Measurement of the secondary structure of adsorbed protein by circular dichroism. 1. Measurements of the helix content of adsorbed melittin. A new circular dichroism (CD) technique is presented which quantifies, in situ, the changes in protein and peptide secondary structure upon adsorption at the quartz/liquid interface. Far-UV CD spectra of adsorbed proteins were recorded from several quartz interfaces contained in a specially constructed cell. Adsorbed, oriented alpha-helical spectra were recorded from hydrophilic and hydrophobic quartz using the bee venom peptide, melittin, which can be induced into an alpha-helical, tetrameric conformation in solution. The hydrophobic quartz provides a model system for oil-in-water emulsions and cell membranes. Surface concentrations were determined by radio-counting and were dependent on the nature of the surface. The characterization of these spectra has been partly achieved using far-UV CD spectra obtained from melittin adsorbed onto hydrophilic colloidal silica particles, where orientation effects are eliminated. Analysis of these spectra reveals considerable denaturation of the helical structures upon adsorption. Surface concentrations from the silica were determined from adsorption isotherms. The surface orientation of adsorbed melittin was dependent on the state of aggregation and hence degree of helicity of the molecule. These results support a model for the mode of action of melittin in lysing membranes.


Purification and characterization of a low-Km 3':5'-cyclic adenosine phosphodiesterase from post-meiotic male mouse germ cells. We describe the purification and the study of the kinetic and hydrodynamic properties of a 'low Km' cAMP phosphodiesterase specifically expressed in haploid male germ cells of the mouse. The enzyme has been purified approx. 13,000-fold with respect to the activity in total cell homogenate. The purified enzyme hydrolyzed specifically cAMP with a Km of 3.3 microM and with a Vmax of 10.5 mumol of cAMP hydrolyzed/min per mg of protein. The hydrolytic activity was neither stimulated nor inhibited by cGMP, whereas it was inhibited by RO 20-1724 and Rolipram. The enzyme showed a Stokes radius of 3.8 nm and a sedimentation coefficient of 3.1 S, corresponding to a native molecular mass of 50 kDa and a frictional ratio of 1.53. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of sucrose gradient fractions of the purified enzyme showed a major band of 43 kDa copeaking with enzyme activity.


Hydrogen exchange in Pseudomonas cytochrome c-551. Hydrogen exchange rates were measured or estimated for 75 amide protons in in ferrocytochrome c-551 from Pseudomonas aeruginosa (82 residues total) at neutral pH and 300 K. Rate constants span at least eight orders of magnitude. Rate constants or limiting estimates were determined by a combination of methods relying upon 1H-NMR spectroscopy, including the direct observation in one- or two-dimensional spectra of the decrease in proton intensity for samples dissolved in deuterium oxide, or, in a few favorable cases, saturation transfer from the solvent protic water. The heme ligand residues and the thioether bridge residues were slowly exchanging backbone amides, but the slowest exchanging backbone amides were found in two clusters. One was composed of Ile-48 and Lys-49 in the last turn of what is termed the 40's helix in the protein. The second was composed of Leu-74, Ala-75, Lys-76 and Val-78 in the C-terminal alpha helix.


Adrenocorticotropin hormone response to diazepam in healthy young men. The effects of diazepam (DZ) infusions on changes in adrenocorticotropin hormone (ACTH) are a source of debate. In this study, 66 healthy young adult men were evaluated for changes in plasma ACTH after intravenous infusions with placebo, 0.12 mg/kg and 0.20 mg/kg of DZ. After the higher DZ dose, 85% of the subjects demonstrated a decrease in ACTH of 20% or more, with the nadir occurring between 30 and 60 min postinfusion and values returning to baseline levels by 180 min. These results support the conclusion that at the clinically relevant doses used here, DZ infusions are associated with a significant decrease in ACTH.


Decreased platelet imipramine binding in Tourette syndrome children with obsessive-compulsive disorder. [3H]Imipramine binding to blood platelets was assessed in eight untreated Tourette syndrome (TS) children, nine drug-free TS children with obsessive-compulsive disorder (OCD), and nine age-matched and gender-matched control subjects. The density of [3H]imipramine binding sites in TS + OCD patients was significantly lower compared with TS-OCD patients (28%) as well as when compared with controls (31%). This alteration was not accompanied by differences in the affinity of the binding site to the ligand. The decreased density of the platelet serotonin "transporter" might implicate the involvement of the serotonergic system in the pathophysiology of OCD in TS patients, but not in TS per se.


Alpha-2-adrenoceptor sensitivity in early alcohol withdrawal. Growth hormone (GH), blood pressure, and pulse rate responses to clonidine (100 micrograms IV) were studied three times during the first week of alcohol withdrawal in 19 alcohol-dependent patients. Fifteen healthy men were used as controls. The results suggest reduced sensitivity of the alpha-2-adrenoceptors involved in GH secretion for at least 1 week after the end of alcohol intake. In contrast, very short-lasting subsensitivity was found in the alpha-2-adrenoceptors regulating blood pressure.


Outbreak of pseudoinfection with Tsukamurella paurometabolum traced to laboratory contamination: efficacy of joint epidemiological and laboratory investigation. From January 1988 to May 1989, one hospital in South Carolina reported 12 isolates of Tsukamurella paurometabolum from 10 patients. There were no common risk factors among the patients. Case-control studies revealed that the positive specimens were significantly more likely to have been processed in the TB/fungal room, to have been tissue samples, and to have been handled by one technician. Typing on the basis of biochemical, antimicrobial resistance, Southern blot, and ribotype profiles showed that the isolates from the outbreak were essentially identical and that they were distinguishable from each of two isolates obtained after the outbreak and from two type strains. These findings support the hypothesis of a common-source outbreak of pseudoinfection. There are reasons to believe that T. paurometabolum is present both in the environment and as a culture contaminant more often than has been recognized and that it is very rarely the true cause of infection in humans. Typing results show differences between one type strain and all of the other isolates studied in terms of colonial morphology, biochemistry, antimicrobial susceptibility, and ribotyping; these differences suggest that the nomenclature of T. paurometabolum may require further clarification.


Successful treatment of Paecilomyces varioti infection in a patient with chronic granulomatous disease and a review of Paecilomyces species infections. An 8-year-old boy who had chronic granulomatous disease developed a soft tissue infection of the right heel after riding on a motor scooter. Infection was insidious, and minor heel pain and fevers occurred only on the day interferon-gamma was injected. Soft tissue biopsy showed hyphal elements, and Paecilomyces varioti grew in culture. The infection was treated with amphotericin B for 7 weeks (total dose, 40 mg/kg) followed by 1 year of therapy with itraconazole (100 mg twice daily). Complete cure was achieved during the follow-up period of 10 months.


Osteogenic response to porous hydroxyapatite ceramics under the skin of dogs. Soft tissue reactions to two materials of different microstructure made with porous and dense hydroxyapatite ceramic granules, were compared in this study. Each was implanted subcutaneously in the abdomen of 10 dogs. The implants were removed 1, 3 and 6 months after implantation, and examined by light microscopy. Heterotopic bone formation was only evident around porous hydroxyapatite ceramic granules in 6 of the 10 animals after 3 months, and in all cases after 6 months, but never around dense granules. These findings suggested that cellular differentiation and induction of osteogenesis may be influenced by the physical and chemical factors in the micromilieu provided by porous hydroxyapatite ceramic granules.


Crystallographic changes in calcium phosphates during plasma-spraying. Coating hydroxyapatite (HA) onto metal implant surfaces using the plasma-spraying technique has been investigated in several laboratories as a means of improving the mechanical properties of the bulk ceramic. This study describes crystallographic changes which can occur during the plasma-spraying of calcium phosphate powders. A precipitated calcium-deficient apatite and a high temperature near-stoichiometric HA were each sprayed onto metal substrate in an argon plasma using several hydrogen gas flow conditions at various temperatures. The surfaces were examined by X-ray diffraction and scanning electron microscopy. The plasma-sprayed products were identified as a mixture of calcium phosphates including HA, beta-tricalcium phosphate (beta-TCP) and calcium oxide. Stoichiometric HA when plasma-sprayed showed the least (5%) degradation. Since beta-TCP is more resorbable than HA in vivo, varying the HA/beta-TCP ratio on the plasma-sprayed surface may provide a method to control surface dissolution of the coating.


Verapamil inhibits in-vitro leucotriene B4 release by rectal mucosa in active ulcerative colitis. Increased mucosal eicosanoid synthesis occurs in active ulcerative colitis; suppression of the synthesis of pro-inflammatory leucotrienes could be therapeutically useful. Neutrophil 5-lipoxygenase is calcium-dependent. In this study, the effect of the calcium channel antagonist, verapamil, on the release of eicosanoids by colitic rectal mucosal biopsies has been examined. Verapamil in therapeutic concentration (5 micrograms/ml, 10(-5) M) reduced leucotriene B4 release from actively inflamed rectal mucosa by 30% (from 60 (5.0 S.E.M.) ng/g wet weight/20 min without, to 42 (5.7 S.E.M.) with verapamil, P less than 0.05), but had no effect on leucotriene B4 release by rectal biopsies taken from patients with quiescent ulcerative colitis (39 (2.8 S.E.M.) ng/g wet weight/20 min without, and 43 (5.0 S.E.M.) with verapamil). Verapamil did not affect mucosal prostaglandin E2 release. The results suggest that, in active ulcerative colitis, verapamil inhibits mucosal 5-lipoxygenase activity and warrants therapeutic evaluation.


Effect of acute and chronic acid suppression on plasma gastrin release in the rat. The mechanisms by which administration of the H+,K(+)-ATPase inhibitor B 831-78 or intragastric perfusion with NaHCO3 induces plasma gastrin release were studied in the rat. Experiments were performed after a washout of residual intragastric contents in fasted animals provided with chronic gastric fistulae. Acute and chronic administration of B 831-78 elevated plasma gastrin dose-dependently up to 5-6 times above control levels, while the increase was only twofold with intragastric NaHCO3 infusion despite similar neutralization of gastric acidity. The profound hypergastrinaemia induced by the H+,K(+)-ATPase inhibitor, after both acute and chronic treatment, was completely prevented or reversed by intragastric perfusion with physiological amounts of acid (0.15 N HCl, 2.5 ml/h). The hypergastrinaemia was, however, largely resistant to high doses of atropine (4.3 mumol/kg) and of the M1 selective muscarinic antagonist telenzepine (10 mumol/kg). In contrast, the modest increase in plasma gastrin induced by gastric perfusion with NaHCO3 was completely suppressed by the high atropine dose and was attenuated by small doses of atropine or telenzepine (0.01 mumol/kg and 1 mumol/kg). These results demonstrate that, in the rat, blockade of the H+,K(+)-ATPase can potently induce gastrin release in the absence of a meal. Moreover, they suggest that interruption of the negative feedback between acid and gastrin release is the main mechanism through which this class of drugs releases gastrin in the rat. Since a similar degree of gastrin release cannot be achieved by alkalinization of gastric contents, additional hormonal or neural regulatory factors may contribute to the drug-induced hypergastrinaemia.


