murguía, Jesús

    2014-01-01

    Carnitine palmitoyltransferase IB (CPT1B) and adrenoceptor beta-3 (ADRB3) are critical regulators of fat metabolism. CPT1B transports free acyl groups into mitochondria for oxidation, and ADRB3 triggers lipolysis in adipocytes, and their respective polymorphisms E531K and W64R have been identified as indicators of obesity in population studies. It is therefore important to understand the effects of these mutations on ADRB3 and CPT1B function in adipose and skeletal muscle tissue, respectively. This study aimed to analyze the rate of lipolysis of plasma indicators (glycerol, free fatty acids, and beta hydroxybutyrate) and fat oxidation (through the non-protein respiratory quotient). These parameters were measured in 37 participants during 30 min of aerobic exercise at approximately 62% of maximal oxygen uptake, followed by 30 min of recovery. During recovery, mean respiratory quotient values were higher in K allele carriers than in non-carriers, indicating low post-exercise fatty acid oxidation rates. No significant differences in lipolysis or lipid oxidation were observed between R and W allele carriers of ADRB3 at any time during the aerobic load. The substitution of glutamic acid at position 531 by lysine in the CPT1B protein decreases the mitochondrial beta-oxidation pathway, which increases the non-protein respiratory quotient value during recovery from exercise. This may contribute to weight gain or reduced weight-loss following exercise.

  3. Synthesis of 8-thiabicyclo[3.2.1]octanes and their binding affinity for the dopamine and serotonin transporters.

    PubMed

    Pham-Huu, Duy-Phong; Deschamps, Jeffrey R; Liu, Shanghao; Madras, Bertha K; Meltzer, Peter C

    2007-01-15

    Cocaine is a potent stimulant of the central nervous system. Its reinforcing and stimulant properties have been associated with inhibition of the dopamine transporter (DAT) on presynaptic neurons. In the search for medications for cocaine abuse, we have prepared 2-carbomethoxy-3-aryl-8-thiabicyclo[3.2.1]octane analogues of cocaine. We report that this class of compounds provides potent and selective inhibitors of the DAT and SERT. The selectivity resulted from reduced activity at the SERT. The 3beta-(3,4-dichlorophenyl) analogue inhibits the DAT and SERT with a potency of IC(50)=5.7 nM and 8.0 nM, respectively. The 3-(3,4-dichlorophenyl)-2,3-unsaturated analogue inhibits the DAT potently (IC(50)=4.5 nM) and selectively (>800-fold vs SERT). Biological enantioselectivity of DAT inhibition was limited for both the 3-aryl-2,3-unsaturated and the 3alpha-aryl analogues (2-fold), but more robust (>10-fold) for the 3beta-aryl analogues. The (1R)-configuration provided the eutomers.

  4. Impaired activation of platelets lacking protein kinase C-theta isoform.

    PubMed

    Nagy, Bela; Bhavaraju, Kamala; Getz, Todd; Bynagari, Yamini S; Kim, Soochong; Kunapuli, Satya P

    2009-03-12

    Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-theta is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC-theta-selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PAR-induced aggregation, dense and alpha-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-theta, platelet aggregation and secretion were also impaired. PKC-mediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-theta RACK peptide, which may contribute to the lower levels of granule secretion when PKC-theta function is lost. Furthermore, the level of JON/A binding to activated alpha(IIb)beta(3) receptor was also significantly decreased in PKC-theta(-/-) mice compared with wild-type littermates. PKC-theta(-/-) murine platelets showed significantly lower agonist-induced thromboxane A(2) (TXA(2)) release through reduced extracellular signal-regulated kinase phosphorylation. Finally, PKC-theta(-/-) mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl(3) in vivo thrombosis model compared with wild-type mice. In conclusion, PKC-theta isoform plays a significant role in platelet functional responses downstream of PAR and GPVI receptors. PMID:19164598

  5. Solution structure of {alpha}-conotoxin PIA, a novel antagonist of {alpha}6 subunit containing nicotinic acetylcholine receptors

    SciTech Connect

    Chi, Seung-Wook; Lee, Si-Hyung; Kim, Do-Hyoung; Kim, Jae-Sung; Olivera, Baldomero M.; McIntosh, J. Michael; Han, Kyou-Hoon . E-mail: khhan600@kribb.re.kr

    2005-12-30

    {alpha}-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing {alpha}6 and {alpha}3 subunits. {alpha}-conotoxin PIA displays 75-fold higher affinity for rat {alpha}6/{alpha}3{beta}2{beta}3 nAChRs than for rat {alpha}3{beta}2 nAChRs. We have determined the three-dimensional structure of {alpha}-conotoxin PIA by nuclear magnetic resonance spectroscopy. The {alpha}-conotoxin PIA has an '{omega}-shaped' overall topology as other {alpha}4/7 subfamily conotoxins. Yet, unlike other neuronally targeted {alpha}4/7-conotoxins, its N-terminal tail Arg{sup 1}-Asp{sup 2}-Pro{sup 3} protrudes out of its main molecular body because Asp{sup 2}-Pro{sup 3}-Cys{sup 4}-Cys{sup 5} forms a stable type I {beta}-turn. In addition, a kink introduced by Pro{sup 15} in the second loop of this toxin provides a distinct steric and electrostatic environment from those in {alpha}-conotoxins MII and GIC. By comparing the structure of {alpha}-conotoxin PIA with other functionally related {alpha}-conotoxins we suggest structural features in {alpha}-conotoxin PIA that may be associated with its unique receptor recognition profile.

