Direct demonstration of actin filament annealing in vitro

Direct electron microscopic examination confirms that short actin filaments rapidly anneal end-to-end in vitro, leading over time to an increase in filament length at steady state. During annealing of mixtures of native unlabeled filaments and glutaraldehyde-fixed filaments labeled with myosin subfragment-1, the structural polarity within heteropolymers is conserved absolutely. Annealing does not appear to require either ATP hydrolysis or the presence of exogenous actin monomers, suggesting that joining occurs through the direct association of filament ends. During recovery from sonication the initial rate of annealing is consistent with a second-order reaction involving the collision of two filament ends with an apparent annealing rate constant of 10(7) M-1s-1. This rapid phase lasts less than 10 s and is followed by a slow phase lasting minutes to hours. Annealing is calculated to contribute minimally to filament elongation during the initial stages of self-assembly. However, the rapid rate of annealing of sonicated fixed filaments observed in vitro suggests that it may be an efficient mechanism for repairing breaks in filaments and that annealing together with polymer-severing mechanisms may contribute significantly to the dynamics and function of actin filaments in vivo.     

Cardiovascular effects of prostaglandin I2 and prostaglandin F2 alpha in the unanesthetized bullfrog, Rana catesbeiana

The cardiovascular effects of prostaglandin (PG)I2 and PGF2 alpha were compared in the unanesthetized American bullfrog (Rana catesbeiana). Control mean arterial pressure (MAP) and heart rate (HR) were 25.7 +/- 1.1 mm Hg and 35.1 +/- 1.1 beats/min, respectively. Intravenous injections of PGI2 decreased MAP and increased HR in a dose-dependent fashion over the range of concentrations tested (0.03, 0.3, 3, and 10 micrograms/kg-body weight [bw]. Neither atropine (1 mg/kg-bw) nor verapamil (1 mg/kg-bw) treatment altered the MAP or HR responses to PGI2 (3 micrograms/kg-bw). However, propranolol (5 mg/kg-bw) significantly blunted the hypotensive effects without affecting the increase in HR. Prostaglandin F2 alpha (tested at 0.3, 3, 30, and 100 micrograms/kg-bw) increased both MAP and HR. Mean arterial pressure increased with concentrations greater than 0.3 microgram/kg-bw and reached peak effects at 30 micrograms/kg-bw. Prostaglandin F2 alpha increased HR at doses greater than 0.3 microgram/kg-bw. Neither the pressor nor positive chronotropic effects of PGF2 alpha (30 micrograms/kg-bw) were affected by atropine or propranolol. However, verapamil significantly attenuated the pressor effects without affecting the increase in HR. These results demonstrate that both prostaglandins have qualitatively similar effects on HR, but opposite effects on MAP. Prostaglandin I2 is a hypotensive prostaglandin, while PGF2 alpha is hypertensive. The pressor effects of PGF2 alpha are partially dependent on calcium influx. The positive chronotropic effects of both prostaglandins are independent of the autonomic nervous system, suggesting a different mechanism of action.     

Mutations that prevent cyclic nucleotide binding to binding sites A or B of type I cyclic AMP-dependent protein kinase

Cyclic nucleotide binding and activation properties of cAMP-dependent protein kinases from five independent mutants of S49 mouse lymphoma cells were studied. These mutants were all hemizygous for expression of mutant regulatory (R) subunits of the type I kinase with lesions that altered the electrostatic charge of R subunit: lesions in three of the mutants mapped to cAMP-binding site A, and those in two of the mutants mapped to cAMP-binding site B. A nucleotide mismatch assay using 32P-labeled cRNA and ribonuclease A confirmed and refined localization of the mutations to single amino acid residues implicated in cAMP binding. R subunits from all mutants retained the ability to bind cAMP, but binding behaved as if it were entirely to nonmutated sites: 1) relative affinities of 11 adenine-modified derivatives of cAMP for mutant enzymes were identical to their relative affinities for the site of wild-type kinase that corresponded to the nonmutated site of the mutant; 2) the potencies of these analogs as activators of mutant kinases were strictly correlated with their binding affinities (for wild-type enzyme activation potencies were correlated with mean affinities of the analogs for cAMP-binding sites A and B); 3) combinations of analogs with strong preferences for opposite cAMP-binding sites in wild-type kinase showed no synergism in activating mutant kinases; 4) dissociation of cAMP from mutant kinases was monophasic; and 5) high salt accelerated dissociation of cAMP from kinases with site B lesions but retarded dissociation from those with site A lesions.     

