Orexin-1 receptors in the rostral ventromedial medulla are involved in the modulation of capsaicin evoked pulpal nociception and impairment of learning and memory.

AIM: To investigate the role of rostral ventromedial medulla orexin-1 receptors in the modulation of orofacial nociception as well as nociception-induced learning and memory impairment in adult male rats. METHODOLOGY: Pulpal nociception was induced by intradental application of capsaicin (100 µg) into the incisors of rats. Orexin-1 receptors agonist (orexin-A, 10, 25 and 50 pmol L-1  rat-1 ) and antagonist (SB-334867-A, 40 and 80 nmol L-1  rat-1 ) were microinjected into the rostral ventromedial medulla prior to capsaicin administration. Total time spent on nocifensive behaviour was recorded by direct visualization of freely moving rats whilst learning and memory were evaluated by the Morris water maze test. One-way analysis of variance and repeated-measures were used for the statistical analysis. RESULTS: Capsaicin-treated rats had a significant increase of nocifensive behaviours (P < 0.001), as well as learning and memory impairment (P < 0.001). However, intraventromedial medulla prior micro-injection of orexin-A (50 pmol L-1  rat-1 ) significantly reduced the nociceptive behaviour (P < 0.001). This effect was blocked by pre-treatment with SB334867-A (80 nmol L-1  rat-1 ). Orexin-A (50 pmol L-1  rat-1 ) also inhibited nociception-induced learning and memory deficits. Moreover, administration of SB-334867-A (80 nmol L-1  rat-1 ) plus orexin-A (50 pmol L-1  rat-1 ) had no effect on learning and memory deficits induced by capsaicin. CONCLUSIONS: The data suggest that rostral ventromedial medulla orexin-A receptors are involved in pulpal nociceptive modulation and improvement of learning and memory deficits induced by intradental application of capsaicin.

Role of haem oxygenase in the renoprotective effects of soluble epoxide hydrolase inhibition in diabetic spontaneously hypertensive rats.

We have shown previously that inhibition of sEH (soluble epoxide hydrolase) increased EETs (epoxyeicosatrienoic acids) levels and reduced renal injury in diabetic mice and these changes were associated with induction of HO (haem oxygenase)-1. The present study determines whether the inhibition of HO negates the renoprotective effect of sEH inhibition in diabetic SHR (spontaneously hypertensive rats). After 6 weeks of induction of diabetes with streptozotocin, SHR were divided into the following groups: untreated, treated with the sEH inhibitor t-AUCB {trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid}, treated with the HO inhibitor SnMP (stannous mesoporphyrin), and treated with both inhibitors for 4 more weeks; non-diabetic SHR served as a control group. Induction of diabetes significantly increased renal sEH expression and decreased the renal EETs/DHETEs (dihydroxyeicosatrienoic acid) ratio without affecting HO-1 activity or expression in SHR. Inhibition of sEH with t-AUCB increased the renal EETs/DHETEs ratio and HO-1 activity in diabetic SHR; however, it did not significantly alter systolic blood pressure. Treatment of diabetic SHR with t-AUCB significantly reduced the elevation in urinary albumin and nephrin excretion, whereas co-administration of the HO inhibitor SnMP with t-AUCB prevented these changes. Immunohistochemical analysis revealed elevations in renal fibrosis as indicated by increased renal TGF-ß (transforming growth factor ß) levels and fibronectin expression in diabetic SHR and these changes were reduced with sEH inhibition. Co-administration of SnMP with t-AUCB prevented its ability to reduce renal fibrosis in diabetic SHR. In addition, SnMP treatment also prevented t-AUCB-induced decreases in renal macrophage infiltration, IL-17 expression and MCP-1 levels in diabetic SHR. These findings suggest that HO-1 induction is involved in the protective effect of sEH inhibition against diabetic renal injury.

A Kinetic Process to Determine the Interaction Type Between Two Compounds, One of Which Is a Reaction Product, Using Alkaline Phosphatase Inhibition as a Case Study.