  6. Identification and characterization of cis elements in the STAT3 gene regulating STAT3 alpha and STAT3 beta messenger RNA splicing.

    PubMed

    Shao, H; Quintero, A J; Tweardy, D J

    2001-12-15

    Signal transducer and activator of transcription 3 (STAT3) is an oncogene and a critical regulator of multiple cell-fate decisions, including myeloid cell differentiation. Two isoforms of STAT3 have been identified: alpha (p92) and beta (p83). These differ structurally in their C-terminal transactivation domains, resulting in distinct functional activities. The cis genetic elements that regulate the ratio of alpha to beta messenger RNA (mRNA) are unknown. In this study, cloning, sequencing, and splicing analysis of the human and murine STAT3 genes revealed a highly conserved 5' donor site for generation of both alpha and beta mRNA and distinct branch-point sequences, polypyrimidine tracts, and 3' acceptor sites (ASs) for each. The beta 3' AS was found to be located 50 nucleotides downstream of the alpha 3' AS in exon 23. Two additional cryptic 3' ASs (delta and epsilon) were also identified. Thus, we identified for the first time the cis regulatory sequences responsible for generation of STAT3 alpha and STAT3 beta mRNA.

  7. Isolation and identification in bovine cerebral cortex of n-butyl beta-carboline-3-carboxylate, a potent benzodiazepine binding inhibitor.

    PubMed Central

    Peña, C; Medina, J H; Novas, M L; Paladini, A C; De Robertis, E

    1986-01-01

    A substance having benzodiazepine-binding inhibitory activity has been extracted from 18 kg of gray matter of bovine cerebral cortex and purified to homogeneity. This substance inhibits competitively [3H]flunitrazepam and ethyl beta-[3H]carboline-3-carboxylate binding with high affinity (Ki, 3 nM), but it is inactive upon 3H-labeled Ro 5-4864, [3H]quinuclidinyl benzylate, [3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol, and upon high-affinity [3H]muscimol binding. This inhibitor has been identified as n-butyl beta-carboline-3-carboxylate (beta-CCB) by fast atom bombardment mass spectroscopy (Mr, 268) and electron bombardment fragmentography, ultraviolet and fluorescence spectra, coelution in HPLC with standard beta-CCB, and by the exact correspondence in Ki with beta-CCB on the displacement of [3H]flunitrazepam binding. The possible artificial formation of beta-CCB has been discarded by a series of control experiments including addition of tryptophan to the starting homogenate, extraction from liver, isolation and purification by an alternative procedure avoiding organic solvents, and by the impossibility of making beta-CCB from beta-carboline-3-carboxylic acid or its methyl ester in the conditions of our extraction and purification procedures. PMID:3014522

  8. Vascular endothelial cells express a functional fas-receptor due to lack of hemodynamic forces.

    PubMed

    Freyberg, M A; Kaiser, D; Graf, R; Friedl, P

    2001-10-01

    The fas system is present in atherosclerotic lesions. However, its role in the initiation and progression is still unclear. Here we show that in endothelial cells (EC) the expression of the fas receptor is regulated by flow conditions. The EC of the vascular system are regularly exposed to a range of hemodynamic forces with great impact on cellular structures and functions. Recently it was reported that in endothelial cells the lack of hemodynamic forces as well as irregular flow conditions trigger apoptosis by induction of a mechanosensitive autocrine loop of thrombospondin-1 and the alpha(V)beta(3) integrin/integrin-associated protein complex. Here we show that EC cultivated under regular laminar flow conditions are devoid of the fas-receptor whereas cultivation under static conditions as well as under turbulence leads to its expression. Stimulation of the fas-receptor by its ligand increases the amount of apoptotic cells by twofold; the increase can be prevented by blocking the fas-receptor. The availability of the expressed fas receptor for stimulation by its ligand hints at a role as a tool for progression of atherosclerosis. PMID:11483857

  9. Quantitative trait analysis reveals transforming growth factor-beta2 as a positive regulator of early hematopoietic progenitor and stem cell function.

