A prospective controlled clinical trial was undertaken to evaluate the effectiveness of stannous fluoride-releasing elastomeric modules (Fluor-I-Ties) and chain (Fluor-I-Chain) in the prevention of enamel decalcification during fixed appliance therapy. Forty-nine patients (782 teeth) were included in the experimental group, where the fluoride-releasing elastomerics were used. Forty-five patients (740 teeth) who received non fluoride-releasing elastomerics formed the control group. All patients had their elastomerics replaced at each visit. Enamel decalcification incidence and distribution were recorded using an index by direct clinical observation. In the control group enamel decalcification occurred in 73 per cent of patients and in 26 per cent of all teeth. In the experimental group the corresponding incidence was 63 and 16 per cent, respectively. The overall reduction in score per tooth produced by the fluoride-releasing elastomerics was 49 per cent, a highly significant difference (P < 0.001). A significant difference was seen in all but the occlusal enamel zones. The majority (over 50 per cent) of lesions occurred gingivally. The teeth most severely affected were the maxillary lateral incisors and mandibular second premolars. There was no difference in treatment duration between groups. (+info)
Probing essential water in yeast pyrophosphatase by directed mutagenesis and fluoride inhibition measurements.
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The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed. (+info)
ACAPs are arf6 GTPase-activating proteins that function in the cell periphery.
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The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs. In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by Arf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery. (+info)
Visualization of a functional Galpha q-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by receptors or AlF4-.
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To investigate how G protein alpha subunit localization is regulated under basal and activated conditions, we inserted green fluorescent protein (GFP) into an internal loop of Galpha(q). alpha(q)-GFP stimulates phospholipase C in response to activated receptors and inhibits betagamma-dependent activation of basal G protein-gated inwardly rectifying K(+) currents as effectively as alpha(q) does. Association of alpha(q)-GFP with the plasma membrane is reduced by mutational activation and eliminated by mutation of the alpha(q) palmitoylation sites, suggesting that alpha(q) must be in the inactive, palmitoylated state to be targeted to this location. We tested the effects of activation by receptors and by AlF(4)(-) on the localization of alpha(q)-GFP in cells expressing both alpha(q)-GFP and a protein kinase Cgamma-red fluorescent protein fusion that translocates to the plasma membrane in response to activation of G(q). In cells that clearly exhibit protein kinase Cgamma-red fluorescent protein translocation responses, relocalization of alpha(q)-GFP is not observed. Thus, under conditions associated with palmitate turnover and betagamma dissociation, alpha(q)-GFP remains associated with the plasma membrane. These results suggest that upon reaching the plasma membrane alpha(q) receives an anchoring signal in addition to palmitoylation and association with betagamma, or that during activation, one or both of these factors continues to retain alpha(q) in this location. (+info)
Phospholipase D in rat myometrium: occurrence of a membrane-bound ARF6 (ADP-ribosylation factor 6)-regulated activity controlled by betagamma subunits of heterotrimeric G-proteins.
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Both protein kinase C and protein tyrosine kinases have been shown to be involved in phospholipase D (PLD) activation in intact rat myometrium [Le Stunff, Dokhac and Harbon (2000) J. Pharmacol. Exp. Ther. 292, 629-637]. In this study we assessed the involvement of monomeric G-proteins in PLD activation in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membrane-bound, were found in myometrial preparations. One form was stimulated by oleate and insensitive to guanosine 5'-[gamma-thio] triphosphate (GTP[S]). The second required ammonium sulphate to be detected and was stimulated by GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodetected in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membrane fraction. A synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 [myrARF6((2-13))] totally abolished PLD activation in the presence of ammonium sulphate and GTP[S], whereas myrARF1((2-17)) and the inhibitory GDP/GTP-exchange factor, Rho GDI, did not. These data are consistent with a membrane-bound ARF6-regulated PLD activity. Finally, the stimulation of PLD by ARF6 was inhibited by AlF(-)(4) and this inhibition was counteracted by the fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1 (495-689) and by the QEHA peptide (from adenylate cyclase ACII), which act as G-protein betagamma-subunit scavengers. It is concluded that G-protein subunits betagamma are involved in a pathway modulating PLD activation by ARF6, illustrating cross-talk between heterotrimeric and monomeric G-proteins. (+info)
Analysis of myo-inositol hexakisphosphate hydrolysis by Bacillus phytase: indication of a novel reaction mechanism.
