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New beginnings for matrix metalloproteinase inhibitors: identification of high-affinity zinc-binding groups.

In an effort to identify promising non-hydroxamate inhibitors of matrix metalloproteinases (MMPs), several new zinc-binding groups (ZBGs) based on pyrone, pyrothione, hydroxypyridinone, and hydroxypyridinethione chelators have been examined. Structural studies with tris(pyrazolyl)borate model complexes show that these ligands bind to the MMP active site zinc(II) ion in a bidentate fashion, similar to that found with hydroxamate-based inhibitors. Fluorescence- and colorimetric-based enzyme assays have been used to determine the IC50 values for these ZBGs against MMP-3; mixed O,S-donor ligands were found to be remarkably potent, with IC50 values as much as 700-fold lower than that found for acetohydroxamic acid. Inhibitory activity was found to parallel metal binding affinity as determined in titrations with model complexes. These results demonstrate that MPIs based on new ZBGs are feasible and may indeed improve the overall performance of inhibitors designed against these important medicinal targets.

Hydroxamic Acids↗

[The protective effects of zinc metallothionein against erythrocyte membrane damage induced by methylmercury].

The influences of zinc metallothionein(ZnMT) induced by Zn on mice erythrocyte membrane configuration, membrane sulfhydryl content, membrane fluidity, membrane permeability and so on after methylmercury (MeHg) treatment were studied, the protection of ZnMT against the membrane damage of MeHg was studied. The results showed that Zn could induce the production of ZnMT in the liver of mice. With the increase of Zn content, the level of ZnMT increased and had dose-effect relationship between them (r = 0.996). ZnMT could protect membrane configuration and -SH and prevent the damage of MeHg on the membrane fluidity and permeability. The results also showed that the dose of Zn administered to mice should be appropriate. High doses of Zn could cause damages.

Animals↗

In vitro antioxidant activities of mouthrinses and their components.

OBJECTIVES: Several forms of periodontal diseases (PD) are often associated with activated phagocytosing leukocytes and contemporary free radical production. Host antioxidant defenses could benefit from mouthrinses used as adjuncts to counteract plaque-associated bacteria. The aim of the present study was to determine possible antioxidant activity (AA) of a number of antiseptic mouthrinses and of their stated active principles (AP), regardless of their efficacy as antimicrobial agents. MATERIAL AND METHODS: The antioxidant activities of 11 mouthrinses and their active principles were tested with a specific spectrophotometric method. Comet assay was used to test whether pure chemical antioxidant activity actually corresponded to prevention of in vitro DNA fragmentation. RESULTS: Methylsalicylate-containing mouthrinses were the most effective. Several compounds, and some vehicles, behaved as antioxidants. Fibroblast DNA fragmentation was limited by preincubation with methylsalicylate-containing mouthrinse but was unaffected by treatment with chlorexidine. CONCLUSION: The results described herein indicate that several mouthrinses possess AA; such a property could be ascribed to either AP or vehicles or both. All the data were obtained in systems in vitro and the demonstration of in vivo AA is necessary. These findings could be useful in the treatment of some forms of PD and should be considered when arranging new mouthrinse formulations.

Allantoin↗

Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms.

Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We have developed two protein-mediated protection schemes that permit independent labeling of multiple thiols. These schemes exploit metal coordination or disulfide bond formation to reversibly protect cysteines in a Cys(2)His(2) zinc finger domain. We constructed a variety of N- and C-terminal fusions of these domains with maltose-binding protein, which were labeled with two or three different fluorophores. Multiple modifications were made by reacting an unprotected cysteine in MBP first, deprotecting the zinc finger, and then reacting the zinc finger cysteines. The fusion proteins were orthogonally labeled with two different fluorophores, which exhibited intramolecular fluorescene resonance energy transfer (FRET). These conjugates showed up to a threefold ratiometric change in emission intensities in response to maltose binding. We also demonstrated that the metal- and redox-mediated protection methods can be combined to produce triple independent modifications, and prepared a protein labeled with three different fluorophores that exhibited a FRET relay. Finally, labeled glucose-binding protein was covalently patterned on glass slides using thiol-mediated immobilization chemistries. Together, these experiments demonstrated that reversible thiol protection schemes provide a rapid, straightforward method for producing multiple, site-specific modifications.

