Membrane expression of platelet calpain

Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.     

Behavioural Effects of the Methanolic Root Bark Extract of Securinega Virosa in Rodents

Securinega virosa is used traditionally as sedative in children and in mental illnesses. In this study, the behavioral effects of methanolic root bark extract of S. virosa were investigated in mice. The results revealed that the extract significantly (P<0.05) and dose-dependently reduced the onset and prolonged the duration of sleep. The extract significantly (P<0.05) decreased exploratory activity and reduced the rate of apomorphine-induced stereotyped climbing at the doses tested (6.25–25mg/kg). It also produced a significant and dose-dependent motor coordination deficit in mice at the doses tested (P<0.01). The intraperitoneal median lethal dose in mice was 774.6mg/kg while the preliminary phytochemical screening revealed the presence of alkaloids, tannins, saponins and flavonoids. These results suggest that methanolic root bark extract of S. virosa contains biologically active principles that are sedative in nature and lend pharmacological credence to the ethnomedical use of the plant.     

The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Belgian Diabetes Registry

Susceptibility to autoimmune insulin-dependent (type 1) diabetes mellitus is determined by a combination of environmental and genetic factors, which include variation in MHC genes on chromosome 6p21 (IDDM1) and the insulin gene on chromosome 11p15 (IDDM2). However, linkage to IDDM1 and IDDM2 cannot explain the clustering of type 1 diabetes in families, and a role for other genes is inferred. In the present report we describe linkage and association of type 1 diabetes to the CTLA-4 gene (cytotoxic T lymphocyte associated-4) on chromosome 2q33 (designated IDDM12). CTLA-4 is a strong candidate gene for T cell- mediated autoimmune disease because it encodes a T cell receptor that mediates T cell apoptosis and is a vital negative regulator of T cell activation. In addition, we provide supporting evidence that CTLA-4 is associated with susceptibility to Graves' disease, another organ- specific autoimmune disease.      

von Willebrand factor-collagen binding activity is increased in newborns and infants

ABSTRACT. von Willebrand factor (vWF) antigen (vWF:Ag) and vWF-collagen binding activity (vWF:CBA) were measured in plasma by parallel quantitative ELISAs in normal newborns and infants ( n =71). The medians for vWF:Ag (IUjml) and vWF:CBA (Ujml), respectively, were 1.46 and 1.91 for 2-7 day-old (n = 43), 1.22 and 1.40 for 2-4 week-old (n = 14), 1.22 and 1.15 for 2-6-month-old (n = 14) infants and 0.98 and 1.08 (n = 36) in normal adults. Elevated levels of vWF:Ag, but particularly vWF:CBA were seen for up to 4 weeks of life reaching adult levels between 2 and 6 months of life. The elevated levels of the vWF parameters indicate that caution should be exercised when interpreting laboratory data and diagnosing von Willebrand disease in newborns and young infants and warrant the use of age-specific reference ranges. The efficient haemostasis observed during early neonatal life may in part be due to the increased ability of vWF to interact with collagen.     

Echocardiogram‐gated multislice spiral CT in functionally univentricular heart following long‐lasting Waterston–Cooley anastomosis

Waterston–Cooley anastomosis may be carried out in patients with tricuspid atresia to provide pulmonary perfusion. It is associated with several complications, including preferential blood flow to the right lung, hypoplasia of the left pulmonary artery, obstruction of the anatomosis or rupture of pulmonary aneurysms. We study a patient with thrombosis in the pulmonary arteries following surgical construction of a Waterston shunt in childhood. Imaging findings and clinical symptoms are discussed with emphasis on echocardiogram‐gated multislice spiral CT.     

Interval between insulin injection and breakfast in diabetes

The relationship between the insulin-breakfast interval, postprandial increase in blood glucose, and glycaemic control was studied in 58 children with diabetes. Patients recorded insulin-breakfast intervals in a home diary over a seven day period, and during a 24 hour period at the weekend provided eight serial capillary dried blood spots for glucose analysis. The highest mean blood glucose value occurred two hours after breakfast and showed a significant correlation with fructosamine concentrations. Weekend insulin-breakfast intervals ranged from 2-30 minutes, with 70% reporting intervals of less than 15 minutes. There was a significant correlation between the weekend insulin-breakfast interval and the after breakfast increase in blood glucose with a mean increment of 0.4 mmol/l in the 30 minute group and 7.2 mmol/l in the 2 minute group. Over the whole study period, children with mean insulin-breakfast intervals of two to 12 minutes had a mean fructosamine concentration of 376 mumol/l compared with 341 mumol/l in those with intervals of 15-35 minutes. This study has shown that the interval between insulin injection and breakfast significantly influences the morning postprandial rise in blood glucose and consequently short term glycaemic control. It is therefore important that patients are encouraged to leave an interval of about 30 minutes between insulin injection and breakfast.     

Differential formation of beta-catenin/lymphoid enhancer factor-1 DNA binding complex induced by nitric oxide in mouse colonic epithelial cells differing in adenomatous polyposis coli (Apc) genotype

Increased cytoplasmic beta-catenin levels and the associated nuclear beta-catenin/T-cell factor (Tcf)-lymphoid enhancer factor (LEF) complex formation have been frequently found in colon cancer. In this context, overproduction of nitric oxide (NO) attributable to inflammatory stimuli in diseases such as ulcerative colitis and Crohn's disease may-contribute to colonic carcinogenesis. Therefore, we examined the modulation by NO of cytoplasmic beta-catenin levels and the formation of the nuclear beta-catenin/LEF-1 DNA binding complex in conditionally immortalized mouse colonic epithelial cells that differed in adenomatous polyposis coli (Apc) genotype, namely young adult mouse colon (YAMC; Apc+/+) and immortal mouse colon epithelium (IMCE; ApcMin/+). Unlike most colon cancer cell lines, this pair of cell lines has either nondetectable or low basal level of beta-catenin when they are cultured under nonpermissive and nonproliferative conditions. Using electrophoretic mobility shift assays, we found that NO-releasing agents (E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide and S-nitroso-N-acetylpenicillamine greatly enhanced the formation of beta-catenin/LEF-1 DNA binding complex in a concentration- and time-dependent fashion in YAMC and IMCE cells. Significantly, IMCE cells showed a markedly greater amount of nuclear beta-catenin/LEF-1 DNA binding complex in response to NO. Super shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Western blot analysis of the soluble cytoplasmic fractions demonstrated that these NO donors caused differential accumulation of cytoplasmic beta-catenin in YAMC and IMCE. In conclusion, this study indicates that the defective beta-catenin degradation machinery attributable to ApcMin/+ mutation in IMCE cells not only affects basal levels but also contributes to NO-induced dysregulation of cytoplasmic beta-catenin and nuclear beta-catenin/LEF-1 DNA binding complex formation.