Charge movement and SR calcium release in frog skeletal muscle can be related by a Hodgkin-Huxley model with four gating particles. Charge movement currents (IQ) and calcium transients (delta[Ca2+]) were measured simultaneously in frog skeletal muscle fibers, voltage clamped in a double vaseline gap chamber, using Antipyrylazo III as the calcium indicator. The rate of release of calcium from the SR (Rrel) was calculated from the calcium transients using the removal model of Melzer, W., E. Rios, and M. F. Schneider (1987. Biophys. J. 51:849-863.). IQ and delta [Ca2+] were calculated for 100 ms depolarizing test pulses to membrane potentials from -30 to +20 mV. To eliminate an inactivating component of Rrel, each test pulse was preceded by a large, fixed prepulse to +20 mV. The resulting Rrel records, which represent the noninactivating component of Rrel, were compared with integral of IQdt.(Q), the total charge that moves. The voltage dependence of the steady state Rrel was steeper then that of Q and shifted to the right. During depolarization, the Rrel waveform was similar to that of Q but was delayed by several ms, while, during repolarization, Rrel preceded Q. All of these results could be explained with a Hodgkin-Huxley type model for E-C coupling in which four voltage sensors in the t-tubule membrane which give rise to IQ must all be in their activating positions for the calcium release channel in the SR membrane to open. his model is consistent with the structural architecture of the triadic junction in which four dihydropyridine receptors (the voltage sensors for E-C coupling) in the t-tubule membrane are closely associated with each ryanodine receptor(the calcium release channel) in the SR membrane [Block, B. A., T. Imagawa, K. P. Campbell, and C. Franzini-Armstrong. 1988. J.Cell. Biol. 107:2587-2600.]). Some aspects of this work have appeared in abstract form (Simon, B. J., and D. Hill. 1991. Biophys. J.59:64a. ([Abstr.]).


Large tetraalkyl ammonium cations produce a reduced conductance state in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel. The purified Ca(2+)-release/ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) functions as a calcium-activated cation-selective channel under voltage clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effect of large tetraalkyl ammonium (TAA) cations, (CnH2n+1)4N+ (n = 4 and 5) on monovalent cation conduction. These cations modify the conductance of the receptor channel at positive holding potentials from the cytosolic side of the channel. Under these conditions, openings are resolved as a mixture of normal full amplitude events and events of reduced conductance. The amplitude of the reduced conductance state is a fixed proportion of the normal open state. As a proportion of all open events, the occurrence of the tetrabutyl ammonium (TBA+) related subconductance state increases with concentration and increasingly positive holding potential. The TBA+ related subconductance state displays similar conduction properties to the unmodified channel; with a linear current-voltage relationship, a similar affinity for K+ and voltage-dependent block by TEA+. A method was used to quantify the voltage dependence of the occurrence of the TBA+ effect, which yielded an effective gating charge of 1.66. A second method based on kinetic analysis of the voltage dependence of transitions between the full open state and the TBA+ related subconductance state produced a similar value. In addition, this analysis revealed that the bulk of the voltage-dependence resided in the off rate. TBA+ related subconductance events, expressed as a proportion of all open events, saturated with increasing TBA+ concentration. Kinetic analysis revealed that this could be entirely accounted for by changes in the on rate. Tetrapentyl ammonium (TPeA+) causes a qualitatively similar effect with a subconductance state of lower amplitude. The voltage-dependence of the effect was comparable to that displayed by TBA+. These findings are interpreted as a form of partial block in which more than one large TAA cation binds at the extremity of the voltage drop to produce an electrostatic barrier for ion translocation.


Molecular basis for the inhibition of 1,4-dihydropyridine calcium channel drugs binding to their receptors by a nonspecific site interaction mechanism. The "membrane bilayer" pathway (Rhodes, D. G., J. G. Sarmiento, and L. G. Herbette. 1985. Mol. Pharmacol. 27:612-623.) for 1,4-dihydropyridine calcium channel drug (DHP) binding to receptor sites in cardiac sarcolemmal membranes has been extended to include the interaction of amphiphiles within the lipid bilayer. These studies focused on the ability of the Class III antiarrhythmic agents bretylium and clofilium to nonspecifically inhibit DHP-receptor binding in canine cardiac sarcolemma. Clofilium was found to inhibit nimodipine binding with an inhibition constant of approximately 5 microM, whereas bretylium had no effect on nimodipine binding. Small angle x-ray diffraction was then used to examine the differential ability of these two Class III agents to inhibit DHP-receptor binding. The time-averaged locations of bretylium, clofilium, and nimodipine in bovine cardiac phosphatidylcholine (BCPC) bilayers (supplemented with 13 mol% cholesterol) were determined to a resolution of 9 A. The location of bretylium as dominated by its phenyl ring in BCPC bilayers was found to be at the hydrocarbon core/water interface, similar to that of the dihydropyridine ring of nimodipine. The location of clofilium as dominated by its phenyl ring was found to be below the hydrocarbon/core water interface within the hydrocarbon chain region of the bilayer, similar to that of the phenyl ring of nimodipine. The location of the dihydropyridine ring portion of nimodipine has previously been shown by neutron diffraction to be located at the hydrocarbon core/water interface of native sarcoplasmic reticulum, consistent with the small angle x-ray data from model membranes in this paper. Therefore, we speculate that the nonspecific inhibition arises from the interaction of clofilium's phenyl ring with the site on the calcium channel receptor where the phenyl ring portion of nimodipine must interact. The DHP-receptor binding pathway would then involve both nonspecific (membrane) and specific (protein) binding components, both of which are necessary for receptor binding.


Fast release of 45Ca2+ induced by inositol 1,4,5-trisphosphate and Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle: evidence for two types of Ca2+ release channels. The kinetics of Ca2+ release induced by the second messenger D-myoinositol 1,4,5 trisphosphate (IP3), by the hydrolysis-resistant analogue D-myoinositol 1,4,5 trisphosphorothioate (IPS3), and by micromolar Ca2+ were resolved on a millisecond time scale in the junctional sarcoplasmic reticulum (SR) of rabbit skeletal muscle. The total Ca2+ mobilized by IP3 and IPS3 varied with concentration and with time of exposure. Approximately 5% of the 45Ca2+ passively loaded into the SR was released by 2 microM IPS3 in 150 ms, 10% was released by 10 microM IPS3 in 100 ms, and 20% was released by 50 microM IPS3 in 20 ms. Released 45Ca2+ reached a limiting value of approximately 30% of the original load at a concentration of 10 microM IP3 or 25-50 microM IPS3. Ca(2+)-induced Ca2+ release (CICR) was studied by elevating the extravesicular Ca2+ while maintaining a constant 5-mM intravesicular 45Ca2+. An increase in extravesicular Ca2+ from 7 nM to 10 microM resulted in a release of 55 +/- 7% of the passively loaded 45Ca2+ in 150 ms. CICR was blocked by 5 mM Mg2+ or by 10 microM ruthenium red, but was not blocked by heparin at concentrations as high as 2.5 mg/ml. In contrast, the release produced by IPS3 was not affected by Mg2+ or ruthenium red but was totally inhibited by heparin at concentrations of 2.5 mg/ml or lower. The release produced by 10 microM Ca2+ plus 25 microM IPS3 was similar to that produced by 10 microM Ca2+ alone and suggested that IP3-sensitive channels were present in SR vesicles also containing ruthenium red-sensitive Ca2+ release channels. The junctional SR of rabbit skeletal muscle may thus have two types of intracellular Ca2+ releasing channels displaying fast activation kinetics, namely, IP3-sensitive and Ca(2+)-sensitive channels.


Effects of tryptophan mutation on the deprotonation and reprotonation kinetics of the Schiff base during the photocycle of bacteriorhodopsin. The rates of deprotonation and reprotonation of the protonated Schiff base (PSB) are determined during the photocycle of nine bacteriorhodopsin mutants in which Trp-10, 12, 80, 86, 137, 138, 182 and 189 are individually substituted by either phenylalanine or cysteine. Of all the mutants, the replacement of Trp-86, Trp-182, and Trp-189 by phenylalanine and Trp-137 by cysteine is found to significantly alter the rate of the deprotonation, but not that of the reprotonation process. As compared with ebR, the Trp-86 mutation dramatically increases the rate of deprotonation of the PSB while the Trp-182 mutation greatly decreases this rate. Temperature dependence studies on the rate constants of the deprotonation demonstrate that the different energetic and entropic effects of the mutation are responsible for the observed different kinetic behavior of the Trp-86 and Trp-182 mutants as compared with that of ebR. In the case of Trp-86 mutant, a large decrease in both energy and entropy of activation suggests that the mutation of this tryptophan residue opens up the protein structure as a result of eliminating the hydrogen-bonding group on its side chain by a phenylalanine substitution. A correlation is observed between the proton pumping yield and the relative amplitudes of the slow deprotonation component but not with rate constants of the rise or decay process at constant pH. These results are best discussed in terms of the heterogeneity model (with parallel cycle) rather than back reaction model.


Regulation of water flow by actin-binding protein-induced actin gelatin. Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by actin filaments corresponds to the gel point measured viscometrically and because gelsolin, which noncovalently severs actin filaments, solates actin gels and restores water flow in a solution of actin cross-linked by ABP. Since ABP gels actin filaments in the periphery of many eukaryotic cells, such actin networks may contribute to physiological cell volume regulation.


Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects. The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.


Lipid surface charge does not influence conductance or calcium block of single sodium channels in planar bilayers. We have studied the effects of membrane surface charge on Na+ ion permeation and Ca2+ block in single, batrachotoxin-activated Na channels from rat brain, incorporated into planar lipid bilayers. In phospholipid membranes with no net charge (phosphatidylethanolamine, PE), at low divalent cation concentrations (approximately 100 microM Mg2+), the single channel current-voltage relation was linear and the single channel conductance saturated with increasing [Na+] and ionic strength, reaching a maximum (gamma max) of 31.8 pS, with an apparent dissociation constant (K0.5) of 40.5 mM. The data could be approximated by a rectangular hyperbola. In negatively charged bilayers (70% phosphatidylserine, PS; 30% PE) slightly larger conductances were observed at each concentration, but the hyperbolic form of the conductance-concentration relation was retained (gamma max = 32.9 pS and K0.5 = 31.5 mM) without any preferential increase in conductance at lower ionic strengths. Symmetrical application of Ca2+ caused a voltage-dependent block of the single channel current, with the block being greater at negative potentials. For any given voltage and [Na+] this block was identical in neutral and negatively charged membranes. These observations suggest that both the conduction pathway and the site(s) of Ca2+ block of the rat brain Na channel protein are electrostatically isolated from the negatively charged headgroups on the membrane lipids.


