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PubMed1996

Mechanisms of stimulation of proximal tubule chloride transport by formate and oxalate.

We have previously demonstrated that formate and oxalate stimulate volume absorption in the rat proximal tubule, consistent with Cl-/formate and Cl-/oxalate exchange process across the apical membrane. To sustain Cl- absorption by these processes requires mechanisms for recycling formate and oxalate from lumen to cell. The aims of the present study were to characterize these mechanisms of formate and oxalate recycling. Proximal tubules and peritubular capillaries were simultaneously microperfused in the rat kidney in situ. Serum formate concentration was determined to be 56.5 +/- 7.7 microM. Addition of 5, 50, and 500 microM formate to both luminal and capillary perfusates significantly increased net Cl- absorption (Jcl) by 26, 26, and 46%, respectively. Jcl was stimulated 38% by 1 microM oxalate added to the perfusates. Removal of sulfate completely prevented the stimulation of Jcl by 1 microM oxalate but had no effect on the stimulation of Jcl by formate. Luminal addition of the Na+/H+ exchange inhibitor ethylisopropylamiloride completely blocked the stimulation of Jcl by 50 microM formate but had no effect on stimulation by oxalate. We conclude that physiological concentrations of formate and oxalate markedly stimulate Cl- and fluid absorption in the rat proximal convoluted tubule. Whereas formate recycling most likely involves Na+/H+ exchange in parallel with H(+)-coupled formate entry, oxalate recycling involves sodium-sulfate cotransport in parallel with sulfate/oxalate exchange.

Amiloride↗
PubMed2001

Essential role of NHE3 in facilitating formate-dependent NaCl absorption in the proximal tubule.

The absorption of NaCl in the proximal tubule is markedly stimulated by formate. This stimulation of NaCl transport is consistent with a cell model involving Cl(-)-formate exchange in parallel with pH-coupled formate recycling due to nonionic diffusion of formic acid or H(+)-formate cotransport. The formate recycling process requires H(+) secretion. Although Na(+)-H(+) exchanger isoform NHE3 accounts for the largest component of H(+) secretion in the proximal tubule, 40-50% of the rates of HCO absorption or cellular H(+) extrusion persist in NHE3 null mice. The purpose of the present investigation is to use NHE3 null mice to directly test the role of apical membrane NHE3 in mediating NaCl absorption stimulated by formate. We demonstrate that formate stimulates NaCl absorption in the mouse proximal tubule microperfused in vivo, but the component of NaCl absorption stimulated by formate is absent in NHE3 null mice. In contrast, stimulation of NaCl absorption by oxalate is preserved in NHE3 null mice, indicating that oxalate-stimulated NaCl absorption is independent of Na(+)-H(+) exchange. The virtually complete dependence of formate-induced NaCl absorption on NHE3 activity raises the possibility that NHE3 and the formate transporters are functionally coupled in the brush border membrane.

Absorption↗
PubMed1989

Oxalate:formate exchange. The basis for energy coupling in Oxalobacter.

In the Gram-negative anaerobe, Oxalobacter formigenes, the generation of metabolic energy depends on the transport and decarboxylation of oxalate. We have now used assays of reconstitution to study the movements of oxalate and to characterize the exchange of oxalate with formate, its immediate metabolic derivative. Membranes of O. formigenes were solubilized with octyl-beta-D-glucopyranoside in the presence of 20% glycerol and Escherichia coli phospholipid, and detergent extracts were reconstituted by detergent dilution. [14C]Oxalate was taken up by proteoliposomes loaded with unlabeled oxalate, but not by similarly loaded liposomes or by proteoliposomes containing sulfate in place of oxalate. Oxalate transport did not depend on the presence of sodium or potassium, nor was it affected by valinomycin (1 microM), nigericin (1 microM), or a proton conductor, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (5 microM) when potassium was at equal concentration on either side of the membrane. Such data suggest the presence of an overall neutral oxalate self-exchange, independent of common cations or anions. Kinetic analysis of the reaction in proteoliposomes gave a Michaelis constant (Kt) for oxalate transport of 0.24 mM and a maximal velocity (Vmax) of 99 mumol/min/mg of protein. A direct exchange of oxalate and formate was indicated by the observations that formate inhibited oxalate transport and that delayed addition of formate released [14C]oxalate accumulated during oxalate exchange. Moreover, [14C]formate was taken up by oxalate-loaded proteoliposomes (but not liposomes), and this heterologous reaction could be blocked by external oxalate. Further studies, using formate-loaded proteoliposomes, suggested that the heterologous exchange was electrogenic. Thus, for assays in which N-methylglucamine served as both internal and external cation, formate-loaded particles took up oxalate at a rate of 2.4 mumol/min/mg of protein. When external or internal N-methylglucamine was replaced by potassium in the presence of valinomycin, there was, respectively, a 7-fold stimulation or an 8-fold inhibition of oxalate accumulation, demonstrating that net negative charge moved in parallel with oxalate during the heterologous exchange. The work summarized here suggests the presence of an unusually rapid and electrogenic oxalate2-:formate1- antiport in membranes of O. formigenes. Since a proton is consumed during the intracellular decarboxylation that converts oxalate into formate plus CO2, antiport of oxalate and formate would play a central role in a biochemical cycle consisting of (a) oxalate influx, (b) oxalate decarboxylation, and (c) formate efflux.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Transport, Active↗
PubMed2005

