Rhinotracheitis in turkey poults.

Rhinotracheitis in turkey poults. A severe upper respiratory disease of young turkeys is described that resulted in high morbidity and mortality. Death was due to asphyxiation produced by occlusion of the trachea or nostrils. The postmortem lesions were tracheitis, pulmonary edema, swollen livers and spleens, and a drastic reduction in bursa size. Bursal necrosis and loss of tracheal epithelium were found in tissue sections from clinically affected birds. Antibody to infectious bursal disease was found by agar-gel precipitin and virus-neutralization tests of serum samples from affected flocks. A geometric mean titer of 31.5 for Newcastle disease was found in unvaccinated flocks of five-week-old turkeys.


Oxidation-reduction studies of the Mo-(2Fe-2S) protein from Desulfovibrio gigas. Potentiometric titration followed by e.p.r. measurements were used to determine the midpoint reduction potentials of the redox centres of a molybdenum-containing iron-sulphur protein previously isolated from Desulfovibrio gigas, a sulphate-reducing bacterium (Moura, Xavier, Bruschi, Le Gall, Hall & Cammack (1976) Biochem. Biophys. Res. Commun. 728 782-789; Moura, Xavier, Bruschi, Le Gall & Cabral (1977) J. Less Common Metals 54, 555-562). The iron-sulphur centres could readily be distinguished into three types by means of g values, temperature effect, oxidation-reduction potential values and reduction rates. The type-I Fe-S centres are observed at 77 K. They show mid-point potential values of -260mV (Fe-S type IA) and -440 mV (Fe-S type IB). Centres of types IA and IB appear to have similar spectra at 77 K and 24 K. The Fe-S type-II centres are only observed below 65 K and have a midpoint potential of -28mV. Long equilibration times (30 min) with dye mediators under reducing conditions were necessary to observe the very slow equilibrating molybdenum signals. The potential values associated with this signal were estimated to be approx. -415 mV for Mo(VI)/Mo(V) and-530mV for Mo(V)/Mo(IV).


Evidence for an essential arginine recognition site on adenosine 3':5'-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle. On the basis of the chemical and structural features of the amino acid sequences in the vicinities of phosphorylatable hydroxyamino acid residues in several of the well-known protein substrates for skeletal-muscle cyclic AMP-dependent protein kinase, it is hypothesized that the phosphorylatable residue at position i and arginine residue at position i-3 of these protein substrates are located on a peptide turn on the hydrophilic protein surface. It is further hypothesized that there is an arginine-recognition site near the active centre on the protein kinase. This site is essential for the function of cyclic AMP-dependent protein kinase, for, not only does it recognize specifically the exposed arginine residue of the protein substrate, but, more importantly, via the interaction with arginine-(i--3), it may help to steer the topologically adjacent serine-i into proper orientation on the nearby active centre for phosphorylation. Model-building and kinetic data that provide support for the proposed hypotheses are presented.


rac-Glycerol 1:2-cyclic phosphate 2-phosphodiesterase, a new soluble phosphodiesterase of mammalian tissues. 1. A soluble phosphodiesterase is present in mammalian tissues which rapidly hydrolyses enantiomorphs of rac-glycerol 1:2-cyclic phosphate, producing rac-glycerol 1-phosphate. 2. The enzyme has been purified up to 1700-fold by a combination of acetone precipitation and chromatography on DEAE-Sephadex A-50, Sephadex G-150 and hydroxyapatite. 3. The Km with glycerol cyclic phosphate as substrate is 7.2 mM, and the pH optimum broad (6.9--7.5). The molecular weight (by gel filtration) of the enzyme is approx. 35500. 4. The phosphodiesterase has no requirement for Ca2+ or Mg2+, but is stimulated by reducing agents (cysteine, dithiothreitol) and Fe2+. 5. The purified phosphodiesterase preparation also hydrolysed 3':5'-cyclic AMP, producing 5'-AMP exclusively, and 2':3'-cyclic AMP, forming 3'-AMP and 2'-AMP in the ratio 7:3. Bis-(p-nitrophenyl) phosphate was slowly hydrolysed, but other phosphodiesters tested were not attacked. 6. The phosphodiesterase is inhibited by theophylline and o-phenanthroline. It is inhibited by Pi and by a variety of phosphomonoesters, of which certain aromatic primary phosphates are particularly effective.


