Phosphatase inhibition promotes antiapoptotic but not proliferative signaling pathways in erythropoietin-dependent HCD57 cells.

Erythropoietin (EPO) allows erythroid precursors to proliferate while protecting them from apoptosis. Treatment of the EPO-dependent HCD57 murine cell line with 70 micromol/L orthovanadate, a tyrosine phosphatase inhibitor, resulted in both increased tyrosine protein phosphorylation and prevention of apoptosis in the absence of EPO without promoting proliferation. Orthovanadate also delayed apoptosis in primary human erythroid progenitors. Thus, we investigated what survival signals were activated by orthovanadate treatment. Expression of Bcl-X(L) and BAD phosphorylation are critical for the survival of erythroid cells, and orthovanadate in the absence of EPO both maintained expression levels of antiapoptotic Bcl-X(L) and induced BAD phosphorylation at serine 112. Orthovanadate activated JAK2, STAT1, STAT5, the phosphatidylinositol-3 kinase (PI-3 kinase) pathway, and other signals such as JNK and p38 without activating the EPO receptor, JAK1, Tyk2, Vav, STAT3, and SHC. Neither JNK nor p38 appeared to have a central role in either apoptosis or survival induced by orthovanadate. Treatment with cells with LY294002, an inhibitor of PI-3 kinase activity, triggered apoptosis in orthovanadate-treated cells, suggesting a critical role of PI-3 kinase in orthovanadate-stimulated survival. Mitogen-activated protein kinase (MAPK) was poorly activated by orthovanadate, and inhibition of MAPK with PD98059 blocked proliferation without inducing apoptosis. Thus, orthovanadate likely acts to greatly increase JAK/STAT and PI-3 kinase basal activity in untreated cells by blocking tyrosine protein phosphatase activity. Activated JAK2/STAT5 then likely acts upstream of Bcl-X(L) expression and PI-3 kinase likely promotes BAD phosphorylation to protect from apoptosis. In contrast, MAPK/ERK activity correlates with only EPO-dependent proliferation but is not required for survival of HCD57 cells.

Protein kinase B (c-Akt), phosphatidylinositol 3-kinase, and STAT5 are activated by erythropoietin (EPO) in HCD57 erythroid cells but are constitutively active in an EPO-independent, apoptosis-resistant subclone (HCD57-SREI cells).

We found that erythropoietin (EPO) and stem cell factor (SCF) activated protein kinase B (PKB/Akt) in EPO-dependent HCD57 erythroid cells. To better understand signals controlling proliferation and viability, erythroid cells that resist apoptosis in the absence of EPO were subcloned and characterized (HCD57-SREI cells). Constitutive activations of PKB/Akt, STAT5a, and STAT5b were noted in these EPO-independent cells. PI3-kinase activity was an upstream activator of PKB/Akt because the PI3-kinase inhibitor LY294002 blocked both constitutive PKB/Akt and factor-dependent PKB/Akt activity. The LY294002 study showed that proliferation and viability of both HCD57-SREI and HCD57 cells correlated with the activity of PKB/Akt; however, PKB/Akt activity alone did not protect these cells from apoptosis. Treatment of HCD57 cells with SCF also activated PKB/Akt, but did not protect from apoptosis. This result suggested that PKB/PI3-kinase activity is necessary but not sufficient to promote viability and/or proliferation. Constitutive STAT5 activity, activated through an unknown pathway not including JAK2 or EPOR, may act in concert with the constitutive PI3-kinase/PKB/Akt pathway to protect the EPO-independent HCD57-SREI cells from apoptosis and promote limited proliferation.

Human tryptase fibrinogenolysis is optimal at acidic pH and generates anticoagulant fragments in the presence of the anti-tryptase monoclonal antibody B12.

Human tryptase is uniquely regulated by its association with heparin and resists inhibition by biological protease inhibitors. The effects of pH and B12, an IgG anti-tryptase mAb, on cleavage of the synthetic substrate tosyl-Gly-Pro-Lys-p-nitroanilide and of the biological substrate fibrinogen by tryptase were examined. Tosyl-Gly-Pro-Lys-pnitroanilide cleavage was optimal at neutral pH and was inhibited by the B12 mAb at acidic and neutral pH values. At pH 7.5, inhibition was reversible and noncompetitive. In contrast, the optimal pH for tryptase to cleave fibrinogen was acidic. B12 dramatically enhanced the rate and extent that tryptase cleaved all three fibrinogen subunits at pH 6.0 to 6.5, but inhibited these activities at neutral pH. Major fibrinogen cleavage fragments generated at acidic pH by the B12:tryptase complex were identical with those made by plasmin. Thus, at acid pH, tryptase alone destroyed the ability of fibrinogen to clot, while the B12:tryptase complex increased the rate of fibrinogenolysis and also generated the anticoagulant, fragment D. The acidic pH optimum for tryptase fibrinogenolysis may direct this activity to tissue sites of inflammation. A putative biological equivalent to B12 would limit tryptase fibrinogenolytic activity at sites of neutral pH, such as blood, but would augment activity at acidic sites.

Complications of laser therapy in the gynecological patient: a review of four patients.

Complications of gynecological laser treatment for perineal disease have been seen with increasing frequency. This may be the result of more women undergoing therapy with this method. Four women presenting with unstable perineal scarring are discussed.

Acceptance of lack of closure: is it an index of advanced reasoning?

Lunzer reported data suggestive of a stage of cognitive development manifest between 9 and 11 years of age characterized by the ability to avoid drawing premature inferences when faced with ambiguity (i.e., accept lack of closure [ALC]). The present study sought to test this hypothesis. Inference tasks emphasizing ALC, memory, and hypothetico-deductive reasoning were administered to 67 males and 74 females (5-12 years in age). Although use of ALC increased with age, considerable use was evidenced on a simple task among 7-8-year-olds. On tasks hypothesized to place increasing demands on working memory, longer tasks were found to be more difficult. Marked improvement due to memory aids suggested that task difficulty results from limitations in working memory as predicted by Pascual-Leone's theory. Tasks requiring hypothetico-deductive reasoning were found to be most difficult. Performance was related to subject's spontaneous use of ALC. Lack of appropriate strategies was hypothesized to prevent solution rather than lack of logical competence. In conclusion, the relationship of ALC to age appears to be mediated by memory development rather than logical development.

A systematic approach to thermal conductivity detector design I. Use of computer-aided data acquisition for the evaluation of cell contributions to peak shape.

The design of thermal conductivity detectors for open tubular gas chromatography was systematically investigated with the aid of modern digital data acquisition techniques. Two important factors in such a design are sensitivity and peak broadening. The latter factor was the principle thrust of this work and contributions to peak shape from both cell design and volume were analyzed by the method of statistical moments. Peak variance (the mean square displacement along the x-axis of a chromatographic peak) was shown to correlate directly with cell volume in cells of the same design but both variance and asymmetry were found to be design dependent.

Formal operations and field independence in a heterogeneous sample.

25 sixth grade students from diverse socioeconomic backgrounds were administered two Piagetian formal operational tasks (bending rods and balance beam) and a test of field independence. A Pearson product-moment correlation coefficient of .92 (p less than .001) was found between the two formal operational tasks. Correlation between the formal operational tasks and field independence were .81 and .77 (p less than .001) respectively. These values support the hypothesis of a unified stage of formal reasoning and also support the hypothesis that a degree of field independence is required for the development of formal stage reasoning.

Vascular responses of the spleen to nerve stimulation during normal and reduced blood flow.

1. The splenic artery flow, the splenic weight and the arterial blood pressure were recorded in cats anaesthetized with sodium pentobarbitone.2. Oscillations in splenic artery flow and splenic weight were observed. Following occlusion and release of the splenic artery, there was a brief increase in flow to above the pre-occlusion level and the oscillations in flow were greatly increased in amplitude. It is suggested that the brief increase is a consequence of the reduction of arterial pressure and that the oscillations are due to synchronization of rhythmic activity of smooth muscle within the spleen.3. Stimulation of the splenic nerves resulted in decreases in splenic artery flow and splenic weight. The size of the responses varied with the frequency of stimulation and maximum responses in both flow and weight were obtained at about 3 impulses/sec.4. After stimulation for 10 min, the splenic weight response was maintained while the flow response showed some recovery towards the control level.5. When the splenic artery flow was reduced to about half the control level for periods up to 2 hr, the flow and weight responses to stimulation of the splenic nerves remained unchanged; the significance of this after a haemorrhage is discussed.6. Intravenous administration of atropine or propranolol did not affect the responses to nerve stimulation. After phenoxybenzamine, nerve stimulation caused a smaller decrease in splenic weight, while the splenic artery flow increased to above the control level. This increase was unaffected by atropine but abolished by propranolol.

The effect of haemorrhage on hepatic artery and portal vein flows in the anesthetized cat.

1. In cats anaesthetized with pentobarbitone, the hepatic artery and portal vein flows and pressures were recorded simultaneously.2. Removal of blood from the animal did not cause a decrease in the hepatic artery flow unless the arterial pressure fell below 80 mm Hg. In contrast, the portal vein flow fell markedly.3. After restoration of the blood, the hepatic artery flow increased to above the pre-haemorrhage level, while portal vein flow returned only partly towards the control level.4. It is concluded that haemorrhage causes a vasodilation in the hepatic artery vascular bed and a vasoconstriction in the vascular beds drained by the portal vein. By this means, the oxygen supply to the liver is maintained as far as possible.

