Osteopontin is involved in urotensin II-induced migration of rat aortic adventitial fibroblasts

Recent studies suggest that both osteopontin and urotensin II (UII) play critical roles in vascular remodeling. We previously showed that UII could stimulate the migration of aortic adventitial fibroblasts. In this study, we examined whether osteopontin is involved in UII-induced migration of rat aortic adventitial fibroblasts and examined the effects and mechanisms of UII on osteopontin expression in adventitial fibroblasts. Migration of adventitial fibroblasts induced by UII could be inhibited significantly by osteopontin antisense oligonucleotide (P < 0.01) but not sense or mismatch oligonucleotides (P > 0.05). Moreover, UII dose- and time-dependently promoted osteopontin mRNA expression and protein secretion in the cells, with maximal effect at 10⁻⁸ mol/l at 3 h for mRNA expression or at 12 h for protein secretion (both P < 0.01). Furthermore, the UII effects were significantly inhibited by its receptor antagonist SB710411 (10⁻⁶ mol/l), and Ca²⁺ channel blocker nicardipine (10⁻⁵ mol/l), protein kinase C (PKC) inhibitor H7 (10⁻⁵ mol/l), calcineurin inhibitor cyclosporine A (10⁻⁵ mol/l), mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10⁻⁵ mol/l) and Rho kinase inhibitor Y-27632 (10⁻⁵ mol/l). Thus, osteopontin is involved in the UII-induced migration of adventitial fibroblasts, and UII could upregulate osteopontin gene expression and protein synthesis in rat aortic adventitial fibroblasts by activating its receptor and the Ca²⁺ channel, PKC, calcineurin, MAPK and Rho kinase signal transduction pathways.     

Magnetic properties of six-coordinated high-spin cobalt(II) complexes: Theoretical background and its application

In this contribution we study and analyse the influence of the different parameters involved in the magnetic susceptibility of six-coordinated high-spin Co(II) complexes. We propose an empirical expression to fit the magnetic susceptibility of polycrystalline samples of mononuclear Co(II) complexes with an axial distortion, the variable parameters being Δ (axial distortion), α (orbital reduction factor) and λ (spin–orbit coupling). This expression avoids solving the 12 × 12 matrix associated to the distortion of the ⁴T_1g term. In order to take into account the magnetic coupling (J) in the polynuclear Co(II) complexes, a perturbational approach is proposed to describe their magnetic susceptibility in the whole temperature range (2–300 K) as a function of J, Δ, α and λ. This approach is valid in the limit of the weak magnetic coupling as compared to the spin–orbit coupling, |J/λ| < 0.1. The model allows the treatment of each cobalt(II) ion in axial symmetry as an effective spin S_eff = 1/2. That causes a drastic reduction of the matrix size of the polynuclear compounds from 12ⁿ × 12ⁿ to 2ⁿ × 2ⁿ, n being the number of Co(II) ions in the complex. The main advantage of the model is to make possible the fit of the magnetic susceptibility data of those polynuclear Co(II) complexes whose high nuclearity involved intractable matrices.     

Stability constants and multinuclear NMR measurements of phosphonic acid derivatives with aluminum in aqueous solutions

We are reporting the stability constants of the different complexes between phosphonoacetic acid (PAA), phosphonoformic acid (PFA), aminomethylphosphonic acid (AMPA), aminoethylphosphonic acid (AEPA) and methylenediphosphonic acid (MDP) with the aluminum metal ion in aqueous solutions. (In this study the term aluminum is reserved for the 3+ ion.) The affinity of the phosphonic acid derivatives to chelate aluminum has been tested by potentiometric titrations with I = 0.10 M KNO₃ at 25 ± 0.1 °C. The proposed aluminum–ligand complex structures have been confirmed by ³¹P NMR, ¹³C NMR, and ²⁷Al NMR experiments. Both PAA and PFA formed simple one to one complexes. The respective values for PAA are log β₁₁₁ = 13.57, log β₁₁₀ = 10.58, and log β₁₁₋₁ = 5.84. The respective values for PFA are log β₁₁₂ = 15.24, log β₁₁₁ = 13.11, and log β₁₁₀ = 6.88. In contrast to PAA and PFA, the major species formed with AMPA and AEPA consist of a series of dimeric complexes. The ³¹P NMR spectra of these complexes indicate that the amine groups do not co-ordinate to aluminum and remain protonated. In addition to these dimeric complexes, a monoprotonated monomer of Al–AMPA also has been detected. The ²⁷Al NMR experiments indicated that the aluminum is hexacoordinated in all the complexes in this study and the hydroxide ion did not remove aluminium from its complexes. Among the ligands studied, PAA and PFA were able to solubilize aluminum at physiological pH. A comparison between the acidities and the chelating properties of the ligands used is presented.     

