Endothelium-dependent relaxation of rat aorta to a histamine H3 agonist is reduced by inhibitors of nitric oxide synthase, guanylate cyclase and Na+,K+-ATPase

The possible involvement of different effector systems (nitric oxide synthase, guanylate cyclase, β-adrenergic and muscarinic cholinergic receptors, cyclooxygenase and lipoxygenase, and Na+,K+-ATPase) was evaluated in a histamine H3 receptor agonist-induced ((R)α-methylhistamine, (R)α-MeHA) endothelium-dependent rat aorta relaxation assay. (R)α-MeHA (0.1 nM – 0.01 mM) relaxed endothelium-dependent rat aorta, with a pD2 value of 8.22 ± 0.06, compared with a pD2 value of 7.98 ± 0.02 caused by histamine (50% and 70% relaxation, respectively). The effect of (R)α-MeHA (0.1 nM – 0.01 mM) was competitively antagonized by thioperamide (1, 10 and 30 nM) (pA2 = 9.21 ± 0.40; slope = 1.03 ± 0.35) but it was unaffected by pyrilamine (100 nM), cimetidine (1 μM), atropine (10 μM), propranolol (1 μM), indomethacin (10 μM) or nordthydroguaiaretic acid (0.1 mM). Inhibitors of nitric oxide synthase, L-NG-monomethylarginine (L-NMMA, 10 μM) and NG-nitro-L-arginine methylester (L-NOARG, 10 μM) inhibited the relaxation effect of (R)α-MeHA, by approximately 52% and 70%, respectively). This inhibitory effect of L-NMMA was partially reversed by L-arginine (10 μM). Methylene blue (10 μM) and ouabain (10 μM) inhibited relaxation (R)α-MeHA-induced by approximately 50% and 90%, respectively. The products of cyclooxygenase and lipoxygenase are not involved in (R)α-MeHA-induced endothelium-dependent rat aorta relaxation nor are the muscarinic cholinergic and β-adrenergic receptors. The results also suggest the involvement of NO synthase, guanylate cyclase and Na+,K+-ATPase in (R)α-MeHA-induced endothelium-dependent rat aorta relaxation

Response of Net Ecosystem Productivity of Three Boreal Forest Stands to Drought

In 2000-03, continuous eddy covariance measurements of carbon dioxide (CO2) flux were made above mature boreal aspen, black spruce, and jack pine forests in Saskatchewan, Canada, prior to and during a 3-year drought. During the 1st drought year, ecosystem respiration (R) was reduced at the aspen site due to the drying of surface soil layers. Gross ecosystem photosynthesis (GEP) increased as a result of a warm spring and a slow decrease of deep soil moisture. These conditions resulted in the highest annual net ecosystem productivity (NEP) in the 9 years of flux measurements at this site. During 2002 and 2003, a reduction of 6% and 34% in NEP, respectively, compared to 2000 was observed as the result of reductions in both R and GEP, indicating a conservative response to the drought. Although the drought affected most of western Canada, there was considerable spatial variability in summer rainfall over the 100-km extent of the study area; summer rainfalls in 2001 and 2002 at the two conifer sites minimized the impact of the drought. In 2003, however, precipitation was similarly low at all three sites. Due to low topographic position and consequent poor drainage at the black spruce site and the coarse soil with low water-holding capacity at the jack pine site almost no reduction in R, GEP, and NEP was observed at these two sites. This study shows that the impact of drought on carbon sequestration by boreal forest ecosystems strongly depends on rainfall distribution, soil characteristics, topography, and the presence of vegetation that is well adapted to these condition

Uticaj pentobarbitala i pentilenetetrazola na nivo azot oksida u frontalnom korteksu pacova