This study describes the development of a new methodology based on a new integrated equation which allows the determination of the kinetic parameters for two mutually non-exclusive inhibitors when one of which is produced during the time-course reaction. Alkaline phosphatase simultaneously inhibited by phosphate and urea is used to illustrate this methodology, including the evaluation of interaction effects between them. Data analyses were carried out using two integrated velocity equations: exclusive linear mixed inhibition (EMI) and non-exclusive linear mixed inhibition (NEMI). Kinetic parameters are estimated using non-linear regression and results show that (i) the interaction between enzyme and the inhibitors urea and phosphate exhibit a mutually non-exclusive behavior; (ii) more specifically, these inhibitors are non-exclusive only in free enzyme (E) species; (iii) the inhibitors also show an interaction with enzyme classified as facilitation; (iv) phosphate is a competitive inhibitor and urea a mixed inhibitor; (v) the inhibition constant for phosphate is much lower than that determined for urea. In addition, a functional Excel Spreadsheet which can be adapted to any kinetic study is also included as a supplement.

Astaxanthin alleviates renal damage of rats on high fructose diet through modulating NFκB/SIRT1 pathway and mitigating oxidative stress.

This study was conducted to determine the effect of astaxanthin (ASX) treatment on alleviation of renal damage in high fructose induced nephrotoxicity in rats. Treatments were arranged in a 2 × 2 factorial fashion: administrations of fructose (30%, via drinking water) and ASX (1 mg/kg/day, within 0.2 ml olive oil) for 8 weeks. Data were analyzed by two-way ANOVA. The ASX treatment decreased serum urea (p < .01) and blood urea-N concentrations (p < .02) at a lower extent in rats receiving fructose than those not receiving fructose. Moreover, the ASX treatment reversed the increases in malondialdehyde (MDA) (p < .0001) and nuclear factor kappa B (NF-κB) (p < .0003) levels and the decreases in superoxide dismutase (SOD) activity (p < .0001) and sirtuin-1 (SIRT1) level (p < .0004), in the kidney upon high fructose consumption. The data suggest that ASX supplementation alleviates renal damage induced by high fructose consumption through modulating NF-κB/SIRT1 pathway and mitigating oxidative stress.

Thymosin beta4 is cytoprotective in human gingival fibroblasts.

Thymosin beta4 (Tbeta(4)) is a naturally occurring, ubiquitous, non-toxic protein with documented wound-healing, anti-inflammatory, anti-apoptotic, and tissue-repair properties in skin, the ocular surface, and the heart. The ability of Tbeta(4) to demonstrate similar protective properties in cells of the oral cavity was analyzed using an in vitro model of cultured human gingival fibroblasts. Thymosin beta 4 significantly suppressed the secretion of interleukin-8 (IL-8) following stimulation with tumor necrosis factoralpha (TNF-alpha), suggesting that it may suppress the inflammatory response initiated by pro-inflammatory cytokines. By contrast, Tbeta(4) was not effective in protecting fibroblasts from challenge with lipopolysaccharide purified from Porphyromonas gingivalis or Escherichia coli. Thymosin beta 4 was able to protect gingival fibroblasts against the known cytotoxic effects of chlorhexidine digluconate, a mouthrinse containing chlorhexidine digluconate, and carbamide peroxide. Additionally, Tbeta(4) was able to protect gingival fibroblasts from the apoptosis that is induced by stimulation with TNF-alpha or by exposure to chlorhexidine. Because of its multifunctional roles in protecting cells against damage, Tbeta(4) may have significant potential for use as an oral heathcare aid with combined antimicrobial, anti-inflammatory, anti-apoptotic, and cytoprotective properties.

Basis for antagonism by sodium bentazon of tritosulfuron toxicity to white bean (Phaseolus vulgaris L.).