Molecular characterization of Rifr mutations in Pseudomonas aeruginosa and Pseudomonas putida

The rpoB gene encoding for β subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rif^r) phenotype of bacteria. Here we have characterized rpoB/Rif^r system in Pseudomonas aeruginosa and Pseudomonas putida as a test system for studying mutational processes. We found that in addition to the appearance of large colonies which were clearly visible on Rif selective plates already after 24 h of plating, small colonies grew up on these plates for 48 h. The time-dependent appearance of the mutant colonies onto selective plates was caused by different levels of Rif resistance of the mutants. The Rif^r clusters of the rpoB gene were sequenced and analyzed for 360 mutants of P. aeruginosa and for 167 mutants of P. putida. The spectrum of Rif^r mutations characterized for P. aeruginosa grown at 37 °C and that characterized for P. putida grown at 30 °C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30 °C. The strong Rif^r phenotype of P. aeruginosa and P. putida was accompanied only with substitutions of these residues which belong to the putative Rif-binding pocket. Approximately 70% of P. aeruginosa mutants, which were isolated at 37 °C and expressed weak Rif^r phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30 °C and 37 °C can be explained by temperature-sensitive growth of several mutants in the presence of rifampicin. Thus, our results imply that both the temperature for the growth of bacteria and the time for isolation of Rif^r mutants from selective plates are critical when the rpoB/Rif^r test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures.     

Evaluation of the association of mercury(II) with some dicysteinyl tripeptides

The present study was undertaken to gain insight into the associations of mercury(II) with dicysteinyl tripeptides in buffered media at pH 7.4. We investigated the effects of increasing the distance between cysteinyl residues on mercury(II) associations and complex formations. The peptide–mercury(II) formation constants and their associated thermodynamic parameters in 3-(N-morpholino)propanesulfonic acid (MOPS) buffered solutions were evaluated by isothermal titration calorimetry. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides in ammonium formate buffered solutions were characterized by LTQ Orbitrap mass spectrometry. The results from these studies show that n-alkyl dicysteinyl peptides (CP 1–4), and an aryl dicysteinyl peptide (CP 5) can serve as effective “double anchors” to accommodate the coordination sites of mercury(II) to form predominantly one-to-one Hg(peptide) complexes. The aryl dicysteinyl peptide (CP 5) also forms the two-to-two Hg₂(peptide)₂ complex. In the presence of excess peptide, Hg(peptide)₂ complexes are also detected. Notably, increasing the distance between the ligating groups or “anchor points” in CP 1–5 does not significantly affect their affinity for mercury(II). However, the enthalpy change (ΔH) values (ΔH₁ ∼ −91 kJ mol⁻¹ and ΔH₂ ∼ −66 kJ mol⁻¹) for complex formation between CP 4 and 5 with mercury(II) are about one and a half times larger than the related values for CP 1, 2 and 3 (ΔH₁ ∼ −66 kJ mol⁻¹ and ΔH₂ ∼ 46 kJ mol⁻¹). The corresponding entropy change (ΔS) values (ΔS₁ ∼ −129 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −116 J K⁻¹ mol⁻¹) of the structurally larger dicysteinyl peptides CP 4 and 5 are less entropically favorable than for CP 1, 2 and 3 (ΔS₁ ∼ −48 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −44 J K⁻¹ mol⁻¹). Generally, these associations result in a decrease in entropy, indicating that these peptide–mercury complexes potentially form highly ordered structures. The results from this study show that dicysteinyl tripeptides are effective in binding mercury(II) and they are promising motifs for the design of multi-cysteinyl peptides for binding more than one mercury(II) ion per peptide.     

