Activation of the phosphatidylinositol cycle in spreading cells

Metabolites of the phosphatidylinositol cycle were analyzed in BHK-21 (C13) cells spreading on fibronectin-coated culture plates in comparison with attached nonspreading cells 45 min after plating. Among the water-soluble metabolites (glycerophosphoinositol, inositol, inositol monophosphate, inositol bisphosphate, inositol trisphosphate, and inositol tetrakisphosphate), significant elevations were found for inositol monophosphate, inositol bisphosphate, and inositol tetrakisphosphate. In the lipid fraction, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate were significantly elevated. The activation of the phosphatidylinositol cycle in spreading versus nonspreading attached BHK-21 (C13) cells may be involved in the permissive effect of the extracellular matrix on cell proliferation.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

The use of transgenic mice in unraveling the multistep process of tumorigenesis

Summary The development of cancer has long been perceived to be a complex and multistep process in which a normal cell progresses to a fully malignant tumor cell in a step-by-step fashion. At the molecular level it is believed that these steps correspond to the acquisition of activated oncogenes or alternatively the inactivation of tumor suppressor genes. With the ability to stably transfer foreign genetic information into the germ line of animals a new powerful tool to study oncogenes became available.     

On the Recovery of [3H]Noradrenaline from Different Metabolic Compartments of Rat Brain with Respect to the Role of Catechol-O-Methyltransferase

Abstract: Rats were treated with reserpine, desmethylimipramine, or carrier, either alone or in combination with tropolone. Either 10 min (t₁) or 1 h (t₂) after intraventricular injection of [³H]noradrenaline, they were decapitated. The total ³H activity and the recovery of [³H]noradrenaline were determined in tissue extracts from various brain regions. Maximum total ³H activity was measured at t₁ in all tropolone-treated rats; the mean sum of these results served as an estimate of the initial tissue concentration of [³H]noradrenaline. At t₁, 40–50% of the sum of [³H]noradrenaline and its metabolites was recovered unchanged in normal rats; reserpine and DMI reduced the recovery to 18–27%. In all groups, the decline of [³H]noradrenaline was retarded after t₁. Inhibition of catechol-O-methyltransferase by tropolone caused consistently elevated [³H]noradrenaline levels, but did not affect the metabolic rate after t₁ when compared with similarly pretreated, but tropolone-free rats. Thus, if catechol-O-methyltransferase was inhibited during the injection of [³H]noradrenaline, a higher percentage of the amine had been taken up into spaces with a slow noradrenaline turnover. The maximum increase was seen when the neuronal uptake, was inhibited by desmethylimipramine. This supported the hypothesis that an additional extraneuronal space exists, in addition to the known intraneuronal and extraneuronal compartments, which has a slow noradrenaline turnover. The tropolone effect on the noradrenaline recovery possibly shows that there might be a saturable “methylating system,” similar to that described for the periphery, in which catechol-O-methyltransferase is linked to the extraneuronal uptake₂. By affecting the access of noradrenaline to non-neuronal cells it might influence the rate of noradrenaline elimination from the intercellular space.     

Synthesis of tritiated 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid ([3H]DIDS) and its covalent reaction with sites related to anion transport in human red blood cells

Summary The potent and specific inhibitor of anion permeability, 4,4-diisothicyanostilbene-2,2-disulfonic acid (DIDS) was synthesized in tritiated form ([³H]DIDS) from tritiated 5-nitrotoluene-o-sulfonic acid. Its reactions with and effects on red blood cells were compared with those of a reduced form ([³H]H₂DIDS), previously used as a tracer for DIDS. The rate of covalent reaction of [³H]DIDS was substantially faster than that of [³H]H₂DIDS at all temperatures tested. With both agents, the rate of reaction was increased in alkaline media, although the response occurred at a lower pH with [³H]DIDS. On the other hand, the relationship of irreversible membrane binding to the degree of inhibition of sulfate fluxes was linear and virtually the same for both agents, with 100% inhibition associated with the binding of approximately 1.2×10⁶ molecules per cell. About 90% of the binding for each probe was to a particular membrane protein, known as band 3, equivalent to about 1 mole of agent per mole of protein.     

Plasma levels of norethisterone measured by radioimmunoassay and gas chromatography-mass fragmentography

Assays designed to quantitate the plasma levels of norethisterone (NET) are compared. Results from radioimmunoassay were compared with those from gas chromatography-mass fragmentography which were established in 5 subjects after a 1-mg oral dose of the steroid. In general, the levels of steroid measured by radioimmunoassay were usually higher than those measured by gas chromatography-mass fragmentography; there was however a correlation coefficient of .96 (P .001). For concentrations of the steroid above 500 pg/ml, the mean overestimate by radioimmunoassay was less than 30%.     

Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies

A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.