White bean (Phaseolus vulgaris L.) was used to study the antagonism caused by Na-bentazon on the phytotoxic action of the sulfonylurea (SU) herbicide tritosulfuron. After 168 h, uptake and translocation of [14C]tritosulfuron were reduced by 60 and 89%, respectively, when Na-bentazon was added to the mixture. Addition of (NH4)2SO4 or replacement of Na-bentazon with NH4-bentazon completely eliminated the negative effects on [14C]tritosulfuron uptake but not on its translocation. Scanning electron microscopy revealed that a mixture of Na-bentazon plus tritosulfuron plus DASH HC (0.156%) formed a rough layer of grain-like crystals on the leaf surface, whereas the addition of (NH4)2SO4 or replacement of Na-bentazon with NH4-bentazon resulted in amorphous deposits that may be more easily absorbed. The antagonism of tritosulfuron's phytotoxicity by Na-bentazon involves two separate processes, chemical (uptake effect) and biochemical (translocation effect).

Inhibitory effect of sodium ascorbate on ethylurea and sodium nitrite carcinogensis and negative findings in progeny after intestinal inoculation of precursors into pregnant hamsters.

To assess their carcinogenic effects, the ethylnitrosourea (ENU) precursors, ethylurea and sodium nitrite, [were administered to pregnant hamsters as a single intragastic] dose on day 15 of gestation, or introduced into the cecum on day 14. Since sodium ascorbate (NaASC) inhibits the biosynthesis of nitrosamides, identical doses of the precursors were given concomitantly with NaASC. Progeny of mothers treater intragastrically developed significant incidences of neurogenic tumors of the peripheral nervous system, with a predominance in females. The concurrent administration of NaASC with ENU precursors prevented carcinogenic effects in the progency, whereas the simultaneous inoculation of the precursors into the cecum produced no carcinogenic effects in the offspring.

Exploring the Counteracting Mechanism of Trehalose on Urea Conferred Protein Denaturation: A Molecular Dynamics Simulation Study.

To provide the underlying mechanism of the inhibiting effect of trehalose on the urea denatured protein, we perform classical molecular dynamics simulations of N-methylacetamide (NMA) in aqueous urea and/or trehalose solution. The site-site radial distribution functions and hydrogen bond properties indicate in binary urea solution the replacement of NMA-water hydrogen bonds by NMA-urea hydrogen bonds. On the other hand, in ternary urea and trehalose solution, trehalose does not replace the NMA-urea hydrogen bonds significantly; rather, it forms hydrogen bonds with the NMA molecule. The calculation of a preferential interaction parameter shows that, at the NMA surface, trehalose molecules are preferred and the preference for urea decreases slightly in ternary solution with respect to the binary solution. The exclusion of urea molecules in the ternary urea-NMA-trehalose system causes alleviation in van der Waals interaction energy between urea and NMA molecules. Our findings also reveal the following: (a) trehalose and urea induced second shell collapse of water structure, (b) a reduction in the mean trehalose cluster size in ternary solution, and (c) slowing down of translational motion of solution species in the presence of osmolytes. Implications of these results for the molecular explanations of the counteracting mechanism of trehalose on urea induced protein denaturation are discussed.

Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease.

The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to compute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(a)s of approximately 3 and approximately 6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.

Glycerophosphocholine and betaine counteract the effect of urea on pyruvate kinase.

Renal medullary cells contain large quantities of organic osmolytes when the levels of salt and urea in renal medullary interstitial fluid are high. Two of these osmolytes, betaine and glycerophosphocholine (GPC), are methylamines. Methylamines generally counteract the perturbing effects of urea on enzymes and other macromolecules. Betaine was previously shown to counteract the effect of urea on enzymes in vitro and to protect renal cells in tissue culture from harmful effects of high urea. Nevertheless, renal medullary cells in vivo and in tissue culture specifically accumulate GPC rather than betaine, in response to high urea. In the present studies we tested directly whether GPC counteracts the effect of urea on the Km of pyruvate kinase (PK) for ADP and compared the effectiveness in this regard of GPC to that of betaine. We find that urea increases the Km (as previously observed), that betaine and GPC decrease it, and that the increase caused by urea is counteracted by betaine or by GPC. The effects of GPC are slightly less than those of betaine. In addition, other renal medullary organic osmolytes (namely sorbitol, inositol and taurine) were already known to be compatible osmolytes whose accumulation protects renal medullary cells from hypertonicity because they have little effect on enzyme function. In agreement with this generalization we find that high sorbitol or inositol has little or no effect on PK activity, but surprisingly that taurine reduces Vmax and greatly elevates Km. In conclusion, the main finding is direct evidence that GPC is a counteracting osmolyte, which explains its accumulation in response to high urea. However, we do not find that GPC is a more effective counteracting osmolyte than betaine, which leaves unexplained the preference of renal cells for GPC over betaine for counteracting the perturbing effect of urea.

