Conformational preferences of modified nucleoside N(2)-methylguanosine (m(2)G) and its derivative N(2), N(2)-dimethylguanosine (m(2)(2)G) occur at 26th position (hinge region) in tRNA

Conformational preferences of the modified nucleosides N²-methylguanosine (m²G) and N², N²-dimethylguanosine (m₂²G) have been studied theoretically by using quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. Automated complete geometry optimization using semiempirical quantum chemical RM1, along with ab initio molecular orbital Hartree–Fock (HF-SCF), and density functional theory (DFT) calculations has also been made to compare the salient features. Single-point energy calculation studies have been made on various models of m²G26:C/A/U44 and m₂²G26:C/A/U44. The glycosyl torsion angle prefers “syn” (χ = 286°) conformation for m²G and m₂²G molecules. These conformations are stabilized by N(3)–HC2′ and N(3)–HC3′ by replacing weak interaction between O5′–HC(8). The N²-methyl substituent of (m²G26) prefers “proximal” or s-trans conformation. It may also prefer “distal” or s-cis conformation that allows base pairing with A/U44 instead of C at the hinge region. Thus, N²-methyl group of m²G may have energetically two stable s-trans m²G:C/A/U or s-cis m²G:A/U rotamers. This could be because of free rotations around C–N bond. Similarly, N², N²-dimethyl substituent of (m₂²G) prefers “distal” conformation that may allow base pairing with A/U instead of C at 44th position. Such orientations of m²G and m₂²G could play an important role in base-stacking interactions at the hinge region of tRNA during protein biosynthesis process.     

Role of TRPM2 in H(2)O(2)-Induced Cell Apoptosis in Endothelial Cells

Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2)O(2))-induced cytosolic Ca(2+) ([Ca(2+)](i)) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+)](i) rise and whole-cell current change in response to H(2)O(2). Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H(2)O(2)-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H(2)O(2)-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+)](i) and whole-cell currents to H(2)O(2). TRPM2 overexpression also aggravated the H(2)O(2)-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+) overload in response to H(2)O(2) and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.     

EP(2) receptor mediates bronchodilation by PGE(2) in mice.

PGE(2) is an important cyclooxygenase product that modulates airway inflammatory and smooth muscle responses. Signal transduction is mediated by four EP receptor subtypes that cause distinct effects on cell metabolism. To determine the role of EP(2) receptor activation, we produced a mouse lacking the EP(2) receptor by targeted gene disruption. The effect of aerosolized PGE(2) and other agonists was measured using barometric plethysmography and by measurements of lung resistance in mechanically ventilated mice. Inhalation of PGE(2) inhibited methacholine responses in wild-type but not in mice lacking the EP(2) receptor [EP(2)(-/-)]. After airway constriction was induced by methacholine aerosol, PGE(2) reduced the airway constriction enhanced pause in wild-type mice (from 0.88 +/- 0.15 to 0.55 +/- 0.06) but increased it in EP(2)(-/-) mice (from 0.73 +/- 0. 08 to 1.27 +/- 0.19). Similar results were obtained in mechanically ventilated mice. These data indicate that the EP(2) receptor mediates the bronchodilation effect of PGE(2).     

H(2)O(2) modulates purinergic-dependent calcium signalling in osteoblast-like cells

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H(2)O(2) and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H(2)O(2) induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H(2)O(2) doses. The H(2)O(2)-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H(2)O(2) failed to evoke calcium signals and millimolar H(2)O(2) induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H(2)O(2) up to 1mM did not increase the cytosolic calcium concentration. In ROS/P2Y(2) cells, stably expressing the P2Y(2) receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H(2)O(2) induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y(2) cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y(2) receptor.     

Upregulation of MIP-2 (CXCL2) expression by 15-deoxy-Delta(12,14)-prostaglandin J(2) in mouse peritoneal macrophages

A peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), has been reported to possess anti-inflammatory activity in activated monocytes/macrophages. In this study, we investigated the effect of 15d-PGJ(2) on the lipopolysaccharide (LPS)-induced expression of chemokine mRNAs, especially macrophage inhibitory protein (MIP)-2 (CXCL2), in mouse peritoneal macrophages. The inhibitory actions of the natural PPARgamma ligands, 15d-PGJ(2) and prostaglandin A1 (PGA1), on the expression of RANTES (regulated upon activation, normal T expressed and secreted; CCL5), MIP-1beta (CCL4), MIP-1alpha (CCL3), IFN-gamma-inducible protein 10 kilodaltons (IP-10; CXCL10) and monocyte chemoattractant protein-1 (MCP-1; CCL2) mRNA in LPS-treated cells were stronger than those of the synthetic PPARgamma ligands troglitazone and ciglitazone. However, 15d-PGJ(2) enhanced the expression of LPS-induced MIP-2 (CXCL2) mRNA. A specific PPARgamma antagonist (GW9662) had no effect on the inhibitory action of 15d-PGJ(2) and PGA1 in LPS-induced chemokine mRNA expression and on the synergistic action of 15d-PGJ(2) in LPS-induced MIP-2 (CXCL2) expression. Moreover, LPS itself reduced the expression of PPARgamma. Although the synergistic effect of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) mRNA expression was remarkable, the production of MIP-2 (CXCL2) in cells treated with 15d-PGJ(2) and LPS did not increase compared to the production in cells treated with LPS alone. The synergistic action of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) mRNA expression was dependent on the activation of nuclear factor-kappaB (NF-kappaB), and 15d-PGJ(2) increased the phosphorylation of p38 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in cells stimulated with LPS. These results suggest that the synergistic effect of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) expression is PPARgamma-independent, and is mediated by the p38 and SAPK/JNK pathway in mitogen-activated protein kinase signaling pathways, which activates NF-kappaB. Our data may give more insights into the different mechanisms contrary to the anti-inflammatory effect of 15d-PGJ(2) on the expression of chemokine genes.     

H(2)O(2) opens BK(Ca) channels via the PLA(2)-arachidonic acid signaling cascade in coronary artery smooth muscle

H(2)O(2) is a reactive oxygen species that contracts or relaxes vascular smooth muscle, but the molecular basis of these effects remains obscure. We previously demonstrated that H(2)O(2) opens the large-conductance, calcium- and voltage-activated (BK(Ca)) potassium channel of coronary myocytes (2) and now report physiological and biochemical evidence that the effect of H(2)O(2) on coronary smooth muscle involves the phospholipase A(2) (PLA(2))/arachidonic acid (AA) signaling cascades. H(2)O(2) stimulation of BK(Ca) channel activity was inhibited by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA(2). Furthermore, H(2)O(2) stimulated release of [(3)H]AA from coronary myocytes, and exogenous AA mimicked the effect of H(2)O(2) on BK(Ca) channels. Inhibitors of protein kinase C activity attenuated the effect of H(2)O(2) on BK(Ca) channels, [(3)H]AA release, or intact coronary arteries. In addition, the effect of H(2)O(2) or AA on BK(Ca) channels was inhibited by blockers of lipoxygenase metabolism. In contrast, inhibitors of cyclooxygenase or cytochrome P-450 had no effect. We propose that H(2)O(2) relaxes coronary arteries by stimulating BK(Ca) channels via the PLA(2)/AA signaling cascade and that lipoxygenase metabolites mediate this response.     

Changes in gene expression induced by H(2)O(2) in cardiac myocytes

Oxidative stress induces cardiac myocyte apoptosis. At least some effects are probably mediated through changes in gene expression. Using Affymetrix arrays, we examined the changes in gene expression induced by H(2)O(2) (0.04, 0.1, and 0.2mM; 2 and 4h) in rat neonatal ventricular myocytes. Changes in selected upregulated genes were confirmed by ratiometric RT-PCR. p21(Cip1/Waf1) was one of the only two genes upregulated in all conditions studied. Of the heat shock proteins, only Hsp70/70.1 was induced by H(2)O(2) with no change in the expression of Hsp25, Hsp60 or Hsp90. Heme oxygenase 1 was also potently upregulated, but not heme oxygenases 2 or 3. Of the intercellular adhesion proteins, syndecan-1 was significantly upregulated in response to H(2)O(2), with little change in the expression of other syndecans and no change in expression of any of the integrins studied. Thus, oxidative stress, exemplified by H(2)O(2), selectively promotes the expression of specific gene family members.     

H(2)O(2) mediates Ca(2+)- and MLC(20) phosphorylation-independent contraction in intact and permeabilized vascular muscle

One purpose of the current study was to establish whether vasoconstriction occurs in all vessel types in response to H(2)O(2). Isometric force was measured in pulmonary venous and arterial rings, and isobaric contractions were measured in mesenteric arteries and veins in response to H(2)O(2). A second purpose was to determine whether H(2)O(2)-induced contraction is calcium independent. The addition of H(2)O(2) to calcium-depleted (using the Ca(2+) ionophore ionomycin in zero calcium EGTA buffer) muscle caused contraction. Furthermore, permeabilized muscle contracted in response to H(2)O(2) even in zero Ca(2+). The final purpose was to determine whether the 20-kDa regulatory myosin light chain (MLC(20)) phosphorylation plays a role in H(2)O(2)-induced contraction. Pulmonary arterial strips were freeze-clamped at various time points during H(2)O(2)-induced contractions, and the relative amounts of phosphorylated MLC(20) were measured. H(2)O(2) caused dose-dependent contractions that were independent of MLC(20) phosphorylation. ML-9, a myosin light chain kinase inhibitor, had no effect on the H(2)O(2) contractile response. In conclusion, H(2)O(2) induces Ca(2+)- and MLC(20) phosphorylation-independent contraction in pulmonary and systemic arterial and venous smooth muscle.     