Influence of urea and calcium dosage on the effectiveness of bacterially induced carbonate precipitation on limestone

Bacterially induced carbonate precipitation has been explored for the protection and consolidation of ornamental stone. Attempts to improve the efficiency of this biodeposition process were primarily focused on the microbial aspects, i.e. type of microorganism and metabolic pathway. In this study, the influence of the chemical parameters, i.e. concentration of calcium salts and urea, on the effectiveness of the biodeposition treatment has been examined. The amount of calcium carbonate that can be precipitated in the stone is conditioned both by the amount of cells retained in the stone and the concentration of urea and calcium used. From sonication experiments, a good consolidation was observed for limestone prisms treated with a calcium dosage of 17 g Ca²⁺ m^?2 with no improvement at higher concentrations. For limestone prisms of 4 cm × 2 cm × 1 cm, the biodeposition treatment resulted in a 63% lower weight loss upon sonication compared to untreated specimens. The waterproofing effect was observed to increase with increasing calcium dosages. While for a calcium dosage of 17 g Ca²⁺ m^?2 the water absorption was similar to that of untreated specimens, concentrations of 67 g Ca²⁺ m^?2 resulted in a 50% decrease of the rate of water absorption. For calcium dosages higher than 34 g Ca²⁺ m^?2 a significant change in the visual aspect (ΔE > 6) of the treated stones could be observed. Overall, the urea/calcium chloride-based biodeposition treatment attained a protective performance comparable with that of the commonly used ethylsilicates.     

The effect of nitrogen containing bisphosphonates,zoledronate and alendronate,on the production of pro-angiogenic factors by osteoblastic cells

Bisphosphonates (BPs) have been shown to influence angiogenesis. This may contribute to BP-associated side-effects such as osteonecrosis of the jaw (ONJ) or atypical femoral fractures (AFF). The effect of BPs on the production of angiogenic factors by osteoblasts is unclear. The aims were to investigate the effect of (1) alendronate on circulating angiogenic factors; vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) in vivo and (2) zoledronate and alendronate on the production of VEGF and ANG-1 by osteoblasts in vitro. We studied 18 post-menopausal women with T score ⩽ −2 randomized to calcium/vitamin D only (control arm, n = 8) or calcium/vitamin D and alendronate 70 mg weekly (treatment arm, n = 10). Circulating concentrations of VEGF and ANG-1 were measured at baseline, 3, 6 and 12 months. Two human osteoblastic cell lines (MG-63 and HCC1) and a murine osteocytic cell line (MLO-Y4) were treated with zoledronate or alendronate at concentrations of 10⁻¹²–10⁻⁶ M. VEGF and ANG-1 were measured in the cell culture supernatant. We observed a trend towards a decline in VEGF and ANG-1 at 6 and 12 months following treatment with alendronate (p = 0.08). Production of VEGF and ANG-1 by the MG-63 and HCC1 cells decreased significantly by 34–39% (p < 0.01) following treatment with zoledronate (10⁻⁹–10⁻⁶ M). Treatment of the MG-63 cells with alendronate (10⁻⁷ and 10⁻⁶) led to a smaller decrease (25–28%) in VEGF (p < 0.05). Zoledronate (10⁻¹⁰–10⁻⁶ M) suppressed the production of ANG-1 by MG-63 cells with a decrease of 43–49% (p < 0.01). Co-treatment with calcitriol (10⁻⁸ M) partially reversed this zoledronate-induced inhibition. BPs suppress osteoblastic production of angiogenic factors. This may explain, in part, the pathogenesis of the BP-associated side-effects.     

Influence of temperature on calcium sensitivity in the ventricular myocardium of the South American lungfish: Effects of extracellular calcium and adrenaline

The mechanical properties of ventricular myocardium of the South American lungfish, Lepidosiren paradoxa, acclimated to 25 °C, were evaluated in vitro at 15, 25 and 35 °C. The inotropism (Fc—% of initial values) of ventricle strips was examined in response to adrenaline (from 10⁻⁸ to 10⁻⁵ M) and extracellular calcium (from 2.5 to 14.5 mM) in all experimental temperatures. At 15 and 25 °C, Fc rose when extracellular Ca²⁺ or adrenaline were increased, while Fc remained unchanged at 35 °C. These results suggest that at lower temperatures Ca²⁺ availability is a limiting step to cardiac performance and can be ameliorated by adrenergic stimulation. In contrast, since inotropic agents failed to increase cardiac inotropism at 35 °C, lungfish myocytes seem to show a high temperature sensitivity, which increases Ca²⁺-buffering capacity and/or Ca²⁺ transportation from and to cytosol as well as myofilaments Ca²⁺ sensitivity.     

DNA binding and photocleavage properties and apoptosis-inducing activities of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)2Cl]Cl and its heterometallic derivatives

The interactions of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)₂Cl]Cl (phen = 1,10-phenanthroline, (Py-3′)TPP = 5-(3′-pyridyl-10,15,20-triphenylporphyrin) (1) and its heterometallic derivatives, [Ni(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (2) and [Cu(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (3), with calf thymus DNA have been investigated by spectroscopic and viscosity measurements in this study. The results showed that these synthetic complexes can bind to double strand helix DNA in groove binding mode, and the intrinsic binding constants of complexes 1, 2 and 3, as calculated according to the decay of the Soret absorption, are (1.35 ± 0.5) ×10⁵ M^?1 (s = 4.2), (1.29 ± 0.5) × 10⁵ M^?1 (s = 5.6) and (1.22 ± 0.5) × 10⁵ M^?1 (s = 6.2) (s is the binding-site size), respectively, which are consistent with those obtained from ethidium bromide-quenching experiments. Further investigations on the photocleavage properties of these complexes on plasmid pBR 322 DNA showed that complexes 1, 2 and 3 could cleave single chain DNA and convert DNA molecules from supercoiled form to the nicked form. As determined by MTT assay, the complexes were also identified as potent antiproliferative agents against A375 human melanoma cells, MCF-7 human breast adrenocarcinoma cells, Colo201 human colon adenocarcinoma cells and HepG2 human liver cancer cells. Complex 1 inhibits the growth of A375 cells through induction of apoptotic cell death and G0/G1 cell cycle arrest. Further investigation on intracellular mechanisms indicated that Complex 1 induced depletion of mitochondrial membrane potential (ΔΨ_m) in A375 cells through regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Our results suggest that ruthenium porphyrin complexes could be candidates for further evaluation as chemopreventive and chemotherapeutic agents for human cancers.     

Comparison of the physical characteristics of chlorosomes from three different phyla of green phototrophic bacteria

Chlorosomes, the major antenna complexes in green sulphur bacteria, filamentous anoxygenic phototrophs, and phototrophic acidobacteria, are attached to the cytoplasmic side of the inner cell membrane and contain thousands of bacteriochlorophyll (BChl) molecules that harvest light and channel the energy to membrane-bound reaction centres. Chlorosomes from phototrophs representing three different phyla, Chloroflexus (Cfx.) aurantiacus, Chlorobaculum (Cba.) tepidum and the newly discovered “Candidatus (Ca.) Chloracidobacterium (Cab.) thermophilum” were analysed using PeakForce Tapping atomic force microscopy (PFT-AFM). Gentle PFT-AFM imaging in buffered solutions that maintained the chlorosomes in a near-native state revealed ellipsoids of variable size, with surface bumps and undulations that differ between individual chlorosomes. Cba. tepidum chlorosomes were the largest (133 × 57 × 36 nm; 141,000 nm³ volume), compared with chlorosomes from Cfx. aurantiacus (120 × 44 × 30 nm; 84,000 nm³) and Ca. Cab. thermophilum (99 × 40 × 31 nm; 65,000 nm³). Reflecting the contributions of thousands of pigment–pigment stacking interactions to the stability of these supramolecular assemblies, analysis by nanomechanical mapping shows that chlorosomes are highly stable and that their integrity is disrupted only by very strong forces of 1000–2000 pN. AFM topographs of Ca. Cab. thermophilum chlorosomes that had retained their attachment to the cytoplasmic membrane showed that this membrane dynamically changes shape and is composed of protrusions of up to 30 nm wide and 6 nm above the mica support, possibly representing different protein domains. Spectral imaging revealed significant heterogeneity in the fluorescence emission of individual chlorosomes, likely reflecting the variations in BChl c homolog composition and internal arrangements of the stacked BChls within each chlorosome.     

Effects of salt stress on volatile compounds,total phenolic content and antioxidant activities of Salvia mirzayanii

Salvia mirzayanii is a medicinal and aromatic plant belonging to the Lamiaceae family, which is an endemic plant in Iran. In this study, the effects of different salt concentrations on total phenolic content, antioxidant activities and volatile components of the aerial parts of S. mirzayanii were studied. The results showed that total phenolic content increased with the increase in salt concentration. The increase was more pronounced under moderate salinity (3.8 mg GAE g^− 1 DW at 6.8 dS m^− 1 NaCl). Plants grown at 6.8 dS m^− 1 NaCl displayed the highest DPPH˚ scavenging activity with the lowest IC₅₀ value (2.13 mg ml^− 1) compared to the control. The volatile components were identified and analyzed by HS (headspace)-GC–MS using the Combi PAL System technique. The main components of control plants were α-terpinyl acetate, 1,8-cineole and bicyclogermacrene. The proportions of these main compounds were differently affected by salinity stress. The results showed that the synthesis of both total phenolic and some important volatile components was induced by moderate salinity.     

Iron containing keratinolytic metallo-protease produced by Chryseobacterium gleum

Chryseobacterium gleum exhibited complete dissolution of whole chicken-feathers (10 g l^?1, pH 8) after 72 h at 30 °C through synthesis of keratinolytic protease when inoculated at 1% (v/v). This enzyme was purified to 67-fold with yield of 2.25% having a specific activity of 1670 U mg^?1 and ～36 kDa Mw. MALDI-TOF MS of this keratinase showed some similarity with the keratinase peptides of Bacillus subtilis (BOFXJ2). The keratinase action was inhibited by EDTA, iodoacetamide and metal ions like mercury, copper and zinc (1 mM each), while it was enhanced by iron and calcium. Keratinase showed presence of 3 mM of Fe M^?1 as tested by atomic absorption spectroscopy and addition of Fe in its apoenzyme retained about 79% of original residual feather degradation activity which portrayed it to be metalloprotease. Purified keratinase revealed significant degradation (85%) of feather concentrate (20 g l^?1) to 3.9 μM ml^?1 of free amino groups in 24 h at an initial pH of 8.0, 30 °C and 120 rpm shaking. This keratinase activity can be controlled precisely by presence of chemical or metal ions which could be of use in biotechnology industry while the culture can be used in poultry waste management.     

The use of transgenic mouse models to reveal the functions of Ca2 + buffer proteins in excitable cells

BackgroundCytosolic Ca^2 + buffers are members of the large family of Ca^2 +-binding proteins and are essential components of the Ca^2 + signaling toolkit implicated in the precise regulation of intracellular Ca^2 + signals. Their physiological role in excitable cells has been investigated in vivo by analyzing the phenotype of mice either lacking one of the Ca^2 + buffers or mice with ectopic expression.Scope of ReviewIn this review, results obtained with knockout mice for the three most prominent Ca^2 + buffers, parvalbumin, calbindin-D28k and calretinin are summarized.Major ConclusionsThe absence of Ca^2 + buffers in specific neuron subpopulations, and for parvalbumin additionally in fast-twitch muscles, leads to Ca^2 + buffer-specific changes in intracellular Ca^2 + signals. This affects the excitation–contraction cycle in parvalbumin-deficient muscles, and in Ca^2 + buffer-deficient neurons, properties associated with synaptic transmission (e.g. short-term modulation), excitability and network oscillations are altered. These findings have not only resulted in a better understanding of the physiological function of Ca^2 + buffers, but have revealed that the absence of Ca^2 + signaling toolkit components leads to protein-and neuron-specific adaptive/homeostatic changes that also include changes in neuron morphology (e.g. altered spine morphology, changes in mitochondria content) and network properties.General SignificanceThe complex phenotype of Ca^2 + buffer knockout mice arises from the direct effect of these proteins on Ca^2 + signaling and moreover from the homeostatic mechanisms induced in these mice. For a better mechanistic understanding of neurological diseases linked to disturbed/altered Ca^2 + signaling, a global view on Ca^2 + signaling is expected to lead to new avenues for specific therapies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Amperometric biosensor based on a site-specific immobilization of acetylcholinesterase via affinity bonds on a nanostructured polymer membrane with integrated multiwall carbon nanotubes

Acetylcholinesterase (AChE) was immobilized on chemically modified poly-(acrylonitrile-methyl-methacrylate-sodium vinylsulfonate) membranes in accordance with three different methods, the first of which involved random enzyme immobilization via glutaraldehyde, the second one—site-specific enzyme immobilization via glutaraldehyde and Concanavalin A (Con A) and the third method—modified site-specific enzyme immobilization via glutaraldehyde in the presence of a mixture of multiwall carbon nanotubes and albumin (MWCNs + BSA), glutaraldehyde and Con A. Preliminary tests for the activity of immobilized AChE were carried out using these three methods. The third method was selected as the most efficient one for the immobilization of AChE and the prepared enzyme carriers were used for the construction of amperometric biosensors for the detection of acetylthiocholine (ATCh).A five level three factorial central composite design was chosen to determine the optimal conditions for the enzyme immobilization with three critical variables: concentration of enzyme, Concanavalin A and MWCNs. The design illustrated that the optimum values of the factors influencing the amperometric current were C_E: 70 U mL⁻¹; C_{Con A}: 1.5 mg mL⁻¹ and C_MWCN: 11 mg mL⁻¹, with an amperometric current 0.418 μA. The basic amperometric characteristics of the constructed biosensor were investigated. A calibration plot was obtained for a series of ATCh concentrations ranging from 5 to 400 μM. A linear interval was detected along the calibration curve from 5 to 200 μM. The correlation coefficient for this concentration range was 0.995. The biosensor sensitivity was calculated to be 0.065 μA μM⁻¹ cm⁻². The detection limit with regard to ATCh was calculated to be 0.34 μM. The potential application of the biosensor for detection and quantification of organophosphate pesticides was investigated as well. It was tested against sample solutions of Paraoxon. The biosensor detection limit was determined to be 1.39 × 10⁻¹² g L⁻¹ of Paraoxon, as well as the interval (10⁻¹¹ to 10⁻⁸ g L⁻¹) within which the biosensor response was linearly dependant on the Paraoxon concentration. Finally the storage stability of the enzyme carrier was traced for a period of 120 days. After 30-day storage the sensor retained 76% of its initial current response, after 60 days—68% and after 120 days—61%.     

Molecular characterization of a novel Na+/H+ antiporter cDNA from Eucalyptus globulus

Environmental stress factors such as salt, drought and heat are known to affect plant productivity. However, high salinity is spreading throughout the world, currently affecting more than 45 million ha. One of the mechanisms that allow plants to withstand salt stress consists on vacuolar sequestration of Na⁺, through a Na⁺/H⁺ antiporter. We isolated a new vacuolar Na⁺/H⁺ antiporter from Eucalyptus globulus from a cDNA library. The cDNA had a 1626 bp open reading frame encoding a predicted protein of 542 amino acids with a deduced molecular weight of 59.1 KDa. Phylogenetic and bioinformatic analyses indicated that EgNHX1 localized in the vacuole. To assess its role in Na⁺ exchange, we performed complementation studies using the Na⁺ sensitive yeast mutant strain Δnhx1. The results showed that EgNHX1 partially restored the salt sensitive phenotype of the yeast Δnhx1 strain. However, its overexpression in transgenic Arabidopsis confers tolerance in the presence of increasing NaCl concentrations while the wild type plants exhibited growth retardation. Expression profiles of Eucalyptus seedlings subjected to salt, drought, heat and ABA treatment were established. The results revealed that Egnhx1 was induced significantly only by drought. Together, these results suggest that the product of Egnhx1 from E. globulus is a functional vacuolar Na⁺/H⁺ antiporter.     