Purification and properties of retinoic acid-binding protein from chick-embryo skin. Retinoic acid-binding protein, which is considered to mediate the biological function of retinoic acid in epithelial differentiation and in the possible control of tumorigenesis, was reproducibly purified from chick-embryo skin by using DEAE-Sephadex and Sephadex G-100 column chromatography and isoelectric focusing. About 1mg of protein was isolated from 60g of skin. The purified protein-ligand complex was found to be homogeneous by electrophoresis on polyacrylamide gels. The binding protein has mol.wt. 17800 and pI 4.5. The binding of [3H]retinoic acid to the protein was completely inhibited by mercury compounds. The inhibition is reversible on treatment without dithithreitol; about 50% of the retinoic acid-binding capacity of the mercury-compound-treated protein is restored by chromatography on Sephadex G-25. iodoacetamide treatment of the protein irreversibly inhibits about 50% of retinoic acid binding.


Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat. 1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.


The oxidation of Pseudomonas cytochrome c-551 oxidase by potassium ferricyanide. Stopped-flow kinetics were made of the reaction between ascorbate-reduced Pseudomonas cytochrome oxidase and potassium ferricyanide under both N2 and CO atmospheres. Under N2 three kinetic processes were observed, two being dependent on ferricyanide concentration, with second-order rate constants of 9.6 X 10(4)M-1.s-1 and 1.5 X 10(4)M-1.s-1, whereas the other was concentration-independent, with a first-order rate constant of 0.17 +/- 0.03s-1. Measurements of their kinetic difference spectra have allowed the fastest and second-fastest phases of the reaction to be assigned to direct bimolecular reactions of ferricyanide with the haem c and haem d, moieties of the enzyme respectively. Under CO, the second-order rate constant for the reaction of the haem c was, at 1.3 X 10(5)M-1.s-1, slightly enhanced over the rate in a N2 atmosphere, but the reaction velocity of the haem d1 component was greatly decreased, being apparently limited to that of the rates of CO dissociation from the molecule (0.15s-1 and 0.03s-1). The results are compared with those obtained during a previous study of the reaction of reduced Pseudomonas cytochrome oxidase with oxidized azurin.


Alterations in response of rat white adipocytes to insulin, noradrenaline, corticotropin and glucagon after adrenalectomy. Correction of these changes by adenosine deaminase. 1. Adipocytes isolated from rats 6--9 days after adrenalectomy had significantly increased sensitivity to insulin action against noradrenaline-stimulated lipolysis. In the presence of adenosine deaminase there was no significant difference in insulin sensitivity between cells from adrenalectomized and sham-operated rats. 2. Adipocytes from adrenalectomized rats had decreased lipolytic responses to all concentrations of noradrenaline and glucagon tested and a decreased lipolytic response to low but not high concentrations of corticotropin. There was no difference in lipolytic response to theophylline after adrenalectomy. Adenosine deaminase corrected the differences in response to noradrenaline and glucagon resulting from adrenalectomy. 3. In the presence of adenosine deaminase rates of lipolysis, after stimulation by high concentrations of noradrenaline, glucagon, corticotropin or theophylline, were the same in cells from adrenalectomized or sham-operated rats. 4. These findings and previously reported effects of adenosine and adrenalectomy on adipocyte function are discussed. It is proposed that changes in adipocyte hormone responsiveness after adrenalectomy may result from changes in adenosine metabolism or release.


Properties of mitochondria isolated from cyanide-sensitive and cyanide-stimulated cultures of Acanthamoeba castellanii. 1. Mitochondria isolated from cultures of Acanthamoeba castellanii exhibit respiratory control and oxidize alpha-oxoglutarate, succinate and NADH with ADP:O ratios of about 2.4, 1.4 and 1.25 respectively. 2. Mitochondria from cultures of which the respiration was stimulated up to 50% by 1mm-cyanide (type-A mitochondria) and from cyanide-sensitive cultures (type-B mitochondria) had similar respiratory-control ratios and ADP:O ratios. 3. State-3 rates of respiration were generally more cyanide-sensitive than State-4 rates, and the respiration of type-A mitochondria was more cyanide-resistant than that of type-B mitochondria. 4. Salicylhydroxamic acid alone had little effect on respiratory activities of either type of mitochondria, but when added together with cyanide, irrespective of the order of addition, inhibition was almost complete. 5. Oxidation of externally added NADH by type-A mitochondria was mainly via an oxidase with a low affinity for oxygen (K(m)[unk]15mum), which was largely cyanide-sensitive and partially antimycin A-sensitive; this electron-transport pathway was inhibited by ADP. 6. Cyanide-insensitive but salicylhydroxamic acid-sensitive respiration was stimulated by AMP and ADP, and by ATP after incubation in the presence of MgCl(2). 7. Addition of rotenone to mitochondria oxidizing alpha-oxoglutarate lowered the ADP:O ratios by about one-third and rendered inhibition by cyanide more complete. 8. The results suggest that mitochondria of A. castellanii possess branched pathways of electron transport which terminate in three separate oxidases; the proportions of electron fluxes via these pathways vary at different stages of growth.


