The ATPase reaction cycle of yeast DNA topoisomerase II. Slow rates of ATP resynthesis and P(i) release.

DNA topoisomerase II catalyzes the transport of one DNA duplex through a transient break in a second duplex using a complex ATP hydrolysis mechanism. Two key rates in the ATPase mechanism, ATP resynthesis and phosphate release, were investigated using 18O exchange and stopped-flow phosphate release experiments, respectively. The 18O exchange results showed that the rate of ATP resynthesis on the topoisomerase II active site was slow compared with the rate of phosphate release. When topoisomerase II was bound to DNA, phosphate was released slowly, with a lag. Since each of the preceding steps is known to occur rapidly, phosphate release is apparently a rate-determining step. The length of the lag phase was unaffected by etoposide, indicating that inhibiting DNA religation inhibits the ATPase reaction cycle at some step following phosphate release. By combining the 18O exchange and phosphate release results, the rate constant for ATP resynthesis can be calculated as approximately 0.5 s(-1). These data support the mechanism of sequential hydrolysis of two ATP by DNA topoisomerase II.

The lipophilicity, pharmacokinetics, and cellular uptake of different chemically-modified tetracyclines (CMTs).

CMTs are analogs of tetracyclines, which are chemically modified to eliminate their antimicrobial efficacy but which retain their inhibitory activity against matrix metalloproteinases. These compounds have been found to inhibit connective tissue breakdown in animal models of diseases such as periodontitis, arthritis and cancer. Because CMTs exhibit different in vivo efficacy in these various models of disease, the current study compared their pharmacokinetics and other properties as follows: Adult male Sprague-Dawley rats were administered by oral gavage a single dose of 5mg of different CMTs suspended in 1 ml 2% carboxymethyl-cellulose, and blood samples were collected from 1-48 hours after dosing. The sera were extracted, then analyzed by HPLC using a C-18 reverse-phase column. The results showed that the peak concentrations (C(max)) in rat sera 1-12 hours after oral administration of CMTs -1, -2,-3, -4,-5,-6,-7,-8 and doxycycline were 5.5, 0.7, 4.6, 6.2, 0.8, 0.7, 9.0 (note: the 3 peaks detected for CMT-7 were combined), 15.0 and 0.9 microg/ml, respectively. Their in vivo half-lives (t(1/2)) were 11, 5, 22, 11, 32, 15, 37, 38, and 17 hours, respectively. Of the anticollagenase CMTs tested, CMT-8 showed the greatest C(max) and t(1/2)values, followed by CMTs-3, -1, -4, and perhaps -7; CMTs-2, -5, and -6 exhibited much lower levels in serum. The relative lipophilicities of the 8 CMTs and doxycycline were tested by examining their extractability in octanol. The results showed that CMT-2, -5, and -6 had the lowest partition coefficients using this organic solvent, while CMT-3 was the most lipophilic. The lipophilicity of the different CMTs was also positively correlated (r(2)=0.767, P<0.05) to peak serum concentrations (C(max)), but not to their serum half-lives (r(2)=0.25,P=0.49). This property of the different CMTs was also found to be positively correlated to their ability to enter into human whole blood cells in vitro (r2=0.95, P<0.001). Since CMT-8, as well as CMTs-3 and -1, consistently exhibited the greatest in vivo efficacy in animal models of tissue breakdown, this may reflect, at least in part, their favorable pharmacokinetics and tissue uptake.

Synthesis and evaluation of 6-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine as a PET tracer for nicotinic acetylcholine receptors.

Both ABT-594 ((R)-2-chloro-5-(2-azetidinylmethoxy)pyridine) and A-85380 (3-[2(S)-2-azetidinylmethoxy]pyridine), novel nicotinic agonists that possess potent non-opioid analgesic properties, have high affinity for neuronal nicotinic acetylcholine receptors (nAChR) but do not elicit the pronounced toxicity of epibatidine. 6-[(18)F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[(18)F]fluoro-A-85380), a F-18 labeled analogue of these two compounds, is therefore a promising radioligand for positron emission tomography (PET) studies in humans. The use of trimethylammonium as a leaving group in nucleophilic aromatic substitution reactions has proven to be a versatile and efficient strategy, and offers several advantages over other leaving groups. Here, we report the synthetic strategy for the preparation of a precursor, as a trimethylammonium iodide salt, and its use in the radiosynthesis to 6-[(18)F]fluoro-A-85380. Preliminary compartative PET studies of 6-[(18)F]fluoro-A-85380 and 2-[(18)F]fluoro-A-85380 were carried out in baboon to examine their suitability as tracers for studying nAChR system.

