Effects of polyamines and analogs on staphylococcal nuclease.

The promoting activity of polyamine analogs (IV approximately XV) on staphylococcal nuclease with DNA as the substrate was compared with that of natural polyamines (I APPROXIMATELY III): I. NH2(CH2)3NH(CH2)4NH(CH2)3NH2(spermine); II. NH2(CH2)3NH(CH2)3NH(CH2)3NH2(thermine); III. NH2(CH2)4NH2 (putrescine); IV. CN(CH2)2NH(CH2)4NH(CH2)2CN; V. HOOC(CH2)2NH(CH2)4NH(CH2)2COOH; VI. C2H5OOC(CH2)2NH(CH2)4NH(CH2)2COOC2H5; VII. HO(CH2)3NH(CH2)4HH(CH2)3OH; VIII. CH3COHH(CH2)3NH(CH2)4NH(CH2)3NHCOCH3; IX. C2H5NH(CH2)3NH(CH2)4NH(CH2)3NHC2H5; X. NH2(CH2)3S(CH2)4S(CH2)3NH2; XI. NH2(CH2)3NH(CH2)2O(CH2)2NH(CH2)3NH2; XII. NH2(CH2)3NCH3(CH2)4HCH3(CH2)3NH2; XIII. CN(CH2)2NCH3(CH2)4NCH3(CH2)2CN; XIV. (CH3)2N(CH2)3NCH3(CH2)4NCH3(CH2)3N(CH3)2; XV. NH2(CH2)2O(CH2)2NH2 Replacement of the terminal groups by CN, COOH, COOEt, NHAc, NHEt, or N(CH3)2 remarkably decreased the activity. The compound VII with terminal hydroxyl groups had a lower promoting activity at low concentrations, but revealed higher activity at higher concentrations and, in contrast to spermine, no inhibition at all even at very high concentrations. Replacement of both internal amino groups by sulfur or NCH3 decreased the activity. The introduction of an ether bond into the internal methylene groups (compound XI) highly decreased the activity. Based upon these findings the possible relationship between structure and activity is discussed.     

Mg(2+) block unmasks Ca(2+)/Ba(2+) selectivity of alpha1G T-type calcium channels

We have examined permeation by Ca(2+) and Ba(2+), and block by Mg(2+), using whole-cell recordings from alpha1G T-type calcium channels stably expressed in HEK 293 cells. Without Mg(o)(2+), inward currents were comparable with Ca(2+) and Ba(2+). Surprisingly, three other results indicate that alpha1G is actually selective for Ca(2+) over Ba(2+). 1) Mg(2+) block is approximately 7-fold more potent with Ba(2+) than with Ca(2+). With near-physiological (1 mM) Mg(o)(2+), inward currents were approximately 3-fold larger with 2 mM Ca(2+) than with 2 mM Ba(2+). The stronger competition between Ca(2+) and Mg(2+) implies that Ca(2+) binds more tightly than Ba(2+). 2) Outward currents (carried by Na(+)) are blocked more strongly by Ca(2+) than by Ba(2+). 3) The reversal potential is more positive with Ca(2+) than with Ba(2+), thus P(Ca) > P(Ba). We conclude that alpha1G can distinguish Ca(2+) from Ba(2+), despite the similar inward currents in the absence of Mg(o)(2+). Our results can be explained by a 2-site, 3-barrier model if Ca(2+) enters the pore 2-fold more easily than Ba(2+) but exits the pore at a 2-fold lower rate.     

Studies of antibacterial activity of binuclear rhodium (II) complexes with heterocyclic nitrogen ligands

Binuclear rhodium (II) complexes, [Rh2(OOCPh)2(phen)2(H2O)2] (OOCPh)2 (1), [Rh2(OOCPh)2(bpy)2(H2O)2] (OOCPh)2 (2), [Rh2(OOCBu(n))2 (bpy)2(H2O)2] (OOCBu(n)2 (3), and [Rh2(OOCPr(n)2 (phen)2(H2O)2] (OOCPr(n)2 (4) (Phen = 1,10-phenanthroline and bpy = 2,2'-bipyridine), have been synthesized and characterized using NMR, IR and electronic spectra. Activity of these compounds against Gram-positive bacteria decreases in the order: 1?2?3 > 4. Complex 1 is active against many Staphylococcus strains resistant to commonly used antibiotics. The complexes 1-4 are much less active agents against Gram-negative bacteria.     