    PubMed

    Langer, Jessica C; Henckaerts, Els; Orenstein, Jonathan; Snoeck, Hans-Willem

    2004-01-01

    Elucidation of pathways involved in mouse strain-dependent variation in the hematopoietic stem cell (HSC) compartment may reveal novel mechanisms relevant in vivo. Here, we demonstrate genetically determined variation in the proliferation of lin-Sca1++kit+ (LSK) primitive hematopoietic progenitor cells in response to transforming growth factor-beta (TGF-beta) 2, the dose response of which was biphasic with a stimulatory effect at low concentrations. In contrast, the dose responses of TGF-beta1 or -beta3 were inhibitory and did not show mouse strain-dependent variation. A quantitative trait locus (QTL) for the effect of TGF-beta2 was identified on chromosome 4 overlapping with a QTL regulating the frequency of LSK cells. These overlapping QTL were corroborated by the observation that the frequency of LSK cells is lower in adult Tgfb2+/- mice than in wild-type littermates, indicating that TGF-beta2 is a genetically determined positive regulator LSK number in vivo. Furthermore, adult Tgfb2+/- mice have a defect in competitive repopulation potential that becomes more pronounced upon serial transplantation. In fetal TGF-beta2-deficient HSCs, a defect only appears after serial reconstitution. These data suggest that TGF-beta2 can act cell autonomously and is important for HSCs that have undergone replicative stress. Thus, TGF-beta2 is a novel, genetically determined positive regulator of adult HSCs.

  10. The combination of epidermal growth factor and transforming growth factor-beta induces novel phenotypic changes in mouse liver stem cell lines.

    PubMed

    Isfort, R J; Cody, D B; Stuard, S B; Randall, C J; Miller, C; Ridder, G M; Doersen, C J; Richards, W G; Yoder, B K; Wilkinson, J E; Woychik, R P

    1997-12-01

    Mouse liver stem cell (oval cell) lines were investigated in order to determine the role which two families of growth and differentiation factors (GDFs), epidermal growth factor (EGF) family and transforming growth factor beta (TGF-beta) family, play in liver regeneration. EGF family members, including EGF, amphiregulin, betacellulin, heparin-binding epidermal growth factor, and TGF-alpha, were mitogenic for oval cell lines while TGF-beta family members, including TGF-beta1, TGF-beta2 and TGF-beta3, inhibited mitogenesis and induced apoptosis in oval cell lines. Surprisingly, the combination of EGF family members and TGF-ss family members resulted in neither proliferation nor apoptosis but instead in a novel cellular response, cellular scattering in tissue culture and morphological differentiation in Matrigel. Analysis of the signal transduction pathways activated by exposure of oval cell lines to either EGF, EGF+TGF-beta, or TGF-beta indicated that novel combinations of intracellular signals result following stimulation of the cells with the combination of EGF+TGF-beta. These data reveal that the dynamics of synergistic GDF action following tissue injury and regeneration results in a new level of complexity not obvious from the study of individual GDFs.

  11. Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion.

    PubMed

    Andreola, Fausto; Calvisi, Diego F; Elizondo, Guillermo; Jakowlew, Sonia B; Mariano, Jennifer; Gonzalez, Frank J; De Luca, Luigi M

    2004-01-01

    Aryl hydrocarbon receptor (AHR)-null mice display a liver fibrosis phenotype that is associated with a concomitant increase in liver retinoid concentration, tissue transglutaminase type II (TGaseII) activity, transforming growth factor beta (TGF beta) overexpression, and accumulation of collagen. To test the hypothesis that this phenotype might be triggered by the observed increase in liver retinoid content, we induced the condition of retinoid depletion by feeding AHR-null mice a vitamin A- deficient diet with the purpose to reverse the phenotype. Liver retinoid content decreased sharply within the first few weeks on the retinoid-deficient diet. Analysis of TGF beta 1, TGF beta 2, and TGF beta 3 expression revealed a reduction to control levels in the AHR -/- mice accompanied by parallel changes in TGaseII protein levels. In addition, we observed an increase in the TGF beta receptors, TGF beta RI and TGF beta RII, as well as in Smad4, and their reduction to wild-type mouse liver levels in AHR -/- mice fed the retinoid-deficient diet. Reduction of peroxisomal proliferator-activated receptor gamma (PPAR gamma) messenger RNA (mRNA) and protein levels in AHR -/- mice was consistent with the presence of hepatic stellate cell (HSC) activation and liver fibrosis. Vitamin A deficiency normalized PPAR gamma expression in AHR -/- mice. In conclusion, livers from AHR -/- mice fed the vitamin A-deficient diet showed a decrease in collagen deposition, consistent with the absence of liver fibrosis.