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Phytic acid (myo-inositol hexakisphosphate, InsP(6)) hydrolysis by Bacillus phytase (PhyC) was studied. The enzyme hydrolyses only three phosphates from phytic acid. Moreover, the enzyme seems to prefer the hydrolysis of every second phosphate over that of adjacent ones. Furthermore, it is very likely that the enzyme has two alternative pathways for the hydrolysis of phytic acid, resulting in two different myo-inositol trisphosphate end products: Ins(2,4,6)P(3) and Ins(1,3,5)P(3). These results, together with inhibition studies with fluoride, vanadate, substrate and a substrate analogue, indicate a reaction mechanism different from that of other phytases. By combining the data presented in this study with (1) structural information obtained from the crystal structure of Bacillus amyloliquefaciens phytase [Ha, Oh, Shin, Kim, Oh, Kim, Choi and Oh (2000) Nat. Struct. Biol. 7, 147-153], and (2) computer-modelling analyses of enzyme-substrate complexes, a novel mode of phytic acid hydrolysis is proposed. (+info)
Methoxyflurane anesthesia in pediatric patients: evaluation of anesthetic metabolism and renal function.
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Serum ionic fluoride concentrations during and following low-dose (6.0 mg/100 ml, 3 hours) methoxyflurane anesthesia and elective operation were measured in 13 pediatric patients (mean age 10.2 years; mean weight 34.5 kg). Peak measured serum ionic fluoride concentration was 21.6 plus or minus 3.3 mumol/1 24 hours after anesthesia. In a previously reported study of adult patients (47.5 years; 71.9 kg), the peak measured serum ionic fluoride concentration was 43.9 plus or minus 5.7 mumol/1 24 hours after low-does (6.8 mg/100 ml,3 hours) methoxyflurane anesthesia. Possible explanations for lower serum ionic fluoride concentrations in pediatric patients comared with adults include 1) slower metabolism of nethoxyflurand; 2) increased renal clearance of ionic fluoride from the blood; 3) greater sorage of ionic fluride in bone; 4) more rapid methoxyflurane elimiantion in the postoperative period. Serum uric acid increased (4.4 to 6.4 mg/100 ml, not significant) 24 hours after anesthesia and operation, while blood urea nitrogen and serum creatinine and osmolality were unchanged postoperatively. (+info)
Arabidopsis RopGAPs are a novel family of rho GTPase-activating proteins that require the Cdc42/Rac-interactive binding motif for rop-specific GTPase stimulation.
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The plant-specific Rop subfamily of Rho GTPases, most closely related to the mammalian Cdc42 and Rac GTPases, plays an important role in the regulation of calcium-dependent pollen tube growth, H(2)O(2)-mediated cell death, and many other processes in plants. In a search for Rop interactors using the two-hybrid method, we identified a family of Rho GTPase-activating proteins (GAP) from Arabidopsis, termed RopGAPs. In addition to a GAP catalytic domain, RopGAPs contain a Cdc42/Rac-interactive binding (CRIB) motif known to allow Cdc42/Rac effector proteins to bind activated Cdc42/Rac. This novel combination of a GAP domain with a CRIB motif is widespread in higher plants and is unique to the regulation of the Rop GTPase. A critical role for CRIB in the regulation of in vitro RopGAP activity was demonstrated using point and deletion mutations. Both types of mutants have drastically reduced capacities to stimulate the intrinsic Rop GTPase activity and to bind Rop. Furthermore, RopGAPs preferentially stimulate the GTPase activity of Rop, but not Cdc42 in a CRIB-dependent manner. In vitro binding assays show that the RopGAP CRIB domain interacts with GTP- and GDP-bound forms of Rop, as well as the transitional state of Rop mimicked by aluminum fluoride. The CRIB domain also promotes the association of the GAP domain with the GDP-bound Rop, as does aluminum fluoride. These results reveal a novel CRIB-dependent mechanism for the regulation of the plant-specific family of Rho GAPs. We propose that the CRIB domain facilitates the formation of or enhanced GAP-mediated stabilization of the transitional state of the Rop GTPase. (+info)