The preparation,characterisation and in vitro cytotoxicity of potentially chemotherapeutic heterobimetallic complexes containing early and late transition metals

The reactions of phosphine Ph(2)P(CH(2))(2)SO(3)Na with Cp(2)M'Cl(2) (M'=Ti, Zr) in aqueous solution give the metallophosphines, Cp(2)Ti(OSO(2)(CH(2))(2)PPh(2))(2) (Cp=cyclopentadienyl) and CpZr(OH)(OSO(2)(CH(2))(2)PPh(2))(2). These react with CODM"Cl(2) (M"=Pd, Pt) (COD=1,5-cyclooctadiene) in dichloromethane to give heterobimetallic complexes Cp(2)Ti(OSO(2)(CH(2))(2)PPh(2))(2)M"Cl(2) and CpZr(OH)(OSO(2)(CH(2))(2) PPh(2))(2)M"Cl(2) respectively. The compounds are characterised by infrared and NMR spectroscopies and elemental analysis. Electrospray mass spectra of the complexes are reported and compared to those of Cp(2)M'Cl(2) in water and dimethylsulfoxide (DMSO). For zirconocene dichloride and its product heterobimetallic complexes, the addition of ethylenediamine tetraacetic acid disodium salt (Na(2)H(2)EDTA) was found to be an effective ionisation enhancement agent for the electrospray mass spectral studies. Cytotoxicity studies for the previously reported Cl(2)Pt(PPh(2)(CH(2))(2)SO(3)H)(2).3.5H(2)O (Wedgwood et al., Inorg. Chim. Acta 290 (1999) 189), and the compounds Cp(2)Ti(OSO(2)(CH(2))(2) PPh(2))(2).1.5H(2)O and Cp(2)Ti(OSO(2)(CH(2))(2)PPh(2))(2)PtCl(2).4H(2)O reported here, have been evaluated by colony formation assay against cisplatin-sensitive and -resistant cell lines L929 and L929/R to highlight potential chemotherapeutic activity. The compound Cl(2)Pt(PPh(2)(CH(2))(2)SO(3)H)(2).3.5H(2)O overcomes cisplatin resistance.     

Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract

Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.     

Biosynthetic studies on the alpha-glucosidase inhibitor acarbose: the chemical synthesis of isotopically labeled 2-epi-5-epi-valiolone analogs

2-epi-5-epi-valiolone is a cyclization product of the C(7) sugar phosphate, sedoheptulose 7-phosphate, involved in the biosynthesis of the aminocyclitol moieties of acarbose, validamycin, and pyralomicin. As part of our investigation into the pathway from 2-epi-5-epi-valiolone to the valienamine moiety of acarbose, we prepared 1-epi-5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-6], 5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-17], 1-epi-2-epi-5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-12] and 2-epi-5-epi-(6-(2)H(2))valiolamine [(6-(2)H(2))-11]. Compounds (6-(2)H(2))-6 and (6-(2)H(2))-17 were synthesized from 2,3,4,6-tetra-O-benzyl-D-glucopyranose in 10 and seven steps, respectively, whereas (6-(2)H(2))-12 and (6-(2)H(2))-11 were synthesized from 2,3,4,6-tetra-O-benzyl-D-mannopyranose in eight and 10 steps, respectively.     