A study of regulation of gluconeogenesis and the supply of cytosolic reducing equivalents for lactate formation in rat kidney-cortical-tubule fragments incubated with pyruvate. 1. Tubule fragments were isolated after treatment of rat kidney cortex with collagenase. The formation of glucose and lactate on incubation with 5mM-pyruvate was then measured under various conditions. 2. When tubule fragments were isolated from fed rats in the absence of Ca2+ and then incubated with various Ca2+ concentrations, an incubation period of 15--30 min was necessary to establish a metabolic steady state. Under these conditions glucose formation was increased by Ca2+, adrenaline or 3':5'-cyclic AMP to a greater extent than was lactate formation. Data show that appreciable lactate formation could not have resulted from glycolytic metabolism of glucose formed by gluconeogenesis during incubation. 3. When tubule fragments were isolated from fed rats in the presence of 1.27 mM-Ca2+ and adjustments made to the Ca2+ concentration at the commencement of incubation, metabolic steady state was rapidly established. Under these conditions lactate formation was almost insensitive to Ca2+ concentration (0.16--4.5 mM), whereas glucose formation varied with Ca2+ concentration in a sigmoidal manner. 3':5'-Cyclic AMP decreased this sigmoidicity. 4. Ca2+ depletion of the tissue before incubation appeared to change permanently the relationship between extracellular Ca2+ concentration and the measured rates of metabolic processes. 5. Under conditions of metabolic steady state, glucose formation by tubule fragments from fed rats was less sensitive than lactate formation to inhibition by 3-mercaptopicolinate or 2-n-butylmalonate. Lactate formation by tubule fragments prepared from 48 h-starved rats was more sensitive to these inhibitors. 6. Estimates were made of the rate of futile cycling of C3 species through pyruvate kinase. This was greater in the starved than in the fed state, was decreased by 3':5'-cyclic AMP in both the fed and the starved state, but was unaffected by Ca2+. 7. These results suggested that formation of lactate and glucose is less tightly linked in kidney cortex than in liver. A considerable amount of the supply of reducing equivalents for lactate formation did not appear to be associated with an energy-dependent translocation from mitochondria to cytosol involving a pyruvate leads to oxaloacetate leads to phosphoenolpyruvate leads to pyruvate cycle.


The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential. 1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0' + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N'-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.


The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential. 1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S(14)CN(-), and the transmembrane pH gradient component from the uptake of [(14)C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN'N'-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (DeltaG(p)=DeltaG(0)'+RTln[ATP]/ [ADP][P(i)]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO(3) the protonmotive force was not detectable (<60mV) but DeltaG(p) was not altered. This result may indicate either that there is no relationship between the protonmotive force and DeltaG(p), or that for an unidentified reason the equilibration of SCN(-) or methylamine with the membrane potential and the pH gradient is prevented by NO(3) (-) in this system.


Properties of a cytochrome c-enriched light particulate fraction isolated from the photosynthetic bacterium Rhodopseudomonas spheroides. Differential centrifugation of suspensions of French-press-disrupted Rhodopseudomonas spheroides yielded a light particulate fraction that was different in many properties from the bulk membrane fraction. It was enriched in cytochrome c and had a low cytochrome b content. When prepared from photosynthetically grown cells this fraction had a very low specific bacteriochlorophyll content. The cytochrome c of the light particles differed in absorption maxima at 77K from cytochrome c2 attached to membranes; there was pronounced splitting of the alpha-band, as is found in cytochrome c2 free in solution. Potentiometric titration at A552--A540 showed the presence of two components that fitted an n = 1 titration; one component had a midpoint redox potential of +345mV, like cytochrome c2 in solution, and the second had E0' at pH 7.0 of +110 mV, and they were present in a ratio of approx. 2:3. Difference spectroscopy at 77K showed that the spectra of the two components were very similar. More of a CO-binding component was present in particles from photosynthetically grown cells. Light membranes purified by centrifugation on gradients of 5--60% (w/w) sucrose retained the two c cytochromes; they contained no detectable succinate-cytochrome c reductase or bacteriochlorophyll and very little ubiquinone, but they contained NADH-cytochrome c reductase and some phosphate. Electrophoresis on sodium dodecyl sulphate/polyacrylamide gels showed that the light membranes of aerobically and photosynthetically grown cells were very similar and differed greatly from other membrane fractions of R. spheroides.


