ABSTRACT
The 5' untranslated leader sequence from the encephalomyocarditis virus was used to engineer bicistronic or tricistronic expression vectors encoding two subunits (P2X2 and P2X3) of an ATP-gated cation channel. Human embryonic kidney (293) and chinese hamster ovary (CHO-K1) cells were transfected with the vector, and stable cell lines were generated by single cell subcloning. Selection was made in a 96-well format on the basis of a sustained increase in intracellular calcium (fluorescence of Fluo3-loaded cells) evoked by the ATP analog alpha beta methylene ATP. A high proportion of transformants expressed heteromeric receptors containing both P2X2 and P2X3 subunits, as evidenced by a nondesensitizing current in response to alpha beta methylene ATP. The method is fast and simple and could be generally useful for the stable expression of heteromultimeric channel proteins.
Subject(s)
Calcium Channels/genetics , Cloning, Molecular/methods , Encephalomyocarditis virus , Genetic Vectors , Receptors, Purinergic P2/genetics , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , CHO Cells , Calcium Channels/biosynthesis , Cell Line , Cricetinae , Gene Expression , Humans , Receptors, Purinergic P2/biosynthesis , Receptors, Purinergic P2X2 , Receptors, Purinergic P2X3 , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/geneticsABSTRACT
We have demonstrated that anti-CD40 antibody stimulates the heterotypic adhesion of B cells to endothelial cells. This has been shown by using normal B lymphocytes and B-cell lines in a quantitative adhesion assay. When B cells, B-cell lines and Epstein-Barr virus (EBV)-transformed B cells from a patient with leucocyte adhesion deficiency (LAD) were stimulated with anti-CD40 antibody, they were found to adhere to both untreated and interleukin-1 (IL-1)-stimulated human umbilical vein endothelial cells (HUVEC), and to the lung carcinoma line A549. To identify the adhesion receptors responsible for this anti-CD40-induced adhesion, cells were pretreated with blocking antibodies prior to assay. Our results indicate that anti-CD40-stimulated adhesion of tonsillar B cells, B-cell lines RPMI-8866, JY, and an EBV-transformed LAD-cell line were predominantly dependent on the very late antigen-4 (VLA-4)-vascular cell adhesion molecule (VCAM) interaction. Anti-CD40-induced adhesion appears to be dependent on the activation of protein tyrosine kinase and protein kinase C and on the presence of divalent cations.
Subject(s)
Antigens, CD/immunology , Antigens, Differentiation, B-Lymphocyte/immunology , B-Lymphocytes/immunology , Lymphocyte Function-Associated Antigen-1/immunology , Receptors, Very Late Antigen/immunology , Antibodies/immunology , Binding, Competitive/immunology , CD40 Antigens , Cell Adhesion/immunology , Cell Adhesion Molecules/analysis , Cells, Cultured , Endothelium, Vascular/immunology , HumansABSTRACT
In a prospective, randomized, assessor-blind multicentre study two antithrombotic subcutaneous regimens were compared in patients undergoing total hip replacement. Group 1 (154 patients) received 750 anti-Xa units of a new low molecular weight heparinoid (Lomoparan) subcutaneously twice a day and group 2 (155 patients) received 5000 units heparin and 0.5 mg dihydroergotamine (heparin-DHE 5000) twice a day. The incidence of deep vein thrombosis, assessed by routine bilateral venography on day 10 (+/- 1), was 17 and 32 per cent in groups 1 and 2 respectively (risk reduction 47 per cent; P = 0.007). One patient in each group developed a symptomatic pulmonary embolism confirmed by lung scanning. Major bleeding complications occurred in one patient in each group and no significant difference was observed between the two groups with respect to minor bleeding complications. Subcutaneous Lomoparan appears to be as safe as heparin-DHE 5000 at the above doses with regard to bleeding complications, and is more efficacious with respect to venous thrombosis.