Human trabecular bone-derived osteoblasts support human osteoclast formation in vitro in a defined, serum-free medium.

While it has been assumed that osteoblasts in the human support osteoclast formation, in vitro evidence of this is currently lacking. We tested the ability of normal human trabecular bone-derived osteoblasts (NHBCs) to support osteoclast formation from human peripheral blood mononuclear cells (PBMC) in response to treatment with either 1alpha,25-dihydroxyvitamin D3 (1,25D) or parathyroid hormone (PTH), using a serum-replete medium previously used to support human osteoclast formation on a stroma of murine ST-2 cells. Under these conditions, NHBC did not support osteoclast formation, as assessed by morphological, histochemical, and functional criteria, despite our previous results demonstrating a link between induction of RANKL mRNA expression and NHBC phenotype in these media. We next tested a defined, serum-free medium (SDM) on NHBC phenotype, their expression of RANKL and OPG, and their ability to support osteoclast formation. SDM, containing dexamethasone (DEX) and 1,25D, induced phenotypic maturation of NHBC, based on the expression of STRO-1 and the bone/liver/kidney isoform of alkaline phosphatase (AP). PTH as a single factor did not induce phenotypic change. 1,25D and DEX induced the greatest ratio of RANKL:OPG mRNA, predictive of supporting osteoclast formation. Consistent with this, co-culture of NHBC with CD14+ PBMC, or bone marrow mononuclear cell (BMMC), or CD34+ BMMC precursors in SDM + 1,25D + DEX, resulted in functional osteoclast formation. Osteoclast formation also occurred in PTH + DEX stimulated co-cultures. Interestingly, SDM supplemented with recombinant RANKL (25-100 ng/ml) and M-CSF (25 ng/ml), did not induce osteoclast formation from any of the osteoclast precursor populations in stromal-free cultures, unlike serum-replete medium. This study demonstrates that under the appropriate conditions, adult human primary osteoblasts can support de novo osteoclast formation, and this model will enable the detailed study of the role of both cell types in this process.

ADP-ribosyl Cyclase↗
PubMed1986

Water soluble complexes of the antiviral drugs, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine and acyclovir: the role of hydrophobicity in complex formation.

We investigated water-soluble complexes of various ligands with the antiviral drugs, 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir) and 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG). For comparison, we also examined the "parent" compounds, guanine and guanosine, as substrates for complex formation. Using the phase-solubility technique, we measured formation constant (K1) values at 23 degrees C in pH 7 buffer. For a single substrate, formation constants with different ligands varied in the order: caffeine greater than pyridoxine approximately cytidine greater than nicotinamide greater than sucrose. With caffeine as the ligand, formation constants with different substrates varied in the order: guanine greater than guanosine approximately acyclovir greater than DHPG. The largest formation constant observed was 58 M-1 (for guanine-caffeine), and the smallest formation constant was 0.29 M-1 (for DHPG-sucrose). Examining the literature for formation constant data on compounds related to DHPG, and comparing literature data with our own, reveals a significant correlation between formation constants and ligand hydrophobicity. For 41 substrate-ligand pairs, least squares linear regression analysis of log K1 values versus various parameters reflecting donor-acceptor abilities (e.g., substrate and ligand HOMO and LUMO values, or substrate oxidation potentials) failed to significantly correlate. We conclude that ligand hydrophobicity is a general determinant of water soluble complex formation, but not necessarily the exclusive or dominant controlling factor for all complexes. Charge-transfer interactions are not important determinants of complex formation for the substrate-ligand combinations that we have considered.