  12. A crucial function of PDGF in TGF-beta-mediated cancer progression of hepatocytes.

    PubMed

    Gotzmann, J; Fischer, A N M; Zojer, M; Mikula, M; Proell, V; Huber, H; Jechlinger, M; Waerner, T; Weith, A; Beug, H; Mikulits, W

    2006-05-25

    Polarized hepatocytes expressing hyperactive Ha-Ras adopt an invasive and metastatic phenotype in cooperation with transforming growth factor (TGF)-beta. This dramatic increase in malignancy is displayed by an epithelial to mesenchymal transition (EMT), which mimics the TGF-beta-mediated progression of human hepatocellular carcinomas. In culture, hepatocellular EMT occurs highly synchronously, facilitating the analysis of molecular events underlying the various stages of this process. Here, we show that in response to TGF-beta, phosphorylated Smads rapidly translocated into the nucleus and activated transcription of target genes such as E-cadherin repressors of the Snail superfamily, causing loss of cell adhesion. Within the TGF-beta superfamily of cytokines, TGF-beta1, -beta2 and -beta3 were specific for the induction of hepatocellular EMT. Expression profiling of EMT kinetics revealed 78 up- and 235 downregulated genes, which preferentially modulate metabolic activities, extracellular matrix composition, transcriptional activities and cell survival. Independent of the genetic background, platelet-derived growth factor (PDGF)-A ligand and both PDGF receptor subunits were highly elevated, together with autocrine secretion of bioactive PDGF. Interference with PDGF signalling by employing hepatocytes expressing the dominant-negative PDGF-alpha receptor revealed decreased TGF-beta-induced migration in vitro and efficient suppression of tumour growth in vivo. In conclusion, these results provide evidence for a crucial role of PDGF in TGF-beta-mediated tumour progression of hepatocytes and suggest PDGF as a target for therapeutic intervention in liver cancer.

  13. A rotary molecular motor that can work at near 100% efficiency.

    PubMed Central

    Kinosita, K; Yasuda, R; Noji, H; Adachi, K

    2000-01-01

    A single molecule of F1-ATPase is by itself a rotary motor in which a central gamma-subunit rotates against a surrounding cylinder made of alpha3beta3-subunits. Driven by the three betas that sequentially hydrolyse ATP, the motor rotates in discrete 120 degree steps, as demonstrated in video images of the movement of an actin filament bound, as a marker, to the central gamma-subunit. Over a broad range of load (hydrodynamic friction against the rotating actin filament) and speed, the F1 motor produces a constant torque of ca. 40 pN nm. The work done in a 120 degree step, or the work per ATP molecule, is thus ca. 80 pN nm. In cells, the free energy of ATP hydrolysis is ca. 90 pN nm per ATP molecule, suggesting that the F1 motor can work at near 100% efficiency. We confirmed in vitro that F1 indeed does ca. 80 pN nm of work under the condition where the free energy per ATP is 90 pN nm. The high efficiency may be related to the fully reversible nature of the F1 motor: the ATP synthase, of which F1 is a part, is considered to synthesize ATP from ADP and phosphate by reverse rotation of the F1 motor. Possible mechanisms of F1 rotation are discussed. PMID:10836501

  14. ND-309, a novel compound, ameliorates cerebral infarction in rats by antioxidant action.

    PubMed

    Tian, Jingwei; Li, Guisheng; Liu, Zhifeng; Zhang, Shumin; Qu, Guiwu; Jiang, Wanglin; Fu, Fenghua

    2008-09-19

    Extract of danshen (Salvia miltiorrhiza Bunge.) has been clinically prescribed in China to treat patients with stroke. The novel compound designated ND-309, namely isopropyl-beta-(3,4-dihydroxyphenyl)-alpha-hydroxypropanoate is a new metabolite of danshen in rat brain. The present study was conducted to investigate whether ND-309 has a protective effect on brain injury after focal cerebral ischemia, and to determine the possible mechanism. Adult male SD rats were subjected to middle cerebral artery occlusion (MCAO) by bipolar electro-coagulation. Behavioral tests were used to evaluate the damage to central nervous system. The cerebral infarct volume and edema were assessed to evaluate the brain patho-physiological changes. Spectrophotometric or spectrofluorometric assay methods were used to determine the generation of reactive oxygen species (ROS), activities of superoxide dismutase (SOD) and glutathione-peroxidase (GSH-Px), contents of malondialdehyde (MDA) and adenosine triphosphate (ATP), as well as respiratory control ratio of the brain mitochondria. The results showed that treatment with ND-309 significantly decreased neurological deficit scores, reduced infarct volume and the edema compared with the model group. Meanwhile, ND-309 significantly increased the brain ATP content, improved mitochondrial energy metabolism, attenuated the elevation of MDA content, the decrease in SOD, GSH-Px activity and the generation of ROS in brain mitochondria. All of these findings indicate that ND-309 has the protective potential against cerebral ischemia injury and its protective effects may be due to the amelioration of cerebral energy metabolism and its antioxidant property.

  15. Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry

    NASA Technical Reports Server (NTRS)

    Wang, N.; Ingber, D. E.

    1995-01-01

    We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

  16. Endothelial Cells Organize Fibrin Clots into Structures That Are More Resistant to Lysis

    NASA Astrophysics Data System (ADS)

    Gray Jerome, W.; Handt, Stefan; Hantgan, Roy R.

    2005-06-01

    Acute myocardial infarction is a major cause of death and disability in the United States. Introducing thrombolytic agents into the clot to dissolve occlusive coronary artery thrombi is one method of treatment. However, despite advances in our knowledge of thrombosis and thrombolysis, survival rates following thrombolytic therapy have not improved substantially. This failure highlights the need for further study of the factors mediating clot stabilization. Using laser scanning confocal microscopy of clots formed from fluorescein-labeled fibrinogen, we investigated what effect binding of fibrin to the endothelial surface has on clot structure and resistance to lysis. Fluorescent fibrin clots were produced over human umbilical vein endothelial cells (HUVEC) and the clot structure analyzed. In the presence of HUVEC, fibrin near the endothelial surface was more organized and occurred in tighter bundles compared to fibrin just 50 [mu]m above. The HUVEC influence on fibrin architecture was blocked by inhibitory concentrations of antibodies to [alpha]V or [beta]3 integrin subunits. The regions of the clots associated with endothelial cells were more resistant to lysis than the more homogenous regions distal to endothelium. Thus, our data show that binding of fibrin to integrins on endothelial surfaces produces clots that are more resistant to lysis.

  17. The role of GABAbeta2 subunit-containing receptors in mediating the anticonvulsant and sedative effects of loreclezole.

    PubMed

    Groves, James O; Guscott, Martin R; Hallett, David J; Rosahl, Thomas W; Pike, Andrew; Davies, Amy; Wafford, Keith A; Reynolds, David S

    2006-07-01

    The majority of inhibitory neurotransmission in the brain is mediated by the gamma-aminobutyric acid (GABA) type A (GABA(A)) receptor. The anticonvulsant loreclezole largely acts by potentiating GABA(A) receptors containing beta2 and beta3 subunits. We used a genetically modified mouse containing a loreclezole-insensitive beta2 subunit (beta2N265S) to determine the role of this subunit in mediating the sedative and anticonvulsive effects of loreclezole. Sedation was assessed by measuring spontaneous locomotor activity and beam walking performance, and anticonvulsant efficacy was determined by pentylenetetrazole (PTZ) and amygdala kindling-induced seizures. The beta2N265S mice did not exhibit loreclezole-mediated sedation as shown by normal locomotor activity and beam walking performance. However, loreclezole also failed to provide significant protection against PTZ-induced seizures in the beta2N265S mice. Reduced efficacy against amygdala-kindled seizures, both acutely and over a 13-day chronic dosing study, was also observed in beta2N265S mice. These results suggest that the majority of the sedative effects and a significant proportion of the anticonvulsant efficacy of loreclezole are mediated via beta2-containing GABA(A) receptors. PMID:16882014

  18. Interleukin-4 improves the migration of human myogenic precursor cells in vitro and in vivo

    SciTech Connect

    Lafreniere, J.F.; Mills, P.; Bouchentouf, M.; Tremblay, J.P. . E-mail: Jacques-P.Tremblay@crchul.ulaval.ca

    2006-04-15

    Different molecules are available to recruit new neighboring myogenic cells to the site of regeneration. Formerly called B cell stimulatory factor-1, IL-4 can now be included in the list of motogenic factors. The present report demonstrates that human IL-4 is not required for fusion between mononucleated myoblasts but is required for myotube maturation. In identifying IL-4 as a pro-migratory agent for myogenic cells, these results provide a mechanism which partly explains IL-4 demonstrated activity during differentiation. Among the different mechanisms by which IL-4 might enhance myoblast migration processes, our results indicate that there are implications of some integrins and of three major components of the fibrinolytic system. Indeed, increases in the amount of active urokinase plasminogen activator and its receptor were observed following an IL-4 treatment, while the plasminogen activator inhibitor-1 decreased. Finally, IL-4 did not modify the amount of cell surface {alpha}5 integrin but increased the presence of {beta}3 and {beta}1 integrins. This integrin modulation might favor myogenic cell migration and its interaction with newly formed myotubes. Therefore, IL-4 co-injection with transplanted myoblasts might be an approach to enhance the migration of transplanted cells for the treatment of a damaged myocardium or of a Duchenne Muscular Dystrophy patient.

  19. Energy of Cohesion, Compressibility, and the Potential Energy Functions of the Graphite System

    NASA Technical Reports Server (NTRS)

    Girifalco, L. A.; Lad, R. A.

    1956-01-01

    The lattice summations of the potential energy of importance in the graphite system have been computed by direct summation assuming a Lennard-Jones 6-12 potential between carbon atoms. From these summations, potential energy curves were constructed for interactions between a carbon atom and a graphite monolayer, between a carbon atom and a graphite surface, between a graphite monolayer and a semi-infinite graphite crystal and between two graphite semi-infinite crystals. Using these curves, the equilibrium distance between two isolated physically interacting carbon atoms was found to be 2.70 a, where a is the carbon-carbon distance in a graphite sheet. The distance between a surface plane and the rest of the crystal was found to be 1.7% greater than the interlayer spacing. Theoretical values of the energy of cohesion and the compressibility were calculated from the potential curve for the interaction between two semi-infinite crystals. They were (delta)E(sub c) = -330 ergs/sq cm and beta =3.18x10(exp -12)sq cm/dyne, respectively. These compared favorably with the experimental values of (delta)E(sub c) = -260 ergs/sq cm and beta = 2.97 X 10(exp -2) sq cm/dyne.

  20. Synthesis and antiviral properties of (+/-)-5'-noraristeromycin and related purine carbocyclic nucleosides. A new lead for anti-human cytomegalovirus agent design.

    PubMed

    Patil, S D; Schneller, S W; Hosoya, M; Snoeck, R; Andrei, G; Balzarini, J; De Clercq, E

    1992-09-01

    (+/-)-5'-Noraristeromycin (3) has been prepared in three steps beginning with the 2,3-O-isopropylidene derivative of (+/-)-(1 alpha, 2 beta, 3 beta, 4 alpha)-4-amino-1,2,3-cyclopentanetriol (7). Also prepared from the same starting material were the related hypoxanthine (4), guanine (5), and 2,6-diaminopurine (6) analogues. Compounds 3-6 were evaluated for antiviral activity against a large number of viruses with marked activity being observed for 3 towards vaccinia virus, human cytomegalovirus, vesicular stomatitis virus, parainfluenza (type 3) virus, measles virus, respiratory syncytial virus, reovirus (type 1), and the arenaviruses Junin and Tacaribe. None of the compounds showed cytotoxicity to the host cell monolayers used in the antiviral studies. Both 3 and 6 have been found to be inhibitors of S-adenosyl-L-homocysteine hydrolase (AdoHcy hydrolase), which likely accounts for their antiviral activity. Inhibition of AdoHcy hydrolase represents a new approach to human cytomegalovirus drug design that should be pursued. Also, the activity of 3 should be further scrutinized for the treatment of pox-, rhabdo-, paramyxo-, reo-, and arenavirus infections. PMID:1326633

  1. Structure of steady state accretion shocks with several cooling functions: Closed integral-form solution

    NASA Technical Reports Server (NTRS)

    Wu, Kinwah; Chanmugam, G.; Shaviv, G.

    1994-01-01

    We present, for the first time, a closed integral-form solution to the accretion shock structures for the case where the cooling is due to optically thin bremsstrahlung emission and a series of power-law cooling functions of density and temperature. Our results can provide useful checks on numerical calculations and simple accurate estimates for valuable parameters such as the shock height. For the case where the cooling rate j = (2/3)Arho(exp 2)(P/rho)(exp 1/2)(1 + epsilon (sub s)(P/P(sub s)(exp alpha)(rho(sub s)/rho)(exp beta)), we find that a substantial amount of the accretion energy is released at the base of the accretion shock in the form of bremsstrahlung radiation. This implies that for a cyclotron-dominated shock (qualitatively given by alpha = 2.0, beta = 3.85, and epsilon(sub s) is much greater than 1), bremsstrahlung cooling still plays a crucial role in determining the shock structure. Our results are shown to be consistent with detailed numerical calculations.

  2. The solubility and colloidal behaviour of neptunium (IV).

    PubMed

    Moriyama, H; Pratopo, M I; Higashi, K

    1989-07-15

    The solubility and colloidal particle formation of Np(IV) in 10(-1) M Na2S2O4 solution in the pH range 2-10 were studied by sequential filtration with decreasing filter pore size. With the help of spectrophotometry and an extraction technique, the species remaining in the final filtrate were found to be Np(V) at pH less than 6 and Np(IV) at pH greater than 8; particle species were attributed to Np(IV). The solubility data obtained at pH less than 6 were interpreted using the reaction: Np(OH)4(am)----NpO2+ + e- + 2H2O, log K = -6.7 +/- 0.4 At pH greater than 8, the solubility increased rapidly with increasing pH in the presence of air, and this was interpreted as being due to the formation of the negatively charged species Np(CO3)4x - 2x. The formation constants beta 3 and beta 4 were estimated to be similar to those reported for Pu(IV). The size distribution of colloidal Np(IV) was also obtained. Some simple models used to describe the observed size distributions and to estimate the equilibrium size distributions of colloidal Np(IV) were examined.

  3. Pseudo-symmetry characterization and refinement of a trigonal crystal form of naphthalene 1,2-dioxygenase.

    PubMed

    Carredano, E; Kauppi, B; Choudhury, D; Ramaswamy, S

    2000-03-01

    Two trigonal crystal structures of naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4 have been refined at 2.6 A resolution. The space group is R3, with four heterodimers in the asymmetric unit. The crystallographic threefold axis coincides with the symmetry axis of the active molecule, a mushroom-shaped alpha(3)beta(3) hexamer. The crystal is formed by symmetrical contacts between the hexamers on three different interaction surfaces, one on the beta-subunit and the other two on the alpha--subunits. Nickel ions mediate one of the alpha-subunit interactions. The two other types of packing contacts sustain two interlaced and almost independent crystal patterns with significantly different temperature factors. The space group of the individual crystal patterns is R32, with the corresponding twofold axes parallel to each other. The interactions between the crystal patterns separate the two parallel twofolds, eliminating the twofold symmetry for the whole crystal. The differences in temperature factors among the molecules in the asymmetric unit have been refined and are different for the two refined structures. An analysis of the structure factors of the pseudo-equivalent reflections showed that their differences lie in their phases and not in their amplitudes, suggesting that R(merge) is not an appropriate indicator for revealing the correct point group. PMID:10713518