Chemical and biological characterization of technetium(I) and Rhenium(I) tricarbonyl complexes with dithioether ligands serving as linkers for coupling the Tc(CO)(3) and Re(CO)(3) moieties to biologically active molecules

The organometallic precursor (NEt(4))(2)[ReBr(3)(CO)(3)] was reacted with bidendate dithioethers (L) of the general formula H(3)C-S-CH(2)CH(2)-S-R (R = -CH(2)CH(2)COOH, CH(2)-C&tbd1;CH) and R'-S-CH(2)CH(2)-S-R' (R' = CH(3)CH(2)-, CH(3)CH(2)-OH, and CH(2)COOH) in methanol to form stable rhenium(I) tricarbonyl complexes of the general composition [ReBr(CO)(3)L]. Under these conditions, the functional groups do not participate in the coordination. As a prototypic representative of this type of Re compounds, the propargylic group bearing complex [ReBr(CO(3))(H(3)C-S-CH(2)CH(2)-S-CH(2)C&tbd1;CH)] Re2 was studied by X-ray diffraction analysis. Its molecular structure exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The potentially tetradentate ligand HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH was reacted with the trinitrato precursor [Re(NO(3))(3)(CO)(3)](2-) to yield a cationic complex [Re(CO)(3)(HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH)]NO(3) Re8 which shows the coordination of one hydroxy group. Re8 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction analysis. Ligand exchange reaction of the carboxylic group bearing ligands H(3)C-S-CH(2)CH(2)-S-CH(2)CH(2)-COOH and HOOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH with (NEt(4))(2)[ReBr(3)(CO)(3)] in water and with equimolar amounts of NaOH led to complexes in which the bromide is replaced by the carboxylic group. The X-ray structure analysis of the complex [Re(CO)(3)(OOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH)] Re6 shows the second carboxylic group noncoordinated offering an ideal site for functionalization or coupling a biomolecule. The no-carrier-added preparation of the analogous (99m)Tc(I) carbonyl thioether complexes could be performed using the precursor fac-[(99m)Tc(H(2)O)(3)(CO)(3)](+), with yields up to 90%. The behavior of the chlorine containing (99m)Tc complex [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 in aqueous solution at physiological pH value was investigated. In saline, the chromatographically separated compound was stable for at least 120 min. However, in chloride-free aqueous solution, a water-coordinated cationic species Tc1a of the proposed composition [(99m)Tc(H(2)O)(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))](+) occurred. The cationic charge of the conversion product was confirmed by capillary electrophoresis. By the introduction of a carboxylic group into the thioether ligand as a third donor group, the conversion could be suppressed and thus the neutrality of the complex preserved. Biodistribution studies in the rat demonstrated for the neutral complexes [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 and [(99m)TcCl(CO)(3)(CH(2)-S-CH(2)CH(2)-S-CH(2)-C&tbd1;CH)] Tc2 a significant initial brain uptake (1.03 +/- 0.25% and 0.78 +/- 0.08% ID/organ at 5 min. p.i.). Challenge experiments with glutathione clearly indicated that no transchelation reaction occurs in vivo.     

Intracellular-produced hydroxyl radical mediates H2O2-induced Ca2+ influx and cell death in rat beta-cell line RIN-5F

The melastatin-related transient receptor potential channel TRPM2 is a Ca(2+)-permeable channel that is activated by H(2)O(2), and the Ca(2+) influx through TRPM2 mediates cell death. However, the responsible oxidants for TRPM2 activation remain to be identified. In the present study, we investigated the involvement of hydroxyl radical on TRPM2 activation in TRPM2-expressing HEK293 cells and the rat beta-cell line RIN-5F. In both cell types, H(2)O(2) induced Ca(2+) influx in a concentration-dependent manner. However, the addition of hydroxyl radical, which was produced by mixing FeSO(4) and H(2)O(2), to the cells, did not increase intracellular Ca(2+) concentration. Interestingly, when H(2)O(2) was added to the cells under intracellular Fe(2+)-accumulated conditions, Ca(2+) influx was markedly enhanced compared to H(2)O(2) alone. In addition, the H(2)O(2)-induced Ca(2+) influx was reduced by hydroxyl radical scavengers and an iron chelator. Under intracellular Fe(2+)-accumulated conditions, H(2)O(2)-induced RIN-5F cell death through TRPM2 activation was also markedly enhanced. Hydroxyl radical scavengers and an iron chelator suppressed the RIN-5F cell death by H(2)O(2). These results strongly suggest that the intracellular hydroxyl radical plays a key role in the activation of TRPM2 during H(2)O(2) treatment, and TRPM2 activation mediated by hydroxyl radical is implicated in H(2)O(2)-induced cell death in the beta-cell line RIN-5F.     