VO(2) kinetics and the O(2) deficit in heavy exercise.

The purpose of this study was to examine a new method for calculating the O(2) deficit that considered the O(2) uptake (VO(2)) kinetics during exercise as two separate phases in light of previous research in which it was shown that the traditional O(2) deficit calculation overestimated the recovery O(2) consumption (ROC). Eight subjects completed exercise transitions between unloaded cycling and 25% (heavy, H) or 50% (very heavy, VH) of the difference between the lactic acid threshold (LAT) and peak VO(2) for 8 min. The O(2) deficit, calculated in the traditional manner, was significantly greater than the measured ROC for both above-LAT exercises: 4.03 +/- 1.01 vs. 2.63 +/- 0.80 (SD) liters for VH and 2.36 +/- 0.91 vs. 1.74 +/- 0.63 liters for H for the O(2) deficit vs. ROC (P < 0.05). When the kinetics were viewed as two separate components with independent onsets, the calculated O(2) deficit (2.89 +/- 0.79 and 1.71 +/- 0.70 liters for VH and H, respectively) was not different from the measured ROC (P < 0.05). Subjects also performed the same work rate for only 3 min. These data, from bouts terminated before the slow component could contribute appreciably to the overall VO(2) response, show that the O(2) requirement during the transition is less than the final steady state for the work rate, as evidenced by symmetry between the O(2) deficit and ROC. This new method of calculating the O(2) deficit more closely reflects the expected O(2) deficit-ROC relationship (i.e., ROC >/= O(2) deficit). Therefore, estimation of the O(2) deficit during heavy exercise transitions should consider the slow component of VO(2) as an additional deficit component with delayed onset.     

H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury

Non-phagocytic NAD(P)H oxidases have been implicated as major sources of reactive oxygen species in blood vessels. These oxidases can be activated by cytokines, thereby generating O(2), which is subsequently converted to H(2)O(2) and other oxidant species. The oxidants, in turn, act as important second messengers in cell signaling cascades. We hypothesized that reactive oxygen species, themselves, can activate the non-phagocytic NAD(P)H oxidases in vascular cells to induce oxidant production and, consequently, cellular injury. The current report demonstrates that exogenous exposure of non-phagocytic cell types of vascular origin (smooth muscle cells and fibroblasts) to H(2)O(2) activates these cell types to produce O(2) via an NAD(P)H oxidase. The ensuing endogenous production of O(2) contributes significantly to vascular cell injury following exposure to H(2)O(2). These results suggest the existence of a feed-forward mechanism, whereby reactive oxygen species such as H(2)O(2) can activate NAD(P)H oxidases in non-phagocytic cells to produce additional oxidant species, thereby amplifying the vascular injury process. Moreover, these findings implicate the non-phagocytic NAD(P)H oxidase as a novel therapeutic target for the amelioration of the biological effects of chronic oxidant stress.     

Synthesis and structural characterization of cadmium(II) complexes with 2-(2-pyridyl)imino-N-(2-thiazolin-2-yl)thiazolidine (PyTT)

We have carried out the synthesis of the cadmium coordination compounds [Cd(NO₃)₂(PyTT)(H₂O)] (1) and [CdCl₂{(μ-Cl)₂CdCl(μ-Cl)(μ-PyTT)Cd}₂]_n (2), together with their structural determination by means of X-ray diffraction. The compounds were also characterized through elemental analysis and infrared spectroscopy. The first complex presents a distorted pentagonal bipyramidal geometry with the axial positions occupied by one oxygen atom from a water molecule and a second one from a nitrate ion which acts as a monodentate ligand, whereas the equatorial plane contains three nitrogen atoms from the organic moiety and two oxygen atoms coming from the other nitrate group, which is bidentate. The structure of the second complex consists of parallel sheets linked by van der Waals forces, each one made up of structural units [CdCl₂{(μ-Cl)₂CdCl(μ-Cl)(μ-PyTT)Cd}₂], which possesses two PyTT ligands, 10 bridging chloro ligands and 5 cadmium(II) centres belonging to three environment types: octahedral CdN₂Cl₄, octahedral CdCl₆, on which a centre of symmetry is located, and tetrahedral CdNCl₃, present in a 2:1:2 ratio.     

Seven in Absentia Homolog 2 (Siah2) Protein Is a Regulator of NF-E2-related Factor 2 (Nrf2)

Under pathological conditions such as ischemia-reperfusion, Nrf2 acts as a key regulator of cellular oxidative response. Provided that Nrf2 is sensitive to hypoxia during ischemia, Nrf2 may affect reactive oxygen species metabolism during reoxygenation. In this study, hypoxia suppressed Nrf2 protein, and its hypoxic suppression was not recovered with knockdown of the Nrf2 repressor Keap1. Moreover, an Nrf2 mutant lacking the Keap1 binding domain was suppressed under hypoxia, suggesting that Keap1 does not contribute to hypoxic Nrf2 suppression. HIF-1α and Siah2 are both key regulators of hypoxic responses. Hypoxia induced the Siah2 protein. Although inhibition or knockdown of Siah2 prevented the suppression of Nrf2, knockdown of HIF-1α did not. Moreover, Siah2 interacted with Nrf2 through a binding motif, suggesting that Siah2 contributes to the suppression of Nrf2. Some cytosolic kinases also play important roles in Nrf2 regulation. In this study, PKC phosphorylates serine residues of Nrf2 during hypoxia. Knockdown of Siah2 rescued hypoxic decreases in an Nrf2 mutant that mimicked phosphorylation at serine 40 or lacked this phosphorylation site, suggesting that Siah2 contributes to the degradation of Nrf2 irrespective of its phosphorylation status. Moreover, knockdown of Siah2 attenuated ubiquitination of the Nrf2 mutant, suggesting that association of Siah2 with Nrf2 causes proteasome-mediated degradation of Nrf2.     

Cellular localization of THIK-1 (K(2P)13.1) and THIK-2 (K(2P)12.1) K channels in the mammalian kidney.

K(+)-channels fulfill several important functions in the mammalian kidney such as volume regulation, recirculation and secretion of K(+) ions, and maintaining the resting potential. In this study we used immunocytochemical methods, in situ hybridization, and nephron segment-specific RT-PCR to obtain a detailed picture of the cellular localization of two tandem pore domain potassium (K(2P)) channels, THIK-1 (K(2P)13.1, KCNK13) and THIK-2 (K(2P)12.1, KCNK12). Monospecific antibodies against C-terminal domains of rat THIK-1 and THIK-2 proteins (GST-fusion proteins) were raised in rabbits, freed from cross-reactivity, and affinity purified. All antibodies were validated by Western blot analysis, competitive ELISA, and preabsorption experiments. The expression of THIK channels in specific nephron segments was confirmed by double staining with marker proteins. Results indicate that in rat and mouse THIK-1 and THIK-2 were expressed in the proximal tubule (PT), thick ascending limb (TAL), connecting tubule (CNT), and cortical collecting duct (CCD). In human kidney THIK-1 and THIK-2 were localized in PT, TAL and CCD. Immunostaining of rat tissue revealed an intracellular expression of THIK-1 and THIK-2 throughout the identified nephron segments. However in mouse kidney THIK-2 was identified in basolateral membranes. Overall, the glomerulus, thin limbs and medullary collecting ducts were devoid of THIK-1 and THIK-2 signal. In summary, THIK-1 and THIK-2 are abundantly expressed in the proximal and distal nephron of the mammalian kidney.     

Partikelgestalt des Spargel-Virus 2 (asparagus virus 2) (Kurze Mitteilung)

Zwei Kernpolyedervirus‐Isolate der Kohleule aus Deutschland (MbKPV‐D) und Moldawien (MbKPV‐Ki) wurden im Biotest geprüft und der Genotyp mit Hilfe der Restriktionsenzym‐Fragmentanalyse (REN) untersucht. Beide Isolate ergaben eine gleiche biologische Aktivität. Die REN‐Profile zeigten weitestgehende Übereinstimmung in der DNA‐Struktur. Geringe Unterschiede wurden in den Profilen der Eco RI‐ und Hind III‐ Schnitte gefunden. Die erhaltenen REN‐Profile stimmen im wesentlichen mit den für ein niederländisches MbKPV‐Isolate beschriebenen Bandenmustern überein.     

Stereospecific formation of alpha-hydroxycarboxylato oxo peroxo complexes of vanadium(V). Crystal structure of (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O

An overview of structurally characterized alpha-hydroxycarboxylatodioxo- and alpha-hydroxycarboxylatooxoperoxovanadates(V) is presented and the geometric parameters of the V2O2 bridging core are discussed. The first case of a stereospecific formation of oxoperoxovanadates(V) is reported: The crystal structures of the isomeric compounds (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O (lact = C3H4O3(2-), the anion of the lactic acid) differ mainly in the arrangement of the V2O2 core and in mutual orientation of the V=O bonds. The complexes with achiral ligands adopt the same structural type as the complexes formed from a racemic mixture of a chiral ligand, while the structure obtained using an enantiopure L,L-hydroxycarboxylate is different.