Effect of structure and size on the electrical properties of nanocrystalline WO3 films.

Nanocrystalline WO3 films were grown by reactive magnetron sputter-deposition by varying the substrate temperature in the range of 303(RT)-673 K. The structure and electrical transport properties of WO3 films were evaluated using X-ray diffraction and dc electrical conductivity measurements. The effect of ultramicrostructure and grain-size was significant on the electrical properties of WO3 films. DC conductivity variation of the WO3 films measured in the temperature range of 120-300 K reveals their semiconducting nature. The temperature dependent electrical conductivity curves exhibit two distinct regions indicative of two different types of electrical transport mechanisms. Analysis of the conductivity indicates that the small polaron and variable-range-hopping mechanisms are operative in 180-300 K and 120-180 K temperature regions, respectively. The density of localized states at the Fermi level, N(EF), has been calculated and it was found to be ∼1×10(19) eV(-1) cm(-3) for all the films.

L-type Ca2+ channel modulation by dihydropyridines potentiates kappa-opioid receptor agonist induced acute analgesia and inhibits development of tolerance in rats.

The effect of 1,4-dihydropyridine (DHP) calcium channel blockers (CCBs), nimodipine (NIM) and lercanidipine (LDP) on the analgesic response of selective kappa-opioid receptor agonists, U50,488H, PD117,302 and U69,593 was determined in male Sprague-Dawley rats using the tail-flick test. The effect of NIM on development of tolerance to U50,488H-induced analgesia and the status of brain DHP-sensitive Ca(2+) channel (L-type) binding sites in both U50,488H-naive and tolerant rats was determined using the highly selective DHP radioligand, [(3)H]PN200-110. Tolerance was induced by injecting U50,488H (25 mg/kg, i.p.) twice daily for 4 days. Intraperitoneal (i.p.) injection of kappa-opioid receptor agonists produced a dose-dependent acute analgesic response. NIM (1 mg/kg; i.p.) and LDP (0.3 mg/kg; i.p.) used in the study produced no tail-flick analgesia. Administration of NIM and LDP (15 min prior) significantly potentiated the analgesia produced by three kappa-opioid receptor agonists. Tolerance developed completely to the analgesic effect induced by U50,488H (25 mg/kg, i.p.) administered on the 5th day. NIM (1 mg/kg, i.p.) twice daily for 4 days not only completely inhibited the development of tolerance to analgesic response but also significantly potentiated it (supersensitivity). There was a significant up-regulation of DHP binding sites (B(max): +41%) in whole brain membranes of tolerant rats when compared to vehicle treated naive rats, implicating increased influx of Ca(2+) through L-type channels in kappa-opioid tolerance. U50,488H (25 mg/kg, i.p.) and NIM (1 mg/kg, i.p.) twice daily for 4 days also resulted in an equivalent up-regulation of DHP binding sites (+36%) as that of U50,488H alone. These results strongly suggest a functional role of L-type Ca(2+) channels in the regulation of pain sensitivity, mechanism of kappa-opioid analgesia and expression of tolerance.

Potentiation of kappa-opioid receptor agonist-induced analgesia and hypothermia by fluoxetine.

The effect of fluoxetine, a selective 5-HT reuptake inhibitor on the analgesic and hypothermic response of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methane sulphonate (U-50,488H) and (+/-)-trans-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzo[b] thiophene-4-acetamide (PD 117302), kappa-opioid receptor agonists, was determined in female Sprague-Dawley rats using the tail-flick method and telethermometer, respectively. Intraperitoneal injections of U-50,488H (U50) and PD 117302 (PD117) produced a dose-dependent analgesic and hypothermic response. Fluoxetine (10 mg/kg, i.p.) by itself did not produce an analgesic response. The analgesic response to U50 (10, 20, and 40 mg/kg, i.p.) and PD117 (7.5, 15, and 22.5 mg/kg, i.p.) was potentiated by fluoxetine injected intraperitoneally 60 min prior to the injection of kappa-opioid agonists. Similarly, the hypothermic response of U50 (20 and 40 mg/kg, i.p.) and PD117 (7.5, 15, and 22.5 mg/kg, i.p.) was potentiated by fluoxetine. The results indicate that selective kappa-opioid receptor agonists-induced analgesia and hypothermia is potentiated by fluoxetine, suggesting the role of extracellular 5-HT in the kappa-opioid receptor-mediated analgesia and hypothermia.