Binding Sites↗

Dinuclear zinc(II) complexes of tetraiminodiphenol macrocycles and their interactions with carboxylate anions and amino acids. Photoluminescence, equilibria, and structure.

The reaction equilibria [H(4)L](2+) + Zn(OAc)(2) right harpoon over left harpoon [Zn(H(2)L)](2+) + 2HOAc (K(1)) and [Zn(H(2)L)](2+) + Zn(OAc)(2) right harpoon over left harpoon [Zn(2)L](2+) + 2HOAc (K(2)), involving zinc acetate and the perchlorate salts of the tetraiminodiphenol macrocycles [H(4)L(1)(-)(3)](ClO(4))(2), the lateral (CH(2))(n)() chains of which vary between n = 2 and n = 4, have been studied by spectrophotometric and spectrofluorimetric titrations in acetonitrile. The photoluminescence behavior of the complexes [Zn(2)L(1)](ClO(4))(2), [Zn(2)L(2)(H(2)O)(2)](ClO(4))(2), [Zn(2)L(2)(mu-O(2)CR)](ClO(4)) (R = CH(3), C(6)H(5), p-CH(3)C(6)H(4), p-OCH(3)C(6)H(4), p-ClC(6)H(4), p-NO(2)C(6)H(4)), and [Zn(2)L(3)(mu-OAc)](ClO(4)) have been investigated. The X-ray crystal structures of the complexes [Zn(2)L(2)(H(2)O)(2)](ClO(4))(2), [Zn(2)L(3)(mu-OAc)](ClO(4)), and [Zn(2)L(2)(mu-OBz)(OBz)(H(3)O)](ClO(4)) have been determined. The complex [Zn(2)L(2)(mu-OBz)(OBz)(H(3)O)](ClO(4)) in which the coordinated water molecule is present as the hydronium ion (H(3)O(+)) on deprotonation gives rise to the neutral dibenzoate-bridged compound [Zn(2)L(2)(mu-OBz)(2)].H(2)O. The equilibrium constants (K) for the reaction [Zn(2)L(2)(H(2)O)(2)](2+) + A(-) right harpoon over left harpoon [Zn(2)L(2)A](+) + 2H(2)O (K), where A(-) = acetate, benzoate, or the carboxylate moiety of the amino acids glycine, l-alanine, l-histidine, l-valine, and l-proline, have been determined spectrofluorimetrically in aqueous solution (pH 6-7) at room temperature. The binding constants (K) evaluated for these systems vary in the range (1-8) x 10(5).

Amino Acids↗

Retrograde tracing of zinc-enriched (ZEN) neuronal somata projecting to the olfactory bulb.

Zinc ions seem to be important to several neurological functions and have been connected to the pathophysiology of epilepsy, neuronal cell death after seizure or stroke, and Alzheimer's disease. Both epilepsy and Alzheimer's disease are clinical conditions believed to involve the olfactory bulb. The mammalian olfactory bulb is densely innervated by zinc-enriched (ZEN) neurons, and the distribution of the ZEN terminals in the mouse olfactory bulb has previously been described. The aim of this study was to describe the origins of ZEN terminals projecting into the main olfactory bulb of the rat. Selective labeling of ZEN terminals was accomplished by intracerebral infusion of sodium selenide, whereby zinc selenium clusters are created in the ZEN terminals. Some of these clusters move by retrograde axonal transport to the somata where they can be silver-enhanced by autometallography (AMG). After infusion of sodium selenide into the main olfactory bulb, retrogradely labeled ZEN somata were found (1) ipsilaterally in all anterior olfactory nuclei, taenia tecta, piriform cortex and lateral entorhinal cortex, and (2) contralaterally in anterior olfactory nuclei except the external division. The ipsilateral anterior olfactory nucleus had the densest population of ZEN somata, and it was found that these somata originated mainly from pyramidal neurons in layers II and III of each area. The olfactory-related centrifugal afferents to the main olfactory bulb are discussed.