Structure and dynamics of Escherichia coli chemosensory receptors. Engineered sulfhydryl studies. Cysteine residues introduced by site-directed mutagenesis have been used to probe the conformation and dynamics of two receptors in the E. coli chemotaxis pathway. (a) Thermal motions of the polypeptide backbone were investigated in the periplasmic D-galactose and D-glucose receptor, a globular protein of known structure. Disulfide bond formation between pairs of engineered sulfhydryls were used to trap collisions during the relative motions of surface alpha-helices I and X. Motions with amplitudes ranging from 4.5 to 15.2 A were detected on timescales ranging from 10(-4) to 10(-1) s, respectively. These results suggest that thermal backbone motions may have larger amplitudes than previously thought. (b) Conformational features of the transmembrane aspartate transducer have been investigated. Engineered sulfhydryls were used to ascertain the location and orientations of two putative transmembrane alpha-helices in the primary structure, to investigate the packing of these helices, to determine the oligomer and surface structures, and to detect thermal and ligand-induced dynamics of the polypeptide backbone. A model for the folded conformation of the transducer oligomer is reviewed.


Does the Na channel conduct ions through a water-filled pore or a condensed-state pathway? Many investigators assert that the ion-conducting pathway of the Na channel is a water-filled pore. This assertion must be reevaluated to clear the way for more productive approaches to channel gating. The hypothesis of an aqueous pore leaves the questions of voltage-dependent gating and ion selectivity unexplained because a column of water can neither serve as a switch nor provide the necessary selectivity. The price of believing in an aqueous pore therefore is a futile search for separate ad hoc mechanisms for gating and selectivity. The fallacy is to assume that only water is available to carry ions rapidly, ignoring the role of the glycoprotein, which can form an elastomeric phase with water. The elastomer is a state of matter, neither liquid nor solid, in which the molecules of a liquid are threaded together with cross-linked polymer chains; it supports fast ion motion (Owen, 1989). An alternative hypothesis for channel gating, based on condensed-state materials science, already exists (Leuchtag, 1988, 1991a). The ferroelectric-superionic transition hypothesis (FESITH) postulates that the Na channel exists in a metastable ordered (closed) state at resting potential and, on threshold depolarization, undergoes a reversible order-disorder phase transition to a less-ordered, ion-conducting (open) state. The ordered state is ferroelectric; the disordered state is a fast ion conductor selective for Li+ and Na+. The basis of the voltage dependence is elevation of transition temperature with electric field, well established in ferroelectrics. FESITH is consistent with single-channel transitions, gating currents, heat and cold block, and other phenomena observed at channel or membrane level. An implication of FESITH, the Curie-Weiss law, has been shown to explain existing data on membrane capacitance versus temperature in squid axon (Leuchtag, 1991c). Only on the basis of a clear understanding of function can we expect new structural data on the Na-channel glycoprotein to generate realistic structure-function models at the molecular level.


Molecular dissection of functional domains of the E1E2-ATPase using sodium and calcium pump chimeric molecules. Proposed models for the catalytic subunit of the E1E2-ATPases (ion pumps) predict that the first four transmembrane domains (M1 - M4) reside in the NH2 terminal one-third of the molecule, and the remainder (M5 - M10) in the COOH terminal one-third. The amino-acid sequences for the 5'-(p-fluorosulfonyl)-benzoyl-adenosine (FSBA) binding region residing just before M5 segment are very well conserved among distinct ion pumps. Taking advantage of these models, we have constructed a set of chicken chimeric ion pumps between the (Na++ K+)-ATPase alpha-subunit and the Ca(2+)-ATPase using the FSBA-binding site as an exchange junction, thereby preserving overall topological structure as E1E2 ATPases. From various functional assays on these chimeric ion pumps, including ouabain-inhibitable ATPase activity, Ca2+ binding, Ca2+ uptake, and subunit assembly based on immuno-coprecipitation, the following conclusions were obtained: (a) A (Na++ K+)-ATPase inhibitor, ouabain, binds to the regions before M4 in the alpha-subunit and exerts its inhibitory effect. (b) The regions after M5 of the (Na++ K+)-ATPase alpha-subunit bind the beta-subunit, even when these regions are incorporated into the corresponding domains in the Ca(2+)-ATPase. (c) The corresponding domains of the Ca(2+)-ATPase, the regions after M5, bind 45Ca even when it is incorporated into the corresponding position of the (Na++ K+)-ATPase alpha-subunit.


cAMP activates the sodium pump in cultured cells of the elasmobranch rectal gland. The inorganic ion content of rectal gland cells cultured from Squalus acanthias was studied by electron probe analysis in order to determine the effect of stimulation by cAMP. Cell sodium was reduced by 30% (P less than 0.01) at 8 min after exposure to dibutyryl cAMP and theophylline and remained low at 25 and 33 min. Chloride content also fell significantly with stimulation. Although cAMP may activate several transport sites, the results are consistent with a direct effect of stimulation to increase the activity of the sodium pump in shark rectal gland.


Effect of atrial natriuretic factor on renal cGMP production in rats with adriamycin-induced nephrotic syndrome. Adriamycin-induced nephrotic syndrome in the rat is associated with a blunted natriuretic response to infusion of atrial natriuretic factor. To study the mechanism of renal hyporesponsiveness to the peptide in rats with experimental nephrosis, we evaluated the effects of the hormone on renal production of cGMP, the second messenger of the hormone. Baseline GFR and sodium excretion were lower in nephrotic as compared with normal controls. Infusion of synthetic rat atrial natriuretic factor (10 micrograms/kg/h) increased fractional sodium excretion by 7.3 +/- 2.4% in control rats but only by 1.4 +/- 0.5% in adriamycin-treated rats (P less than 0.05). However, the increments in urinary nucleotide excretion rate (UcGMP x V/GFR), in response to atrial natriuretic factor infusion, were comparable in control and nephrotic rats (control, 114.7 +/- 16.1 pmol/mL; adriamycin, 95.5 +/- 12.0 pmol/mL; P was not significant). The in vitro generation of cGMP in response to incremental doses of the hormone (10(-11) to 10(-6) M + 1 mM 3-isobutyl methyl xanthine) was of similar magnitude in isolated glomeruli derived from control (2.4 +/- 0.25 to 9.1 +/- 1.0 pmol/mg of protein) and nephrotic rats (2.9 +/- 0.2 to 10.3 +/- 1.0 pmol/mg of protein) and was not impaired in suspensions of medullary tissue derived from nephrotic rats (control, 8.4 +/- 0.6 to 14.2 +/- 1.2 pmol/mg of protein; adriamycin, 7.3 +/- 0.7 to 22.0 +/- 2.4 pmol/mg of protein).(ABSTRACT TRUNCATED AT 250 WORDS)


Cell surface receptors and ectoenzymes in mesangial cells. Mesangial cells possess a variety of receptors for hormones and autacoids. They are also equipped with ectoenzymes whose function may be to control the availability of autacoids and hormones at their receptor sites. Several examples are considered. Receptors for angiotensin II (AII) are present both on murine and human mesangial cells. One single group of receptors has been demonstrated in each of these preparations. Mesangial cell AII receptors are linked to phospholipase C via a G protein. They belong to the AT1 subtype because (125I)AII is displaced from its binding sites preferentially by AT1 antagonists such as DUP 753 and EXP 3,174, whereas AT2 antagonists are much less potent. AT1 antagonists suppress the biological effects of AII in mesangial cells, including the stimulation of intracellular calcium concentration and the increase of prostaglandin synthesis and of (3H)leucine incorporation. Mesangial cells also have receptors for atrial natriuretic factor, but the distribution between B receptors with guanylate cyclase activity and clearance (C) receptors varies with the species. Both types are present in murine mesangial cells, whereas only C receptors are found in human mesangial cells. In contrast, human epithelial cells possess both B and C receptors. Ecto-5'-nucleotidase activity results in the production of adenosine, which acts on mesangial cells through A1 and A2 receptors. This enzyme is markedly induced in rat mesangial cells by interleukin-1, whose effect is mediated in part by prostaglandin E2 and cAMP. Various other cAMP-stimulating agents also induce 5'-nucleotidase expression in rat mesangial cells. Ectopeptidases are present in all glomerular cell types but essentially in epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Cellular signaling by endothelin peptides: pathways to the nucleus. Endothelins (ET) are potent regulatory peptides that evoke diverse responses in glomerular mesangial cells. These include short-term actions, such as contraction and secretion, and long-term, adaptive responses, such as cell growth. Although much attention has been focused on the second messenger cascades, which govern short-term effects, the pathways of cytosolic and nuclear signaling, which effect long-term changes, remain unclear. Several distal signaling events by ET receptors have been characterized in rat mesangial cells. These include activation of a cytosolic protein kinase, mitogen-activated protein kinase and an inducible transcription factor, activator protein-1 (AP-1). This review focuses on the activation of mitogen-activated protein kinase and activator protein-1 by ET and discusses the potential role of these third and fourth messengers in controlling long-term cellular adaptations. Characterization of these and other cytosolic and nuclear signals should provide important insights into the pleiotropic actions of ET peptides.


Regulation of cellular functions by extracellular matrix. Multicellular organisms are formed by specialized cells assembled in tissues. Individual cells contact and interact with other cells and with the extracellular matrix--a network of secreted proteins and carbohydrates that fills the intercellular spaces. The extracellular matrix helps cells to bind together and regulates a number of cellular functions, such as adhesion, migration, proliferation, and differentiation. It is formed by macromolecules, locally secreted by resident cells. The two main classes of macromolecules are polysaccharide glycosaminoglycans, usually covalently linked to proteins in the form of proteoglycans, and fibrous proteins of two functional types, structural (collagen, elastin) and adhesive (fibronectin, laminin, vitronectin, etc.). Receptors for extracellular matrix macromolecules are present in virtually all of the cells studied. They belong to the superfamily of integrins, alpha beta heterodimers, which, in most cases, recognize the Arg-Gly-Asp sequence of extracellular matrix proteins. On the exterior side of the cell, integrins link an extracellular matrix macromolecule, whereas in the cytosol, they bind the cytoskeleton, thereby forming a membrane bridge between extracellular and intracellular fibers. This structure enables the cell to adhere to the substratum. Similar to hormone- or growth factor-receptor binding, the interaction of the integrin with its specific ligand induces immediate signal transduction and influences cellular activities.