Use of competitive inhibition for driving sensitivity and dynamic range of urea ENFETs.

An urea biosensor based on urease-BSA (bovine serum albumin) membrane immobilised on the surface of an ion-sensitive field effect transistor (ISFET) has been studied in a mix buffer solution composed of potassium phosphate, Tris, citric acid and sodium tetraborate. In this mix buffer, the biosensor showed a dynamic larger than the one observed in a phosphate or Tris buffer. Investigation of the individual effect of each component of the buffer solution on the biosensor response has shown that tetraborate anion acts as a strong competitive inhibitor for the hydrolysis reaction of urea catalysed by urease. The biosensor response was investigated in a phosphate buffer with different concentrations of tetraborate anion. The results showed that the apparent constant of Michaelis-Menten, K(m(app)), increases from 4.3 to 79.3 mM, for experiments realised without and with 0.5 mM sodium tetraborate, respectively. The mean value, determined graphically, for the inhibition constant, K(i), was 29 microM. The graphical representation of biosensor calibration curves in semilogarithmic co-ordinates showed that the linear range of the biosensor can be extended up to three orders of magnitude, allowing an urea detection in a concentration range 0-100 mM.

Evaluation of the ability of d-penicillamine to protect rats against the neurotoxicity induced by zinc pyridinethione, acrylamide, 2,5-hexanedione and p-bromophenylacetylurea.

Dietary exposure of rats to three different concentrations of zinc pyridinethione (ZPT; 166, 332, 498 ppm) caused delayed onset failure in a treadmill test and, at the higher concentrations (332 and 498 ppm), death. Daily treatment with d-penicillamine (d-PEN) increased the latency period for treadmill failure and lethality. Comparable levels of toxicity were achieved only after d-PEN treated rats had consumed 2-3 times more ZPT than rats not treated with d-PEN. In contrast to ZPT, administration of d-PEN did not affect the onset of treadmill failure associated with acrylamide, p-bromophenylacetylurea or 2,5-hexanedione. Thus, d-PEN provided protection which was selective for ZPT.

N-acetyl-beta-D-glucosaminidase assay in urine: urea inhibition.

Urea inhibition of urinary N-acetyl-beta-D-glucosaminidase (NAG) was examined with human isoenzymes and total enzyme in urine. The fluorometric substrate 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide (MUNAG) and the colorimetric substrate m-cresolsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide (MCPNAG) were used to determine substrate kinetics and apparent urea inhibition kinetics. Apparent KmS of 0.47 (MUNAG) and 0.35 mmol/L (MCPNAG) for human NAG isoenzyme A and 0.46 (MUNAG) and 0.30 mmol/L (MCPNAG) for human NAG isoenzyme B were determined. Apparent Kis of approximately 100 mmol/L for urea inhibition of both isoenzymes in either substrate and apparent Kis of 79 (MUNAG) and 91 mmol/L (MCPNAG) for NAG in dilute urine samples were found. The potential for urea inhibition at physiological concentrations of urea and at normal assay dilutions therefore exists. Inhibition at these dilutions was minimal when substrate concentrations of 2 x Km or greater were used, but the substrate MUNAG at 1/2 x Km gave the highest sensitivity and lower blanks and allowed better quantitation of low activity samples. Within-run CVs of 3.2% with MUNAG and 10% with MCPNAG were found for low activity samples.