  4. Unique insecticide specificity of human homomeric rho 1 GABA(C) receptor.

    PubMed

    Ratra, Gurpreet S; Erkkila, Brian E; Weiss, David S; Casida, John E

    2002-03-24

    Several convulsants and major insecticides block the gamma-aminobutyric acid (GABA)-gated chloride channel in brain on binding to the GABA(A) receptor. The GABA(C) receptor, important in retina and present in brain, is also coupled to a chloride channel and is therefore a potential target for toxicant action examined here in radioligand binding and electrophysiological experiments. Human homomeric rho 1 GABA(C) receptor expressed in human embryonic kidney cells (HEK293) undergoes specific and saturable high-affinity binding of 4-n-[3H]propyl-4' -ethynylbicycloorthobenzoate ([3H]EBOB) using a cyano analog (CNBOB) to determine non-specific binding. This GABA(C) rho 1 receptor is very sensitive to CNBOB and lindane relative to alpha-endosulfan, tert-butylbicyclophosphorothionate, picrotoxinin and fipronil (IC(50) values of 23, 91, 800, 1080, 4000 and >10000 nM, respectively, in displacing [3H]EBOB). A similar potency sequence (except for picrotoxinin) is observed for inhibition of GABA-induced currents of rho 1 receptor expressed in Xenopus oocytes. The present study does not consider rho 2 homomeric and rho 1 rho 2 heteromeric GABA(C) receptors which are known to be more sensitive than rho 1 to picrotoxinin. The inhibitor sensitivity and specificity of this rho 1 GABA(C) receptor differ greatly from those of human homomeric beta 3 and native GABA(A) receptors.

  5. GABA receptor antagonists and insecticides: common structural features of 4-alkyl-1-phenylpyrazoles and 4-alkyl-1-phenyltrioxabicyclooctanes.

    PubMed

    Sammelson, Robert E; Caboni, Pierluigi; Durkin, Kathleen A; Casida, John E

    2004-06-15

    Fipronil [5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulfinylpyrazole] is one of the most important insecticides. Structure-activity studies described here reveal that fipronil retains its very high binding potency at the human beta3 and house fly gamma-aminobutyric acid (GABA) receptors and toxicity to house flies on replacing the pyrazole trifluoromethylsulfinyl moiety with tert-butyl or isopropyl and the phenyl trifluoromethyl substituent with ethynyl, trifluoromethoxy, bromo or chloro. Among the compounds studied, those with other alkyl groups at the 4-position of the pyrazole, as well as phenyl substitution without one or both of the 2,6-dichloro groups, are less effective. 5-Amino-4-tert-butyl-3-cyano-1-(2,6-dichloro-4-ethynylphenyl)pyrazole is highly effective and almost isosteric with 4-tert-butyl-3-cyano-1-(4-ethynylphenyl)-2,6,7-trioxabicyclo[2.2.2]octane (the most potent 4-alkyl-1-phenyltrioxabicyclooctane) as a noncompetitive GABA antagonist and insecticide. These findings are interpreted as three binding subsites in the GABA receptor: a hydrophobic site undergoing steric interaction with the tert-butyl or equivalent group; a hydrogen bonding site to pyrazole N-2; a pi bonding site to the face of the phenyl moiety; with supplemental enhancement by the 3-cyano and 4-ethynyl substituents.

  6. Fecal steroid monitoring for assessing gonadal and adrenal activity in the golden eagle and peregrine falcon.

    PubMed

    Staley, Airica M; Blanco, Juan M; Dufty, Alfred M; Wildt, David E; Monfort, Steven L

    2007-08-01

    We examined the efficacy of noninvasive monitoring of endocrine function via fecal steroid immunoassays in the golden eagle and peregrine falcon. High-pressure liquid chromatography analyses of fecal glucocorticoid metabolites (fGCM) revealed that minor percentages of immunoreactive fGCM co-eluted with [(3)H]corticosterone in both sexes of the eagle (2.5-2.7%) and falcon (7.5-11.9%). In contrast, most fecal estrogen metabolites in eagle and falcon females co-eluted with radiolabeled estradiol-17beta ([(3)H]; 57.6, 64.6%, respectively) or estrone ([(3)H]; 26.9, 4.1%, respectively). Most fecal progestin metabolite immunoreactivity in the female eagle (24.8%) and falcon (21.7%) co-eluted with progesterone ([(14)C]). Most fecal androgen metabolite immunoreactivity in eagle (55.8%) and falcon (63.7%) males co-eluted with testosterone ([(14)C]). Exogenous adrenocorticotropin hormone induced increased fGCM excretion above pre-treatment in both species, but only significantly (P < 0.05) in the eagle. Both species showed increased fGCM after saline administration, suggesting the detection of 'handling stress.' Both species exhibited enterohepatic and renal recirculation of administered steroids as demonstrated by biphasic and triphasic excretion patterns. Thus, noninvasive fecal hormone monitoring is a valid and promising tool for assessing gonadal and adrenal status in rare and threatened birds-of-prey. PMID:17464481

  7. Switch in gap junction protein expression is associated with selective changes in junctional permeability during keratinocyte differentiation.