Acyclovir↗
PubMed1994

Experiments on the initiation of calcium fluoride formation with reference to the solubility of dental enamel and brushite.

As calcium fluoride formation following topical application of fluoride may be responsible for at least some of the caries-reducing effect of fluoride, the concentration of fluoride necessary to induce its formation were examined. The aim was to determine the degree of supersaturation with respect to calcium fluoride necessary for inducing its spontaneous precipitation, with close reference to its possible formation and retention on dental hard tissue when topical fluoride solutions are used clinically. Powdered enamel or brushite were suspended for 4 h in aqueous solutions buffered at pH 7.2 and 5.0. After the equilibration, ion concentrations were determined and degrees of saturation with respect to apatite and brushite were calculated. In aqueous solutions at pH 7.2 and 5.0 with similar concentrations as those found in the equilibrated suspensions the fluoride concentration was adjusted to from 5 to 500 parts/10(6). After 2 h of gentle agitation the supernatant was analysed, the precipitate (if any) isolated and examined by X-ray diffraction. Initiation of spontaneous calcium fluoride formation required a calcium fluoride ion-activity product of 10(-7.6) or more, 300 parts/10(6) fluoride were necessary to initiate calcium fluoride formation in neutral solution saturated with enamel, and the increased solubility of enamel apatite at low pH allowed calcium fluoride formation from solutions with as low as 100 parts/10(6) fluoride. When phosphate was present in the solution a competing apatite formation could mask the calcium fluoride formation. In neutral solutions saturated with respect to brushite, spontaneous fluorapatite formation was initiated by 100 pats/10(6) fluoride. With 200 parts/10(6) fluoride a further competing formation of calcium fluoride occurred.

Calcium Fluoride↗
PubMed1996

Metabolite complex formation of orphenadrine with cytochrome P450. Involvement of CYP2C11 and CYP3A isozymes.

Expression and inhibition of cytochrome P450 (CYP) isozymes capable of forming an orphenadrine metabolite complex were studied in microsomes of untreated and inducer-treated male and female rats. High levels of complex-forming isozymes were found in microsomes of untreated male as compared to female rats. Treatment of male rats with several P450 inducers did not considerably increase the extent of in vitro complex formation. In female rats, however, phenobarbital or dexamethasone treatments led to pronounced induction. The isozyme specificity of complex formation was investigated by several approaches including: 1. inhibition by orphenadrine of isozyme-specific P450 activities, such as hydroxylation of testosterone, O-dealkylation of pentoxy-and ethoxyresorufin and complex formation with triacetyloleandomycin (TAO), 2. inhibition of orphenadrine complex formation by metyrapone, TAO, and cimetidine, and 3. correlation of complex levels with immunochemically, enzymatically, or spectroscopically determined amounts of P450 isozymes. Our data suggest that CYP2C11, a CYP3A isozyme and an unidentified P450 species are involved in complex formation with orphenadrine, but exclude the involvement of CYP1A1/2 and CYP2B1/2. The capability of CYP2C11 to form a metabolite complex with orphenadrine is strongly suggested for the following reasons: 1. Efficient inhibition of testosterone 2 alpha- and 16 alpha-hydroxylation by complex formation with orphenadrine in microsomes of untreated male rats, 2. high expression of orphenadrine-complexing isozymes in untreated male compared to female rats, 3. specific inhibition of in vitro complex formation by cimetidine, 4. suppression of complex-forming isozymes by 3-methylcholanthrene and beta-naphthoflavone, and 5. concomitant induction of complex-forming isozymes, immunodetectable CYP2C11, and testosterone 2 alpha-hydroxylase by stanozolol. That at least one, but not all, CYP3A isozymes is involved in complex formation is concluded from inhibition experiments with TAO that show that orphenadrine complexation can be significantly inhibited in microsomes of dexamethasone-treated, but not in microsomes of untreated rats. Furthermore, complex formation with TAO is not inhibited by orphenadrine in microsomes of phenobarbital (PB)-treated rats. In PB-treated female rats, a further unidentified complex-forming isozyme can be detected that is not inhibited by complex formation with TAO.

Animals↗
PubMed1989

Synacrosomal formation after cell fusion of round spermatids of Xenopus laevis.

Cell fusion was induced by hypotonic medium in pairs of spermatids which were derived from single secondary spermatocytes. In a pair of fused spermatids, a single acrosome (synacrosome) eventually formed whenever the cell fusion was induced during the course of acrosomal formation. Direct observation of the process of synacrosomal formation was made on pairs of fused spermatids which had completed acrosomal formation. Two patterns occurred, namely, fusion of two acrosomes or enlargement of one with diminution of the other. The total volume of the two acrosomes before synacrosomal formation almost equaled the volume of the coalesced synacrosomes in fused spermatids. Neither colchicine nor cytochalasin B prevented synacrosomal formation in spermatids which were fused after each had completed acrosomal formation. These results indicate that neither microtubules nor microfilaments seem to play a role in the formation of a synacrosome in pairs of fused spermatids. However, cycloheximide did inhibit acrosomal formation when present during the early stage of acrosome differentiation in pairs of spermatids which had been fused just after second meiotic division. This fact indicates that acrosomal formation is mediated by some protein(s) which are synthesized during the initial period of acrosomal formation.

Acrosome↗
PubMed2007

Relevance of PCDD/PCDF formation for the evaluation of POPs destruction technologies--review on current status and assessment gaps.

One important criterion for assessment of a POPs destruction technology is the potential formation of new POPs and other toxic by-products, in particular whether the highly toxic PCDDs/PCDFs are formed and under which operation conditions their formation is relevant. For incineration processes the formation mechanisms of PCDDs/PCDFs have been investigated thoroughly and strategies and technologies were developed to minimize their formation and emission. A detailed assessment of non-combustion technologies with respect to PCDD/PCDF formation is, however, lacking to date. A comparison of reaction conditions for PCDD/PCDF formation from precursor formation studies and actual applied conditions of a broad range of POPs destruction technologies in the present paper indicates that the operation conditions for a number of destruction technologies have the potential to generate high concentrations of PCDDs/PCDFs if dioxin precursors are present and that also PCDD/PCDF de novo formation can take place. Therefore a strategy and regulations for a more profound assessment and monitoring of the fate of PCDD/PCDF formation and emission is essential for the evaluation of POP destruction technologies and for a sound risk management of POPs. The present paper aims to provide a critical impulse in this respect, discusses the relevant formation pathways with respect to POPs destruction technologies and proposes a basic framework on how evaluations may be performed.

Benzofurans↗
PubMed2005

Microbubbles during radiofrequency catheter ablation: composition and formation.

OBJECTIVES: The purpose of this study was to measure tissue temperatures associated with microbubble formation during radiofrequency (RF) ablation. BACKGROUND: Microbubble formation visualized by echocardiography has been used to indicate excessive tissue heating during RF pulmonary vein isolation. However, little is known about the tissue temperatures associated with microbubble formation. METHODS: Optical fluorometric thermometry probes were used to record tissue temperatures in isolated porcine atrium overlying either lung or esophageal tissue in a saline bath. RF energy was delivered through an irrigated ablation electrode during echocardiographic monitoring for microbubble formation. RESULTS: The maximal recorded tissue temperatures were 81.0 +/- 5.0 degrees C and 88.3 +/- 8.1 degrees C at the time of intermittent (type 1) microbubble formation for lung and esophageal preparations, respectively. During continuous (type 2) microbubble formation, the temperatures were 91.4 +/- 8.2 degrees C and 99.2 +/- 7.8 degrees C, respectively (both P < .001 vs type 1). Tissue temperatures averaged >100 degrees C at the time of "pops." The maximal recorded temperature occurred up to 4 mm deep in the tissues and frequently occurred external to the atrial tissue. The total RF lesion volumes for lung and esophageal preparations were related to the pattern of microbubble formation but not to total power delivered. After generation of type 1 bubbles, up to 60% reductions in RF energy were needed to restore target tissue temperatures of 65 degrees C. Gas chromatographic analysis of the microbubbles was consistent with steam formation. CONCLUSIONS: Microbubble formation during RF ablation represents excessive tissue heating to the point of steam formation. Maximal tissue heating may occur in the adjacent lung and esophagus during cooled ablation.

Animals↗
PubMed2006

Modeling dichloroacetic acid formation from the reaction of monochloramine with natural organic matter.

A kinetic model was developed to predict dichloroacetic acid (DCAA) formation in chloraminated systems. Equations describing DCAA formation were incorporated into an established comprehensive monochloramine-natural organic matter (NOM) reaction model. DCAA formation was theorized to be proportional to the amount of NOM oxidized by monochloramine and described by a single dimensionless DCAA formation coefficient, theta(DCAA) (M(DCAA)/M(DOC(ox)). The applicability of the model to describe DCAA formation in the presence of six different NOM sources was evaluated. DCAA formation could be described by considering a single NOM source-specific value for theta(DCAA) over a wide range of experimental conditions (i.e., pH, NOM, free ammonia, and monochloramine concentrations). DCAA formation appears to be directly proportional to the amount of active chlorine (monochloramine and free chlorine) that reacted with the NOM under these experimental conditions. Values of theta(DCAA) for all six NOM sources, determined by nonlinear regression analysis, varied from 6.51 x 10(-3) to 1.15 x 10(-2) and were linearly correlated with specific ultraviolet absorbance at 280 nm (SUVA(280)). The ability to model monochloramine loss and DCAA formation in the presence of NOM provides insight into disinfection by-product (DBP) formation pathways under chloramination conditions. The subsequent model and correlations to SUVA has the potential to aid the water treatment industry as a tool in developing strategies that minimize DBP formation while maintaining the microbial integrity of the water distribution system.

Chloramines↗
PubMed1999

Oxidative DNA damage mediated by copper(II), iron(II) and nickel(II) fenton reactions: evidence for site-specific mechanisms in the formation of double-strand breaks, 8-hydroxydeoxyguanosine and putative intrastrand cross-links.

The role of metal ion-DNA interactions in the Fenton reaction-mediated formation of putative intrastrand cross-links, 8-hydroxydeoxyguanosine (8-OHdG) and single- and double-strand breaks was investigated. Salmon sperm DNA and pBluescript K+ plasmid were incubated with hydrogen peroxide and either copper(II), iron(II), or nickel(II), which differ in both their affinity for DNA and in the spectrum of oxidative DNA damage they induce in Fenton reactions. EDTA was included in these incubations according to two different strategies; the first (strategy 1) in which DNA and metal ions were mixed prior to the addition of EDTA, the second (strategy 2) in which EDTA and metal ions were mixed prior to the addition of DNA. The formation of the putative intrastrand cross-links, monitored by 32P-postlabelling, was not affected by the addition of between 10 microM and 5 mM EDTA to the copper(II) Fenton reaction according to strategy 1. In contrast, the level of cross-links declined significantly upon inclusion of 20 microM EDTA and above when added according to strategy 2. Similarly, formation of these lesions declined in the iron(II) Fenton reaction more dramatically upon addition of 5 mM EDTA when added according to strategy 2 compared to strategy 1, while the yield of cross-links formed in the nickel(II) Fenton reaction declined equally with both strategies with up to 25 mM EDTA. The formation of single- and double-strand breaks was investigated in plasmid DNA by agarose gel electrophoresis and subsequent densitometry. The formation of linear DNA in the iron(II) Fenton reaction decreased dramatically upon inclusion of EDTA according to strategy 2, while no such decline was observed using strategy 1. In contrast, the formation of linear DNA in the copper(II) Fenton reaction decreased upon inclusion of EDTA according to both strategies. A decrease in the formation of open-circular DNA was also observed upon inclusion of EDTA according to both strategies; however this decrease occurred at a lower EDTA concentration in strategy 2 (100 microM) compared to strategy 1 (200 microM), and the level of open-circular DNA reached a lower level (8. 5% compared to 24.2%). The nickel(II) Fenton reaction generated only open-circular DNA, and this was completely inhibited upon addition of 25 microM EDTA according to both strategies. There was less formation of 8-OHdG in the copper(II) and iron(II) Fenton reactions when EDTA was added according to strategy 2 than according to strategy 1. These results suggest that a site-specific mechanism is involved in the formation of double-strand breaks and, to a lesser extent, 8-OHdG and the putative intrastrand cross-links, while the formation of single-strand breaks is more likely to involve generation of hydroxyl radicals in solution.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

The effect of macrophage-colony stimulating factor and other humoral factors (interleukin-1, -3, -6, and -11, tumor necrosis factor-alpha, and granulocyte macrophage-colony stimulating factor) on human osteoclast formation from circulating cells.

Macrophage-colony stimulating factor (M-CSF) is an essential requirement for human osteoclast formation, but its effect on the proliferation and differentiation of circulating osteoclast precursor cells is unknown. Other growth factors and cytokines are also known to support/stimulate osteoclast formation from mouse marrow precursors, but it is not certain whether these factors similarly influence human osteoclast formation. In this study, human monocytes were cocultured with osteoblast-like UMR-106 cells on coverslips and dentine slices for up to 21 days in the presence of 1,25 dihydroxyvitamin D(3) (10(-7) mol/L), dexamethasone (10(-8) mol/L), and various concentrations of either M-CSF or other humoral factors (interleukin [IL]-1beta, IL-3, IL-6, and IL-11; tumor necrosis factor-alpha [TNF-alpha]; and granulocyte macrophage [GM]-CSF). The effect on osteoclast formation was assessed by tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor staining and lacunar bone resorption. The results of time-course and proliferation studies showed that M-CSF stimulated both the proliferative and differentiation stages of human osteoclast formation from circulating osteoclast precursors in a dose-dependent manner. A high concentration of M-CSF (100 ng/mL) did not inhibit osteoclast formation. IL-3 and GM-CSF were also capable of stimulating human osteoclast formation, although these growth factors were much less potent than M-CSF. IL-3- and GM-CSF-stimulated osteoclast formation was inhibited by an antibody specific for human M-CSF. Osteoclast formation and lacunar resorption was not seen when either TNF-alpha, IL-1beta, IL-6 (+ soluble IL-6 receptor), or IL-11 was substituted for M-CSF during coculture. These results confirm that M-CSF is essential for human osteoclast formation from circulating mononuclear precursors, and also shows that IL-3 and GM-CSF may support osteoclast differentiation via the stimulation of M-CSF production by human monocytes.

Adult↗
PubMed1994

Formation of pseudouridine in U5 small nuclear RNA.

The formation of pseudouridine (psi) on U5 small nuclear RNA (U5 snRNA) was studied using an in vitro modification system. Labeled U5 RNA, synthesized in vitro and therefore unmodified, was incubated in reactions containing S100 and/or nuclear extracts (NE) from HeLa cells, and the levels of psi were determined. There are three psi residues found in human U5 RNA, at positions 43, 46, and 53. Incubation of unmodified U5 RNA in reactions containing either S100 or NE supports psi formation at positions 43 and 46, which are found in a loop in the predicted secondary structure of U5 RNA. However, psi formation at position 53, which is found in a stem, is dependent on the presence of NE during the incubation. The order of extract addition does not have a significant effect on the formation of psi at position 53 as long as NE is present. The most efficient psi formation was observed with a combination of S100 and NE which allowed for efficient small nuclear ribonucleoprotein particle (snRNP) assembly and psi formation. When 9S and 20S U5 snRNPs were isolated by velocity sedimentation gradient centrifugation after incubation in the combined extracts, there was little difference in the psi levels at any of the positions for the two distinct particles. Mutations in the U5 RNA sequence do affect psi formation. U5 RNAs that have mutated Sm binding sites or are truncated prior to the Sm binding site have very low levels of psi formation at positions 43 and 46 and no detectable psi formation at position 53. A deletion of five nucleotides from 39 to 43 abolishes psi formation at positions 43 and 46, but the modification of position 53 is unaffected.

Base Sequence↗
PubMed2003

The strength of dehalogenase-substrate hydrogen bonding correlates with the rate of Meisenheimer intermediate formation.

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA by using an active site aspartate as the nucleophile. Formation of the corresponding Meisenheimer complex (EMc) is followed by chloride ion expulsion which forms the arylated intermediate (EAr). This is then hydrolyzed to the product. In this paper, we explore the relationship between active site polarizing forces acting on the benzoyl carbonyl and the rate of formation of the Meisenheimer complex. The polarizing forces at the C[double bond]O group were modulated by introducing site-selected mutations (A112V, Y65D, G113A, G113S, G113N, and F64P), near the C[double bond]O binding site. Using either the substrate, 4-CBA-CoA, or the substrate analogue, 4-methylbenzoyl-CoA (4-MBA-CoA), Raman difference spectroscopy provided the position of the C[double bond]O stretching frequency (nu(C)[double bond](O)) for a total of 10 enzyme-ligand complexes. In turn, the values of the C[double bond]O frequencies could be converted to differences in effective hydrogen bonding strengths between members of the series, based on earlier model studies [Clarkson, J., Tonge, P. J., Taylor, K. L., Dunaway-Mariano, D., and Carey, P. (1997) Biochemistry 36, 10192-10199]. Catalysis in the F64P, G113A, G113S, and G113N dehalogenase mutants was very slow with k(cat) values ranging from 8 x 10(-3) to 7.6 x 10(-6) s(-1). The EAr intermediate did not accumulate to a detectable level on these enzymes during a single turnover. Catalysis in the Y65D and A112V dehalogenase mutants were almost as efficient as catalysis in wild-type dehalogenase with k(cat) values of 0.1-0.6 s(-1). In wild-type dehalogenase, 22% of the bound substrate accumulated as the EAr intermediate during a single turnover (k(obs) for EAr formation = 24 s(-(1)); in the Y65D mutant, the level of accumulation is 17% (k(obs) for EAr formation = 3 s(-1)), and in the A112V mutant, the level is 23% (k(obs) for EAr formation = 17 s(-1)). The k(obs) for EAr formation in wild-type dehalogenase and the more active dehalogenase mutants (Y65D and A112V) was taken to be an estimate of the k for EMc formation, and the k(obs) for EP formation in a single turnover was taken to be an estimate of the k for EMc formation in the severely impaired mutants (F64P, G113A, G113S, and G113N). A plot of the log k(obs) for EMc formation versus the C[double bond]O stretching frequency of bound 4-CBA-CoA (or 4-MBA-CoA) is a straight line (R(2) = 0.9584). Throughout the series, nu(C)[double bond](O) varied by 61 cm(-1), corresponding to the change in hydrogen bonding enthalpy of 67 kJ/mol. The results show that changes in polarizing forces at the benzoyl carbonyl are transmitted to the benzoyl (4) position and correlate with the rate of aromatic nucleophilic addition five chemical bonds away. Interestingly, the relationship between effective polarizing forces and reactivity seen here for dehalogenase is similar to that reported for the addition-elimination reaction involving the hydrolysis of a series of acyl serine proteases.

Binding Sites↗
PubMed2003

Effects of ginsenosides on carbachol-stimulated formation of inositol phosphates in rat cortical cell cultures.

We examined the effect of ginseng total saponins (GTS) on phosphoinositide metabolism stimulated by activation of muscarinic receptor using rat cortical cultures. Carbachol stimulated formation of [3H]inositol phosphates ([3H]InsPs) by 3.3-fold over basal level in [3H]inositol-prelabeled cells. Pretreatment of GTS inhibited formation of [3H]InsPs evoked by carbachol by 70%-90%. Addition of GTS alone had no effect on the basal formation of [3H]InsPs. The inhibitory effect of the GTS on carbachol-stimulated formation of [3H]InsPs was dose- and time-dependent. IC50 was 6.0 +/- 2.8 microg/ml. We also examined the effect of GTS on [3H]InsP1, [3H]InsP2, or [3H]InsP3 formation evoked by carbachol. Although GTS had no effect on the basal [3H]InsP1, [3H]InsP2, or [3H]InsP3 formation, pretreatment of GTS inhibited [3H]InsP1, [3H]InsP2, or [3H]InsP3 formation evoked by carbachol, respectively. Addition of individual ginsenosides such as ginsenoside Rb1, Rc, Rd, Re, or Rg2 had no effect on the basal formation of [3H]InsPs, whereas pretreatment of ginsenoside Rb2, Rc, Rd, Re, Rf, Rg1 or Rg2 inhibited formation of [3H]InsPs evoked by carbachol by 79%-89%. The results suggest that the inhibitory effect of GTS and its individual ginsenosides on carbachol-stimulated formation of [3H]InsPs in cortical neurons could be one pharmacological action of Panax ginseng.

Animals↗
PubMed1989

Plasma insulin-like growth factors and bone formation in uremic hyperparathyroidism.