Afferent Pathways↗

[Biochemical evaluation of damage due to lead: importance and significance of erythrocyte zinc-protoporhyrin IX and urinary amino acid determination].

The early detection of lead intoxitation needs practical, simple, reproducible and diagnostically valid screening test. The determination of ALA-D (delta-amino-levulinic acid-dehydratase) in erythrocytes is one of the most reliable test for the evaluation of the occupational exposure to lead. However this test is difficult to standardize, sensible to lead contamination of laboratory glassware and the activity of enzyme decreases rapidly if stored. The determination of erythrocytes ZPP (zinco-protoporphyrin IX) was proposed as useful, alternative test. The protoporphyrin IX is a metabolic intermediate in heme biosynthesis; in erythrocytes is present as free form and zinc-boundend compound. The ZPP give high values only in lead intoxication and sideropenic anemia. The ALA-D and ZPP in erythrocytes were measured and compared in a group of workers exposed to lead. We have shown a good correlation between these two biochemical parameters. Aminoacid excretion in urine from workers exposed to lead was measured and compared with other biochemical parameters of intoxication. All lead workers examined had excessive urinary CP (coproporphyrin) and ALA (delta-amino-levulinic acid) excretion. An abnormal excretion of glycine was present in eight workers (32%), whereas in other four (15%) the glycinuria was at limit of normal values. An abnormal excretion of lysine was present in six workers (21%). The last data appear very interesting because the action of lead in lysine metabolism was not known.

Aminolevulinic Acid↗

Catalytic asymmetric vinylation and dienylation of ketones.

A solution to the long-standing problem of catalytic asymmetric vinylation of ketones is reported. Vinylzinc reagents are generated via hydrozirconation of terminal alkynes followed by transmetalation to zinc. In the presence of our catalyst, which is formed in situ from a bis(sulfonamide) diol ligand (1) and titanium tetraisopropoxide, the vinylzinc reagent undergoes 1,2-addition to a variety of ketones and enones with enantioselectivities (typically >90%) and high yields. This method is tolerant of functional groups, including alkyl, aryl and vinyl halides, esters, silyl protected alcohols, sulfides, and alkenes. Thus, enantioenriched tertiary allylic alcohols bearing a variety of functional groups can be prepared. It has also been found that 2,2-disubstituted vinylzinc reagents, substitution patterns not accessible through hydrozirconation, can be added to ketones with high enantioselectivities to generate trisubstituted allylic alcohols. Furthermore, we have developed an asymmetric addition of dienyl groups to ketones in the presence of our catalyst. This method enables the synthesis of dienols in high yields with enantioselectivities as high as 94%.

Alkenes↗

Synthesis, electrochemistry, absorption and electro-polymerization of aniline-ethynyl metalloporphyrins.

Free-base, nickel(II), zinc(II) and iron(III) mono(4-aniline-ethynyl)biphenylporphines (AEBPPs) were synthesized and their electro-polymerization reactions were studied. Electrochemical and UV-Visible experiments showed that AEBPPs were electro-polymerized onto Pt and indium tin oxide (ITO) surfaces without significantly changing their redox and spectral properties. This suggests that the pi-conjugations of these porphyrins were not greatly affected upon electro-polymerization. SEM images indicated the formation of porphyrin films on the electrode surfaces, while EDS spectra of the metallo-AEBPP films confirmed deposits of the corresponding metals. Upon closer inspection, the AFM images revealed nano-scale fine structure of these porphyrin films. Both SEM and AFM images of the porphyrin films showed similar trends of porphyrin accumulation.