    PubMed Central

    Brissette, J L; Kumar, N M; Gilula, N B; Hall, J E; Dotto, G P

    1994-01-01

    Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of alpha 1 (connexin 43) and beta 2 (connexin 26) gap junction proteins is down-modulated, whereas that of beta 3 (connexin 31) and beta 4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucifer yellow is significantly reduced, whereas that of small metabolites, such as nucleotides and amino acids, proceeds unimpeded. Thus, a switch in gap junction protein expression in differentiating keratinocytes is accompanied by selective changes in junctional permeability that may play an important role in the coordinate control of the differentiation process. Images PMID:8022804

  8. In vivo beta-adrenergic induction of the unmasking of the uncoupling protein in rat brown fat.

    PubMed

    Goubern, M; Chapey, M F; Laury, M C; Portet, R

    1993-09-01

    1. In 28 degrees C adapted rats (WA) both cold stress and norepinephrine (NE) led to a 4-fold increase of uncoupling protein dependent proton conductance which was abolished by propranolol (PRO). 2. In 4-day warm re-exposed rats (after 10 days at 5 degrees C) (WR) the same uncoupling by cold stress was observed but the NE effect was lower. Uncoupling by cold stress was not abolished by PRO. 3. In WR rats, uncoupling was not due to the involvement of an alpha-adrenergic pathway. 4. Both beta-agonist isoproterenol and beta 3-agonists BRL 35135A and ICI D7114 led to high levels of unmasking. 5. Interscapular brown adipose tissue surgical denervation, which abolished cold stress unmasking both in WA and, WR rats, indicates a mediation by direct sympathetic innervation. 6. Depending on the thermal history of the rat, the possibility that unmasking by cold stress could be mediated by different types of beta-receptors is discussed. PMID:7903611

  9. Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP

    PubMed Central

    1991-01-01

    A new cellular model for the study of brown adipocyte development and differentiation in vitro is presented. Preadipocytes isolated from brown adipose tissue (BAT) of the djungarian dwarf hamster Phodopus sungorus are able to proliferate and differentiate in vitro into true brown adipocytes able to express the BAT marker protein the uncoupling protein (UCP). Whereas basal UCP expression is very low, its mRNA levels as well as the UCP detected by immunoblotting are highly increased by beta-adrenergic stimulation. The novel, atypical beta- adrenergic compound D7114 (ICI Pharmaceuticals, Macclesfield, Cheshire, England) was found to increase the number of adipocytes as well as UCP mRNA and UCP content of mitochondria, indicating the involvement of an atypical or beta 3 receptor. Insulin was found to play an important role in brown adipocyte differentiation and mitochondrial development, whereas T3 seemed to be implicated more directly in UCP expression. In a defined, serum-free medium a synergistic stimulatory action of insulin and T3 on UCP expression was found, which seems to involve a pathway different from that of beta-adrenergic UCP stimulation. PMID:1684582

  10. Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP.

    PubMed

    Klaus, S; Cassard-Doulcier, A M; Ricquier, D

    1991-12-01

    A new cellular model for the study of brown adipocyte development and differentiation in vitro is presented. Preadipocytes isolated from brown adipose tissue (BAT) of the djungarian dwarf hamster Phodopus sungorus are able to proliferate and differentiate in vitro into true brown adipocytes able to express the BAT marker protein the uncoupling protein (UCP). Whereas basal UCP expression is very low, its mRNA levels as well as the UCP detected by immunoblotting are highly increased by beta-adrenergic stimulation. The novel, atypical beta-adrenergic compound D7114 (ICI Pharmaceuticals, Macclesfield, Cheshire, England) was found to increase the number of adipocytes as well as UCP mRNA and UCP content of mitochondria, indicating the involvement of an atypical or beta 3 receptor. Insulin was found to play an important role in brown adipocyte differentiation and mitochondrial development, whereas T3 seemed to be implicated more directly in UCP expression. In a defined, serum-free medium a synergistic stimulatory action of insulin and T3 on UCP expression was found, which seems to involve a pathway different from that of beta-adrenergic UCP stimulation. PMID:1684582

  11. 3':5'-cyclic AMP and hormonal control of puparium formation in the fleshfly Sarcophaga bullata.

    PubMed

    Fraenkel, G; Blech