Independent transposition of multiple Ac elements in the same transgenic tomato cell. To effectively use transposable elements for the genetic manipulation of plant species lacking well characterized endogenous elements, it is important to evaluate the behavior of known transposable elements following their introduction into heterologous host species. One critical parameter concerns the timing of transposition in relation to the development of the transgenic host since this will affect the frequency with which transposition events are captured in the gametes. In order to examine whether different elements in the same cell are differentially active during development, we used Southern hybridizations to assess the activity of Activator (Ac) elements in progeny plants derived from a tomato transformant carrying five Ac's at two loci. All of the elements at one locus transposed in the primary transformant at a developmental stage resulting in the transmission of newly transposed elements to the next generation. In contrast, one or more of the Ac's at the second locus were not active at this stage and were transmitted to the next generation at the original donor T-DNA insertion site. These elements were, however, transpositionally active in somatic tissue. These results demonstrated that individual transposable elements in the same transformed cell can be differentially activated during development.


Generation of superoxide anion radical by alpha-terthienyl in the anal gills of mosquito larvae Aedes aegypti: a new aspect in alpha-terthienyl phototoxicity. The present study documents that the secondary plant metabolites, especially alpha-terthienyl, exert phototoxic action through inhibition of certain enzymes and generation of singlet oxygen. Some of the reports have emerged exhibiting involvement of free radical generation in vitro by alpha-terthienyl. We provide evidence for the generation of a free radical viz., superoxide anion radical, by alpha-terthienyl employing spin-trapping techniques, probably due to the extension of the latter reaction. On the basis of this observation the phototoxic action of alpha-terthienyl on Aedes aegypti larvae is explained.


Cerebellar form of progressive multifocal leukoencephalopathy in a patient with chronic renal failure. Progressive multifocal leukoencephalopathy (PML) developed in a 64-year-old woman who had undergone hemodialysis treatment due to chronic renal failure (CRF) for 6 yr. Her initial symptom was ataxia, and computed tomographic (CT) scan and magnetic resonance imaging (MRI) suggested a demyelinating disease of the cerebellum. Her cell-mediated immunity was impaired. At autopsy, the cerebellar disease was confirmed as PML by ultrastructural and immunohistochemical studies. Moreover, the JC type of papova virus infection was verified by Southern blot analysis.


Four cases with intrafamilial clustering of hepatitis C virus infection. Four hepatitis C patients with intrafamilial clustering of hepatitis C virus (HCV) infection are reported. Antibodies to C100-3 antigen, capsid protein of HCV and GOR epitope were tested to detect histories of HCV infection. Transmission of HCV from mother to children, from father to children, and from wife to husband was implicated. Of all family members studied, three were positive for all antibodies, one for only antibody to capsid protein, two for antibodies to capsid protein and GOR epitope but negative for antibody to C100-3 antigen and one vice versa.


A case of Goodpasture's syndrome associated with anti-myeloperoxidase antibodies. A case of Goodpasture's syndrome with anti-myeloperoxidase (MPO) antibodies is reported. Histological examination revealed crescentic glomerulonephritis and alveolar hemorrhage with linear deposition of IgG along the glomerular capillary walls and alveolar capillary walls by immunofluorescence microscopy. Not only anti-glomerular basement membrane (GBM) antibodies but also anti-MPO antibodies, an anti-neutrophil cytoplasmic antibody, were simultaneously detected in the serum. Although it is generally accepted that crescentic glomerulonephritis in Goodpasture's syndrome is mediated by anti-GBM antibodies, this case suggested that anti-MPO antibodies might also participate in the pathogenesis of crescentic glomerulonephritis and probably alveolar hemorrhage of Goodpasture's syndrome, especially with vasculitis.


Malignant fibrous histiocytoma of the liver: a case report and review of the literature. Primary malignant fibrous histiocytoma (MFH) of the liver is reported in a 79-year-old man. The tumor, measuring 8.0 x 8.0 x 6.0 cm, was located in the left lobe of the liver and consisted of spindle cells in a storiform pattern intermingled with bizarre giant cells. Immunohistochemically, most of the tumor cells expressed vimentin. Cytoplasmic immunoreactivity for alpha 1-antichymotrypsin was documented in the giant cells. However, epithelial expression could not be demonstrated.


Effects of graded exercise on blood pressure, heart rate, and plasma hormones in cardiac transplant recipients before and during antihypertensive therapy. The effects of graded supine ergometry on blood pressure, heart rate, and plasma hormones were studied in 14 hypertensive heart transplant recipients before and after 2 weeks and 6 months of enalapril (20 mg/day) plus furosemide (20-80 mg/day) alone or combined with verapamil (120-360 mg/day). Each time, measurements were obtained at rest and at 25 and 50 W exercise. Antihypertensive therapy normalized blood pressure, while heart rate and the blood pressure response to exercise remained unaltered. Pretreatment resting plasma renin activity and catecholamine levels were normal, while atrial natriuretic factor and cyclic guanosine monophosphate concentrations were elevated. All hormones increased significantly with exercise. During treatment, plasma renin activity increased and atrial natriuretic factor and cyclic guanosine monophosphate levels decreased significantly, with a blunted exercise response; concentration of catecholamines increased significantly, with augmented exercise response. Thus, the chosen regimen allowed effective, lasting BP control in hypertensive transplant patients but was associated with significant changes in plasma hormones. Whereas the rise in plasma renin activity may be attributed to converting enzyme inhibition, the decreases in atrial natriuretic factor and cyclic guanosine monophosphate and increases in catecholamine levels seem to indicate marked changes in resting and particularly exercise hemodynamics during antihypertensive therapy.


Elevated TNF receptor plasma concentrations in patients with rheumatoid arthritis. Two types of tumor necrosis factor receptors have been characterized, both capable of transmitting the signal and exerting the biological functions of TNF and lymphotoxin. We measured the plasma concentrations of two types of TNF binding proteins (sTNFR-A and sTNFR-B) in patients with rheumatoid arthritis (RA) and spondylarthropathies (SpA) using an enzyme-linked binding assay. In normal controls (n = 43), mean plasma concentrations were 1030 +/- 55 and 1461 +/- 59 pg/ml for sTNFR types A and B, respectively. In 67 patients with moderate RA, mean levels were 1422 +/- 82 pg/ml (type A) and 2088 +/- 109 pg/ml (type B); in 34 patients with severe RA, 2588 +/- 279 pg/ml and 4494 +/- 550 pg/ml, respectively, were measured (P less than 0.0001 compared to normal controls). Concentrations of both type A and type B sTNFR were highly correlated in severe RA (R2 = 0.7) but not in SpA or normal controls. T lymphocytes in synovial fluid of patients with RA expressed predominantly type A TNF receptors on their surface; in some patients a weaker expression of type B receptors was also detectable. Soluble TNF binding proteins in patients with RA were able to neutralize TNF in a cytotoxicity assay, demonstrating their ability to act as "TNF-inhibiting factors". We conclude that both types of TNF receptors are parameters of disease activity in RA and may also act as TNF antagonists.


The diagnostic significance of antibody specificity indices in multiple sclerosis and herpes virus induced diseases of the nervous system. The antibody specificity index (ASI) indicates the cerebrospinal fluid (CSF)/serum difference of antibody amounts per weight unit IgG (normal less than 1.5). It has proven to be the most sensitive inflammation parameter in CSF analysis so far, more sensitive than the Western blot, the "oligoclonal" response, and the empirical differentiation of CSF immunoglobulins. By this diagnostic criterion, several benign viral meningitis cases were found to be caused by the varicella/zoster virus. The diagnostic relevance of local zoster antibody synthesis was greatest in ganglionitis cases, e.g., in zoster oticus sine herpete (facial paresis) and acute radicular syndromes of the elderly. The diagnostic significance of the local immune response against measles, rubella, and zoster antigens (MRZ response) was ascertained further. Together with oligoclonal gamma-globulin fractionation, there is now only 1 out of 100 multiple sclerosis (MS) patients left who has been found to have a normal CSF.


Lymphadenopathy in connection with human herpes virus type 6 (HHV-6) infection. Two siblings and their mother developed afebrile generalized lymphadenopathy. The lymph nodes were movable and painless. During the course of the illness, the mother and one child developed an uncharacteristic rash. Increased titers of human herpes virus type 6 (HHV-6) antibodies were found in all three family members and in an unrelated patient with lymphadenitis colli. The enlarged lymph nodes decreased in size within several weeks. We speculate these symptoms to be caused by an infection with this lymphotropic virus.


Cytokines, receptors, and inhibitors.

Cytokines, receptors, and inhibitors. Cytokines are endogenous mediators in inflammatory and immunologic host defense reactions. In various diseases cytokines produced in excess cause systemic or local toxic effects. Cytokines therefore are tightly controlled by regulation of their biosynthesis and release and by counteracting mechanisms which limit their activities. Two new cytokine inhibitory mechanisms have recently been discovered. First, the generation of soluble receptors which compete with cellular receptors for cytokine binding has been recognized as a general phenomenon. Second, a receptor antagonist polypeptide binding to the receptor but not eliciting biological activity has been discovered in the IL-1 system. These polypeptides, when expressed in various recombinant forms, are not only research tools but may find also direct clinical use.


Inhibition of angiotensin II and platelet-derived growth factor-induced vascular smooth muscle cell proliferation by calcium entry blockers. Structural changes within the blood vessel wall such as hyperplasia and hypertrophy of vascular smooth muscle cells are important factors in the pathogenesis of hypertension. Humoral growth factors such as angiotensin II (AII) and platelet-derived growth factor BB (PDGF-BB) may participate in the remodelling of the blood vessel wall. Whether and by which mechanisms antihypertensive treatment is capable of influencing the structural blood vessel alterations to date remains unclear. In the present study, the effect of nifedipine and diltiazem on AII- and PDGF-BB-induced vascular smooth muscle cell proliferation was examined. Nifedipine and diltiazem at a concentration of 10 microM did not affect baseline DNA synthesis in isolated vascular smooth muscle cells in culture. AII (final concentration 100 nM) and PDGF-BB (50 ng/ml) stimulated DNA synthesis by approximately 9.0- and 4.6-fold, respectively. Both AII- and PDGF-BB-induced DNA synthesis was significantly blunted by diltiazem and nifedipine in a concentration of 10 microM, while no significant influence was seen with concentrations from 10 nM up to 1 microM. In contrast, no significant influence of these drugs could be observed on fetal calf serum 5%-induced DNA synthesis. The findings indicate that calcium antagonists possess antimitogenic potential and that they may thus contribute to the regression of structural changes of the blood vessels associated with hypertension.


