"Clotspeed," a mathematical simulation of the functional properties of prothrombinase

Prothrombinase is a Ca2+-dependent, 1:1, enzymatic complex of Factor Xa and Factor Va that assembles on the surface of negatively charged phospholipid vesicles or platelets. It catalyzes the proteolytic conversion of prothrombin to the blood-clotting enzyme thrombin. Experimentally determined kinetic parameters, plus Kd and n values for the interaction of substrate, cofactor (Factor Va), and serine protease (Factor Xa) for both phospholipid and each other, were used to develop a model that simulates the functional properties of the enzymatic complex. Through the use of a desk-top computer and a program designated "Clotspeed," the distribution of enzymatic components and substrate between the bulk fluid and phospholipid is determined for a given set of initial concentrations of reaction components. Simulated reaction rates are then calculated from the calculated distributions, fractional binding, and local and bulk concentration of reactants. Predicted behavior includes formal Michaelis-Mentenlike properties for the reaction, increasing apparent Km with increased levels of phospholipid, and apparent inhibition by excess substrate, enzyme, and phospholipid. Inhibition by excess enzyme and phospholipid was demonstrated experimentally in quantitative agreement with predicted results. The model is useful in that it rationalizes well the seemingly unusual properties of prothrombinase in straightforward physical terms, provides a means of rationally choosing experimental conditions to both further test and refine the model, and explores the properties not only of prothrombinase but also other blood-clotting or surface-bound enzymatic complexes.     

Cytogenetic analysis of chorionic villi: a technical assessment

Summary Eighty-five samples of chorionic villi from women undergoing prenatal diagnosis at 8 to 12 weeks' gestation were subjected to cytogenetic analysis. Samples were prepared by a direct technique that permits limited analysis within two hours and by a short-term culture technique that permits detailed structural analysis within one week. An adequate number of cell divisions for cytogenetic analysis was obtained from 96% of living fetuses. Using both the direct technique and short-term culture, satisfactory banded chromosomal preparations were made in 93% of cases. Eleven of 12 pregnancies (92%) shown by ultrasound to be dead shortly before sampling, had cytogenetic abnormalities. Further studies are needed to develop banding definition equivalent to that available on cultured amniocytes.     

Epinemin: a new protein associated with vimentin filaments in non-neural cells

In this report I describe a new protein, defined by a monoclonal antibody, which is associated with vimentin filaments in a variety of cultured cells and in skeletal muscle. By immunofluorescence it is absent in smooth muscle, in cells without vimentin, and in neural vimentin containing cells. This protein has a molecular weight of 44,500, a pl of 5, a two-dimensional tryptic peptide fingerprint pattern different from vimentin, is unrelated to actin by Cleveland peptide analysis and by light and electron microscopy, and is not recognized by either a polyclonal antivimentin antibody (Frank, E.D., and L. Warren, 1981, Proc. Natl. Acad. Sci. USA, 78:3020-3024) or a monoclonal antibody against all classes of intermediate filaments (Pruss, R.M., R. Mirsky, M.C. Raff, R. Thorpe, A.J. Dowding, and B.H. Anderton, 1981, Cell, 27:419-428). The protein is resistant to nonionic detergent extraction, is soluble in high salt and can thus be removed from vimentin filaments, but fragments with vimentin in either low salt or anionic detergent and collapses with vimentin in colchicine-treated cells. By light microscopy, the distribution of the protein is indistinguishable from vimentin filaments and appears uniform along them. In contrast, immunoferritin electron microscopy reveals that the molecule is distributed in an intermittent pattern on vimentin filaments. Adopting the terminology of Granger and Lazarides (1980, Cell, 30:263-275), the molecule is called epinemin, meaning "upon filaments."     