Levels of nitric oxide (NO) in the rats frontal cortex were continuously monitored before and after intraperitoneal administration of an antiepileptic drug-pentobarbital (20 and 40 mg/kg) or convulsant drug - pentylenetetrazol (50 mg/kg). Pentobarbital decreased the levels of NO in a dose dependent manner However, NO levels had a tendency to increase following the administration of pentylenetetrazol. It is suggested that central NO participates in the modulation of neuronal excitability, supporting the idea that NO is an important excitatory factor involved in the regulation of seizure susceptibility. Also, our results on anaesthetized rats suggests that endogenous NO may be involved in the mechanism of action of antiepileptic and analeptic drugs and this further suggest that NO levels in the human brain may decrease during antiepileptic therapy and increase during epileptic attacks or administration of excitatory drugs. The aim of the present study was to determine the possible role of NO levels in the brain during neuronal excitability and seizures.Nivo azot oksida (NO) u frontalnom korteksu pacova meren je kontinuirano kako pre, tako i nakon intraperitonealne primene antiepileptika pentobarbitala (u dozi od 20 i 40 mg/kg) ili konvulzivnog agensa pentilenetetrazola (u dozi od 50 mg/kg). Rezultati ovih eksperimenta su ukazali da pentobarbital smanjuje nivo NO u frontalnom korteksu pacova, dok koncentracija NO ima tendeciju rasta nakon primene pentilenetetrazola. Osim toga, dokazano je da endogeni NO ima važnu ekscitatornu ulogu u centralnim mehanizmima nastanka epilepsije. Takođe, naši rezultati su ukazali da kod anestetisanih životinja endogeni nivo NO ima uticaja na dejstvo kako antikonvulzivnih, tako i prokonvulzivnih lekova. Nivo NO u mozgu pacova je bio snižen tokom terapije antiepilepticima, a povišen tokom epileptičkih napada ili primene lekova iz grupe centralnih stimulansa

Global gene disruption in human cells to assign genes to phenotypes

Insertional mutagenesis in a haploid background can disrupt gene function[superscript 1]. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones[superscript 1] enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites[superscript 2]. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT

Preliminary Investigation of the Corrosion Behavior of Proprietary Micro-alloyed Steels in Aerated and Deaerated Brine Solutions

The corrosion performance of fairly new generation of micro-alloyed steels was compared in different concentrations of aerated and deaerated brines. Electrochemical polarization, weight loss and surface analyses techniques were employed. The results showed a threshold of corrosion rate at 3.5 wt.% NaCl in both aerated and deaerated solutions. The average corrosion current density for steel B, for example, increased from 1.3 µA cm¯² in 1 wt.% NaCl to 1.5 µA cm¯² in 3.5 wt.% NaCl, but decreased to 1.4 µA cm¯² in 10 wt.% deaerated NaCl solutions. The aerated solutions exhibited an average of over 80% increase in corrosion current density in the respective concentrations when compared with the deaerated solution. These results can be attributed to the effects of dissolved oxygen (DO) which has a maximum solubility in 3.5 wt.% NaCl. DO as a depolarizer and electron acceptor in cathodic reactions accelerates anodic metal dissolution. The difference in carbon content and microstructures occasioned by thermo-mechanical treatment contributed to the witnessed variation in corrosion performance of the steels. Specifically, the results of the various corrosion techniques corroborated each other and showed that the corrosion rate of the micro-alloyed steels can be ranked as CR[Steel A] < CRₓ₆₅ < CR[Steel B] < CR[Steel C]

A Global Overview of the Genetic and Functional Diversity in the Helicobacter pylori cag Pathogenicity Island

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAI–carrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown

Role of Cajal Bodies and Nucleolus in the Maturation of the U1 snRNP in Arabidopsis

Background: The biogenesis of spliceosomal snRNPs takes place in both the cytoplasm where Sm core proteins are added and snRNAs are modified at the 59 and 39 termini and in the nucleus where snRNP-specific proteins associate. U1 snRNP consists of U1 snRNA, seven Sm proteins and three snRNP-specific proteins, U1-70K, U1A, and U1C. It has been shown previously that after import to the nucleus U2 and U4/U6 snRNP-specific proteins first appear in Cajal bodies (CB) and then in splicing speckles. In addition, in cells grown under normal conditions U2, U4, U5, and U6 snRNAs/snRNPs are abundant in CBs. Therefore, it has been proposed that the final assembly of these spliceosomal snRNPs takes place in this nuclear compartment. In contrast, U1 snRNA in both animal and plant cells has rarely been found in this nuclear compartment. Methodology/Principal Findings: Here, we analysed the subnuclear distribution of Arabidopsis U1 snRNP-specific proteins fused to GFP or mRFP in transiently transformed Arabidopsis protoplasts. Irrespective of the tag used, U1-70K was exclusively found in the nucleus, whereas U1A and U1C were equally distributed between the nucleus and the cytoplasm. In the nucleus all three proteins localised to CBs and nucleoli although to different extent. Interestingly, we also found that the appearance of the three proteins in nuclear speckles differ significantly. U1-70K was mostly found in speckles whereas U1A and U1C in,90 % of cells showed diffuse nucleoplasmic in combination with CBs and nucleolar localisation. Conclusions/Significance: Our data indicate that CBs and nucleolus are involved in the maturation of U1 snRNP. Difference

