RESUMEN
A fraction of FVIII:Ag in commercial recombinant FVIII (rFVIII) cannot bind VWF whereas all the FVIII:Ag in plasma-derived FVIII (pd-FVIII) concentrates does. To compare the FVIII:C activities of the fractions of rFVIII:Ag that can and cannot bind VWF. The FVIII:Ag contents of the rFVIII Kogenate, and Advate and a pd-FVIII-pd-VWF (Fanhdi) were measured by ELISA. The FX activation was initiated by adding 1.0 IU of FVIII:C of each FVIII-containing product to a coagulant phospholipids suspension containing 1.0 nm FIXa, 100 nm FX, 1 µm hirudin and 2 mm calcium chloride and measured after 1, 5 and 10 min. The same approach was followed after adding 2.0 IU of pd-VWF to 1.0 IU of FVIII:C of Kogenate or Advate. The FVIII:Ag content/IU of FVIII:C of Kogenate, Advate and Fanhdi were 1.80 ± 0.05, 1.31 ± 0.9 and 0.84 ± 1.5 IU respectively. Only Kogenate and Advate effectively enhanced FX activation 1 min after adding each FVIII:C to the coagulant suspension containing FIXa and FX. Thus, the FXa initially generated by FIXa readily activated FVIII:C in control Kogenate and Advate to thereby effectively enhance FX activation while the VWF in Fanhdi continued to suppress FX activation for up to 10 min. Addition of pd-VWF to Kogenate or Advate effectively decreased their enhancements of FX activation to the same level as Fanhdi over 10 min. The FVIII:Ag fraction in Kogenate and Advate that cannot bind VWF appears to be inactive as it has no measureable FVIII:C activity in the presence of added VWF in vitro.
Asunto(s)
Factor VIII/metabolismo , Factor de von Willebrand/metabolismo , Pruebas de Coagulación Sanguínea , Ensayo de Inmunoadsorción Enzimática , Factor IXa/metabolismo , Factor Xa/metabolismo , Unión ProteicaRESUMEN
Regulation of the sterol-synthesizing mevalonate pathway occurs in part through feedback-regulated endoplasmic reticulum degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). In yeast, the Hmg2p isozyme of HMG-R is regulated in this manner. We have tested the involvement of ubiquitination in the regulated degradation of Hmg2p, by using both genetic and direct biochemical approaches. Hmg2p degradation required the UBC7 gene, and Hmg2p protein was directly ubiquitinated. Hmg2p ubiquitination was dependent on UBC7 and was specific for the degraded yeast Hmg2p isozyme. Furthermore, Hmg2p ubiquitination was regulated by the mevalonate pathway in a manner consistent with regulation of Hmg2p stability. Thus, regulated ubiquitination appeared to be the mechanism by which Hmg2p stability is controlled in yeast. Finally, our data indicated that the feedback signal controlling Hmg2p ubiquitination and degradation was derived from farnesyl diphosphate, and thus implied conservation of an HMG-R degradation signal between yeast and mammals.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/metabolismo , Enzimas Ubiquitina-Conjugadoras , Ubiquitinas/metabolismo , Hidrólisis , Ligasas/genética , Ligasas/metabolismo , Ácido Mevalónico/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
In one hundred consecutive patients with non-purulent pleural exudates without apparent cause, the final diagnosis was tuberculosis in 58, malignancy in 20, pyogenic infection in four, cardiomyopathy in two, pulmonary infarction in one. The aetiology remained unknown in 15. The technique of "semi-open" pleural biopsy was performed under local anaesthesia. It accurately detected 70% of cancer and 69% of tuberculosis cases with a 9% complication rate and no mortality. Tuberculosis was seen at all ages but mainly between 20 and 39 yrs, where it represented 75% of cases. In this age group, malignancy was relatively rare: 10% of cases. In our environment of limited facilities, early chemotherapy trial for tuberculosis is justified for unknown pleural exudates in patients below the age of 40 yrs.