Enhancement of the repair of carcinogen-induced DNA damage in the hamster pancreas by dietary selenium

We measured single strand breaks (SSB) in pancreas DNA produced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters fed purified diets containing added sodium selenite (Se) at 0.0, 0.1 and 5.0 ppm. There were fewer SSB in those given the 5.0 ppm Se diet throughout the experiment. One hour after dosing with BOP (20 mg/kg), there were 2.26 ± 0.47, 2.83 ± 0.43 and 1.74 ± 0.43 SSB per 10⁸ daltons (mean ± S.E.M.) respectively in the three groups. The SSB were repaired faster in the 5.0 ppm Se-fed group. The approximate half-lives of the SSB were 33, 30 and 8 days, respectively. In the hamsters fed 5.0 ppm Se there was a small, statistically significant increase in pancreatic DNA synthesis. Autoradiographic analysis indicated that this was repair synthesis. In a second experiment, hamsters were fed one of the three diets prior to and for 2 days after administration of a single dose of BOP (20 mg/kg). They were then fed the 5.0 ppm Se diet for 5 days. The number of SSB was compared with those in hamsters fed their original diet for 7 days after BOP dosing. There was a statistically significant difference in the number of SSB in the hamsters fed 0.1 ppm Se before and for 2 days after BOP. In these hamsters there were 1.21 ± 0.24 SSB per 10⁸ daltons compared with 3.19 ± 0.4 (mean ± S.E.M.). These results suggest high levels of dietary Se stimulate the repair of carcinogen-induced DNA damage.     

The concentration and control of cytoplasmic free inorganic pyrophosphate in rat liver in vivo

The concentration of cytoplasmic free pyrophosphate was calculated in freeze-clamped livers of rats from the measured concentration of reactants and K(eq.) of the UDP-glucose pyrophosphorylase reaction (UDP-alpha-d-glucose 1-phosphate uridylyltransferase, EC 2.7.7.9). The K(eq.) of the UDP-glucose pyrophosphorylase reaction was redetermined at 38 degrees C, pH7.0, I=0.25mol/l and free [Mg(2+)]=1mm, and was 4.55 in the direction of glucose 1-phosphate formation. The activity of UDP-glucose pyrophosphorylase in rat liver was between 46 and 58mumol of glucose 1-phosphate formed/min per g fresh wt. in the four dietary conditions studied. A fluorimetric assay with enzymic cycling was developed for the measurement of glucose 1-phosphate in HClO(4) extracts of rat liver. The calculated free cytoplasmic PP(i) concentration in nmol/g fresh wt. of liver was 2.3+/-0.3 in starved, 3.8+/-0.4 in fed, 4.9+/-0.6 in meal-fed and 5.2+/-0.4 in sucrose-re-fed animals. These values agree well with recently determined direct measurements of total PP(i) in rat liver and suggest that there is not a large amount of bound or metabolically inert PP(i) in rat liver. The cytoplasmic [ATP]/[AMP][PP(i)] ratio is 10(3) times the cytoplasmic [ATP]/[ADP][P(i)] ratio and varies differently with dietary state. The reaction PP(i)+H(2)O-->2P(i) catalysed by inorganic pyrophosphatase (EC 3.6.1.1) does not attain near-equilibrium in vivo. PP(i) should be considered as one of the group of small inorganic ions which is metabolically active and capable of exerting a controlling function in a number of important metabolic reactions.     

Epilepsy and Oral Contraception

A controlled cross-over trial in 20 epileptic women, receiving regular anticonvulsant therapy showed that an oral contraceptive with a low oestrogen/ progestogen content had no significant effect on the average frequency of fits compared with identical dummy tablets.     

The binding of o-aminoazotoluene to deoxyribonucleic acid, ribonucleic acid and protein in the C57 mouse

1. (3)H-labelled o-aminoazotoluene was synthesized from [G-(3)H]o-toluidine on a semi-micro scale. 2. An association of (3)H with DNA, RNA and protein from the liver, kidney and spleen of female C57b mice was demonstrated after the administration of a single dose of [(3)H]o-aminoazotoluene. 3. This association is judged to represent covalent binding as a result of experiments involving solvent extraction, examination of the acid hydrolysates of the DNA and RNA and administration of [(3)H]water with unlabelled o-aminoazotoluene. 4. Examination of the extents of binding at various times after the administration of a single dose of [(3)H]o-aminoazotoluene showed that there was a peak of binding to liver DNA in the female mice at about 16hr. that was not present in the male mice. 5. The extent of binding to DNA, RNA and protein at 16hr. in the female C57b mouse liver was greater than that in the spleen and kidney.