[The biological properties of production strain 205 of the tick-borne encephalitis virus. Its virulence, thermostability and resistance to the action of urea]. / Izuchenie biologicheskikh svoistv proizvodstvennogo shtamma 205 virusa kleshchevogo éntsefalita. Virulentnost', termostabil'nost' i ustoichivost' k deistviiu mocheviny.

The characterization of such properties of tick-borne encephalitis virus strain 205 and its clones as virulence, thermal stability and sensitivity to 2.5 M urea was obtained. The following combination of the genetic markers of strain 205 was established: mNic+, mNsc+, T50+, Ur. The clones possessed pronounced virulence, but showed different heat sensitivity (T50+, T50 +/-) and resistance to the action of urea (Ur and U +/-). At the early terms of observation, the clone isolated from a large viral plaque was more antigen-active.

Development of urease-chitosan membrane.

Urease was covalently immobilized on glutaraldehyde-pretreated chitosan membrane. The optimum immobilization conditions were determined with respect to glutaraldehyde pretreatment of membranes (concentration and pH of glutaraldehyde solution, time of membrane-glutaral-dehyde reaction) and to reaction of glutaraldehyde-pretreated membranes with urease (concentration and pH of urease solution). The obtained membrane has high enzymatic activity, and can be applied for enzymatic removal of urea e.g. in the treatment of chronic or acute uraemia.

Why is glycine not a part of the osmoticum in the urea-rich cells?

Kidney cells of animals including human and marine invertebrates contain high amount of the protein denaturant, urea. Methylamine osmolytes are generally believed to offset the harmful effects of urea on proteins in vitro and in vivo. In this study we have investigated the possibility of glycine to counteract the effects of urea on three proteins by measuring thermodynamic stability, ΔGD° and functional activity parameters (K(m) and k(cat)). We discovered that glycine does not counteract the effects of urea in terms of both protein stability and functional activity. We also observed that the glycine alone is compatible with enzymes function and increases protein stability in terms of T(m) (midpoint of thermal denaturation) to a great extent. Our study indicates that a most probable reason for the absence of a stabilizing osmolyte, glycine in the urea-rich cells is due to the fact that this osmolyte is non-protective to macromolecules against the hostile effects of urea, and hence is not chosen by evolutionary selection pressure.

Effect of transient receptor potential vanilloid 1 (TRPV1) receptor antagonist compounds SB705498, BCTC and AMG9810 in rat models of thermal hyperalgesia measured with an increasing-temperature water bath.

The transient receptor potential vanilloid 1 (TRPV1) receptor is activated by noxious heat, various endogenous mediators and exogenous irritants. The aim of the present study was to compare three TRPV1 receptor antagonists (SB705498, BCTC and AMG9810) in rat models of heat hyperalgesia. The behavioural noxious heat threshold, defined as the lowest temperature evoking nocifensive reaction, was measured with an increasing-temperature water bath. The effects of TRPV1 receptor antagonists were assessed in thermal hyperalgesia induced by the TRPV1 agonist resiniferatoxin (RTX), mild heat injury (51 degrees C, 20s) or plantar incision in rats. The control heat threshold was 43.2+/-0.4 degrees C. RTX induced an 8-10 degrees C decrease in heat threshold which was dose-dependently inhibited by oral pre-treatment with any of the TRPV1 receptor antagonists with a minimum effective dose of 1mg/kg. The mild heat injury-evoked 7-8 degrees C heat threshold drop was significantly reversed by all three antagonists injected i.p. as post-treatment. The minimum effective doses were as follows: SB705498 10, BCTC 3 and AMG9810 1mg/kg. Plantar incision-induced heat threshold drop (7-8 degrees C) was dose-dependently diminished by an oral post-treatment with any of the antagonists with minimum effective doses of 10, 3 and 3mg/kg, respectively. Assessment of RTX hyperalgesia by measurement of the paw withdrawal latency with a plantar test apparatus yielded 30 mg/kg minimum effective dose for each antagonist. In conclusion, measurement of the noxious heat threshold with the increasing-temperature water bath is suitable to sensitively detect the effects of TRPV1 receptor antagonists in thermal hyperalgesia models.