The concentration levels of Cd, Pb, Hg, Cu, Zn and Se in blood of the population in the Czech Republic.

Knowledge of normal levels of concentrations of trace elements (Cd, Pb, Hg, Cu, Zn, and Se) in the population serves, among others, in design of regulations concerning health protection, determination of exposure limits and prevention of diseases caused by deficiency of trace elements. Concentrations of the named elements in whole blood of the Czech population were determined by means of atomic absorption spectrometry. The blood was collected during 1996-1998 from 1,216 blood donors (896 males and 320 females, average age 33 years) and 758 children (397 boys and 361 girls, average age 9.9 years). Mineralisation in a microwave digestion system was used in sample preparation. The accuracy of results was checked by means of the Control material Seronorm Whole Blood 404107 and Seronorm Serum 704121, Nycomed. Values of concentrations of the trace elements in blood found for adult (medians) were 0.7 microgram Cd.l-1, 800 micrograms Cu.l-1, 0.78 microgram Hg.l-1, 41 micrograms Pb.l-1, 76 micrograms Se.l-1, and 5,800 micrograms Zn.l-1, respectively. Statistically significant differences between men and women have been found in the concentrations of Cu, Hg, Pb, and Zn. In the juvenile population following medians of concentrations have been found: 0.15 microgram Cd.l-1, 1,047 micrograms Cu.l-1, 0.46 microgram Hg.l-1, 34 micrograms Pb.l-1, 69 micrograms Se.l-1, and 8,180 micrograms Zn.l-1. Statistically significant differences between boys and girls were found only in Pb and Zn concentrations. Concentrations of the studied elements correspond to the published values concerning population not exposed professionally.

Alphabeta protomers of Na+,K+-ATPase from microsomes of duck salt gland are mostly monomeric: formation of higher oligomers does not modify molecular activity.

The distance that separates alphabeta protomers of the Na(+), K(+)-ATPase in microsomes and in purified membranes prepared from duck nasal salt glands was estimated by measuring fluorescence resonance energy transfer between anthroylouabain bound to a population of alphabeta protomers and either N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain or 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain bound to the rest. Energy transfer between probes bound in the microsomal preparation was less than in the purified membranes. The efficiency of energy transfer between anthroylouabain and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain was 29.2% in the microsomes compared with 62.6% in the purified preparation. Similar results were obtained with 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain as acceptor. We calculate that either the protomer bound probes were on the average 13 A farther apart in the microsomes than in the purified membranes, or that 53% of the protomers are monomeric in the microsome preparation. Microsomes prepared in the presence of phalloidin (a toxin that binds to F actin and stabilizes the actin-based cytoskeleton) showed less quench than those prepared in its absence. The data support the hypothesis that protomers are kept apart by their association with the cytoskeleton. The turnover rate while hydrolyzing ATP is the same in the microsomal and purified preparations; higher oligomer formation has no significant effect on the enzyme reaction mechanism.

Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives.

Controlled modification of the carboxylic acid moieties of hyaluronic acid with mono- and polyfunctional hydrazides leads to biochemical probes, biopolymers with altered physical and chemical properties, tethered drugs for controlled release, and crosslinked hydrogels as biocompatible scaffoldings for tissue engineering. Methods for polyhydrazide synthesis, for prodrug preparation, for hydrogel crosslinking, and for monitoring biodegradation are described.

Culture, gender, and suicidal behavior in Sri Lanka.

