Human liver RNA-programmed in vitro synthesis of a polypeptide related to human apolipoprotein B

AbstractIn an in vitro synthesizing system programmed with RNA from human liver a polypeptide with an estimated Mr of 80000 (80 kDa)±1400 (mean±SD, n=5) was synthesized. This polypeptide could be precipitated with antiserum to a narrow density cut of LDL (d=1.030-1.055) or antiserum against the high-Mr form of apoB (apoB 100 [4]). The synthesized protein is immunologically related to a 75 kDa protein isolated from LDL. We suggest that the 80 kDa protein represents a primary translation product of apoB synthesized in human liver

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

Human arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed. © 1989 Tissue Culture Association, Inc

931-115 Phase I Studies on Inogatran, a New Selective Thrombin Inhibitor

Inogatran is a new, synthetic, active site inhibitor of thrombin with a molecular weight of 439 dalton. Inogatran (pINN) selectively, rapidly and competitively binds thrombin with a Ki value of 15nmol/l. In vitro it doubles the plasma thrombin time at a concentration of 23nmol/1 and the activated partial thromboplastin time (APTT) at 1.1μmol/1. Thrombin induced platelet aggregation is inhibited at an IC50 of 17 nmol/1 Inogatran was studied in healthy male human volunteers with regard to tolerability, pharmacokinetics and effects on haemostasis. It was given intravenously as a bolus in doses from 0.002 to 1.1μmol/kg (n=2-4 at each dose). The highest peak plasma concentration observed was 7μmol/1 corresponding to an APTT prolongation of 3 times. The drug was also given as a constant infusion over 4h at a dose of 0.73μmol/kg per h (n=16) which resulted in a mean plasma concentration at steady state of 1.9μmol/l and an APTT prolongation of 2.3 times. Finally, it was given as a bolus with radiolabeled compound in a total dose of 25μmol (n=16). The drug was well tolerated and without side effects with the exception of slightly increased bleeding tendency at the blood sampling site. Inogatran had a volume of distribution of 0.26ml/kg and a total plasma clearance of 6.1ml/min per kg, resulting in a half life of about one hour. The drug was not metabolised and it was excreted unchanged with the elimination evenly distributed between urine and faeces. Ex vivo the thrombin time was linearly correlated to the plasma concentration while the APTT-concentration curve was non-linear. At the highest plasma concentrations a slight prolongation of the capillary bleeding time was seen in some subjects. Markers of thrombin activity (thrombin-antithrombin complex and prothrombin fragments 1–2) decreased during the constant infusion of the drug. There was no effect on fibrinolysis (PAI-l and t-PA activities) or protein C. It is concluded that inogatran is a safe and effective anticoagulant with favourable pharmacokinetics for i.v. us