Bone formation in uremia is considered to be regulated in part by parathyroid hormone (PTH). However, while low levels of immunoreactive PTH are usually associated with low rates of bone formation in uremia, elevated PTH levels do not always correlate with increased bone formation. In an attempt to identify other factors that may regulate bone formation in uremic patients, we measured plasma immunoreactive insulin-like growth factors (IGF-I and IGF-II) in 15 patients who did not have aluminum-associated reductions in bone formation. Plasma levels of IGF-I but not PTH, were significantly higher in patients with high rates of bone formation when compared to patients with low or normal bone formation (P less than 0.02). While the bone formation rate at the tissue level correlated significantly with plasma PTH (r = 0.53, P less than 0.05) and IGF-I (r = 0.67, P less than 0.01), only for plasma IGF-I were there significant correlations with bone apposition (r = 0.57, P less than 0.05) and bone formation rate at the BMU level (r = 0.62, P less than 0.02), parameters which reflect mineralization activity at the cellular level. Among the static histologic parameters, osteoblastic osteoid correlated only with plasma PTH (r = 0.76, P less than 0.001), while osteoclast number correlated with both PTH (r = 0.56, P less than 0.05) and IGF-I (r = 0.67, P less than 0.01). There were no correlations between IGF-II levels and bone histology. From these data we suggest that IGF-I may promote bone formation in uremic patients with hyperparathyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1998

Unilateral injection of calcitonin gene-related peptide (CGRP) induces bilateral oedema formation and release of CGRP-like immunoreactivity in the rat hindpaw.

The contribution of calcitonin gene-related peptide (CGRP) to bilateral oedema formation in the rat hindpaw following an unilateral challenge with CGRP was investigated. Rats were injected into the left hindpaw with either saline, CGRP or a CGRP antagonist (CGRP8-37). All injections were given in a double blind fashion and in a volume of 100 microl. CGRP and CGRP8-37 were administered in concentrations of 75, 150 or 300 pmol. Volumes of the right and left hindpaw were measured every hour for 5 h by plethysmometry. Injection of CGRP 300 pmol into the left hindpaw resulted in a bilaterally increased hindpaw volume after 5 h as compared with the groups given saline. No changes were found in hindpaw volumes following the injection of either 75 or 150 pmol of CGRP or 75, 150 or 300 pmol of CGRP8-37 as compared with saline injection. To elucidate whether or not the bilateral oedema formation was related to a release of endogenous CGRP, microdialysis of the contralateral hindpaw was carried out, and concentrations of CGRP-like immunoreactivity (-LI) were determined by radioimmunoassay and high performance liquid chromatography. Injection of CGRP 300 pmol into the left hindpaw increased the release of CGRP-LI into the right hindpaw perfusate after 4 and 5 h. No changes in CGRP-LI were detected in the right hindpaw perfusate following challenge with saline or CGRP8-37. To study the contribution of the nervous system to the contralateral release of CGRP-LI, sciatic nerve ligated and intact sham-operated rats were used. Sciatic nerve ligation but not sham-operation on the non-injected side abolished the increased release of CGRP-LI following contralateral administration of CGRP 300 pmol. To study the spinal cord mechanisms resulting in the bilateral oedema formation following unilateral challenge with 300 pmol of CGRP, intrathecal pretreatment with either 10 nmol bicuculline (GABA(A) receptor antagonist) or 10 nmol CGRP8-37 was carried out. Bicuculline but not CGRP8-37 abolished the bilateral oedema formation induced by CGRP 300 pmol. In order to study the mechanisms by which administration of CGRP 300 pmol induces oedema, CGRP 300 pmol was administered concomitantly with either 300 pmol of CGRP8-37 (CGRP receptor antagonist), or 3 nmol of promethazine (H1 receptor antagonist), or 3 nmol of s(-)-propranolol (5-HT1 receptor antagonist), or 3 nmol of cyproheptadine (5-HT2 receptor antagonist) or 3 nmol of ICS 205-930 (5-HT3 receptor antagonist). Oedema formation was measured at 1, 5, 7 and 24 h. Injection of CGRP 300 pmol into the left hindpaw induced a bilateral oedema formation which was still significant at 24 h. Concomitant administration of either CGRP8-37, ICS 205-920 or cyproheptadine blocked the oedema formation at 24 h. No effect on oedema formation was found when CGRP 300 pmol was co-administered with either promethazine or s(-)-propranolol (H1 and 5-HT1 receptor antagonists, respectively). The results of the present study show that both the nervous system and local inflammatory processes contribute to bilateral hindpaw oedema formation following unilateral challenge with CGRP 300 pmol. Our results indicate that endogenous release of CGRP following inflammatory response may play an important role in inducing oedema formation.

Animals↗