Ca-dependent slow action potentials in human skeletal muscle

Slow Ca-action potentials (CaAP) were studied in normal human skeletal muscle fibers obtained during surgery (fibers with both ends cut). Control studies also were carried out with intact as well as cut rat skeletal muscle fibers. Experiments were performed in hypertonic Cl-free saline with 10 or 84 mM Ca and K-channel blockers; muscles were preincubated in a saline containing Cs and tetraethylammonium. A current-clamp technique with two intracellular microelectrodes was used. In human muscle, 14.5% of the fibers showed fully developed CaAPs, 21% displayed nonregenerative Ca responses, and 64.5% showed only passive responses; CaAPs were never observed in 10 mM Ca. In rat muscle, nearly 90% of the fibers showed CaAPs, which were not affected by the cut-end condition. Human and rat muscle fibers had similar membrane potential and conductance in the resting state. In human muscle (22-32 degrees C, 84 mM Ca), the threshold and peak potential during a CaAP were +26 +/- 6 mV and +70 +/- 3 mV, respectively, and the duration measured at threshold level was 1.7 +/- 0.5 sec. In rat muscle, the duration was four times longer. During a CaAP, membrane conductance was assumed to be a leak conductance in parallel with a Ca and a K conductance. In human muscle (22-32 degrees C, 84 mM Ca, 40 micron fiber diameter), values were 0.4 +/- 0.1 microS, 1.1 +/- 0.7 microS, and 0.9 +/- 0.4 microS, respectively. Rat muscle (22-24 degrees C, 84 mM Ca) showed leak and K conductances similar to those found in human fibers. Ca-conductance in rat muscle was double the values obtained in human muscle fibers.     

Basic fibroblast growth factor and insulin-like growth factor I are strong mitogens for cultured mouse keratinocytes

Basic fibroblast growth factor (bFGF), but not acidic fibroblast growth factor (aFGF), was found to be mitogenic for cultured mouse keratinocytes. A six-to-nine fold increase in 3H-thymidine (3H-dT) incorporation into the acid insoluble pool and a similar increase of the labeling index can be measured when bFGF, at a concentration between 1 and 10 ng/ml, is added to keratinocytes arrested in serum-free and growth factor-free medium with a Ca++-concentration below 0.1 mM. The half-maximal response is observed between 0.2 and 0.7 ng/ml. In the same culture system, insulin-like growth factor I/somatomedin C (IGF-I) and insulin act as mitogens. IGF-I shows half-maximal stimulation with 2-3 ng/ml, insulin with 100-500 ng/ml. Basic FGF, IGF-I and insulin can be classified as strong stimulators of DNA synthesis in mouse keratinocytes. In this regard they are comparable to epidermal growth factor, which shows a half-maximal stimulation at a concentration between 1.5-2 ng/ml. These results show that in addition to mesenchymal cells, FGF is a growth factor not only for neuroectodermal cells, but ectodermal cells in general. They further support the idea that the growth promoting effect of insulin on keratinocytes may be mediated by the IGF-I receptor.     