Transduction of folate receptor cDNA into cervical carcinoma cells using recombinant adeno-associated virions delays cell proliferation in vitro and in vivo.

Although folate receptors (FRs) mediate folate uptake into cells, the independent role of FRs in cell proliferation remains unclear. We tested the hypothesis that transduction of FR cDNA in sense or antisense orientation using recombinant adeno-associated virus modulated FR expression and altered proliferation of cervical carcinoma cells (which constitutively overexpress FR genes). We determined that the integration of recombinant adeno-associated virions was not site specific. When compared with untransduced cells, sense and antisense FR cDNA-transduced cells exhibited an increase and decrease in FR mRNA and FR expression on the cell surface, respectively. However, when compared with antisense FR cDNA-transduced and untransduced cells, sense FR cDNA-transduced cells exhibited statistically significant (a) increased in total FRs, (b) smaller colonies, (c) lowered cell proliferation in vitro, and (d) less tumor volume with dramatic prolongation of tumor doubling times (225.6 h vs. 96 h) after transplantation into nude mice. Finally, (f) using single cell-derived transduced clones, an inverse relationship between cell proliferation and FR expression was established (r = 0.90, P < 0.001). Thus, transduction of sense/antisense FR cDNA into cervical carcinoma cells modulated expression of FRs and had an impact on cell proliferation in vitro and in vivo.

Evidence that the hydrophobicity of isolated, in situ, and de novo-synthesized native human placental folate receptors is a function of glycosyl-phosphatidylinositol anchoring to membranes.

Although normal human chorionic villi-associated hydrophobic placental folate receptors (PFR) are converted to hydrophilic forms by an endogenous, EDTA-sensitive, Mg(2+)-dependent protease under serum-free conditions (Verma, R. S., and Antony, A. C. (1991) J. Biol. Chem. 266, 12522-12535), it is not known whether hydrophobic PFR are also susceptible to conversion by endogenous phospholipases. We isolated and characterized hydrophobic PFR, and tested the hypothesis that purified, in situ, and de novo-synthesized native PFR were covalently linked to glycosyl-phosphatidylinositol (GPI) anchors. 125I-hydrophobic PFR, but not 125I-hydrophilic PFR, (i) separated into the Triton X-114 micellar phase at 30 degrees C, (ii) efficiently incorporated into phosphatidylcholine-cholesterol liposomes, and (iii) were covalently labeled by the hydrophobic probe 3-(trifluoromethyl)-3-(meta[125I]iodophenyl)diazirine, [125I]TID. (iv) [125I]TID-labeled- and [phenyl-3H]Triton X-100-bound hydrophobic PFR, as well as native PFR in situ, were released as hydrophilic forms by recombinant (r) GPI-specific phospholipase(PL) C (GPI-PLC), and GPI-PLD (but not by PLC), in the absence and presence of a concentration of EDTA known to inhibit endogenous Mg(2+)-dependent protease. (v) Nitrous acid deamination of [125I]TID-labeled hydrophobic PFR as well as (r)GPI-PLC cleavage of [phenyl-3H]Triton-X-100- and [125I] TID-labeled hydrophobic PFR, released hydrophobic radiolabeled moieties which comigrated on thin layer chromatography distinct from free radiolabel. Finally, (vi) biosynthetic studies on chorionic villi cultured in vitro revealed incorporation of radiolabeled precursors into the GPI anchor of hydrophobic PFR. We conclude that native hydrophobic PFR are linked to GPI anchors and are therefore potential substrates for three distinct endogenous enzymes (GPI-PLC, GPI-PLD, and specific Mg(2+)-dependent metalloprotease) in maternal serum and placenta in vivo.

Stimulation of NADH oxidation by xanthine oxidase and polyvanadate in presence of some dehydrogenases and flavin compounds.

The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.

Decavanadate acts like an alpha-adrenergic agonist in redistributing protein kinase C activity.

Perfusion of rat livers with polyvanadate, but not metavanadate, was found to increase in plasma membrane and decrease in cytosol protein kinase C activity, similar to that obtained with phenylephrine, an alpha-adrenergic agonist. The effect was prevented by phenoxybenzamine, but not by propranolol implicating alpha-adrenergic receptor activation. Comparison of crystal structures of decavanadate and nonadrenaline revealed the occurrence of a structural feature of O-O-O(N) with distances of 5.5 A and 2.9 A.

Redistribution of subcellular calcium in rat liver on administration of vanadate.

Mitochondria isolated from the livers of rats administered with sodium meta-, ortho-, or polyvanadate, but not vanadyl sulphate, exhibited enhanced Ca2+ -stimulated respiration and uptake of calcium. These effects were shown also by mitochondria isolated from livers perfused with polyvanadate. The concentration of acid-soluble calcium decreased significantly in the mitochondrial fraction on vanadate treatment, while that in the cytosol showed a corresponding increase. Phenoxybenzamine, an antagonist to alpha-adrenergic receptors, effectively inhibited vanadate-induced Ca2+ mobilization, but surgical sympathectomy was without effect. This is the first demonstration of vanadate mimicking alpha-adrenergic agonists in vivo.

Increase in alpha-glycerophosphate dehydrogenase and other oxidoreductase activities of hepatic mitochondria on administration of vanadate to the rat.

Liver mitochondria isolated from vanadate-administered rats showed increased (20-25%) rates of oxidation of both NAD(+)-linked substrates and succinate. Respiratory control index and ADP/O were unaffected by the treatment. Dormant and uncoupler-stimulated ATPase activity also was not affected by vanadate administration. Membrane-bound, electron-transport-linked dehydrogenase activities (both NAD(+)- and succinate-dependent) increased by 15-20% on vanadate treatment. Mitochondrial alpha-glycerophosphate dehydrogenase activity increased by 50% on vanadate administration. The above effects of vanadate on oxidoreductase activities could be prevented by the prior administration of antagonists to alpha-adrenergic receptors. Substrate-dependent H2O2 generation by mitochondria also showed an increase on vanadate administration.

Vanadate-stimulated NADH oxidation requires polymeric vanadate, phosphate and superoxide.

NADH oxidation, catalyzed by the microsomal enzyme system is stimulated on addition of polymeric vanadate. Maximum stimulation by polymeric vanadate was obtained in the presence of phosphate buffer. The small stimulation obtained by metavanadate (500 microM) increased on acidification followed by neutralization, or on adding a trace amount of polymeric vanadate (1 microM).

Vanadate-stimulated NADH oxidation by xanthine oxidase: an intrinsic property.

Vanadate-dependent oxidation of NADH by xanthine oxidase does not require the presence of xanthine and therefore is not due to cooxidation. Addition of NADH or xanthine had no effect on the oxidation of the other substrate. Oxidation of NADH was high at acid pH and oxidation of xanthine was high at alkaline pH. The specific activity was relatively very high with NADH. Concentration-dependent oxidation of NADH Concentration-dependent oxidation of NADH was obtained in the presence of the polymeric form of vanadate, but not orthovanadate or metavanadate. Both NADH and NADPH were oxidized, as in the nonenzymatic system. Oxidation of NADH, but not xanthine, was inhibited by KCN, ascorbate, MnCl2, cytochrome c, mannitol, Tris, epinephrine, norepinephrine, and triiodothyronine. Oxidation of NADH was accompanied by uptake of oxygen and generation of H2O2 with a stoichiometry of 1:1:1 for NADH:O2:H2O2. A 240-nm-absorbing species was formed during the reaction which was different from H2O2 or superoxide. A mechanism of NADH oxidation is suggested wherein Vv and O2 receive one electron each successively from NADH followed by VIV giving the second electron to superoxide and reducing it to H2O2.