Influence of various nutrients and their mode of application on plasma cholecystokinin (CCK) bioactivity. CCK is known to be a major endocrine stimulant of the exocrine pancreas. However, the influence of various compositions of nutrients and their mode of administration on plasma CCK is still not clear. We therefore studied plasma CCK after either oral or intraduodenal administration of various solid and liquid diets. Plasma CCK was measured by bioassay. Nine healthy male volunteers received three different isocaloric diets via intraduodenal perfusion (1 kcal/ml; 300 ml/90 min). Diet A consisted of low-molecular oligopeptides (energy: protein 18%, fat 22%, carbohydrates 60%); diet B was a high molecular diet enriched with fat and fiber (protein 15%, fat 30%, carbohydrates 55%, fiber 1 g/100 ml); and diet C was a high molecular diet, poor in fat and fiber-free (protein 14%, fat 7%, carbohydrates 79%). All diets caused a rapid increase in plasma CCK, followed by a slow decrease. The highest CCK plasma values were achieved with diet B (8.4 +/- 1.6 pM); diets A and C led to similar, rather low plasma CCK values (A: 5.0 +/- 0.7 pM). Another five volunteers received the above-described liquid diets orally. Integrated CCK plasma values were similar for all oral liquid diets studied and not different from those seen after intraduodenal administration of the high-molecular high-fat diet B. Oral administration of a solid diet (ten volunteers) comparable to diet B in calories and nutrient composition caused a rather small and delayed increase in plasma CCK.(ABSTRACT TRUNCATED AT 250 WORDS)


Treatment of atopic eczema with evening primrose oil: rationale and clinical results. Recently a defect in the function of the enzyme delta-6-desaturase has been discussed as a major factor in the development of atopic eczema. Delta-6-desaturase is responsible for the conversion of linoleic acid to gamma linolenic acid. Several plants, including evening primrose, are known to be fairly rich in gamma linolenic acid. Hence, substitution of gamma linolenic acid in patients prone to developing atopic eczema seems like a feasible concept. During the last few years different clinical trials have been performed. Controlled trials following a parallel study design showed marked improvement in atopic eczema. Patients treated with the drug showed less inflammation, dryness, scaling and overall severity compared to controls. Although these findings have been supported by meta-analysis, there is still conflicting evidence in trials based on a crossover design alone, demonstrating a decrease in itching. At present, evening primrose oil in doses used for the treatment of atopic eczema is considered safe. However, still more trials addressing both efficacy and safety are needed to make a final decision.


Lipoprotein receptors in oocyte growth.

Lipoprotein receptors in oocyte growth. The transport and removal of lipoproteins from the blood in the laying hen is of particular interest because it is a system in which the massive transport of lipid to one organ (the ovary) coexists with regulatory mechanisms for the control of lipid homeostasis in extraovarian tissues. In order to achieve this dual task, the laying hen expresses dichotomous receptor-mediated pathways. On one hand, very low density lipoproteins (VLDL) and vitellogenin (VTG), which together form over 95% of the lipid in a fully grown oocyte (i.e., an egg yolk), are transported into oocytes via a 95-kDa receptor protein. This receptor, termed oocyte VLDL/VTG receptor, is exclusively produced in growing oocytes and is absent from somatic cells. It shows a high degree of structural similarity to other members of the so-called low density lipoprotein (LDL) receptor family, but in contrast to the LDL receptor, its expression is not suppressed by sterols. On the other hand, somatic cells, but not oocytes, synthesize a 130-kDa receptor that recognizes VLDL-derived, cholesterol-rich lipoproteins. This receptor is the functional analogue to the mammalian LDL receptor in that it mediates, at least in part, extraoocytic lipid homeostasis. The somatic LDL receptor of the chicken recognizes apolipoprotein (apo)B, but not VTG, in accordance with VTG's exclusive routing to growing oocytes. Within oocytes, both apoB of VLDL and VTG undergo limited specific postendocytic proteolytic processing. Recent studies have shown that this breakdown of macromolecular plasma precursor molecules is catalyzed by an endosomal form of cathepsin D and is a key event in the formation of yolk, the major nutrient source for the developing embryo.


Combined and alternating ganciclovir and foscarnet in acute and maintenance therapy of human immunodeficiency virus-related cytomegalovirus encephalitis refractory to ganciclovir alone. A case report and review of the literature. Cytomegalovirus (CMV) causes life-threatening disseminated infections and in particular vision-threatening infections of the retina in patients with the acquired immunodeficiency syndrome. Ganciclovir currently represents the most frequently used therapy for CMV retinitis. However, cases of ganciclovir-resistant CMV strains have been described, in which foscarnet seems to be an effective alternative. Both drugs have serious toxicities, and relapses frequently occur during maintenance therapy. In a patient with CMV encephalitis, we administered a 3-week combination ganciclovir/foscarnet induction therapy (ganciclovir 5 mg/kg every 12 h; foscarnet 60 mg/kg every 8 h), followed by an alternating maintenance administration of both drugs every other day (ganciclovir 5 mg/kg, foscarnet 120 mg/kg) to reduce toxicity and resistance. This regimen was tolerated well and seemed to be more effective than ganciclovir alone in a patient with CMV encephalitis.


Computed tomography of bronchogenic carcinoma: findings and reevaluation after radiotherapy. 493 computed tomograms were performed in 150 patients with bronchogenic carcinomas after radiotherapy. In early controls after termination of the irradiation, tumors with atelectasis could be delineated slightly better. In tumors without atelectasis, however, delineation deteriorated. In 50 late controls the radiation-induced pulmonary injury resulted in further masking of the tumor area, independent of the initial degrees of airway obstruction. Post-radiation changes were always earlier and more extensively visible by CT than by conventional radiography. No radiation effect on delineation of the mediastinal lymph nodes was provable. In 87% of all cases the remission rate of the tumors could be adequately evaluated by CT. In 43% of the late controls a delayed complete remission was seen. Fifteen out of 20 tumor recurrences or progressions were recognizable only by CT. The most important diagnostic criteria after radiotherapy of bronchogenic carcinomas are: 1. Consideration of the irradiation modalities and timing of the CT examination. 2. knowledge about the course of the pulmonary reaction to irradiation and 3. subtle comparisons of serial CT examinations in tumor follow-up.


Determination of the level of the core of 2',5'-oligoadenylates by high performance liquid chromatography. A simple and rapid method for analysis of the core of 2',5'-oligoadenylates, mainly based on the use of high performance liquid chromatography (HPLC), is described. Perchloric acid extracts of tissues or cells were first treated with nuclease P1. Portions of the extracts were then digested with alkaline phosphatase. HPLC analysis of the extracts was performed on a column system composed of an Ultrasphere ODS precolumn (4.6 x 45 mm) and an Ultrasphere Octyl column (4.6 x 250 mm) by stepwise elution using a 50 mM ammonium phosphate buffer, pH 7, containing 3.5 and 7% methanol. Three species of the core of 2',5'-oligoadenylates (dimer, trimer and tetramer) from a number of samples were eluted separately with 7% methanol, and the concentration of each core was directly estimated using constant values calculated with the standard core. The level of the core of 2',5'-oligoadenylates in tissues and cells determined by our method is similar to that reported by other authors who used biological, radiobinding or radioimmunological assays.


Recent advances in antitumor vaccines.

Recent advances in antitumor vaccines. Immunization with anti-idiotypic antibodies can induce cell-mediated and humoral antitumor immunity in animal models. This immunity can sometimes cause tumor destruction. However, more needs to be learned about how best to induce the type of immune response that is responsible for tumor destruction, since the presence of anti-idiotypic antibodies has been shown occasionally to enhance, rather than to inhibit, tumor growth. There is evidence suggesting that immunization of human cancer patients with Ab2 can have therapeutic benefit, and also that patients who mount a vigorous Ab2 response following treatment with an Ab1 may do clinically better than those who do not make any Ab2. Although the generation of Ab2 related to infused antitumor Ab1 does not cause tumor rejection in the majority of patients, and although the clinical data from patients given Ab2 are scarce, the suggestion that Ab2 may cause destruction of human cancers indicates that further work in this area may become rewarding.


Abortive human cytomegalovirus infection in patients after allogeneic bone marrow transplantation. Infection with human cytomegalovirus (HCMV) after allogeneic bone marrow transplantation (BMT) was studied in 12 HCMV seronegative recipients of marrow from seropositive donors by weekly monitoring of cultures, expression of HCMV antigenemia (pp65) in granulocytes, polymerase chain reaction (PCR) on HCMV-DNA in granulocytes and IgM and IgG anti-HCMV antibodies. Eight patients remained negative in all tests as did 33 HCMV seronegative recipients of marrow from seronegative donors. In four patients, a transient expression of HCMV antigen pp65 in granulocytes from peripheral blood, together with a positive PCR on HCMV-DNA from the same samples were found without positive cultures, seroconversion or expression of other HCMV antigens in granulocytes. The data indicate the presence of an abortive HCMV infection in these four patients.


Cytomegalovirus antigenemia assay or PCR can be used to monitor ganciclovir treatment in bone marrow transplant recipients. The antigenemia assay, polymerase chain reaction (PCR) and rapid culture technique on buffy coat cells (DEAFF test) were used to monitor 37 cytomegalovirus (CMV) seropositive bone marrow transplant (BMT) recipients for active CMV infection during the first 3 months after BMT. The antigen assay and PCR demonstrated a comparable sensitivity for the detection of CMV in blood: discordant results were only obtained in the early or late phase of infection when the viral load was low. The antigen assay was more sensitive than the DEAFF test. Only 12 out of 40 antigen-positive samples yielded a positive result with DEAFF test, whereas viremia without antigenemia was never found. The discordance between these two tests increased further during antiviral therapy with ganciclovir. A correlation was observed between the duration of antigenemia during treatment and the recurrence of systemic CMV reactivation. Ten out of 11 patients with antigen-positive leukocytes present for more than 1 week after starting the treatment subsequently exhibited a relapse of active infection, whereas only three out of nine patients who resolved their antigenemia within 1 week did so. In conclusion, the antigen assay and PCR are useful techniques for detection of CMV infection in BMT patients. Test results obtained during therapy give reliable information regarding the viral load and the possibility of recurrence of antigenemia, and can be taken into account when prolonged administration of ganciclovir is considered.


Bilateral median nerve palsy in a cyclist. Cyclists are prone to a number of sport-related musculoskeletal injuries, mainly of the lower limb. Nerve compression injuries are relatively rare, though in the hand ulnar nerve compression is well described. We describe a case of bilateral median nerve compression caused by cycling.


Endoscopy for upper gastrointestinal bleeding at emergency unit. From November 1, 1990 to January 31, 1991, 381 patients visited our emergency unit with the chief complaints of hematemesis (n = 153) and melena (n = 228). Of these patients, 298 (78.2%) received UGI endoscopy at the emergency unit, 29 (7.6%) received examination after they were admitted to wards, 3 (0.8%) received endoscopy at outpatient clinic and 51 (13.4%) did not have endoscopy. The percentages of endoscopic diagnoses in 330 patients who had UGI endoscopy were gastric ulcer (GU) 33.6%, duodenal ulcer (DU) 32.7%, esophageal varices (EV) 17.0%, and others 15.5%. Negative findings were noted in 3 cases and no definite bleeding source was found in 1 patient who had blood retention in stomach. The diagnostic rate of endoscopy was 98.8%. Of the 56 patients with EV, 45 (80.4%) had hematemesis; in contrast, 85 of the 108 DU patients (78.8%) complained of melena only. Of the 219 patients with bleeding GU/DU, 64 (29.2%) had endoscopic therapies and 7 (3.2%) needed operation. Sixty-four (29.2%) of them received UGI endoscopy within 6 hours after arrival. They had significantly higher frequency of stigmata of recent hemorrhage (SRH) and more endoscopic therapies than the remaining patients. The overall mortality rate in our patients was 6.0%; the mortality rate in patients with EV was much higher than that of the patients with GU/DU (19.6% vs. 1.8%, P less than 0.001). Of the 23 expired cases, only 11 died of hypovolemic shock. The remaining 12 patients died of deterioration or complications of their underlying diseases.(ABSTRACT TRUNCATED AT 250 WORDS)


Early CT findings of acute cerebral infarction in the middle cerebral artery territory. For an early and definitive diagnosis of acute cerebral infarction by computed tomography (CT), we retrospectively analyzed the initial CT findings of 14 patients with proven acute middle cerebral artery (MCA) infarction within 4 hours after stroke onset. The following results were obtained: (a) abnormal CT findings could be recognized quite early in 13 patients (92.9%), (b) the most common and earliest finding was loss of defination of the gray-white interface at the lateral margins of the insula. This sign was recognized in 12 patients (85.7%) and could be detected as early as 1 hour after stroke onset, (c) the next common finding was narrowing or blurring of the Sylvian fissure in 10 patients (71.4%) and could be detected 1.5 hours after stroke onset, (d) an obscured outline or partial disappearance of the lentiform nucleus was recognized in 7 patients (50%) and could be detected 1.5 hours after stroke onset, (e) effacement of the cerebral sulci was found in 6 patients (42.9%) and could be detected 2.5 hours after stroke onset, (f) increased density in MCA or its major branches was not seen in our patients.


[MRI of adrenal tumors].

[MRI of adrenal tumors]. Within a two and half years period, we collected a total of twenty three cases of adrenal tumors diagnosed by MRI. They included: one cystic case, twelve cases (13 lesions) of adenoma, two cases (3 lesions) of hyperplasia, four cases of pheochromocytoma, three cases of metastases, and one case of adenocarcinoma. Except for the case of adrenal cyst which was followed for one and a half years, all the other twenty two cases were proved by operation and pathology. The benign adenoma and hyperplasia were small in size, and had relative isointensities to the liver in the T1WI and the T2WI. On the contrary, the malignant tumors and pheochromocytoma, all had inhomogeneous signal intensities, showed relatively lower in signal intensities in T1WI and higher in T2WI as compared with the liver. In T2WI, the tumor to liver signal intensity ratio of adenoma and hyperplasia were less than 1.80, whereas the malignant tumors and pheochromocytoma were larger than 1.80. In comparing fifteen cases with Gd-DTPA intravenous injection, all of the benign adenoma did not show an increase in signal intensity, but the malignant tumors and pheochromocytoma showed increase in signal intensity. We concluded that we could primarily differentiate the nature of adrenal tumors by their change in signal intensities between T1WI and T2WI, by measuring the tumor to liver signal intensity ratio or by Gd-DTPA IV injection. Today, although adrenal gland MRI examination is more time consuming and expensive, it is more valuable for highly clinically suspected adrenal lesions with equivocal results after CT or sonogram study.(ABSTRACT TRUNCATED AT 250 WORDS)


[Intracellular effect of the ventral prostate in zinc deficient rats]. Concentration of androgens, binding-sites of androgen-receptors as well as the activity of 5 alpha-reductase of the ventral prostates have been studied in the zinc-deficient rats. Eight-week-old Sprague-Dawley rats were housed individually in cages and divided into two groups, I. Experimental group, redivided into 2 sub-groups: (a) maintained on a zinc-deficient diet for 3 and 6 weeks, (b) zinc-deficient diet for 3 weeks then re-fed normal diet, II. Control group fed with normal diet. The ventral prostates were dissected free and homogenized after the rats sacrificed. Androgens and androgen-receptor were quantified by RIA and "exchange assay" respectively. The result obtained from the zinc-deficient rats were significantly different (P less than 0.05) from those of the control group 1. The zinc-deficient 3 and 6 weeks rats showed the plasma testosterone concentration decreased by 18% and 15%. 2. The cytosolic (fmo1/mg protein) dihydrotestosterone and its binding receptor were decreased by 10-15% and 28-30%, respectively. 3. The nuclear (fmo1/mg DNA) dihydrotestosterone and its receptor protein were ony 2-3% and 3-3.6% of the control group respectively. 4. Studies on the androgen metabolism showed that the metabolite, dihydrotestosterone (fmo1/mg protein) of rats with zinc-deficient 6 weeks was only 48% of the control group. 5. The prostate weight (mg) of zinc-deficient rats was 45-50% of the control group.(ABSTRACT TRUNCATED AT 250 WORDS)


[Brainstem auditory evoked potentials in diabetes mellitus]. It is well-known that diabetic patients develop peripheral and autonomic neuropathy, and recent review has also suggested the occurrence of central pathway abnormality in diabetics. In this article, we conducted the BAEP study on 61 cases of NIDDM and 11 cases of IDDM. Peak latency, interpeak latency (IPL) and peak amplitude of BAEPs were analyzed in each case. For further correlation, the motor and sensory nerve conduction velocities of median nerve, the blood sugar, the serum HbA1c were measured. Two nondiabetic groups, age and sex matched with NIDDM and IDDM groups, were used as control. In NIDDM group, the results showed prolongation of all peak latency and IPL except peak latency of wave II and wave IV in the left side and bilateral IPL III-V. There was no statistically significant amplitude difference between NIDDM and age-matched control group. The result of IDDM group revealed prolongation of all peak latency and IPL, except the right IPL III-V. As for amplitude, waves III and V in the right side and waves I and V in the left side were reduced as compared with the age-matched young control group. There was no statistically significant difference in all peak latencies and IPLs between NIDDM and IDDM groups. In both groups of NIDDM and IDDM, the MNCV and SNCV of median nerve were significantly delayed in conduction. The prolongation of III and V peak latency had a linear correlation with their amplitude reduction. In conclusion, both peripheral and central conduction dysfunction occur in both IDDM and NIDDM patients.


[The interrelationship between the O2-dependent bactericidal mechanism and hyperbilirubinemia of newborn]. In order to investigate the influence of indirect bilirubin to O2-dependent bactericidal mechanism in adult and newborn phagocytes we use the NBT reduction activity of granulocytes and monocytes as index, by infiltrated granulocytes and monocytes in different concentration of indirect bilirubin with or without latex stimulator. The NBT reduction activity of granulocytes infiltrated in 20 mg/dl indirect bilirubin will increase slightly, compared to uninfiltrated; but with latex stimulator, the increment will decrease significantly, this change is reversible, when indirect bilirubin be washed, the stimulating capacity by latex stimulator in granulocyte will be stronger; there were no such change pattern in monocytes. Since there were a big difference in NBT reduction activity of granulocytes and monocytes between newborns, with or without latex stimulator, we can't get a satisfied result. Indirect bilirubin in cells will increase NBT reduction activity of monocytes, but not in granulocytes.


[Autonomic nerve preserving operation for rectal carcinoma: preliminary postoperative urinary function of 15 cases]. The autonomic nerve preserving operation was performed on 15 patients with rectal carcinoma (9 men and 6 women), from August 1990 to February 1991. The average age was 57.2 years ranging from 28 to 70 years. Low anterior resection was performed on 6 patients having middle rectal carcinoma. Seven Colonic anastomosis and two abdominoperineal resections were performed for lower rectal carcinoma. The average operation time was 4 hours and 22 minutes. The average blood loss was 1560 cc. Urinary functions were evaluated pre- and post-operatively. None of the patients needed re-catheterization postoperatively. Urodynamic studies showed no significant differences. We recommend this operation for rectal cancer because it prevented the postoperative autonomic dysfunction without affecting the curability.


[Personality characteristics of patients with fibromyalgia]. Fibromyalgia is a form of nonarticular rheumatism characterized by diffuse musculoskeletal pain. To investigate the personality characteristics of fibromyalgia, Ko's Mental Health Questionnaire was evaluated in 27 fibromyalgia patients and 23 normal controls. The fibromyalgia patients scored significantly higher than normal controls on hypochondriasis, depression, anxiety and compulsion, and significantly lower on independence. The fibromyalgia patients had 52% incidence of depression. Major depression occurred before onset of fibromyalgia at a mean of 5.5 years in 36% of the patients who experienced depression. The longer the duration of fibromyalgia, the higher the incidence of depression, and more numbers of tender points, indicating that the symptoms of fibromyalgia were more severe.


[Small cell lung cancer and long-term survival: our experience in VGH-Taipei from 1971 to 1985]. We diagnosed 627 cases of SCLC from 1971 to 1985 in VGH-Taipei. Sixty-nine cases received no treatment, while 558 cases received treatment including chemotherapy, radiotherapy, surgical intervention or combined modality. The median survival time of the treated group and untreated group was 6 and 1 months respectively (p less than 0.001). Female sex was a positive prognostic factor (p less than 0.05) and median survival time of female and male were 7 and 5 months respectively. Age (older or younger than 65 y/o) was not a significant prognostic factor in our series (p = 0.13). Thirteen cases (2.33%) of patients in the treated group survived longer than 5 years. Among them, 11 cases were limited disease and 2 cases were extensive disease. The characteristics of these 13 patients were analysed to find them in relatively good performance status, less body weight loss, less biochemical abnormality and more limited disease. In them, no secondary tumor was noted during the follow-up period.


[Clinical experience of GDM screening program]. From Jan. 1985 to Jun. 1990, 2561 pregnant women attending our prenatal clinic were screened for gestational diabetes. A 50-gm, 1-hour glucose challenge test was given and 470 patients (18.4%) showed plasma glucose values greater than or equal to 140 mg/dl. These women were subjected to a further 3-hr 100-gm OGTT to diagnose diabetes using O'Sullivans' criteria. Of these 470 cases, 80 (3.1%) of total cases were shown to be diabetic. Strict glycemic control with a fasting plasma sugar under 105 mg/dl and postprandial sugar under 120 mg/dl was conducted using diet and insulin in all cases. We compared the perinatal outcome of GDM pregnant women with normal pregnant women. Statistically, there was no significant difference. This study clearly demonstrates that a GDM screening program should be recommended for every pregnant woman. Prompt glycemic control should be given for all diabetic mothers to optimize the perinatal outcome.


Bilateral putaminal necrosis caused by methanol poisoning: a case report. Bilateral putaminal necrosis is characteristic of methanol poisoning. A 31-year-old male alcoholic had headache, impaired consciousness, neck stiffness, roving eyes with dilated unreactive pupils, papilloedema, abdominal pain, vomiting, and severe metabolic acidosis after a binge. Abnormalities of the cerebrospinal fluid included an initial pressure of 240 mmH2 O, RBC 286/mm3, WBC 8/mm3, and protein 179 mg/dl. Peritoneal dialysis was performed on the 2nd day after drinking. A blood test for methanol was not performed until the 5th day, and its results was negative. However, computed tomography (CT) on the 3rd day showed necrosis and hemorrhage of bilateral putamina and the cerebral cortex, and post-contrast enhancement of meninges. On the 22nd day, a CT revealed further changes: necrosis of bilateral subcortical white matter, and post-contrast gyral enhancement at the otherwise normal-looking areas of the cerebral cortex. We suggest that, in certain situations, the characteristic CT findings are helpful in the diagnosis of methanol poisoning.


Mollaret's meningitis: a case report and literature review. Mollaret's meningitis is a rare syndrome with characteristic features. We present a case with interesting clinical course and cerebrospinal fluid studies. The patient was admitted to Veterans General Hospital-Taipei 6 times between May, 1984 and May, 1991 because of recurrent meningitis (Mollaret's meningitis). No causative etiology was identified although a series of investigations were performed, including studies of the blood and CSF, roentgenogram and nuclear scintinographs. To date, there has been no standard therapeutic modality for this disease. We use aspirin for his symptomatic control with satisfactory result though colchicine has been reported as being effective also.


[Neurogenic pulmonary edema following a grand mal seizure: a case report]. A case of neurogenic pulmonary edema following a grand mal epileptic seizure is presented. This condition is rather rare and the exact pathophysiology remains unknown, but it is believed that the event may be related to transitional increase of intracranial pressure during seizure attack. Pulmonary edema may be a serious complication of epileptic seizure. Rapid recognition and management is very important since the prognosis depends on the severity of pulmonary edema and appropriate treatment.


The 1991 Merck Frosst Award. Multidrug resistance in small cell lung cancer. The two-year survival rate of patients with small cell lung cancer is less than 10%. The major reason for this poor outcome is the development of drug resistance. Panels of small cell lung cancer cell lines have been established, providing models for the study of drug resistance in this tumour. One such model is the doxorubicin-selected H69AR cell line. H69AR displays the typical multidrug resistance phenotype in that it is cross-resistant to anthracyclines, Vinca alkaloids (e.g., vinblastine) and epipodophyllotoxins (e.g., VP-16). However, H69AR cells do not overexpress P-glycoprotein, the membrane drug efflux pump frequently found on multidrug resistant cells. Some alterations in glutathione levels and associated enzyme activities were found but the data do not support the notion that enhanced drug detoxication is involved in H69AR cell resistance. Fewer drug-induced DNA strand breaks, reduced levels of topoisomerase II, and reduced formation of drug-stabilized DNA/topoisomerase II complexes were observed in H69AR cells. These data implicate topoisomerase II in the resistance phenotype of H69AR cells, but cannot explain H69AR cell resistance to the Vinca alkaloids, which do not have topoisomerase II as a target. Monoclonal antibodies against antigens overexpressed on H69AR cells have been derived and four have been characterized. Immunoscreening of an H69AR cDNA expression library has allowed the identification of one of these antigens as p36 (annexin II), a Ca2+/phospholipid binding protein. Chemosensitizers and novel xenobiotics have been examined for their ability to circumvent the drug resistance of H69AR cells. The limited success of these investigations suggests that innovative approaches may be required. In conclusion, the data obtained with H69AR and other models of small cell lung cancer indicate that multiple mechanisms contribute to drug resistance in this disease.


Effects of cAMP, its analogues, and forskolin on lung fluid production by in vitro lung preparations from fetal guinea pigs. Lungs from fetal guinea pigs (62 +/- 1 days of gestation) were supported in vitro for 3 h and fluid production was determined by a dye dilution method, based on Blue Dextran 2000. Twenty untreated lungs produced fluid at 1.41 +/- 0.22 mL.kg-1 body weight.h-1, with no significant changes during later hours. Treatments with analogues of cAMP, cAMP, or forskolin during the middle hour reduced production significantly. Dibutyryl cAMP at 10(-3) M produced reabsorption (117.8 +/- 13.6% reduction, p less than 0.001, n = 10); at 10(-4) M it reduced production (77.3 +/- 11.0% fall, p less than 0.001, n = 10). 8-Bromo-cAMP appeared more effective; at 10(-4) M it caused slight reabsorption (109.0 +/- 8.9% reduction, p less than 0.001, n = 6) and at lower concentrations it decreased production (at 10(-6) M, 67.6 +/- 9.6% fall, p less than 0.001, n = 6; at 10(-7) M, 40.0 +/- 14.3% fall, p less than 0.001, n = 6). At high doses, cAMP itself produced similar effects (at 5 x 10(-3) M, 141.6 +/- 22.8% reduction, p less than 0.001, n = 6); at 10(-4) it was ineffective (n = 3). Forskolin at 10(-6) M induced the strongest reabsorptions seen (159.1 +/- 10.9% reduction, p less than 0.001, n = 6); at lower concentrations it reduced production (at 10(-8) M, 73.8 +/- 5.5% fall, p less than 0.001, n = 6; at 10(-9) M, 29.2 +/- 9.2% fall, p less than 0.05, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)


Cocaine hepatotoxicity during protein undernutrition of retrovirally infected mice. Effects of cocaine administration on lipid peroxidation and liver damage in immunocompromised mice fed different levels of dietary proteins were investigated. Indices of lipid peroxidation and serum aminotransferases as evidence of free radical attack and liver damage were compared in mice fed a low protein (4%) or regular protein diet (20% protein) for 3 weeks and then infected with murine leukemia virus and given daily intraperitoneal injections of increasing progressive doses of 5-45 mg.kg-1.day-1 of cocaine for 11 weeks. Cocaine administration significantly increased hepatic triglycerides, serum aminotransaminases, conjugated dienes, lipid fluorescence, and malondialdehyde levels. These changes were exacerbated by retroviral infection and also by protein undernutrition. Retroviral infection additively increased indices of cocaine-induced lipid peroxidation and hepatic damage. Significant increases in indices of lipid peroxidation and greater liver injury were also detected in similarly treated mice that received the low protein diet compared with well-nourished mice. These results show that immunocompromised mice fed low levels of dietary protein form significantly increased immunogenic lipid peroxidation adducts during cocaine treatment.


Intracellular cyclic AMP concentration modulates gap junction permeability in parturient rat myometrium. We investigated whether cell-to-cell coupling between myometrial cells of parturient rats is influenced by intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration. To evaluate the coupling, we measured input resistance (Ro) and injected Lucifer Yellow (LY) using microelectrode techniques. The intercellular spread of the dye was then observed. Longitudinal muscle strips from rat myometrium were exposed to isoproterenol, forskolin, or dibutyryl cAMP (DB-cAMP) to elevate cAMP. Isoproterenol (10(-11)-10(-6) M) and DB-cAMP (10(-5)-10(-3) M) hyperpolarized the resting membrane potential (Em) and increased Ro in a dose-dependent fashion. Forskolin (10(-6) M) also hyperpolarized Em and increased Ro. When LY was injected into a single cell, LY spread rapidly and extensively to neighboring cells in parturient control tissues, while LY transfer was completely blocked by any of the three agents at high concentrations. The increased Ro and blocked transfer of LY owing to these agents indicate that the cell-to-cell coupling was decreased both electrically and metabolically. Myometrial cells of parturient rats show increased number and size of gap junctions (GJs). The rapid and reversible decrease in coupling is interpreted to reflect the reduced permeability of GJs between the muscle cells because of an elevation of cAMP. Control of GJ permeability by this second messenger may be important for the physiological regulation of intercellular coupling and the extent of synchronizing and coordinating electrical, metabolic, and contractile activity in the uterine wall during pregnancy and parturition.


Characterization of intestinal smooth muscle responses and binding sites for endothelin. The contractile activity of and binding sites for endothelin-1 (ET-1) were investigated in isolated guinea-pig ileal longitudinal smooth muscle (GPILM). ET-1 produced concentration-dependent contractions of GPILM that either slowly subsided in the continued presence of ET-1 or rapidly subsided following washing of the tissue. The ED50 value for ET-1 contractions was 4.2 +/- 1.3 x 10(-9) M. The removal of extracellular calcium or pretreatment with nifedipine produced a complete inhibition of the contractions to ET-1. The IC50 value of nifedipine for inhibition of ET-1 mediated contractions was 3.0 +/- 0.8 x 10(-8) M. ET-1 produced a marked prolonged homologous desensitization of its contractile response but did not affect the responses mediated by carbachol, histamine, serotonin, substance P, and PLA2. High-affinity binding sites for 125I-labelled ET-1 were identified on microsomal membranes prepared from GPILM with Kd and Bmax values obtained by Scatchard analysis of 3.5 +/- 0.6 x 10(-10) M and 2138 +/- 159 fmol/mg protein, respectively. The binding of 125I-labelled ET-1 to GPILM microsomes was characterized by a rapid association (kob value of 0.077 min-1 at a radioligand concentration of 0.45 nM and an extremely slow dissociation (k1 value of 0.011 min-1; t1/2 value of 793 min). The binding was unaffected by the calcium channel antagonists nifedipine, verapamil, and diltiazem (10(-6) M); the receptor antagonists phenoxybenzamine, atropine, and naloxone (10(-6) M) and propranolol; and the peripheral benzodiazepine receptor antagonists Ro 5-4864 and PK 11195 and psychotomimetic drug phencyclidine (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)


Functional sensitivity of the native skeletal Ca(2+)-release channel to divalent cations and the Mg-ATP complex. Sarcoplasmic reticulum (SR) vesicles, prepared from rabbit skeletal muscle, were characterized by functional and binding assays and incorporated into planar lipid bilayers. Single-channel activity was recorded in an asymmetric calcium buffer system and studied under voltage clamp conditions. Under these experimental conditions, a large conductance (100 pS in 50 mM Ca2+ trans) divalent cation selective channel displaying high ruthenium red and low Ca2+ sensitivity was identified. This pathway has been previously described as the Ca(2+)-release channel of the SR of skeletal muscle. We now report that in the presence of a Mg-ATP complex, the Ca2+ sensitivity of the open probability of this channel is increased. Furthermore, we show that micromolar cis Sr2+ concentrations also activated the Ca(2+)-release channel. The open probability of the Sr(2+)-activated channel was increased in the presence of a 2 mM Mg-ATP complex and adenine nucleotides on the cytoplasmic face of the Ca(2+)-release channel. These results were confirmed by isotopic flux measurements using passively 45Ca(2+)-loaded vesicles. In the latter case, the presence of extravesicular AMP-PCP (the nonhydrolysable ATP analog) enhanced the percentage of 45Ca2+ release induced either by Ca2+ or Sr2+ activation. In conclusion our findings emphasize the fact that the divalent cation activation of the Ca(2+)-release channel may be induced by Ca2+ and Sr2+, but not by Ba2+, in the presence of adenine nucleotides. Furthermore, they support the view that in situ Ca2+ and Mg-ATP complexes are involved in modulating the gating mechanism of this specific pathway.


