Selective perturbation of the myosin recovery stroke by point mutations at the base of the lever arm affects ATP hydrolysis and phosphate release

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys(84) and Arg(704), we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release.     

Effect of serotonin-acting agents on the serotonin content of immune cells. A peculiar observation

The effect of the tryptophan hydroxylase inhibitor, PCPA methylester, the serotonin reuptake inhibitor fluoxetine and MAO-A inhibitor clorgyline on the serotonin content of rat immune cells was studied, using labelled antibodies and flow cytometry. Each molecule significantly increased in males the serotonin concentration of peritoneal lymphocytes and the monocyte-macrophage-granulocyte group (mo-gran), however the agents were ineffective towards mast cells. In females fluoxetine and clorgyline increased the serotonin concentration in peritoneal lymphocytes and mo-gran. Fluoxetine also increased the serotonin level in mast cells. Thymus was absolutely resistant to the drugs in both genders. The results call attention (1) to the reverse effect of serotonin-acting agents on immune cells, (2) to the influence of the milieu where the cell is located and (3) the effect of gender.     

Impact of neonatal imprinting with vitamin A or D on the hormone content of rat immune cells

Neonatal single vitamin A (retinol; 3 mg) or vitamin D3 (cholecalciferol; 0.05 mg) treatment (imprinting) was done in male and female rats and 2 months later the endorphin, triiodothyronine (T3) and ACTH content of immune cells (peritoneal lymphocytes, monocyte-granulocyte-macrophage group [mo-gran], mast cells and thymic lymphocytes) were studied immunocytochemically by using flow cytometry and confocal microscopy. The T3 content was significantly decreased in peritoneal lymphocytes and in mo-gran and the endorphin content decreased in thymocytes of male animals, while ACTH was decreased in female lymphocytes and mo-gran. Vitamin D treatment was absolutely ineffective. The imprinting effects of vitamins A and D and their differences are discussed. The results call attention to the possible harmful effect of vitamin treatments during the perinatal critical period.     

How does the unicellular <Emphasis Type="Italic">Tetrahymena</Emphasis> utilise the hormones that it produces? Paying a visit to the realm of atto-and zeptomolar concentrations

Changes in the hormone content of Tetrahymena pyriformis GL were investigated during histamine, serotonin or insulin treatment at concentrations of 10⁻⁶M to 10⁻²¹M for 30 min. The immunologically demonstrable hormone content was studied by using specific antibodies, flow cytometry and confocal microscopy. Histamine at the higher ranges elevated the serotonin content of Tetrahymena, whereas serotonin at the lower ranges (down to 10⁻²¹M) decreased its histamine levels. Insulin did not affect its serotonin content, whereas serotonin increased its insulin content at each concentration studied (down to 10⁻²¹M). Insulin between 10⁻⁶M and 10⁻²¹M increased the hista-mine levels of Tetrahymena, although histamine influenced its insulin level only at 10⁻⁶M. Our results call attention to the presence of hormonal interactions even at “low” levels of phylogeny and to the extreme sensitivity of the hormone receptors of Tetrahymena. These data might explain (1) the requirement of Tetrahymena for (vertebrate) hormone production and hormone receptors and (2) the way that it uses these hormones under natural conditions.This work was supported by the National Research Fund (OTKA-T-037303), Hungary.     

Anorectic estrogen mimics leptin's effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals

Metabolic hormones, such as leptin, alter the input organization of hypothalamic circuits, resulting in increased pro-opiomelanocortin (POMC) tone, followed by decreased food intake and adiposity. The gonadal steroid estradiol can also reduce appetite and adiposity, and it influences synaptic plasticity. Here we report that estradiol (E2) triggers a robust increase in the number of excitatory inputs to POMC neurons in the arcuate nucleus of wild-type rats and mice. This rearrangement of synapses in the arcuate nucleus is leptin independent because it also occurred in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, and was paralleled by decreased food intake and body weight gain as well as increased energy expenditure. However, estrogen-induced decrease in body weight was dependent on Stat3 activation in the brain. These observations support the notion that synaptic plasticity of arcuate nucleus feeding circuits is an inherent element in body weight regulation and offer alternative approaches to reducing adiposity under conditions of failed leptin receptor signaling.     

In vitro effect of biogenic amines on the hormone content of immune cells of the peritoneal fluid and thymus. Is there a hormonal network inside the immune system?

Immune cells contain different hormones and hormone-like molecules, such as insulin, endorphin, triiodothyronine (T3) histamine, serotonin. In earlier in vitro experiments insulin down-regulated histamine, serotonin and T3 content of thymus cells. Now we studied the effect of biogenic amines on the endorphin, T3, serotonin and histamine content of rat peritoneal and thymic cells. Cells were obtained from male rats of 100g body weight. 100 ng/ml serotonin or 300 ng/ml histamine was added for 30 min. After that the cells were prepared for flow cytometric analysis with antibodies to endorphin, T3, histamine and serotonin as primary antibodies and anti-rabbit IgG as secondary antibody. Finishing the measurements the cells were also studied by confocal microscopy. T3 concentration (binding of anti-T3 antibody) increased in peritoneal mast cells after serotonin treatment and in the monocyte-macrophage-granulocyte group after histamine treatment. Thymocytes' T3 content radically decreased after both treatments. Serotonin and histamine treatment also radically reduced the amine content of each other. Endorphin level was resistant to hormonal treatments. The results call attention to a possible hormonal network inside the immune system in which hormones produced by the immune cells themselves can influence each other.     

Arabidopsis PPP family of serine/threonine phosphatases

Serine/threonine-specific phosphoprotein phosphatases (PPPs) are ubiquitous enzymes in all eukaryotes, but their regulatory functions are largely unknown in higher plants. The Arabidopsis genome encodes 26 PPP catalytic subunits related to type 1, type 2A and so-called novel phosphatases, including four plant-specific enzymes carrying large N-terminal kelch-domains, but no apparent homologue of the PP2B family. The catalytic subunits of PPPs associate with regulatory protein partners that target them to well defined cellular locations and modulate their activity. Recent studies of phosphatase partners and their interactions have directed attention again to functional dissection of plant PPP families, and highlight their intriguing roles in the regulation of metabolism, cell cycle and development, as well as their roles in light, stress and hormonal signalling.     

Nitric oxide, mitochondrial hyperpolarization, and T cell activation

T lymphocyte activation is associated with nitric oxide (NO) production, which plays an essential role in multiple T cell functions. NO acts as a messenger, activating soluble guanyl cyclase and participating in the transduction signaling pathways involving cyclic GMP. NO modulates mitochondrial events that are involved in apoptosis and regulates mitochondrial membrane potential and mitochondrial biogenesis in many cell types, including lymphocytes. Mitochondrial hyperpolarization (MHP), an early and reversible event during both activation and apoptosis of Tlymphocytes, is regulated by NO. Here, we discuss recent evidence that NO-induced MHP represents a molecular switch in multiple T cell signaling pathways. Overproduction of NO in systemic lupus erythematosus induces mitochondrial biogenesis and alters Ca(2+) signaling. Thus, whereas NO plays a physiological role in lymphocyte cell signaling, its overproduction may disturb normal T cell function, contributing to the pathogenesis of autoimmunity.     

Poly(ADP-Ribose) polymerase inhibition improves endothelial dysfunction induced by hypochlorite

Reactive oxygen species, such as myeloperoxidase-derived hypochlorite, induce oxidative stress and DNA injury. The subsequent activation of the DNA-damage-poly(ADP-ribose) polymerase (PARP) pathway has been implicated in the pathogenesis of various diseases, including ischemia-reperfusion injury, circulatory shock, diabetic complications, and atherosclerosis. We investigated the effect of PARP inhibition on the impaired endothelium-dependent vasorelaxation induced by hypochlorite. In organ bath experiments for isometric tension, we investigated the endothelium-dependent and endothelium-independent vasorelaxation of isolated rat aortic rings using cumulative concentrations of acetylcholine and sodium nitro-prusside. Endothelial dysfunction was induced by exposing rings to hypochlorite (100-400 microM). In the treatment group, rings were preincubated with the PARP inhibitor INO-1001. DNA strand breaks were assessed by the TUNEL method. Immunohistochemistry was performed for 4-hydroxynonenal (a marker of lipid peroxidation), nitrotyrosine (a marker of nitrosative stress), and poly(ADP-ribose) (an enzymatic product of PARP). Exposure to hypochlorite resulted in a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings, which was significantly improved by PARP inhibition, whereas the endothelium-independent vasorelaxation remained unaffected. In the hypochlorite groups we found increased DNA breakage, lipidperoxidation, and enhanced nitrotyrosine formation. The hypochloride-induced activation of PARP was prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of hypochlorite-induced endothelial dysfunction, which can be prevented by PARP inhibitors.