Function, activity, and membrane targeting of cytosolic phospholipase A(2)zeta in mouse lung fibroblasts

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA(2)alpha(-/-) lung fibroblasts stimulated with A23187 or serum. cPLA(2)alpha(+/+) fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA(2)alpha(-/-) fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA(2) alpha(-/-) fibroblasts was inhibited by the cPLA(2)alpha inhibitors pyrrolidine-2 (IC(50), 0.03 microM) and Wyeth-1 (IC(50), 0.1 microM), implicating another C2 domain-containing group IV PLA(2). cPLA(2) alpha(-/-) fibroblasts contain cPLA(2)beta and cPLA(2)zeta but not cPLA(2)epsilon or cPLA(2)delta. Purified cPLA(2)zeta exhibited much higher lysophospholipase and PLA(2) activity than cPLA(2)beta and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA(2)beta. In contrast to cPLA(2)beta, cPLA(2)zeta expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA(2)zeta exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA(2)alpha and has been identified as the PLA(2) responsible for calcium-induced fatty acid release and prostaglandin E(2) production from cPLA(2) alpha(-/-) lung fibroblasts. In response to ionomycin, EGFP-cPLA(2)zeta translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA(2)alpha translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes.     

Vanadium-induced HB-EGF expression in human lung fibroblasts is oxidant dependent and requires MAP kinases

Vanadium pentoxide (V(2)O(5)) is a transition metal derived from the burning of petrochemicals that causes airway fibrosis and remodeling. Vanadium compounds activate many intracellular signaling pathways via the generation of hydrogen peroxide (H(2)O(2)) or other reactive oxygen species. In this study, we investigated the regulation of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in human lung fibroblasts after V(2)O(5) treatment. V(2)O(5)-induced HB-EGF mRNA expression was abolished by N-acetyl-l-cysteine, suggesting an oxidant-mediated effect. Exogenous H(2)O(2) (>10 microM) mimicked the effect of V(2)O(5) in upregulating HB-EGF expression. Fibroblasts spontaneously released low levels of H(2)O(2) (1-2 microM), and the addition of V(2)O(5) depleted the endogenous H(2)O(2) pool within minutes. V(2)O(5) caused a subsequent increase of H(2)O(2) into the culture medium at 12 h. However, the burst of V(2)O(5)-induced H(2)O(2) occurred after V(2)O(5)-induced HB-EGF mRNA expression at 3 h, indicating that the V(2)O(5)-stimulated H(2)O(2) burst did not mediate HB-EGF expression. Either V(2)O(5) or H(2)O(2) activated ERK-1/2 and p38 MAP kinase. Inhibitors of the ERK-1/2 pathway (PD-98059) or p38 MAP kinase (SB-203580) significantly reduced either V(2)O(5)- or H(2)O(2)-induced HB-EGF expression. These data indicate that vanadium upregulates HB-EGF via ERK and p38 MAP kinases. The induction of HB-EGF is not related to a burst of H(2)O(2) in V(2)O(5) treated cells, yet the action of V(2)O(5) in upregulating HB-EGF is oxidant dependent and could be due to the reaction of V(2)O(5) with endogenous H(2)O(2).     

Mitochondrial Ca(2+) uptake depends on the spatial and temporal profile of cytosolic Ca(2+) signals

Using confocal imaging of Rhod-2-loaded HeLa cells, we examined the ability of mitochondria to sequester Ca(2+) signals arising from different sources. Mitochondrial Ca(2+) (Ca(2+)mit) uptake was stimulated by inositol 1,4,5-trisphosphate (InsP(3))-evoked Ca(2+) release, capacitative Ca(2+) entry, and Ca(2+) leaking from the endoplasmic reticulum. For each Ca(2+) source, the relationship between cytosolic Ca(2+) (Ca(2+)cyt) concentration and Ca(2+)mit was complex. With Ca(2+)cyt < 300 nm, a slow and persistent Ca(2+)mit uptake was observed. If Ca(2+)cyt increased above approximately 400 nm, Ca(2+)mit uptake accelerated sharply. For equivalent Ca(2+)cyt increases, the rate of Ca(2+)mit rise was greater with InsP(3)-evoked Ca(2+) signals than any other source. Spatial variation of the Ca(2+)mit response was observed within individual cells. Both the fraction of responsive mitochondria and the amplitude of the Ca(2+)mit response were graded in direct proportion to stimulus concentration. Trains of repetitive Ca(2+) oscillations did not maintain elevated Ca(2+)mit levels. Only low frequency Ca(2+) transients (<1/15 min) evoked repetitive Ca(2+)mit signals. Our data indicate that there is a lag between Ca(2+)cyt and Ca(2+)mit increases but that mitochondria will accumulate calcium when it is elevated over basal levels regardless of its source. Furthermore, in addition to the characteristics of Ca(2+) signals, Ca(2+) uniporter desensitization and proximity of mitochondria to InsP(3) receptors modulate mitochondrial Ca(2+) responses.     

Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.     

Acute effects of hydrogen peroxide on skeletal muscle microvascular oxygenation from rest to contractions

Reactive oxygen species, such as hydrogen peroxide (H(2)O(2)), exert a critical regulatory role on skeletal muscle function. Whether acute increases in H(2)O(2) modulate muscle microvascular O(2) delivery-utilization (Qo(2)/Vo(2)) matching [i.e., microvascular partial pressure of O(2) (Pmv(O(2)))] at rest and following the onset of contractions is unknown. The hypothesis was tested that H(2)O(2) treatment (exogenous H(2)O(2)) would enhance Pmv(O(2)) and slow Pmv(O(2)) kinetics during contractions compared with control. Anesthetized, healthy young Sprague-Dawley rats had their spinotrapezius muscles either exposed for measurement of blood flow (and therefore QO(2)), VO(2), and Pmv(O(2)), or exteriorized for measurement of force production. Electrically stimulated twitch contractions (1 Hz, ~7 V, 2-ms pulse duration, 3 min) were evoked following acute superfusion with Krebs-Henseleit (control) and H(2)O(2) (100 μM). Relative to control, H(2)O(2) treatment elicited disproportionate increases in QO(2) and VO(2) that elevated Pmv(O(2)) at rest and throughout contractions and slowed overall Pmv(O(2)) kinetics (i.e., ~85% slower mean response time; P < 0.05). Accordingly, H(2)O(2) resulted in ~33% greater overall Pmv(O(2)), as assessed by the area under the Pmv(O(2)) curve (P < 0.05). Muscle force production was not altered with H(2)O(2) treatment (P > 0.05), evidencing reduced economy during contractions (~40% decrease in the force/VO(2) relationship; P < 0.05). These findings indicate that, although increasing the driving force for blood-myocyte O(2) flux (i.e., Pmv(O(2))), transient elevations in H(2)O(2) impair skeletal muscle function (i.e., reduced economy during contractions), which mechanistically may underlie, in part, the reduced exercise tolerance in conditions associated with oxidative stress.     

Modulation of cardiac ryanodine receptor channels by alkaline earth cations

Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.     

Localization of P2X and P2Y receptors in dorsal root ganglia of the cat.

The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X(5) receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y(2) = P2Y(4)>P2X(3)>P2X(2) = P2X(7)>P2X(6)>P2X(1) = P2X(4)>P2Y(1). P2X(3), P2Y(2), and P2Y(4) receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y(2), P2X(1), P2X(3), P2X(4), and P2X(6) receptor staining was detected in small- and medium-diameter neurons. However, P2Y(4), P2X(2), and P2X(7) staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X(3) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y(4) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y(2) receptor-positive neurons also stained for IB(4), CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X(3)-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y(1) receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse.     