In less than 50 years, the rate of suicide among Sri Lankans has risen from a modest level to one of the highest in the world. This article describes the current pattern of suicides, including sex differences and similarities, and reviews some of the institutional practices, material conditions, and social norms that may figure in the increases. A study is reported, which asked how ordinary Sri Lankans account for suicidal behavior and what practices they recommend for assisting suicidal individuals. Gender inflected these accounts, with essentialist accounts associated with women's suicides and contextual accounts associated with men's suicides.

Synthesis and in vitro degradation of new polyvalent hydrazide cross-linked hydrogels of hyaluronic acid.

New polyvalent hydrazide cross-linkers were synthesized, characterized, and used to prepare hydrazide cross-linked hydrogels derived from hyaluronic acid (HA). First, the chemical synthesis and characterization of the di-, tri-, tetra-, penta-, and hexahydrazides are presented. Second, HA concentration, buffer type and concentration, and ratio of HA to carbodiimide to cross-linker were varied to obtain HA-hydrogels with different chemical and physical properties. Third, two new assays are described to monitor the stability of HA-hydrogels toward hyaluronidase (HAse) and other media. These assays were used to evaluate the stability of cross-linked HA-hydrogels to HAse solutions and different pH values. Hydrophobic cross-linkers gave the most stable gels, and the susceptibility of the gels to HAse was independent of cross-linker concentration. HAse does not significantly penetrate the HA-hydrogels and acts primarily at the gel-solution interface. The HA-hydrogels are stable in acid environments and dissolve gradually above pH 7.0.

Reversible labeling of a chemosensitizer binding domain of p-glycoprotein with a novel 1,4-dihydropyridine drug transport inhibitor.

A photoreactive dihydropyridine (DHP), BZDC-DHP (2,6-dimethyl-4-(2-(trifluoromethyl)-phenyl)-1,4-dihydropyridine-3,5- dicarboxylic acid (2-[3-(4-benzoylphenyl)propionylamino]ethyl) ester ethyl ester), and its tritiated derivative were synthesized as novel probes for human p-glycoprotein (p-gp). (-)-[3H]BZDC-DHP specifically photolabeled p-gp in membranes of multidrug-resistant CCRF-ADR5000 cells. In reversible labeling experiments a saturable, vinblastine-sensitive and high-affinity (Kd = 16.3 nM, Bmax = 58 pmol/mg of protein, k(+1) = 0.031 nM-1 min-1, k(-1) = 0.172 min-1) binding component was present in CCRF-ADR5000 membranes but absent in the sensitive parent cell line. Binding was inhibited by cytotoxics and known chemosensitizers with a p-gp characteristic pharmacological profile. For eight chemosensitizers tested, the potency for binding inhibition correlated (r > 0.94) with the potency for drug transport inhibition (measured using rhodamine 123 accumulation). The DHP niguldipine and a structurally related pyrimidine stereoselectively stimulated reversible (-)-[3H]BZDC-DHP binding, suggesting that more than one DHP molecule can bind to p-gp at the same time. Our data demonstrate that DHPs label multiple chemosensitizer domains on p-gp, distinct from the vinblastine interaction site. (-)-[3H]BZDC-DHP represents a valuable tool to characterize the molecular organization of chemosensitizer binding domains on p-gp by both reversible binding and photoinduced covalent modification. It provides a novel simple screening assay for p-gp active drugs.

Gender, politics, and psychology's ways of knowing.

Recent work on the psychology of gender has emphasized comparisons of men and women. Such comparisons rest on a view of gender as an individual difference or psychological attribute. Feminist theorists have challenged this view as limited and inadequate. In place of it, a variety of alternative conceptions of gender are emerging. These conceptions shift the focus of analysis from the individual to interpersonal and institutional arenas. Moreover, they dispute the idea of gender as static, unitary, and separable from other markers of social identity and status. In contrast to Alice Eagly (1995), I assert that the production of knowledge (whether by scientific procedures or other means) is not set apart from society, but rather is always and inevitably embedded within it. Therefore, I call for efforts to uncover the ways in which psychological knowledge is shaped by ongoing societal struggles and cultural politics.

Evidence for NH2- and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The pH kinetic behavior of several rat fructose-2,6-bisphosphatase forms was analyzed. The bisphosphatase maximal velocity of the hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was optimal at pH 5, but decreased to 12% of the optimal value in the pH range 7.0-7.5. This decrease depended on deprotonation of a group with a pK of 5.7. In contrast, the separate bisphosphatase domain, a 30-amino acid COOH-terminal truncated form (CT30) of the liver enzyme, and the skeletal muscle bifunctional enzyme exhibited pH-insensitive maximal velocities which were 5-10-fold higher than that of the bisphosphatase of the liver bifunctional enzyme at pH 7.0-7.5. The pK values of the C-2 and C-6 phosphoryl groups were 6.0 and 5.75, respectively, as determined by 31P NMR. Analysis of log kcat/Km versus pH profiles revealed two pK values, one at 6.1, which probably is a substrate pK, and the other at 8.4, which represents an enzyme group. Protein kinase-catalyzed phosphorylation of the liver isoform activated the bisphosphatase, and the pK of the group seen in the kcat profile was increased from 5.7 to 6.4. However, phosphorylation of the CT30 mutant had no effect on the bisphosphatase. The data indicate that NH2- and COOH-terminal interactions in the liver bifunctional enzyme affect the pH dependence of the fructose-2,6-bisphosphatase and its activation by phosphorylation.

Synthesis and use of fluorescent molecular probes for measuring cell-surface enzymatic oxidation of amino acids and amines in seawater.

A method for investigating cell-surface enzymatic oxidative deamination of amino acids and amines in seawater was developed. This technique used synthetic fluorescent Lucifer Yellow derivatives of the amino acid lysine and the amine cadaverine as molecular probes to investigate oxidation pathways and rates. The probes were chemically stable under the conditions used and did not adsorb to container surfaces. The oxidative deamination of the fluorescent probes added to phytoplankton cultures and the subsequent production of their fluorescent oxidation products could be selectively detected by HPLC at 250 pM levels. This approach allows selective investigation of cell-surface enzymatic oxidation since neither transport of the probes across the cell membrane nor chemical transformation of the probes occurs. Bacteria were also capable of oxidizing the fluorescent amino acid probe.

Inositol 1,4,5-trisphosphate receptors: labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands.

We have photolabeled the inositol 1,4,5-trisphosphate (IP3) receptor and probed the IP3 ligand binding site using two novel photoaffinity ligands, [125I] (azidosalicyl)aminopropyl-IP3 ([125I]ASA-IP3) and [3H] (benzoyldihydrocinnamyl)aminopropyl-IP3 ([3H]BZDC-IP3). Both ligands have high affinity for the IP3 receptor and, when photoactivated, label the IP3 receptor protein with appropriate inositol phosphate selectivity. The high specific activity of [125I]ASA-IP3 allowed identification of a single photolabeling site within the IP3R by two-dimensional peptide analysis. Substantially higher levels of incorporation into the receptor are achieved with [3H]BZDC-IP3 (50-60% efficiency) than with [125I]ASA-IP3 (3%), facilitating the use of [3H]BZDC-IP3 as a better ligand for the high-efficiency labeling and purification of IP3R-labeled peptides. Peptides were generated from photolabeled IP3 receptor by trypsin digestion and purified by high-pressure liquid chromatography (HPLC). A single purified [3H]BZDC-IP3-labeled peptide, corresponding to IP3R amino acids 476-501, was sequenced and shown to match specific sequences in the N-terminal 20% of the IP3 receptor, an area suggested on the basis of mutagenesis studies to contain the IP3 recognition site.

Purification and characterization of inositol-1,3,4-trisphosphate 5/6-kinase from rat liver using an inositol hexakisphosphate affinity column.

The metabolism of inositol 1,3,4-trisphosphate is a pivotal branch point of inositol phosphate turnover; its dephosphorylation replenishes cellular inositol pools, its phosphorylation at the 6-position supports the synthesis of inositol pentakisphosphate, and its phosphorylation at the 5-position produces inositol 1,3,4,5-tetrakisphosphate (Shears, S.B. (1989) J. Biol. Chem. 264, 19879-19886). In order to increase understanding of the control of inositol-1,3,4-trisphosphate kinase activity, the enzyme was highly purified from rat liver by precipitation with polyethylene glycol, MonoQ ion-exchange chromatography, heparin-agarose affinity chromatography, and a novel affinity chromatography procedure that utilized Affi-Gel resin to which InsP6 was coupled (Marecek, J.F., and Prestwich, G.D. (1991) Tetrahedron Lett. 32, 1863-1866). The final purification was about 26,000-fold, with a 6% yield. This final preparation performed both 5- and 6-kinase activities in the ratio of approximately 1:5. The affinity of the enzyme for inositol 1,3,4-trisphosphate was 0.04 microM, the highest yet determined for an inositol phosphate kinase. Both inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6-tetrakisphosphate were competitive inhibitors of the kinase (Ki values of 2-4 microM). The enzyme was determined to have a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinase activity was unaffected by Ca2+/calmodulin, protein kinase A, or protein kinase C.

New tetherable derivatives of myo-inositol 2,4,5- and 1,3,4-trisphosphates.

(+/-)-myo-Inositol 1-(3-aminopropyl hydrogen phosphate) 3,4-bis(disodium phosphate) (5) and (+/-)-myo-inositol 2-(3-aminopropyl hydrogen phosphate) 4,5-bis(disodium phosphate) (11) have been synthesized by conventional procedures. Each derivative has been immobilized on a polymeric resin in order to give a bioaffinity matrix.

Evidence for an InsP3-gated channel protein in isolated rat olfactory cilia.

Stimulation of rat olfactory cilia (ROC) with odorants leads to a transient elevation in the levels of either cAMP or inositol trisphosphate (InsP3). We have characterized the binding of [3H]InsP3 to isolated ROC. Unlabeled InsP3 displaced [3H]InsP3 binding in a dose-dependent manner (dissociation constant = 3.9 +/- 0.65 microM). Binding was stereospecific and dependent on the number of phosphates in the inositol ring. A ciliary protein of 120 kDa molecular mass was labeled specifically upon exposure of cilia membranes to ultraviolet light in the presence of the 125I-labeled InsP3 analogue 1-O-[N-(4-azidosaliciloxy)-3-aminopropyl-1-phospho]-myo-inositol 4,5-bisphosphate. Labeling of this protein displayed the same stereospecificity as binding of [3H]InsP3 to ROC. In addition, ROC membranes incorporated into a phospholipid bilayer at the tip of a patch pipette displayed an increase in conductance upon exposure to micromolar D-myoinositol 1,4,5-trisphosphate in 45% of the trials (n = 88). The InsP3-gated conductance is relatively nonspecific for cations and is distinct from the cAMP-gated conductance. The conductance displayed stereospecificity consistent with the InsP3 binding experiments. The results suggest that the site of action for odorant-stimulated elevations in InsP3 concentration in rat olfactory cilia is at a ciliary InsP3-gated channel.

Photoaffinity labeling and characterization of isolated inositol 1,3,4,5-tetrakisphosphate- and inositol hexakisphosphate-binding proteins.

We have isolated high affinity inositol (1,3,4,5)-tetrakisphosphate (IP4)- and inositol hexakisphosphate (IP6)-binding proteins from detergent-solubilized rat brain membranes using a P1-tethered IP4 derivative linked to an Affi-Gel support. To determine the identity, binding characteristics, and distribution of the individual IP4 recognition sites, we have synthesized an IP4 photoaffinity label probe, 125I-(D,L)-1-O-[N-(4-azidosalicyloxy)-3-aminopropyl-1-phospho]- IP4 (125I-ASA-IP4). Two apparently distinct IP4-binding proteins (IP4BP), isolated with the IP4 affinity column, display high affinity and selectivity for IP4 over inositol trisphosphate (IP3), inositol pentakisphosphate (IP5), and IP6. The first IP4-binding protein (IP4BP1) which has a KD for IP4 of 4 nM, is comprised of a protein at 182 kDa which is specifically photolabeled with high affinity by 125I-ASA-IP4. The second, IP4BP2, has an affinity for IP4 of 1.5 nM and contains proteins at 84 and 174 kDa, both of which are specifically photoaffinity labeled. A putative IP6-binding protein (IP6BP), also isolated with the IP4 affinity column, binds IP6 with a KD of 14 nM and comprises three proteins of 115, 105, and 50 kDa. The 115- and 105-kDa subunits, but not the 50-kDa subunit, specifically incorporate the photolabel. The IP4BP (182, 174, and 84 kDa) and IP6BP (115 and 105 kDa) proteins are specifically photolabeled in the crude membrane, partially purified, and purified fractions. These receptor-binding proteins vary in inositol phosphate specificity and in the effects of pH, Ca2+, and heparin on IP4 photoaffinity labeling. In addition, IP4BP and IP6BP are enriched in the brain but differ in their regional localizations within the brain.