Na,K pump stimulation by intracellular Na in isolated, intact sheep cardiac Purkinje fibers

Regulation of the Na,K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37 degrees C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aNai) was calculated from the voltage difference between an Na+-selective microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aiK) or chloride (aCli) was measured by a third separate microelectrode. Strands were loaded by Na,K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10(-8) M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aNai ranging between 25 and 90 mM and Vm was -30.8 +/- 2.2 mV. The Na,K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 +/- 5.9 mV with Rb+ activation and to -66.0 +/- 7.7 mV with K+ activation. aiNa decreased in both cases to 0.5 +/- 0.2 mM in 5-15 min. The maximum rate of aiNa decline (maximum delta aNai/delta t) was the same with K+ and Rb+ at concentrations greater than 20 mM. The response was abolished by 10(-5) M acetylstrophantidin. Maximum delta aNai/delta t was independent of outside Na+, while aKi was negatively correlated with aNai (aKi = 88.4 - 0.86.aNai). aCli decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aNai. This model provided a functional isolation of the Na,K pump, so that the relation between the pump rate (delta aNai/delta t) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1.K 0.5 was 10.5 +/- 0.6 mM (the aNai that gives delta aNai/delta t = Vmax/2) and n equaled 1.94 +/- 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g-1, giving a maximum Na+ turnover of 300 s-1. The Na,K pump in intact Purkinje strands exhibited typical sigmoidal saturation kinetics with regard to aNai as described by the equation upsilon/Vmax = aNai(1.94)/(95.2 + aNai(1.94)). The maximum sensitivity of the Na,K pump to aiNa occurred at approximately 6 mM.     

The superoxide generating system of B cell lines. Structural homology with the phagocytic oxidase and triggering via surface Ig

EBV-transformed B lymphocyte cell lines (EBV-BLCL) produce superoxide after stimulation with phorbol ester, a capacity unique among nonmyeloid cells. The superoxide producing system of EBV-BLCL (B cell oxidase) was compared with the phagocytic NADPH-oxidase and the relationship of the capacity to produce superoxide to the presence of the EBV-genome was analyzed. The two EBV-transformed B cell lines F1 and HELL generated superoxide in response to PMA (2.3 nmol/10(6) F1 cells x 1 h and 6.27 nmol/10(6) HELL cells x 1 h with 1 microgram/ml of PMA), whereas no superoxide release was detected with the EBV-positive Burkitt lymphoma line WIL-2 and the EBV-negative plasmocytoma line U-266. Also, F1 and HELL showed lucigenin-dependent chemiluminescence (CL) after PMA-treatment, whereas no CL responses were detected from WIL-2 or U-266. Further, F1 and HELL cells contained a low potential cytochrome b-245 (10.9 and 61.0 pmol/mg protein, respectively) and also a 45 kDa diphenylene-iodonium (DPI)-binding peptide, both components of the phagocytic NADPH-oxidase. In contrast, neither the cytochrome b-245 nor the 45 kDa DPI-binding peptide were detected in WIL-2 and U-266. In addition, DPI inhibited O2- production by PMA-stimulated EBV-BLCL and polymorphonuclear granulocytes. Further, F1 line cells showed superoxide dismutase-inhibitable lucigenin-dependent CL when triggered by protein A-bearing staphylococci (Cowan strain I) or by a mAb directed against human IgG in the presence of solid-phase goat anti-mouse-Ig antibody. From a panel of eight EBV-BLCL, only five responded with CL when exposed to protein A-bearing staphylococci, whereas all showed CL when treated with phorbol ester. Inasmuch as all eight EBV-BLCL possessed surface Ig and a "functional" oxidase, their differential response to cross-linking of surface Ig may be determined by differences in signal transduction. Superoxide production by EBV-BLCL appears thus related to expression of an electron transport chain structurally homologous, if not identical, with the "phagocytic" NADPH-oxidase. Apparently, the presence of EBV-genome in B cell lines does not per se lead to expression of this oxidase. This suggests that nontransformed B cells may, at a certain differentiation stage, also express a superoxide-generating chain. From the finding of stimulation of superoxide production of EBV-BLCL via surface Ig it appears possible that also Ag may be able to trigger such B cells to production of superoxide which might have an important role in the physiology of B cells.     

Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution

Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues.