Biochemical characterization of a thrombin inhibitor from the bloodsucking bug Dipetalogaster maximus

From the bloodsucking bug Dipetalogaster maximus, a protein with anticoagulant activity was isolated and biochemically characterized. The isolated protein, named dipetalogastin, possesses an average molecular mass of 11.8 kD. Its N-terminal sequence shows homology to rhodniin, a thrombin inhibitor isolated from the bug Rhodnius prolixus. The in vitro anticoagulant activity of dipetalogastin occurs via the inhibition of thrombin. The anticoagulant and thrombin inhibitory potency of dipetalogastin is comparable to that of recombinant hirudin. Its specific thrombin inhibitory activity is 9,300 antithrombin units/mg protein. Dipetalogastin forms only 1:1 molar complexes with thrombin. It is a tight-binding inhibitor of thrombin possessing a dissociation constant of 125 fM. It does not inhibit factor Xa or alpha-chymotrypsin and only weakly inhibits trypsin.     

Initial experience with bosentan (Tracleer) as treatment for pulmonary arterial hypertension (PAH) due to congenital heart disease in infants and young children

INTRODUCTION: Bosentan, a dual endothelin-receptor antagonist, has been shown to be an effective treatment option in patients with the idiopathic form of pulmonary arterial hypertension (PAH). We used bosentan as compassionate treatment in infants and young children with congenital heart disease (CHD) who had a) PAH preoperatively representing a contraindication to corrective surgery or b) persisting PAH after corrective surgery causing right heart failure and reduced exercise tolerance. METHODS: Seven children with PAH due to CHD (median age 3.8 years; range 1.5 to 6.4 years) received 3 mg/kg/d bosentan (Tracleer) orally. Clinical, echocardiographic and hemodynamic parameters were measured and laboratory tests performed before treatment and during steady state while on treatment. Routine liver function parameters were monitored monthly. RESULTS: Mean bosentan treatment time was 8.6+/-5 months. During bosentan therapy there were no significant adverse events. The clinical status remained stable or improved in all patients: NYHA class decreased from 2.6+/-0.6 to 1.7+/-0.6 (p<0.05). This was associated with a mean reduction of the right ventricular systolic pressure (RVSP) from 96+/-11 mmHg to 71+/-26 mmHg (p<0.05). CONCLUSIONS: Treatment with bosentan in infants and young children with PAH due to congenital heart disease was tolerated without significant side effects and resulted in stabilization of clinical status. A significant reduction in right ventricular systolic pressure (RVSP) could be demonstrated. These results suggest that the dose regimen used is appropriate and safe for the treatment of infants and children with PAH, resulting in a reduction of pathologically increased pulmonary vascular resistance.     

Arterioportal fistula causing acute pancreatitis and hemobilia after liver biopsy

We report the case of a 49-year-old patient who developed hemobilia and acute pancreatitis from an arterioportal fistula after a percutaneous liver biopsy, and we analyze diagnostic testing and management based on a concise review of the available literature. Hemobilia can present as late as 10 days after liver biopsy. Acute pancreatitis is a rare complication of hemobilia. To our knowledge, this is the first documented case of an arterioportal fistula after percutaneous liver biopsy with the late manifestation of hemobilia and acute pancreatitis.     

Correlates of reperfusion ventricular fibrillation in dogs

To elucidate determinants of reperfusion ventricular fibrillation (VF), regional myocardial blood flow, ATP, creatine phosphate (CP), heart rate and blood pressure were compared in 2 groups of anesthetized dogs: those that fibrillated spontaneously upon release of a 15-minute coronary artery occlusion (VF group, n = 8) and those that did not fibrillate when reperfused (No VF group, n = 27). Arterial pressure and heart rate before and during coronary artery occlusion were similar in both groups. Ischemie endo- and epicardial ATP values, measured at the end of the occlusion period, were reduced approximately 20% of nonischemic values in both groups. In contrast, CP (nmohmg protein^?1) within the ischemie zone was significantly lower in the VF group in both the epicardium (14.3 ± 1.6 in the VF group vs 22.8 ± 2.5 in the No VF group, p < 0.01) and the endocardium (9.0 ± 2.0 in the VF group vs 18.7 ± 1.8 in the No VF group, p < 0.01). Furthermore, epi- and endocardial regional myocardial blood flow in the center of the ischemic zone during occlusion was significantly lower in VF dogs than in No VF dogs. Epicardial flow was 0.06 ± 0.03 ml·min^?1·g^?1in VFdogsvs 0.44 ± 0.06 in No VF dogs (p < 0.001) and endocardial flow was 0.03 ± 0.02 ml·min^?1·g^?1 in VF dogs vs 0.23 ± 0.04 ·ml-min^?1·g^?1 in No VF dogs (p < 0.001). These data suggest that low levels of regional myocardial blood flow and CP during coronary artery occlusion are associated with an increased risk of VF on reperfusion. Thus, the severity of ischemia in the center of the ischemie zone may be a determinant of reperfusion VF.     

Decreased VEGF concentration in lung tissue and vascular injury during ARDS.

Endothelial injury is an important prognostic factor in acute respiratory distress syndrome (ARDS). Decreased production of vascular endothelial growth factor (VEGF) in ARDS may favour vascular lesions, since VEGF promotes endothelial survival by inhibiting apoptosis. This study sought to document low VEGF levels in lung tissue from ARDS patients, to determine whether the cause was injury to alveolar type II cells (the main pulmonary source of VEGF) and to evaluate the vascular consequences. Lung specimens were obtained by open biopsy or autopsy from 29 patients with severe ARDS (two survivors) and five controls. As compared with controls, homogenates of lung tissue from ARDS patients contained less VEGF (median (interquartile range) ARDS 8.2 (4.7-12.2) versus controls 28.4 (9.9-47.1) ng x g(-1) protein). Increased immunostaining with surfactant protein B was seen in ARDS lungs. Extensive cellular apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling staining), including endothelial and alveolar type II cells, was demonstrated, and vascular bed density (CD31 immunostaining) decreased in ARDS lungs as compared with controls. VEGF levels were negatively correlated to apoptotic endothelial cell counts. In conclusion, decreased vascular endothelial growth factor levels in lung tissue may participate in the decrease in lung perfusion in acute respiratory distress syndrome.     

Determinants of total body oxygen consumption in adults undergoing cardiac catheterization

Oxygen consumption was measured in 500 patients (151 F, 349 M, ages 12–84 yr.) undergoing routine cardiac catheterization. Sex, age, and heart rate were found to be the strongest predictors of oxygen consumption index (OCI). Males had higher OCI values than females at any age. Older patients of both sexes had lower OCI values than younger patients. Heart rate correlated directly with OCI. Treatment with pro-pranolol correlated inversely with OCI. However, after correction for heart rate, there were no significant differences in OCI values between propranolol treated and non-treated patients. A linear regression equation was developed using combinations of variables to predict OCI, and tables are presented for predicting OCI in those patients in whom this variable cannot be directly measured.     

Localization and movement of newly synthesized cholesterol in rat ovarian granulosa cells

The distribution and movement of cholesterol were studied in granulosa cells from the ovaries of estrogen-stimulated hypophysectomized immature rats cultured in serum-free medium. Plasma membrane cholesterol was distinguished from intracellular cholesterol with cholesterol oxidase, an enzyme that converts cell surface cholesterol to cholestenone, leaving intracellular cholesterol untouched. Using this approach we showed that 82% of unesterified cholesterol was associated with the plasma membrane in granulosa cells cultured for 48 h in serum-free medium in both the presence and absence of added androstenedione and FSH. FSH and androstenedione stimulated a marked increase in steroid hormone (progestin) production. The movement of newly synthesized cholesterol to the plasma membrane also was followed using cholesterol oxidase. Newly synthesized cholesterol reached the plasma membrane too rapidly to be measured in unstimulated cells (t1/2 less than 20 min); however, in cells stimulated by FSH and androstenedione, this rate was considerably slower (t1/2 approximately 2h). Therefore, cholesterol movement to the plasma membrane appears to be regulated by gonadotropins in these cells. We tested whether steroid biosynthesis used all cell cholesterol pools equally. To this end we administered [3H]acetate and [14C]acetate at different times and determined their relative specific contents in various steroids after defined intervals. The relative ages of the steroids (youngest to oldest) were: lanosterol, progestins, intracellular cholesterol, and plasma membrane cholesterol. This finding suggests that progestins use newly synthesized intracellular cholesterol in preference to preexisting intracellular or cell surface cholesterol. A measure of this effect is that the specific activity of secreted hormone was 15- to 30-fold greater than that of intracellular cholesterol. We conclude that the various cholesterol compartments in granulosa cells are discrete. While the major fraction of cholesterol in these steroidogenic cells resides in the plasma membrane, it is not in rapid equilibrium with intracellular cholesterol. Furthermore, steroidogenesis appears to use newly synthesized over preexisting cholesterol, suggesting a shunt pathway.