Blood Platelets and Proteolytic Enzymes

This thesis gives an account of the investigation of the effect of a variety of proteolytic enzymes on platelet aggregation. The discovery of platelets in blood and the recognition of their importance in haemostasis and thrombus formation is described in an historical introduction, which is followed by an account of materials and methods used, including three in vitro tests designed to estimate the ability of platelets to aggregate. Techniques for the preparation, purification and characterization of degradation products formed by proteolytic digestion of fibrinogen are also described. The experimental section of the thesis begins with an account of the assessment in vitro of the effect of fibrinogenolytic agents, particularly streptokinase and trypsin, and of products of fibrinogenolytic activity on platelet aggregation and adhesion. Platelet aggregation was assessed in two ways; by an artificial circulation system - the Chandler tube system, which measures the rate of formation of platelet aggregates in flowing recalcified citrated plasma, and by a turbidimetric method which measures the ability of platelets in response to adenosine-di-phosphate (ADP). As a measure of platelet adhesion, the diminution in platelet count as a result of passage of citrated whole blood or platelet-rich plasma through a column of glass beads was used (modified Hellem technique). Platelet clumping, as estimated by all methods, was enhanced by both streptokinase and trypsin although the concentrations of each enzyme required to produce this effect varied in the different assay systems. Streptokinase, at a concentration producing maximum lytic activity, accelerated the rate of formation of platelet aggregates in the Chandler tube and produced a marked increase in platelet adhesiveness in the Hellem-type technique. An increase in platelet ADP reactivity was only detected in the turbidimetric system after the plasma antiplasmin had been destroyed. Trypsin, at a concentration of 10 ug/ml sample produced a significant increase in the rate of formation of platelet aggregates in the Chandler tube system and an increase in platelet adhesiveness in the Hellem-type technique, but the concentration of enzyme had to be increased to 100 ug/ml sample before an increase in platelet ADP reactivity was detected. Neither streptokinase nor trypsin influence platelet aggregation by a direct action on the platelet; streptokinase produces no significant enhancement and trypsin produces almost total inhibition of aggregation of washed platelets resuspended in buffer. The effect on platelet aggregation of other proteolytic enzymes -urokinase, ficin, and chymotrypsin was also investigated, but none of these produced any enhancement of platelet reactivity to ADP in the turbidimetric system. These enzymes were however examined in less detail than streptokinase and trypsin. Heparin was also studied and at the concentrations of 0.05 units and 10 units per ml plasma was found to cause significant enhancement of ADP induced! aggregation in the turbidimetric system, but when present at a concentration of 500 units per ml plasma caused significant inhibition of ADP reactivity. The enhancement of platelet aggregation by streptokinase and trypsin was associated with marked fibrinogenolysis; several fibrinogen degradation products were prepared and their effect on platelet aggregation and adhesion examined. The degradation products were prepared by incubation of fibrinogen with either trypsin or urokinase-activated plasminogen followed by fractionation of the protein components of the incubation mixture on a DEAE cellulose column. The ultracentrifugal and electrophoretic properties of the breakdown products were determined and the number of peptide bonds broken during formation of two of the products determined. The effect on platelet aggregation and adhesion was examined of three products of tryptic digestion of fibrinogen; the product of least digestion (molecular weight apprximately 83,000) produced no significant effect on platelet ADP reactivity or platelet adhesiveness, the product of prolonged digestion (molecular weight less than 13,000) inhibited ADP induced platelet aggregation while the product of intermediate digestion (molecular weight 25,000) enhanced the rate of formation of platelet aggregates, ADP reactivity and platelet adhesiveness. A product prepared by digestion with urokinase-activated plasminogen enhanced both the rate of formation of platelet aggregates in the Chandler tube and ADP induced platelet aggregation. The two larger fragments produced by trypsin both exhibited a marked 'antithrombin' effect. The three in vitro methods used to study platelet aggregation and adhesion were compared. No mathematical correlations were found between the results obtained with the different methods; however the effect of trypsin and streptokinase could be detected with each method and the observations made with each method could to some extent be influenced by the concentration of ADP in the system. The experimental data presented demonstrate that under certain circumstances the enzymes streptokinase and trypsin can produce enhancement of platelet aggregation

Recreating the Law School to Increase Minority Participation: The Conceptual Law School

That is the situation. Nonetheless, the purpose of this Article is not to criticize the current law school model. It is a model that has, in many respects, served society well, having produced thousands of competent lawyers over the years since it became the dominant model. It is the model that has produced all of the minority lawyers that are currently members of the profession. Moreover, to their credit, faculty and administration at many law schools are very motivated to improve the situation but are constrained in their efforts by the law and other factors. This Article is not meant to be unduly critical of the current law school model. In addition, this is not meant to be yet another article arguing that law schools need to increase skills training. Rather, the simple question put forth is whether a different model for legal education might serve as an alternative to the traditional law school model and, in conjunction with the efforts of the traditional law schools, aid in increasing the numbers of minorities licensed to practice law

It's My Soul

Reflections onmy Soul

A new method for studying human polycystic kidney disease epithelia in culture

A new method for studying human polycystic kidney disease epithelia in culture. Human polycystic kidney disease (PKD) epithelia were successfully grown in culture and expressed abnormal characteristics. Cysts lining epithelia of superficial and deep cysts were microdissected and compared to individual normal human proximal straight tubules (PST) and cortical collecting tubules (CCT) grown in defined media. PKD cyst epithelia differed from normal renal tubular epithelia in growth patterns and structural and functional properties. PKD epithelia grew more rapidly and showed cyst–like areas in otherwise confluent monolayers. Polygonal and elongate cells contained an epithelial–specific cytokeratin antigen and had polarized morphology. An extremely abnormal basement membrane morphology was seen and consisted of some banded collagen and numerous unique blebs or spheroids. These blebs were apparently extruded from intracellular vacuoles and stained with ruthenium red, suggesting a proteoglycan component. Cytochemistry of marker enzymes demonstrated the presence of NaK-ATPase and alkaline phosphatase, but a lack of γ-glutamyl transpeptidase. The response of adenylate cyclase activity to vasopressin, parathyroid hormone, and forskolin was significantly diminished in PKD cells as compared to PST and CCT. These studies suggest a defect in cell growth and basement membrane synthesis in human PKD. Cultured PKD epithelia provide a new tool for the study of the pathogenesis of this disease

MicroRNA-122-dependent and -independent replication of Hepatitis C Virus in Hep3B human hepatoma cells

AbstractThe study of Hepatitis C Virus (HCV) has benefitted from the use of the Huh7 cell culture system, but until recently there were no other widely used alternatives to this cell line. Here we render another human hepatoma cell line, Hep3B, permissive to the complete virus life cycle by supplementation with the liver-specific microRNA miR-122, known to aid HCV RNA accumulation. When supplemented, Hep3B cells produce J6/JFH-1 virus titres indistinguishable from those produced by Huh7.5 cells. Interestingly, we were able to detect and characterize miR-122-independent replication of di-cistronic replicons in Hep3B cells. Further, we show that Argonaute-2 (Ago2) is required for miR-122-dependent replication, but dispensable for miR-122-independent replication, confirming Ago2's role in mediating the activity of miR-122. Thus Hep3B cells are a model system for the study of HCV, and miR-122 independent replication is a model to identify proteins involved in the function of miR-122

Five- to nine-year follow-up results of balloon angioplasty of native aortic coarctation in infants and children