Nitrate/nitrite reductase activity of sulfido/selenido bridged dinuclear ruthenium(III) complexes

Series of dinuclear species [Ru(2)(L)(2)(LH)(2)-mu-S(2)Cl(2)], [Ru(2)(L)(2)(LH)(4)-mu-Se(2)Cl(2)] (L=L(1)H and L(3)H) and [Ru(2)(L(2))(2)(L(2)H)-mu-Se(2)Cl(2)].2H(2)O, where L(1)H, L(2)H and L(3)H represent for 2-mercapto-5-phenyl-1,3,4-oxadiazole, 2-mercapto-benzimidazole and 2-mercapto-benzothiazole, respectively, have been prepared and characterized by their elemental analyses and spectral (IR, UV-visible, 1H NMR and FAB mass) data. The diamagnetism of these complexes are indicative of an exchange coupled dinuclear ruthenium(III) species containing S(2)(2-) and Se(2)(2-) bridges. The complexes along with free heterocycles (L(1)H-L(3)H) as well as RuCl(3) x 3H(2)0 were tested for their nitrate and nitrite reductase activities. The electrochemical behaviour of the complexes showed irreversible oxidation peaks at +(1.04-1.40) V quite comparable to those reported for sulphido bridged complexes.     

Evidence that PAR2-triggered prostaglandin E2 (PGE2) formation involves the ERK-cytosolic phospholipase A2-COX-1-microsomal PGE synthase-1 cascade in human lung epithelial cells

We investigated possible involvement of three isozymes of prostaglandin E synthase (PGES), microsomal PGES-1 (mPGES-1), mPGES-2 and cytosolic PGES (cPGES) in COX-2-dependent prostaglandin E(2) (PGE(2)) formation following proteinase-activated receptor-2 (PAR2) stimulation in human lung epithelial cells. PAR2 stimulation up-regulated mPGES-1 as well as COX-2, but not mPGES-2 or cPGES, leading to PGE(2) formation. The PAR2-triggered up-regulation of mPGES-1 was suppressed by inhibitors of COX-1, cytosolic phospholipase A(2) (cPLA(2)) and MEK, but not COX-2. Finally, a selective inhibitor of mPGES-1 strongly suppressed the PAR2-evoked PGE(2) formation. PAR2 thus appears to trigger specific up-regulation of mPGES-1 that is dependent on prostanoids formed via the MEK/ERK/cPLA(2)/COX-1 pathway, being critical for PGE(2) formation.     

Isoprenoid phenol and quinone precursors of ubiquinones and dihydroubiquinones [ubiquinones(H2)] in fungi

1. Ten moulds and two yeasts were analysed for the presence of 2-polyprenylphenols, 2-polyprenyl(H(2))phenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-2-polyprenyl(H(2))phenols, 6-methoxy-2-polyprenyl-1,4-benzoquinones, 6-methoxy-2-polyprenyl(H(2))-1,4-benzoquinones, 5-demethoxyubiquinones, 5-demethoxyubiquinones(H(2)), ubiquinones and ubiquinones(H(2)). 2. The organisms were found to be of three types: (a) those that contained only ubiquinones (Aspergillus fumigatus and Penicillium brevi-compactum) or ubiquinones(H(2)) (Alternaria solani, Claviceps purpurae and Penicillium stipitatum); (b) those that contained 5-demethoxyubiquinones and ubiquinones (Agaricus campestris, Aspergillus niger, Phycomyces blakesleeanus, Rhodotorula glutinis and Saccharomyces cerevisiae) or 5-demethoxyubiquinones(H(2)) and ubiquinones(H(2)) (Aspergillus quadrilineatus and Neurospora crassa); (c) one that contained 2-decaprenyl(H(2))phenol, 6-methoxy-2-decaprenyl(H(2))phenol, 6-methoxy-2-decaprenyl(X-H(2))-1,4-benzoquinone, 5-demethoxyubiquinone-10(X-H(2)) and ubiquinones(H(2)) (Aspergillus flavus). 3. Studies were made on the biosynthesis of ubiquinones and ubiquinones(H(2)) by Asp. flavus, Phyc. blakesleeanus and S. cerevisiae. These provided evidence that in Phyc. blakesleeanus 5-demethoxyubiquinone-9 is a precursor of ubiquinone-9 and that in S. cerevisiae 5-demethoxyubiquinone-6 is a precursor of ubiquinone-6. In addition they yielded results that may be interpreted as providing evidence that in Asp. flavus 6-methoxy-2-decaprenyl(X-H(2))-1,4-benzoquinone and 5-demethoxyubiquinone-10(X-H(2)) are precursors of ubiquinone-10(X-H(2)).     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.