Nonspecific inhibition of nitric oxide synthesis evokes endothelin-dependent increases in myocardial contractility

The role of endogenous nitric oxide (NO) in modulating myocardial contractility is still unclear, in part because of unknown, secondary effects of blocking NO release. We hypothesized that the nonspecific inhibition of nitric oxide synthase (NOS) enhances endothelin-1 (ET-1) effects, which can play a role in ET-A receptor-dependent myocardial contractile responses. The myocardial contractility was estimated from the slope of the left ventricular end-systolic pressure–diameter relationship in closed-chest, pentobarbital-anesthetized dogs. Group 1 (n = 7) was the saline-treated control, while in groups 2 (n = 7) and 3 (n = 7) N-nitro-l-arginine (NNA, 4 mg kg^?1), a nonselective NOS blocker, was administered with or without pretreatment with the ET-A receptor antagonist ETR-P1/fl peptide (100 nmol kg^?1 iv). Plasma ET-1, nitrite/nitrate (NO_x) and blood superoxide levels were measured, and myocardial ET-1 content and xanthine oxidoreductase (XOR) activity were determined from myocardial biopsies. The infusion of NNA over 120 min decreased the plasma NO_x, significantly elevated the plasma ET-1 and blood superoxide levels, and in parallel greatly increased the left ventricular contractility as compared with the untreated controls [47.5 vs 30 mm Hg mm^?1]. The myocardial ET-1 content decreased simultaneously, while the XOR activity and blood superoxide level were significantly elevated. These effects, including NNA-induced positive inotropy, were significantly suppressed by pretreatment with ETR-P1/fl peptide. These results demonstrate that a diminished NO synthesis leads to a preponderant ET-1 effect, which increases myocardial contractility through an ET-A receptor-dependent mechanism.     

Trispyrazolylmethane piano stool complexes of iron(II) and cobalt(II)

A series of cationic trispyrazolylmethane complexes of the general form [Tm^RM(CH₃CN)₃]²⁺ (Tm = tris(pyrazolyl)methane, 1, R = 3,5-Me₂, M = Fe(II); 2, R = 3-Ph, M = Fe(II); 3, R = 3,5-Me₂, M = Co(II); 4, R = 3-Ph, M = Co(II)) with ‘piano-stool’ structures was prepared by the reaction of the N₃tripodal ligands (Tm^R)with [(CH₃CN)₆M](BF₄)₂ in a 1:1 stoichiometric ratio. Magnetic susceptibility measurements indicate that all four complexes with BF₄⁻ counter anions are paramagnetic, high-spin systems in the solid state with μ_eff at high temperatures of 5.2 (1, S = 2), 5.4 (2, S = 2), 4.9 (3, S = 3/2) and 4.6 (4, S = 3/2) BM, respectively. Comparisons of bond lengths from the metal centre to the Tm^R nitrogen donors, and from the metal centre to the acetonitrile nitrogen donors indicate that the neutral tripodal ligands appear to be more weakly coordinated to the metal centre than are the acetonitrile ligands. Reactions of these tripodal complexes with bidentate phosphine ligands, such as 1,2-diphosphinoethane or 1,2-bis(diallylphosphino)ethane leads to displacement of the tripodal ligand, or to the formation of more thermally stable bis-ligand complexes M(Tm^R)₂ (R = 3,5-dimethyl).     

Overexpression of the TIR-X gene results in a dwarf phenotype and activation of defense-related gene expression in Arabidopsis thaliana

The Arabidopsis genome encodes various proteins with a Toll/interleukin-1 receptor (TIR) domain. Many of these proteins also contain nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains and function as resistance (R) proteins. However, the protein encoded by At2g32140 (a TIR-X gene) contains a TIR domain but lacks NBS and LRR domains. We found that transgenic plants overexpressing At2g32140 displayed a dwarf phenotype and showed increased expression of defense-related genes. In general, the growth defect caused by activation of defense responses is suppressed under high-temperature conditions. However, transgenic plants overexpressing At2g32140 displayed a much stronger dwarf phenotype at 28 °C than at 22 °C. This dwarf phenotype was suppressed under the combination with known salicylic-acid pathway mutants. These findings suggest that At2g32140 encodes a protein involved in the plant defense response.     