Characterization of a novel inositol 1,4,5-trisphosphate receptor in isolated olfactory cilia.

Inositol 1,4,5-trisphosphate (InsP3), a product of G-protein-mediated receptor activation of phosphoinositide turnover, plays the role of a second messenger when olfactory neurons are stimulated with certain olfactory stimuli. In this paper we examine the specific binding of [3H]InsP3 to isolated olfactory cilia, microsomes and brain membranes from the channel catfish (Ictalurus punctatus) and, by photoaffinity labelling with an InsP3 analogue (125I-labelled 1-[3-(4-azidosalicyloxy)-aminopropyl]inositol 1,4,5-trisphosphate (125I-ASA-InsP3)], we tentatively identify the major InsP3-binding protein in catfish olfactory cilia. InsP3 binding to ciliary membranes is specific and saturable, with a Kd of 1.10 +/- 0.31 microM and a maximum number of binding sites (Bmax) of 17.6 +/- 5.8 pmol/mg. The rank order for potency of inhibition of [3H]InsP3 binding is Ins(1,4)P2 less than Ins(1,3,4)P3 less than Ins(1,3,4,5)P4 = Ins(1,4,5)P3 less than Ins(2,4,5)P3. Exposure of cilia membranes to u.v. light in the presence of 125I-ASA-InsP3 results in the labelling of a protein with apparent Mr 107,000. Labelling is specifically prevented by Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(1,3,4,5)P4, but not by Ins(1,4)P2 or Ins(1,3,4)P3. Both specific [3H]InsP3 binding and photoaffinity labelling of the Mr-107,000 protein were displaced by heparin. The Kd and the inhibition of [3H]InsP3 binding and of photoaffinity labelling by inositol phosphates and heparin are consistent with the ability of micromolar concentrations of Ins(1,4,5)P3 [but not Ins(1,3,4)P3] to activate the InsP3-gated currents in patch-clamp experiments with olfactory neurons. These results suggest that InsP3 binding to a Mr-107,000 cilia membrane protein may represent binding to the olfactory InsP3-gated cation channel.

Two pathways for oxygen exchange by heavy meromyosin and their dependence on actin.

In the hydrolysis of MgATP by acto heavy meromyosin (HMM) there are two enzymatic pathways that differ in the properties of their intermediate oxygen exchange; one of these is designated the low exchange pathway (P1); the other is designated the high exchange pathway (P2). A plot of the P1 flux versus the actin concentration gives a sigmoid curve, whereas the corresponding curve for the P2 flux rises in an approximately hyperbolic manner. At low concentrations of actin, where the sigmoid curve of the P1 flux is in a lag phase, the major flux is along P2; but at higher concentrations of actin, as the P1 curve rises sharply, the flux along P1 comes to predominate. Even at the highest levels of actin, at saturating levels for both pathways, the kinetics of exchange along P1 and P2 are significantly different. In addition to these differences in the actin dependence, the flux of P1 relative to P2 is markedly inhibited by KCl. Therefore, which of the two pathways dominates during the hydrolysis of MgATP by HMM is strongly dependent on experimental conditions. The findings suggest that P1 involves the interaction of HMM with two actin units whereas P2 involves the interaction of HMM with one actin unit. The results are discussed in relation to a kinetic scheme based on this proposal.