The effects of stimulation of the hepatic nerves, infusions of noradrenaline and occlusion of the carotid arteries on liver blood flow in the anaesthetized cat.

1. In anaesthetized cats, the hepatic artery, portal vein and inferior vena cava pressures and the hepatic artery and portal vein flows were recorded using pressure transducers and electro-magnetic flowmeters.2. The hepatic nerves were stimulated with maximal stimuli for periods of 2-5 min. The magnitude of the response varied with the frequency of stimulation over the range 1-10 impulses/sec. The resistance to flow increased in both the hepatic artery and the portal vein.3. In the hepatic artery, mean pressure remained virtually constant, while the flow showed an initial marked decrease followed by a return towards the control level. In the portal vein, the flow remained constant while portal pressure showed a maintained increase. These responses were unaffected by previous administration of atropine and propranolol, but were blocked by phenoxybenzamine.4. Infusions of noradrenaline into the hepatic artery produced changes similar to those following stimulation of the nerves. In contrast, when the hepatic arterial pressure was maintained constant, intravenous infusions of noradrenaline produced a maintained decrease in hepatic artery flow.5. The occurrence of autoregulation of the hepatic artery flow at arterial pressures above 80-100 mm Hg was confirmed.6. Occlusion of the carotid arteries caused a rise in arterial pressure with little change in hepatic artery flow, but when the hepatic artery pressure was maintained at the pre-occlusion level the flow showed an abrupt decrease, usually followed by a recovery towards the control level. This decrease was abolished by section of the hepatic nerves and removal of the adrenal glands.7. It is concluded that the increase in hepatic artery resistance during occlusion of the carotid arteries was dependent on the hepatic nerves, the adrenal medullary secretions and an intrinsic autoregulatory mechanism.

The effects of adrenaline and noradrenaline on venous return and regional blood flows in the anaesthetized cat with special reference to intestinal blood flow.

1. In cats, a venous long-circuit technique was used to measure the blood flows in the superior vena cava and the hepatic, renal and iliac segments of the inferior vena cava. The sum of these flows gave the venous return (minus coronary and bronchial flows). In further experiments using an electromagnetic flowmeter, flow in the portal vein and in the superior mesenteric and coeliac arteries was measured.2. Approximately two-thirds of the hepatic blood flow is derived from the portal vein.3. After block of conduction in the cervical region of the spinal cord, the proportions of the venous return coming from each region during the control periods were not significantly altered although the arterial pressure and total venous return were decreased.4. Intravenous infusions of adrenaline caused an increase in venous return which was associated with a marked increase in hepatic blood flow. The increase in hepatic blood flow was due to an increase in flow in the superior mesenteric artery and portal vein. Flow in the coeliac artery remained unchanged. This response was unaffected by block of the cervical region of the spinal cord and by atropine or pentolinium.5. Intravenous infusions of noradrenaline caused little change in venous return or regional blood flows. Small increases in superior mesenteric artery flow were occasionally seen and on cessation of the infusion a large but brief increase occurred. These facts suggest that noradrenaline has a similar action to adrenaline but this is masked by concomitant vasoconstriction.

The effect of haemorrhage on venous return and regional blood flow in the anaesthetized cat.

1. In cats under pentobarbitone anaesthesia, a venous long-circuit technique was used to measure the blood flows in the superior vena cava and the hepatic, renal and iliac segments of the inferior vena cava. The sum of these flows gave the venous return (minus coronary and bronchial flows).2. In these preparations, the mean venous return was 130 ml./kg. Of this 28% came from the superior vena cava and 37% from the hepatic, 23% from the renal and 12% from the iliac segments of the inferior vena cava.3. After haemorrhage, the flows from all the venae cavae segments decreased. The quantitative changes varied with the particular cat, the degree and duration of the haemorrhage and whether the animal had been subjected to a previous haemorrhage.4. The proportion of the reduced venous return draining from the superior vena cava and the hepatic segment increased, that draining from the renal and iliac segments decreased. Vasoconstriction occurred in all vascular beds, but was greatest in the kidney and hind limbs. Thus the blood flow through the head and liver was partially maintained at the expense of that through the kidneys and hind limbs.5. Autoregulation of blood flow in the kidneys was usually seen immediately after the first removal of blood but with the onset of renal vasoconstriction it was reduced or abolished for the remainder of the experiment.