Modulation of the beta-adrenergic response in cultured rat heart cells

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A₂-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A₂ inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A₂ in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclooxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A₂-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.Abbreviations NDGA nordihydroguaiaretic acid - HETE hydroeicosatetraenoic acid - HPETE hydroperoxyeicosatetraenoic acid     

Structure of apo- and monometalated forms of NDM-1--a highly potent carbapenem-hydrolyzing metallo-β-lactamase

The New Delhi Metallo-β-lactamase (NDM-1) gene makes multiple pathogenic microorganisms resistant to all known β-lactam antibiotics. The rapid emergence of NDM-1 has been linked to mobile plasmids that move between different strains resulting in world-wide dissemination. Biochemical studies revealed that NDM-1 is capable of efficiently hydrolyzing a wide range of β-lactams, including many carbapenems considered as "last resort" antibiotics. The crystal structures of metal-free apo- and monozinc forms of NDM-1 presented here revealed an enlarged and flexible active site of class B1 metallo-β-lactamase. This site is capable of accommodating many β-lactam substrates by having many of the catalytic residues on flexible loops, which explains the observed extended spectrum activity of this zinc dependent β-lactamase. Indeed, five loops contribute "keg" residues in the active site including side chains involved in metal binding. Loop 1 in particular, shows conformational flexibility, apparently related to the acceptance and positioning of substrates for cleavage by a zinc-activated water molecule.     

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19(Arf) and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19(Arf)-p53 signaling, and induces senescence in primary cells.     

Gene-based sequence diversity analysis of field pea (Pisum)

Sequence diversity of 39 dispersed gene loci was analyzed in 48 diverse individuals representative of the genus Pisum. The different genes show large variation in diversity parameters, suggesting widely differing levels of selection and a high overall diversity level for the species. The data set yields a genetic diversity tree whose deep branches, involving wild samples, are preserved in a tree derived from a polymorphic retrotransposon insertions in an identical sample set. Thus, gene regions and intergenic "junk DNA" share a consistent picture for the genomic diversity of Pisum, despite low linkage disequilibrium in wild and landrace germplasm, which might be expected to allow independent evolution of these very different DNA classes. Additional lines of evidence indicate that recombination has shuffled gene haplotypes efficiently within Pisum, despite its high level of inbreeding and widespread geographic distribution. Trees derived from individual gene loci show marked differences from each other, and genetic distance values between sample pairs show high standard deviations. Sequence mosaic analysis of aligned sequences identifies nine loci showing evidence for intragenic recombination. Lastly, phylogenetic network analysis confirms the non-treelike structure of Pisum diversity and indicates the major germplasm classes involved. Overall, these data emphasize the artificiality of simple tree structures for representing genomic sequence variation within Pisum and emphasize the need for fine structure haplotype analysis to accurately define the genetic structure of the species.     

Effects of Handling or Immobilization on Plasma Levels of 3,4-Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.     

Class B scavenger receptor types I and II and CD36 targeting improves sepsis survival and acute outcomes in mice

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.     

Homocysteine metabolism in peripheral blood mononuclear cells: evidence for cystathionine beta-synthase activity in resting state

Activated peripheral blood mononuclear cells (PBMC) release homocysteine and possess cystathionine β-synthase (CBS) activity; however, it was thought that there is no CBS in resting state. Previously, we found that nickel decreased intracellular homocysteine concentration in un-stimulated (e.g. resting) PBMC, suggesting that resting PBMC might also have active homocysteine metabolism. Here, we demonstrated that un-stimulated PBMC synthesize (incorporate L-[methyl-14C]methionine to DNA, lipids and proteins), release (increase extracellular homocysteine), and metabolize homocysteine. Intracellular homocysteine concentration varied with incubation time, depending on extracellular concentrations of methionine, homocysteine, and glutathione. Methionine synthase activity was constant and independent of thiol concentrations. In Western blot, CBS protein was clearly identified in freshly isolated PBMC. CBS protein level and activity increased with incubation time, upon stimulation, and similar to intracellular homocysteine, depending on intra- and extracellular homocysteine and glutathione concentrations. According to our knowledge, this is the first evidence that certifies homocysteine metabolism and regulatory role of CBS activity to keep balanced intracellular homocysteine level in resting PBMC. Homocysteine, released by PBMC, in turn can modulate its functions contributing to the development of hyperhomocysteinemia-induced diseases.     

T cell activation-induced mitochondrial hyperpolarization is mediated by Ca2+- and redox-dependent production of nitric oxide

Activation, proliferation, or programmed cell death of T lymphocytes is regulated by the mitochondrial transmembrane potential (Deltapsi(m)) through controlling ATP synthesis, production of reactive oxygen intermediates (ROI), and release of cell death-inducing factors. Elevation of Deltapsi(m) or mitochondrial hyperpolarization is an early and reversible event associated with both T cell activation and apoptosis. In the present study, T cell activation signals leading to mitochondrial hyperpolarization were investigated. CD3/CD28 costimulation of human PBL elevated cytoplasmic and mitochondrial Ca(2+) levels, ROI production, and NO production, and elicited mitochondrial hyperpolarization. Although T cell activation-induced Ca(2+) release, ROI levels, and NO production were diminished by inositol 1,4,5-triphosphate receptor antagonist 2-aminoethoxydiphenyl borane, superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, spin trap 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide, and NO chelator carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, mitochondrial hyperpolarization was selectively inhibited by carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (-85.0 +/- 10.0%; p = 0.008) and, to a lesser extent, by 2-aminoethoxydiphenyl borane. Moreover, NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate diethylenetriamine elicited NO and ROI production, Ca(2+) release, transient ATP depletion, and robust mitochondrial hyperpolarization (3.5 +/- 0.8-fold; p = 0.002). Western blot analysis revealed expression of Ca-dependent endothelial NO synthase and neuronal NO synthase isoforms and absence of Ca-independent inducible NO synthase in PBL. CD3/CD28 costimulation or H(2)O(2) elicited severalfold elevations of endothelial NO synthase and neuronal NO synthase expression, as compared with beta-actin. H(2)O(2) also led to moderate mitochondrial hyperpolarization; however, Ca(2+) influx by ionomycin or Ca(2+) release from intracellular stores by thapsigargin alone failed to induce NO synthase expression, NO production, or Deltapsi(m) elevation. The results suggest that T cell activation-induced mitochondrial hyperpolarization is mediated by ROI- and Ca(2+)-dependent NO production.     

Differential tyrosine phosphorylation of plasma membrane Ca2+-ATPase and regulation of calcium pump activity by carbachol and bradykinin

We investigated the effects of thapsigargin (TG), bradykinin (BK), and carbachol (CCh) on Ca(2+) entry via endogenous channels in human embryonic kidney BKR21 cells. After depletion of Ca(2+) stores by either TG, BK, or CCh, the addition of Ca(2+) gave a much larger rise in Ca(2+) levels in CCh-treated and TG-treated cells than in cells treated with BK. However, in experiments performed with Ba(2+), a cation not pumped by Ca(2+)-ATPases, only a modest difference between CCh- and BK-stimulated Ba(2+) entry levels was observed, suggesting that the large difference in the Ca(2+) response is mediated by a differential regulation of Ca(2+) pump activity by CCh and BK. This hypothesis is supported by the finding that when Ca(2+) is removed during the stable, CCh-induced Ca(2+) plateau phase, the decline of cytosolic Ca(2+) is much faster in the absence of CCh than in its presence. In addition, if Ca(2+) is released from a caged Ca(2+) compound after a UV pulse, the resulting Ca(2+) peak is much larger in the presence of CCh than in its absence. Thus, the large increase in Ca(2+) levels observed with CCh results from both the activation of Ca(2+) entry pathways and the inhibition of Ca(2+) pump activity. In contrast, BK has the opposite effect on Ca(2+) pump activity. If Ca(2+) is released from a caged Ca(2+) compound, the resulting Ca(2+) peak is much smaller in the presence of BK than in its absence. An investigation of tyrosine phosphorylation levels of the plasma membrane Ca(2+)-ATPase (PMCA) demonstrated that CCh stimulates an increase in tyrosine phosphorylation levels, which has been reported to inhibit Ca(2+) pump activity, whereas in contrast, BK stimulates a reduction of PMCA tyrosine phosphorylation levels. Thus, BK and CCh have a differential effect both on Ca(2+) pump activity and on tyrosine phosphorylation levels of the PMCA.