Stimuli responsive polymorphism of C12NO/DOPE/DNA complexes: Effect of pH,temperature and composition

N,N-dimethyldodecylamine-N-oxide (C₁₂NO) is a surfactant that may exist either in a neutral or cationic protonated form depending on the pH of aqueous solutions. Using small angle X-ray diffraction (SAXD) we observe the rich structural polymorphism of pH responsive complexes prepared due to DNA interaction with C₁₂NO/dioleoylphosphatidylethanolamine (DOPE) vesicles and discuss it in view of utilizing the surfactant for the gene delivery vector of a pH sensitive system. In neutral solutions, the DNA uptake is low, and a lamellar L_α phase formed by C₁₂NO/DOPE is prevailing in the complexes at 0.2 ≤ C₁₂NO/DOPE < 0.6 mol/mol. A maximum of ~ 30% of the total DNA volume in the sample is bound in a condensed lamellar phase L_α^C at C₁₂NO/DOPE = 1 mol/mol and pH 7.2. In acidic conditions, a condensed inverted hexagonal phase H_II^C was observed at C₁₂NO/DOPE = 0.2 mol/mol. Commensurate lattice parameters, a_HC ≈ d_LC, were detected at 0.3 ≤ C₁₂NO/DOPE ≤ 0.4 mol/mol and pH = 4.9–6.4 suggesting that L_α^C and H_II^C phases were epitaxially related. While at the same composition but pH ~ 7, the mixture forms a cubic phase (Pn3m) when the complexes were heated to 80 °C and cooled down to 20 °C. Finally, a large portion of the surfactant (C₁₂NO/DOPE > 0.5) stabilizes the L_α^C phase in C₁₂NO/DOPE/DNA complexes and the distance between DNA strands (d_DNA) is modulated by the pH value. Both the composition and pH affect the DNA binding in the complexes reaching up to ~ 95% of the DNA total amount at acidic conditions.     

Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson's disease

Homer1 protein is an important scaffold protein at postsynaptic density and has been demonstrated to play a central role in calcium signaling in the central nervous system. The aim of this study was to investigate the effects of Homer1 knockdown on MPP⁺ induced neuronal injury in cultured dopamine (DA) neurons. We found that down-regulating Homer1 expression with specific small interfering RNA (siRNA) significantly suppressed LDH release, reduced Propidium iodide (PI) or Hoechst staining, increased the number of tyrosine hydroxylase (TH) positive cells and DA uptake, and attenuated apoptotic and necrotic cell death after MPP⁺ injury. Homer1 knockdown decreased intracellular reactive oxygen species (ROS) generation through inhibition of intracellular calcium overload, but did not affect the endogenous antioxidant enzyme activities. Calcium imaging was used to examine the changes of intracellular Ca^2 + concentration ([Ca^2 +]_cyt) and Ca^2 + in endoplasmic reticulum (ER) ([Ca^2 +]_ER), and the results showed that Homer1 siRNA transfection attenuated ER Ca^2 + release up to 120 min after MPP⁺ injury. Furthermore, decrease of [Ca^2 +]_cyt induced by Homer1 knockdown in MPP⁺ treated neurons was further enhanced by NMDA receptor antagonists MK-801 and AP-5, but not canonical transient receptor potential (TRPC) channel antagonist SKF-96365. l-type calcium antagonist isradipine but not nimodipine further inhibited intracellular calcium overload after MPP⁺ insult in Homer1 down-regulated neurons. These results suggest that Homer1 knockdown has protective effects against neuronal injury in in vitro PD model by reducing calcium overload mediated ROS generation, and this protection may be dependent at least in part on the regulatory effects on the function of calcium channels in both plasma membrane and ER.