Functional and Transcriptional Induction of Aquaporin-1 Gene by Hypoxia; Analysis of Promoter and Role of Hif-1α

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O2 transport. It has been reported that this protein contributes to gas permeation (CO2, NO and O2) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5′ proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl2) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases

Role of Cajal Bodies and Nucleolus in the Maturation of the U1 snRNP in Arabidopsis

Background: The biogenesis of spliceosomal snRNPs takes place in both the cytoplasm where Sm core proteins are added and snRNAs are modified at the 59 and 39 termini and in the nucleus where snRNP-specific proteins associate. U1 snRNP consists of U1 snRNA, seven Sm proteins and three snRNP-specific proteins, U1-70K, U1A, and U1C. It has been shown previously that after import to the nucleus U2 and U4/U6 snRNP-specific proteins first appear in Cajal bodies (CB) and then in splicing speckles. In addition, in cells grown under normal conditions U2, U4, U5, and U6 snRNAs/snRNPs are abundant in CBs. Therefore, it has been proposed that the final assembly of these spliceosomal snRNPs takes place in this nuclear compartment. In contrast, U1 snRNA in both animal and plant cells has rarely been found in this nuclear compartment. Methodology/Principal Findings: Here, we analysed the subnuclear distribution of Arabidopsis U1 snRNP-specific proteins fused to GFP or mRFP in transiently transformed Arabidopsis protoplasts. Irrespective of the tag used, U1-70K was exclusively found in the nucleus, whereas U1A and U1C were equally distributed between the nucleus and the cytoplasm. In the nucleus all three proteins localised to CBs and nucleoli although to different extent. Interestingly, we also found that the appearance of the three proteins in nuclear speckles differ significantly. U1-70K was mostly found in speckles whereas U1A and U1C in,90 % of cells showed diffuse nucleoplasmic in combination with CBs and nucleolar localisation. Conclusions/Significance: Our data indicate that CBs and nucleolus are involved in the maturation of U1 snRNP. Difference

Olprinone Attenuates the Acute Inflammatory Response and Apoptosis after Spinal Cord Trauma in Mice

BACKGROUND: Olprinone hydrochloride is a newly developed compound that selectively inhibits PDE type III and is characterized by several properties, including positive inotropic effects, peripheral vasodilatory effects, and a bronchodilator effect. In clinical settings, olprinone is commonly used to treat congestive cardiac failure, due to its inotropic and vasodilating effects. The mechanism of these cardiac effects is attributed to increased cellular concentrations of cAMP. The aim of the present study was to evaluate the pharmacological action of olprinone on the secondary damage in experimental spinal cord injury (SCI) in mice. METHODOLOGY/PRINCIPAL FINDINGS: Traumatic SCI is characterized by an immediate, irreversible loss of tissue at the lesion site, as well as a secondary expansion of tissue damage over time. Although secondary injury should be preventable, no effective treatment options currently exist for patients with SCI. Spinal cord trauma was induced in mice by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, apoptosis, and locomotor disturbance. Olprinone treatment (0.2 mg/kg, i.p.) 1 and 6 h after the SCI significantly reduced: (1) the degree of spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation, (4) pro-inflammatory cytokines, (5) NF-kappaB expression, (6) p-ERK1/2 and p38 expression and (7) apoptosis (TUNEL staining, FAS ligand, Bax and Bcl-2 expression). Moreover, olprinone significantly ameliorated the recovery of hind-limb function (evaluated by motor recovery score). CONCLUSIONS/SIGNIFICANCE: Taken together, our results clearly demonstrate that olprinone treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma