Angiotensin II does not induce apoptosis but rather prevents apoptosis in cardiomyocytes

The ability of angiotensin II (ang II) to produce apoptosis is controversial. Cardiomyocytes, isolated from 7-day embryonic chick hearts and maintained in culture for 72 h, were treated with ang II. There was no evidence of ang II-induced apoptosis consistently demonstrated by six different techniques: electrophoretic separation of fragmented DNA, staining with TUNEL, enzyme-linked immunosorbent assay specific for fragmented DNA, dual staining of cells with fluorescein diacetate and propidium iodide with analysis by flow cytometry, staining of nuclei with propidium iodide and cell microscopy. In contrast, apoptosis was readily induced by camptothecin or staurosporine or serum deprivation. The absence of ang II-induced cell death was also demonstrated in neonatal mouse cardiomyocytes in culture. We further sought to answer the question whether ang II Type 1 receptor blockade by antagonizing the potential beneficial effects mediated through this receptor and producing more ang II binding to the ang II Type 2 receptors, would lead to cardiac apoptosis. There was no evidence of ang II-induced apoptosis in the presence of the ang II Type 1 receptor antagonist losartan in embryonic chick cardiomyocytes. Rather ang II prevented serum deprivation-induced apoptosis. In summary, in these cardiomyocytes ang II does not induce but rather prevents apoptosis.     

Peroxynitrite does not impair pulmonary and systemic vascular responses.

The effects of peroxynitrite (ONOO-) on vascular responses were investigated in the systemic and hindquarters vascular bed and in the isolated perfused rat lung. Intravenous injections of ONOO- decreased systemic arterial pressure, and injections of ONOO- into the hindquarters decreased perfusion pressure in a dose-related manner. Injections of ONOO- into the lung perfusion circuit increased pulmonary arterial perfusion pressure. Responses to ONOO- were rapid in onset, short in duration, and repeatable without exhibiting tachyphylaxis. Repeated injections of ONOO- did not alter systemic, hindquarters, or pulmonary responses to endothelium-dependent vasodilators or other vasoactive agonists and did not alter the hypoxic pulmonary vasoconstrictor response. Injections of sodium nitrate or nitrite or decomposed ONOO- had little effect on vascular pressures. Pulmonary and hindquarters responses to ONOO- were not altered by a cyclooxygenase inhibitor in a dose that attenuated responses to arachidonic acid. These results demonstrate that ONOO- has significant pulmonary vasoconstrictor, systemic vasodepressor, and vasodilator activity; that short-term repeated exposure does impair vascular responsiveness; and that responses to ONOO- are not dependent on cyclooxygenase product release.     

Light alters activity but does not disturb tandem coordination of termite mating pairs

Group-living animals coordinate their movements via local interactions, which can be mediated by visual, tactile, and chemical communication channels. Termite mating pairs form tandems with one male imago following one female imago in a synchronised way to explore the environment and search for a nesting site. Imagoes are the only developmental stage with compound eyes in termites, but the role of vision during tandem runs remains unknown. Here, we investigate the movements during tandem runs of two termite species, Coptotermes formosanus, which swarms during the night, and Reticulitermes speratus, which swarms during the day. We performed the experiments with light and in complete darkness. We found that females and males of both species adjust their speed to each other to form a stable tandem and reunite efficiently upon separation, with or without light. However, the activity was dependent on light conditions in the diurnal R. speratus, in which termites were more active with light. On the other hand, the nocturnal C. formosanus was mostly insensitive to light environments, with termites being slightly more active in darkness. Our results suggest that termites can use light as an environmental cue to start forming mating pairs but not as means to locate mates or coordinate their movements.     

Balloon injury does not induce heme oxygenase-1 expression, but administration of hemin inhibits neointimal formation in balloon-injured rat carotid artery.

It has been shown that antioxidant agents act inhibitorily against neointimal formation after balloon injury, suggesting the role of oxidative stress as a promotor of intimal cell proliferation. Heme oxygenase-1 (HO-1) is an inducible form of heme catabolizing enzyme that is induced by and acts against oxidative tissue injury. In this set of experiments, we showed that HO-1 was present in newly formed neointima; however, arterial HO-1 expression did not increase in response to balloon injury in rat carotid artery. Intraperitoneal administration of hemin, a HO-1 inducer, for 5 consecutive days resulted in about a 4-fold increase of serum bilirubin concentration. In addition, hemin injection increased HO-1 protein expression in the carotid artery, the heart, the kidney, and the liver. In this condition, balloon injury-induced neointimal formation was markedly inhibited. Local application of tin protoporphyrin, a HO inhibitor, blocked this effect, suggesting that induced HO-1 in the carotid artery was responsible for the inhibition of neointimal formation after balloon injury. This study suggests that induction of the endogenous antioxidant gene can suppress neointimal formation after balloon injury.     

Acute tryptophan depletion does not improve endurance cycling capacity in a warm environment

Newsholme’s theory of central fatigue suggests that acute tryptophan depletion should improve endurance exercise capacity in a warm environment by reducing serotonergic activity in the brain. Eight males cycled to volitional exhaustion at 55 % $ \dot{V}{\rm O}_{2} $ peak in 30.1 ± 0.5 °C and 30 ± 7 % relative humidity on two separate occasions, after consuming either an amino acid load to deplete their circulating tryptophan concentration (TD), or a control amino acid load (CON). Blood samples were taken before ingesting the amino acids, before the start of exercise, every 15 min during exercise and at the point of exhaustion. Heart rate (HR), core (Tc) and skin (Tsk) temperatures and ratings of perceived exertion (RPE) and thermal comfort (TC) were also monitored every 10 min during exercise. Plasma tryptophan (P = 0.003) and free tryptophan (P < 0.001) concentrations, and the free tryptophan to branched-chain amino acid ratio (P = 0.004) were all lower on the TD trial than on the CON trial. There was no difference in endurance exercise capacity (TD 99.2 ± 24.4 min as compared to CON 108.4 ± 21.6 min; P = 0.088). There was a tendency for HR (P = 0.053) and Tc (P = 0.069) to be higher on the TD trials. There were no differences for any of the other parameters. Endurance cycling capacity in a warm environment is not improved by acute tryptophan depletion, suggesting tryptophan availability is not a significant factor in the development of fatigue in such situations.     

Early gestation dexamethasone alters baroreflex and vascular responses in newborn lambs before hypertension

Exposure of the early gestation ovine fetus to exogenous glucocorticoids induces alterations in postnatal cardiovascular physiology, including hypertension. To determine whether autonomic function and systemic vascular reactivity are altered by in utero programming before the development of systemic hypertension, we examined arterial baroreflex function and in vivo hemodynamic and in vitro vascular responses to vasoactive agents in 10- to 14-day-old newborn lambs exposed to early gestation glucocorticoids. Dexamethasone (Dex, 0.28 mg.kg-1.day-1) or saline was administered to pregnant ewes by intravenous infusion over 48 h beginning at 27 days gestation (term 145 days), and lambs were allowed to deliver (n=6 in each group). Resting mean arterial blood pressure (MABP; 77+/-1 vs. 74+/-3 mmHg) and heart rate (HR; 249+/-9 vs. 226+/-21 beats/min) were similar in Dex-exposed and control animals, respectively. The arterial baroreflex curve, relating changes in HR to MABP, was significantly shifted toward higher pressure in the Dex-exposed lambs although no change in the sensitivity (gain) of the response was seen. In vivo changes in blood pressure in response to bolus doses of ANG II (20, 50, and 100 ng/kg) and phenylephrine (2, 5, and 10 microg/kg) were similar in the two groups. However, Dex lambs displayed greater decreases in MABP in response to ganglionic blockade with tetraethylammonium bromide (10 mg/kg; -30+/-3 vs. -20+/-3 mmHg, P<0.05) and greater increases in MABP after nitric oxide synthase blockade with NG-nitro-L-arginine (25 mg/kg; 23+/-3 vs. 13+/-2 mmHg, P<0.05) compared with control lambs. By in vitro wire myography, mesenteric and femoral artery microvessel contractile responses to KCl were similar, whereas responses to endothelin (in mesenteric) and norepinephrine (in femoral) were significantly attenuated in Dex lambs compared with controls. Femoral vasodilatory responses to forskolin and sodium nitroprusside were similar in the two groups (n=4). These findings suggest that resetting of the baroreflex, accompanied by increased sympathetic activity and altered nitric oxide-mediated compensatory vasodilatory function, may be important contributors to programming of hypertension.     

Cbp deficiency alters Csk localization in lipid rafts but does not affect T-cell development

The ubiquitously expressed transmembrane adaptor Csk-binding protein (Cbp) recruits Csk to lipid rafts, where the latter exerts its negative regulatory effect on the Src family of protein tyrosine kinases. We have inactivated Cbp in the mouse germ line. In contrast to Csk gene inactivation, which leads to embryonic lethality and impaired T-cell development, Cbp-deficient mice were viable and exhibited normal T-cell development but with an increased thymocyte population. In the absence of Cbp, the amount of Csk that localizes to the lipid rafts was greatly reduced. Interestingly, this altered lipid raft localization of Csk did not lead to any detectable biochemical or functional defect in T cells. The T-cell receptor-induced intracellular calcium flux, cell proliferation, and cytokine secretion were not affected by the absence of Cbp. Peripheral T-cell tolerance to superantigen SEB was also largely intact in Cbp-deficient mice. Thus, Cbp is dispensable for T-cell development and activation.     

Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.     

Leaf habit of tree species does not strongly predict leaf litter decomposition but alters climate-decomposition relationships

Plant and Soil - Leaf habit of tree species (evergreen versus deciduous) is proposed to be an important determinant of leaf litter decomposition, but it remains largely understudied as to how...     

Chronic amphetamine alters D-2 but not D-1 agonist-induced behavioral responses in rats

In two experiments, using different drug doses and periods of drug administration, rats were given amphetamine (AMPH) either continuously (via slow-release pellets), or intermittently (via injections). In both experiments, only the rats pretreated with intermittent AMPH subsequently showed heightened responsivity to the selective D-2 dopamine agonist LY171555 but not to SKF38393 (a D-1 agonist). This altered response to LY171555 was still present 30 days after the AMPH withdrawal, implying that D-2 dopamine receptors at least partially mediate AMPH inverse tolerance effects. The behavioral response to the D-2 agonist was clearly different in animals receiving high versus low doses of AMPH, suggesting that different drug-state learning may have occurred during pretreatment. In a third experiment, in which rats were given repeated daily injections of either the D-1 or the D-2 agonist, only rats pretreated with the D-2 agonist and subsequently injected with the D-2 agonist clearly showed heightened responsivity. These data imply an important role of D-2 receptors in the AMPH inverse tolerance effect.     

Direct oxidation of lymphocytes by chromic acid does not induce blastogenesis

Blastogenic and cytotoxic effects of hexavalent chromium were evaluated by direct, 2 and 20 min oxidation of lymphocytes by 10.0 to 0.0005 mM CrO₃ at 0°C. Oxidized cells exhibited concentration-dependent cytotoxicity and the inhibition of tritiated thymidine incorporation rates. When lymphocytes were oxidized first by 1.0 mM periodic acid (H₅IO₆) and thereafter by 1.0 mM CrO₃, the viability and [³H]-TdR incorporation rates of sequentially oxidized cells were identical to the corresponding indicators of lymphocytes oxidized only by CrO₃. The reversal of the oxidation sequence restored [³H]-TdR incorporation to control levels and increased cell survival. It is therefore concluded that direct interaction of hexavalent CrO₃ with plasma membrane of lymphocytes results in concentration-dependent cytotoxicity and the inhibition of [³H]-TdR incorporation, but it does not induce blastogenesis.     

Overexpression of DRG2 suppresses the growth of Jurkat T cells but does not induce apoptosis

Developmentally regulated GTP-binding protein (DRG) is a new subfamily within the superfamily of GTP-binding proteins. Its expression is regulated during embryonic development. To investigate the effect of the expression of DRG2 on cell growth, we constructed a human Jurkat-T-cell line that overexpresses DRG2. Overexpression of DRG2 suppressed the growth and the aggregation of Jurkat cells but did not induce apoptotic cell death. We used cDNA microarray analysis to examine the global changes in gene expression induced by an overexpression of DRG2. DNA array analyses identified genes that may suppress cell growth at a number of levels in multiple signaling cascades in Jurkat cells and also several prosurvival genes that may protect cells from apoptosis.     

Pioglitazone does not affect vascular or inflammatory responses after endotoxemia in humans.

PPARgamma agonists have been proposed to exert more than metabolic benefits, particularly by anti-inflammatory mechanisms. We hypothesized that pioglitazone might modulate inflammatory and vascular responses to lipopolysaccharide (LPS). In a placebo-controlled parallel-group study in 18 healthy male subjects, the E. coli endotoxin model of inflammation (20 IU/kg i. v.) was employed to test the effect of 60 mg pioglitazone over nine days on inflammatory cytokines. Macrovascular function and microvascular blood flow were assessed by brachial artery ultrasound and retinal blood flow parameters, respectively. Pioglitazone increased brachial artery diameter by 5.6% but had no effect on other outcome parameters under resting conditions. LPS increased cytokine levels to peak concentrations of 91.3+/-22.5 ng/ml (IL-6), 261.4+/-60.0 ng/ml (TNFalpha), and 524.5+/-15.3 ng/ml (VCAM-1). The endotoxin caused microvascular vasodilation and increased retinal white blood cell flux, while baseline brachial artery diameter remained unchanged. Pioglitazone had no effect on inflammatory cytokine or adhesion molecule release but mitigated LPS-induced hypotension (p<0.05). Neither brachial artery function nor microvascular blood flow was altered by pioglitazone. In conclusion, acute immune reactions to LPS are not affected by pioglitazone, which exerts subtle vascular effects alone and during endotoxemia. The anti-inflammatory properties of short-term pioglitazone to endotoxins in healthy subjects are therefore limited.     

Trigger factor depletion or overproduction causes defective cell division but does not block protein export.

Trigger factor is an abundant cytosolic protein of Escherichia coli which can stabilize proOmpA for in vitro translocation across inner membrane vesicles. The gene encoding E. coli trigger factor was isolated and sequenced, allowing construction of strains in which the expression of trigger factor is readily regulated. We found no defect in the in vivo rate of synthesis or secretion of proOmpA in trigger factor-depleted cells. The primary physiological defect in trigger factor-depleted or -overproducing cells is an enrichment of filamented cells. Filamentation of the trigger factor-overproducing strain is suppressed by a multicopy plasmid expressing the essential division gene ftsZ, suggesting that trigger factor has an important role in cell division.     

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 × 10⁶ cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 °C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.     

Generation of nitroxyl by heme protein-mediated peroxidation of hydroxylamine but not N-hydroxy-L-arginine

The chemical reactivity, toxicology, and pharmacological responses to nitroxyl (HNO) are often distinctly different from those of nitric oxide (NO). The discovery that HNO donors may have pharmacological utility for treatment of cardiovascular disorders such as heart failure and ischemia reperfusion has led to increased speculation of potential endogenous pathways for HNO biosynthesis. Here, the ability of heme proteins to utilize H(2)O(2) to oxidize hydroxylamine (NH(2)OH) or N-hydroxy-L-arginine (NOHA) to HNO was examined. Formation of HNO was evaluated with a recently developed selective assay in which the reaction products in the presence of reduced glutathione (GSH) were quantified by HPLC. Release of HNO from the heme pocket was indicated by formation of sulfinamide (GS(O)NH(2)), while the yields of nitrite and nitrate signified the degree of intramolecular recombination of HNO with the heme. Formation of GS(O)NH(2) was observed upon oxidation of NH(2)OH, whereas NOHA, the primary intermediate in oxidation of L-arginine by NO synthase, was apparently resistant to oxidation by the heme proteins utilized. In the presence of NH(2)OH, the highest yields of GS(O)NH(2) were observed with proteins in which the heme was coordinated to a histidine (horseradish peroxidase, lactoperoxidase, myeloperoxidase, myoglobin, and hemoglobin) in contrast to a tyrosine (catalase) or cysteine (cytochrome P450). That peroxidation of NH(2)OH by horseradish peroxidase produced free HNO, which was able to affect intracellular targets, was verified by conversion of 4,5-diaminofluorescein to the corresponding fluorophore within intact cells.