AbstractObjectives. To evaluate the usefulness of balloon angioplasty for relief of native aortic coarctation, we reviewed our experience with this procedure, with special emphasis on follow-up results.Background. Controversy exists with regard to the role of balloon angioplasty in the treatment of native aortic coarctation.Methods. During an 8.7-year period ending September 1993, 67 neonates, infants and children underwent balloon angioplasty for native aortic coarctation. A retrospective review of this experience with emphasis on long-term follow-up forms the basis of this study.Results. Balloon angioplasty produced a reduction in the peak-to-peak coarctation gradient from 46 ± 17 (mean ± SD) to 11 ± 9 mm Hg (p < 0.001). No patient required immediate surgical intervention. At intermediate-term follow-up (14 ± 11 months), catheterization (58 patients) and blood pressure (2 patients) data revealed a residual gradient of 16 ± 15 mm Hg (p > 0.1). When individual results were scrutinized, 15 (25%) of 60 had recoarctation, defined as peak gradient >20 mm Hg. Recoarctation was higher (p < 0.01) in neonates (5 [83%] of 6) and infants (7 [39%] of 18) than in children (3 [8%] of 36), respectively. Two infants in our early experience had surgical resection with excellent results. Three patients had no discrete narrowing but had normal arm blood pressure and had no intervention. The remaining 10 patients had repeat balloon angioplasty with reduction in peak gradient from 52 ± 13 to 9 ± 8 mm Hg (p < 0.001). Reexamination 31 ± 18 months after repeat angioplasty revealed a residual gradient of 3 to 19 mm Hg (mean 11 ± 6). Three (5%) of 58 patients who underwent follow-up angiography developed an aneurysm. Detailed evaluation of the femoral artery performed in 51 (88%) of 58 patients at follow-up catheterization revealed patency of the femoral artery in 44 (86%) of 51 patients. Femoral artery occlusion, complete in three (6%) and partial in four (8%), was observed, but all had excellent collateral flow. Blood pressure, echocardiography-Doppler ultrasound and repeat angiographic or magnetic resonance imaging data 5 to 9 years after angioplasty revealed no new aneurysms and minimal (2%) late recoarctation.Conclusions. On the basis of these data, it is concluded that balloon angioplasty is safe and effective in the treatment of native aortic coarctation; significant incidence of recoarctation is seen in neonates and infants; repeat balloon angioplasty for recoarctation is feasible and effective; and the time has come to consider balloon angioplasty as a therapeutic procedure of choice for the treatment of native aortic coarctation

Synthesis Of Novel 2-aryl-3-(2-morpholinoethyl)-1,3-thiazinan-4-ones Via Ultrasound Irradiation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)This study describes the synthesis of fourteen thiazinanones from a multicomponent reaction of 2-morpholinoehtylamine (as primary amine), arenealdehydes (as carbonyl compound) and the mercaptopropionic acid using both conventional (thermal heating) and ultrasound methodologies. Through thermal heating methodology, the thiazinanones were obtained in 49 to 97% yields for 16 hours and through sonochemistry methodology, the reaction time was reduced for 25 minutes with yields 41 to 88%. The full identification and characterization of unpublished heterocycles were achieved by proton (H-1) and carbon 13 (C-13) nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and infrared. Some of them were also characterized by elemental analysis.27611091115UFPelFAPERGS [11/2068-7]FAPESP [2013/18203-5]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Healthcare and Financial Decision Making and Incident Adverse Cognitive Outcomes among Older Adults

OBJECTIVES To determine if poorer healthcare and financial decision making forecasts adverse cognitive outcomes in old age. Specifically, we hypothesized that poorer decision making would be associated with an increased risk of incident Alzheimer's dementia, an increased risk of incident mild cognitive impairment (MCI), and a more rapid decline in cognition. DESIGN An ongoing prospective observational cohort study of aging (the Rush Memory and Aging Project). SETTING The greater Chicago area. PARTICIPANTS A total of 952 community‐based older adults without dementia at baseline. MEASUREMENTS Participants completed a measure of healthcare and financial decision making at baseline and underwent annual standardized evaluations to track clinical status and cognitive functions (global cognition, episodic memory, semantic memory, working memory, perceptual speed, and visuospatial ability). RESULTS During up to 9 years of follow‐up (M  = 5.2 y; standard deviation = 2.7), 156 participants developed Alzheimer's dementia (16.4% of 952), 253 participants developed MCI (33.2% of 760), and each cognitive measure declined (all P s < .001). In Cox proportional hazards models adjusted for age, sex, and education, poorer decision making was associated with an increased risk of incident Alzheimer's dementia (hazard ratio [HR] = 1.17; 95% confidence interval [CI] = 1.10‐1.24; P  < .001) and incident MCI (HR = 1.16; 95% CI = 1.10‐1.22; P  < .001). Further, in linear mixed‐effects models, poorer decision making among those who were initially free of cognitive impairment was associated with a more rapid decline in global cognition and four of five specific cognitive domains (all P s < .05). CONCLUSION Our results suggest that poorer healthcare and financial decision making heralds adverse cognitive outcomes in old age