Echocardiographic follow-up of right atrial tumoral invasion by hepatocarcinoma: a case report. Hepatocellular carcinoma is known for its tendency to grow in the venous system. We describe a patient suffering from hepatocellular carcinoma with right atrial metastatic tumor. Echocardiography demonstrated a right atrial mass as a result of invasion of the inferior vena cava and next extension into right atrium. The autopsy showed an irregular polypoid mass that occupied the entire right and left atrium, hepatocarcinoma was confirmed by histologic studies. We advocate performing echocardiographic examination in patients with hepatocarcinoma who have cardiac symptoms and signs.


Distinct receptors for Leu- and Met-enkephalin on the metacerebral giant cell of Aplysia. 1. The effects of D-Ala2-Leu-enkephalin (DALEU), D-Ala2-Met-enkephalin (DAMET), and FMRFamide on the metacerebral cell (MCC) of Aplysia were determined in current- and voltage-clamp experiments. 2. Distinct receptors exist on this neuron for the three substances. 3. DALEU elicited a depolarizing response due to an inward current but not accompanied by a significant change in membrane conductance. 4. In contrast, DAMET elicited a hyperpolarizing response due to an outward current, also not associated with a significant change in membrane conductance. 5. Both the DALEU and the DAMET responses increased with hyperpolarization, decreased with depolarization, but did not reverse at potentials less than -30 mV. Neither response was sensitive to naloxone. 6. FMRFamide induced a voltage-dependent outward current that reversed at about -76 mV. This neuron was responsive to much lower concentrations of FMRFamide than either of the enkephalins, and the response to FMRFamide appears to be a conductance increase to K+. 7. These results suggest that the MCC neuron has distinct receptors for Leu- and Met-enkephalin that activate unusual responses of opposite polarity, as well as more usual inhibitory responses to FMRFamide.


Ouabain blocks some rapid concentration-induced clamp acetylcholine responses on Helix neurons. 1. The effects of ouabain, a potent inhibitor of Na(+)-K+ ATPase, were determined on the transmembrane responses of internally dialyzed Helix neurons to rapid acetylcholine (ACh) application using the "concentration clamp" technique. 2. Ouabain selectively depressed "A"-type responses to ACh, which are due to a selective increase in membrane permeability to chloride. In contrast, the "B"-type responses, due primarily to an increase in monovalent cation permeability, was unaffected. 3. The blockade of the Cl- responses was not associated with a change of the reversal potential of the response. Ouabain depressed the maximal response without shifting the dose-response curve. 4. Ouabain caused an increase in the time constant of decay of the ACh current, but the value in the presence of ouabain was not different from that of a lower concentration of ACh determined so as to give a response of the same peak amplitude. Therefore, the effect of ouabain is not on the process of receptor desensitization directly.


The effects of cAMP, Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine responses in Helix neurons. 1. Using internal perfusion and concentration-clamp procedures applied to Helix neurons, the effects of cAMP, Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine Cl-dependent responses were determined. 2. Intracellular cAMP (10(-4) M) depressed those acetylcholine responses which were blocked by ouabain but had no effect on ouabain-insensitive acetylcholine responses. In the presence of elevated intracellular cAMP, ouabain had no further depressant effect on these acetylcholine responses. Both elevated cAMP and ouabain reduced the acetylcholine response without altering the current-voltage curves. 3. An increase in intracellular Ca2+ concentration depressed the amplitude of current induced by application of acetylcholine in neurons with ouabain-sensitive responses and shifted the dose-response relationship to the right. However, elevated Ca2+ did not reduce the maximal response induced by acetylcholine, nor did it prevent the reduction of that response by ouabain. 4. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of protein kinase C activity, caused depression of both the ouabain-sensitive and the ouabain-insensitive acetylcholine responses. The inhibitory effect of TPA was markedly enhanced after addition of ATP to the intracellular medium and was greatly reduced by cooling to 5 degrees C. The blocking effect of ouabain, however, reexamined in the presence of TPA. 5. These observations are consistent with the hypothesis that the depression of acetylcholine induced Cl--responses in Helix neurons is a result of an increase in intracellular cAMP concentration but is unrelated to activation of protein kinase C or increases in intracellular Ca2+.


Characteristics of C6 glioma cells overexpressing a gap junction protein. 1. C6 glioma cells transfected with connexin43 cDNA display a dramatic increase in the level of connexin43 mRNA and protein. 2. This overexpression of connexin43 is evident at the cellular level, as revealed with in situ hybridization and immunocytochemistry. Transfection with connexin43 cDNA also induced actin stress fibers in these glioma cells. 3. Although we observed up to a 50-fold increase in the level of connexin43 mRNA following transfection, virtually all of this mRNA was present in the polysomal fraction. 4. Overexpression of connexin43 mRNA did not appear to compete with other cellular mRNAs for access to the translational machinery. 5. It is likely that the reduced proliferation rate of the transfected cells, reported earlier, is due to enhanced connexin43 expression and intercellular coupling.


Localization of binding sites for atrial natriuretic factor and angiotensin II in the central nervous system of the clawed toad Xenopus laevis. The distribution of binding sites for atrial natriuretic factor (ANF) and angiotensin II (A II) was investigated in the central nervous system (CNS) of the clawed toad Xenopus laevis by means of in vitro autoradiography using [125I]-rat ANF(99-126) or [125I] [Val5] A II and [125I]human A II as labeled ligands. The highest densities of specific ANF-binding were detected in the nucleus habenularis, thalamic regions, hypophyseal pars nervosa and nucleus interpeduncularis. Moderate ANF-binding was found in the bulbus olfactorius, pallium, septum, striatum, lateral forebrain bundle, nucleus infundibularis, hypophyseal pars distalis and tectum. The highest levels of specific A II binding sites were observed in the nucleus praeopticus, nucleus habenularis, hypophyseal pars nervosa and pars distalis, whereas the amygdala contained moderate A II binding. The existence of specific binding sites for ANF and A II in the CNS of Xenopus laevis suggests that both peptides act as neurotransmitters or neuromodulators in the amphibian CNS. The co-localization of dense binding sites for both peptides in the nucleus habenularis, hypophyseal pars nervosa and pars distalis supports the view that ANF and A II have opposite regulatory functions in these regions.


Doxorubicin inhibits human DNA topoisomerase I. Purified human DNA topoisomerase I was assayed quantitatively by enzyme titrations with supercoiled pHC624 DNA in the presence of 0-2.0 microM doxorubicin. Supercoiled and relaxed DNAs were resolved by agarose gel electrophoresis in the presence of ethidium bromide, and the percentage of conversion of supercoiled DNA to relaxed DNA was quantified by scanning microdensitometry. The inhibition of DNA topoisomerase I activity was measured at varying concentrations of doxorubicin. Doxorubicin inhibited enzyme activity at an IC50 value (the concentration required to inhibit 50% of the total activity) of 0.8 microM. Similar inhibition was observed for daunomycin, a structurally related anthracycline antitumor drug. These results indicate that anthracyclines inhibit human DNA topoisomerase I activity at concentrations that cause DNA damage and cytotoxicity in vivo.


The role of deoxycytidine-metabolizing enzymes in the cytotoxicity induced by 3'-amino-2',3'-dideoxycytidine and cytosine arabinoside. The cellular metabolism of 3'-amino-2',3'-dideoxycytidine (3'-NH2-dCyd), a cytotoxic agent previously reported to be a poor substrate for purified Cyd/dCyd deaminase (dCydD), was compared with that of cytosine arabinoside (ara-C) in cells that displayed dCydD activity (HeLa) and in cells that did not (L1210). Growth inhibition induced by 3'-NH2-dCyd was dependent on the levels of anabolic enzymes, particularly dCyd kinase (dCydK), whereas cytotoxicity induced by ara-C was dependent on the expression of both anabolic and catabolic enzyme activities. Competition kinetics using purified enzyme revealed that the binding affinity of ara-C to dCydK was 5-fold that of the amino analog. However, this binding advantage is apparently offset in cells that contain high levels of dCydD, since the Ki values for this enzyme were 0.2 and 23 mM for ara-C and 3'-NH2-dCyd, respectively. This was reflected in the decrease in analog sensitivity observed between the two cell lines, whereby the concentrations of ara-C and 3'-NH2-dCyd required to inhibit growth by 50% were 200 and 7 times higher, respectively, in the dCydD-containing HeLa cells as compared with the dCydD-deficient L1210 cells. The metabolic stability and cytotoxicity of 3'-NH2-dCyd was independent of cell number. An unexpected finding was the extent to which the effectiveness of ara-C could be mitigated by the number of dCydD-containing cells. A completely cytotoxic concentration of ara-C was rendered nontoxic by a 10-fold increase in cell number. This observation was supported by an increase in I-beta-D-arabinofuranosyluracil (ara-U) formation, a decrease in ara-C 5'-triphosphate (ara-CTP) accumulation, and a rise in cell viability with increasing cell number. These findings indicate that unlike ara-C, the effectiveness of 3'-NH2-dCyd is independent of the level of deaminase, which suggests its possible utility in situations in which high levels of deaminase are manifest.