Aniline Compounds↗

Regulatory effects of zinc and copper on the calcium transport system in rat liver nuclei. Relation to SH groups in the releasing mechanism.

In isolated hepatic nuclei, the heavy metals Zn2+ and Cu2+ (10 microM) inhibited Ca2+ uptake and caused a prompt release of Ca2+ from preloaded nuclei in a concentration-dependent manner, with Zn2+ being more effective than Cu2+. The sulfhydryl group reducing agent dithiothreitol (DTT) protected the nuclei from the effects of heavy metals; DDT (1 mM) almost completely blocked Zn(2+)- or Cu(2+)-induced Ca2+ release and inhibition of Ca2+ uptake. The sulfhydryl modifying reagent N-ethylmaleimide (NEM; 0.2 mM) also caused Ca2+ release, but it did not have an appreciable effect on Ca2+ uptake. Furthermore, in the presence of NEM heavy metals did not evoke Ca2+ release. The present study demonstrates that Zn2+ and Cu2+ have a stimulatory effect on Ca2+ release from isolated rat liver nuclei, and that the SH group may play an important role in the Ca(2+)-releasing mechanism in liver nuclei.

Adenosine Triphosphate↗

Consistency in calibrated backscattered electron images of calcified tissues and minerals analyzed in multiple imaging sessions.

Pure metal standards have been used to calibrate the operating envionment in quatitative backscattered electron (BSE) imaging of mineralized tissue, allowing comparisons to be made between various mineralization states of bone at the microscopic level. It has not previuously been documented that calibration procedures produce consistent, reliable results over multiple imaging sessions. In this study, BSE images were obtained from bones, pure metals, and a naturally occurring mineral in multiple imaging sessions over a six day period. The graylevel histogram profile (GHP) from each specimen was analyzed for changes in the shape and relative placement on the graylevel spectrum. Computer controlled calibration and a restrospective calibration method using pure aluminum and pure magnesium-aluminum-zinc demonstrated consistency between imaging sessions. Calibrated weighted mean graylevels (WMGLs) for biological meterials had an average standard deviation of 5.9 graylevels (2.4% variation) during the course of the study. WMGLs for inorganic materials had an average standard deviation of 0.9 graylevels (0.4% variation). A trend towards increased image brightness, due to specimen and/or embedding media degradation, was observed in the biological tissues. No increase in rightness was observed for the inorgtanic specimens. Kurtosis and skewness tests revealed a slight deviation from normality in all specimens, which remained consistent between multiple imaging sessions. These results demonstrate the BSE image analysis of bones and mineral can be calibrated with negligible precision error allowing comparisons between data within and between multiple imaging sessions.

Aluminum↗

Inhibitory effect of benzethonium and zinc chloride mouthrinses on human dental plaque and gingivitis.

In a double-blind study the plaque- and gingivitis-inhibiting capacities of three aqueous mouthrinse solutions of benzethonium chloride, zinc chloride, and benzethonium chloride combined with zinc chloride were compared to those of a placebo mouthrinse. Forth-three adults with clean teeth and healthy gingiva were randomly assigned to the treatment and control groups. All participants received an initial prophylaxis. Assessments of plaque and gingivitis were made before and after a 10-day test period. During this time the subjects suspended oral hygiene procedures and rinsed daily with 20 ml of their respective rinse solutions. According to the measurements made before and after the test period, the mouthrinse containing benzethonium chloride produced 42 to 42.9% less plaque than any of the other three formulations. The differences were statistically significant ( P smaller than 0.05). Gingivitis scores of the four groups did not differ significantly. These findings indicate that benzethonium chloride is a moderately effective antiplaque agent.

Adult↗

Modeling zinc enzyme inhibition with functional thiolate ligands.

The blocking of zinc enzymes by thiolate-containing inhibitors was modeled by treating TpPh,MeZn-OH with functional thiols. The latter were chosen such that they contain an additional donor function (COOH, COOR, NH2, NHR, OH) in a position favorable for chelation. Of them, mercapto carboxylic acid esters were incorporated as thiolates. The corresponding mercapto carboxylic acids, however, used only their carboxylate function for coordination. Various mercapto amines, mercapto alcohols, and mercaptophenol were exclusively converted to thiolate ligands. The two modes of inhibitor attachment, terminal or chelating, were observed equally frequently. As a rule, they occur as alternatives for similar ligands. In case of 2-mercaptophenol they coexist in the crystalline state and in solution. Hydrogen bonding, both intra- and intermolecular, seems to be a decisive factor determining the inhibitor attachments. Its persistence in solution is underlined by the observation that TpPh,MeZn-hydroxythiophenolates are methylated about 2 orders of magnitude slower than TpPh,MeZn-SPh itself.

Enzyme Inhibitors↗

Antilipolytic actions of vanadate and insulin in rat adipocytes mediated by distinctly different mechanisms.

Vanadate, which mimics the biological effects of insulin, also inhibits lipolysis in rat adipocytes. Here we demonstrate that the antilipolytic effect of vanadate differs from that of insulin at least by the five following criteria: 1) vanadate inhibits lipolysis mediated by high (supraphysiological) concentrations of catecholamines; 2) vanadate antagonizes (Bu)2cAMP-mediated lipolysis; 3) vanadate antagonizes isobutylmethylxanthine-dependent lipolysis, 4) vanadate inhibits lipolysis mediated by okadaic acid; and 5) wortmannin, which blocks the antilipolytic effect of insulin, fails to block vanadate-mediated antilipolysis. Vanadate does activate phosphoinositol 3-kinase, and wortmannin blocks this activation. Our working hypothesis assumes that all of the insulin-like effects of vanadate, including antilipolysis, are initiated by the inhibition of protein phosphotyrosine phosphatases (PTPases). Among documented PTPase inhibitors we found that VOSO4 (oxidation state +4), several organic vanadyl compounds (+4), zinc (Zn2+), tungstate (W), and molybdate (Mo) also had antilipolytic activity. The order of potency was vanadyl acetylacetonate > or = VOSO4 > or = NaVO3 > or = vanadyl-dipicolinate > Zn2+ >> W > Mo, and it correlated better with the inhibition of adipose membranal-PTPases in cell-free experiments. We have concluded that the antilipolytic effect of vanadate is 1) mechanistically distinct from that of insulin, 2) independent of phosphoinositol 3-kinase activation, and 3) independent of the lipolytic cascade. We also strongly suggest that the antilipolytic effect of vanadate emanates from inhibiting adipose membranal, rather than cytosolic PTPases, and present preliminary data showing distinct differences in catalysis between these two PTPase categories. Overall, the study indicates that antilipolysis can be manifested via alternative, insulin-independent, signal-transducing pathways.

Adipocytes↗

A fluorescence method to determine picomole amounts of Zn(II) in biological systems.

The formation of complexes between n-(6-methoxy-8-quinolyl)-p-toluensulfonamide (TSQ) and Zn(II) in methanol has been used as an analytical procedure for Zn(II) determination in biological systems. Using 1 ml cuvettes, the limit of detection of the method was approximately 20 pmoles of Zn(II). Linearity between fluorescence and zinc concentration was obtained up to approximately 1 microM Zn(II). Common multivalent cations present in biological systems like Al3+, Cu2+, Fe3+, Ca2+, and Mg2+, interfered with the measurement of Zn(II) only when present in excess of 20, 33, 60, 500 and 30,000 times the Zn (II) concentration, respectively. In human serum and semen, deproteinization of the samples permitted a good correlation between the TSQ method and the total Zn content determined by atomic absorption measurements. In rat spermatids, deproteinization and Zn (II) determination using the TSQ method gives approximately a 20% underestimation of the total Zn (II) content of the cells as compared to atomic absorption spectrophotometry. The method gives low resolution of Zn (II) peaks when tested as an analytical procedure to measure Zn (II) binding to protein fractions eluted during column chromatography.

Aminoquinolines↗

Metallothionein, nitric oxide and zinc homeostasis in vascular endothelial cells.

Recent in vitro studies suggest that the oxidoreductive capacity of metal thiolate clusters in metallothionein (MT) contributes to intracellular zinc homeostasis. We used fluorescence-based techniques to address this hypothesis in intact endothelial cells, focusing on the contributory role of the important redox signaling molecule, nitric oxide. Microspectrofluorometry with Zinquin revealed that the exposure of cultured sheep pulmonary artery endothelial cells to S-nitrosocysteine resulted in the release of N, N,N',N'-tetrakis(2. pyridylmethyl)ethylendiamine (TPEN) chelatable zinc. Cultured sheep pulmonary artery endothelial cells were transfected with a plasmid expression vector suitable for fluorescence resonance energy transfer containing the cDNA of MT sandwiched between two mutant green fluorescent proteins. The exposure of cultured sheep pulmonary artery endothelial cells transfected with this chimera to nitric oxide donors or to agents that increased cytoplasmic Ca(2+) via endogenously generated nitric oxide decreased the efficiency of fluorescence resonance energy transfer in a manner consistent with the release of metal (Zn) from MT. A physiological role for this interaction in intact tissue was supported by the lack of myogenic reflex in resistance arteries of MT knockout mice unless endogenous nitric oxide synthesis was blocked. These data suggest an important role for metal thiolate clusters of MT in nitric oxide signaling in the vascular wall.

Animals↗

pH dependence and compartmentalization of zinc transported across plasma membrane of rat cortical neurons.

In this study, Zn(2+) transport in rat cortical neurons was characterized by successfully combining radioactive tracer experiments with spectrofluorometry and fluorescence microscopy. Cortical neurons showed a time-dependent and saturable transport of (65)Zn(2+) with an apparent affinity of 15-20 microM. (65)Zn(2+) transport was pH dependent and was decreased by extracellular acidification and increased by intracellular acidification. Compartmentalization of newly transported Zn(2+) was assessed with the Zn(2+)-selective fluorescent dye zinquin. Resting cortical neurons showed uniform punctate labeling that was found in cell processes and the soma, suggesting extrasynaptic compartmentalization of Zn(2+). Depletion of intracellular Zn(2+) with the membrane-permeant chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) resulted in the complete loss of punctate zinquin labeling. After Zn(2+) depletion, punctate zinquin labeling was rapidly restored when cells were placed in 30 microM Zn(2+), pH 7.4. However, rapid restoration of punctate zinquin labeling was not observed when cells were placed in 30 microM Zn(2+), pH 6.0. These data were confirmed in parallel (65)Zn(2+) transport experiments.

Animals↗

Thiolate exchange in [TmR]ZnSR' complexes and relevance to the mechanisms of thiolate alkylation reactions involving zinc enzymes and proteins.

The zinc and cadmium thiolate complexes [TmBut]MSCH2C(O)N(H)Ph (M = Zn, Cd) may be obtained via treatment of the respective methyl complex [TmBut]MMe with PhN(H)C(O)CH2SH. The molecular structure of [TmBut]ZnSCH2C(O)N(H)Ph has been determined by X-ray diffraction, thereby demonstrating the presence of an intramolecular N-H S hydrogen bond between the amide N-H group and thiolate sulfur atom. [TmBut]ZnSCH2C(O)N(H)Ph mimics the function of the Ada DNA repair protein by undergoing alkylation with MeI to give [TmBut]ZnI and MeSCH2C(O)N(H)Ph. A series of crossover experiments and 1H NMR magnetization transfer studies establish that thiolate exchange between [TmR]ZnSR' derivatives is facile in this system, an observation that supports the previous suggestion that the alkylation of [TmPh]ZnSCH2C(O)N(H)Ph by MeI may proceed via a sequence that involves dissociation of [PhN(H)C(O)CH2S]-.

Alkylation↗