Evidence for the involvement of superoxide in vitamin K-dependent carboxylation of glutamic acid residues of prothrombin. The formation of vitamin K epoxide and the vitamin K-dependent carboxylation of glutamic acid residues present in synthetic substrates and decarboxyprothrombin are both inhibited by superoxide dismutase. Catalase only inhibits the generation of vitamin K epoxide, suggesting that the carboxylation and epoxidation reactions are not inter-dependent.


Collagen production by cultures containing multinucleated cells derived from synovial fibroblasts. Multinucleate cells are found frequently in rheumatoid synovium. In this study, polyethylene glycol was used to fuse rabbit synovial fibroblasts. Approximately 40% of the cells developed more than one nucleus in a 24 hour period, during which time cell membranes had increased permeability. In cultures containing multinucleate cells, 3H-thymidine incorporation was depressed for 24 hours although 3H-leucine incorporation into TCA precipitable material was unaffected; autoradiography showed that depression of 3H-thymidine continued for at least 4 days. Collagenase production by cultures containing fused cells was increased more than 10-fold over control cultures during a 28 day period.


Correlation between articular cartilage collagenase activity and osteoarthritis. Articular cartilage collagenase activity was determined for 28 sections obtained from twelve femoral heads. For each one square centimeter area, a section was graded by a histologic-histochemical grading system for the severity of the arthritis. Collagenase activity was found primarily in those areas of moderately severe disease, but not in mild or end stage arthritis.


Studies with primaquine in vitro: superoxide radical formation and oxidation of haemoglobin. 1. The production of superoxide radicals from primaquine diphosphate in aqueous solution has been demonstrated, using as indicator the reduction of cytochrome C with inhibition of the reaction by superoxide dismutase. 2. Primaquine-mediated oxidation of haemoglobin to methaemoglobin was reduced by the addition of catalase and increased by superoxide dismutase. Mannitol, a hydroxyl radical scavenger, abolished the increase in methaemoglobin observed in the presence of superoxide dismutase. EDTA reduced the oxidation of haemoglobin with and without superoxide dismutase. 3. Although the oxidation of haemoglobin in the presence of primaquine includes the effects of hydrogen peroxide, superoxide and hydroxyl radicals and metal ions, the results indicate that hydrogen peroxide, rather than the superoxide radical, is the main oxidizing species. The increase in haemoglobin oxidation occurring with superoxide dismutase may result from the augmented rate of hydrogen peroxide formation from superoxide radicals.


Autoimmunity and the immunologic network. Recent developments in autoimmunity suggest that there are three stages in the response to self, which can be called autorecognition, autoimmunity, and autoimmune disease. The first is physiologic and fundamental to a network theory of immunologic control that is based upon recognition of idiotypes and antigens related to the major histocompatibility complex. Many foreign antigens may be recognized immunologically only if they can be imposed upon an existing network of immunologic communication. The existence of anti-receptor autoimmune diseases (such as myasthenia gravis and Grave's disease) leads to the postulate that the immune network may normally function to help regulate hormone and other nonimmune cell surface receptors. Chronic autoimmune diseases may be caused either by genetically determined abnormalities in the immune network or by an antigenic perturbation of the network that results in unresponsiveness and tolerance of an offending agent.


Inhibition of collagen peptidase in HeLa cells and human tumours by compounds including drugs used in cancer therapy. Collagen-peptidase activity in extracts of HeLa cells and human tumours is inactivated by Razoxane (ICRF-159), cyclophosphamide, 5-fluorouracil, thiotepa, aprotinin, EDTA and phenanthroline. As this activity, in association with other enzymes, may contribute to tissue lysis in cancers, chemical intervention may reduce invasiveness and modify the processes of infiltration and metastasis. Accordingly, some drugs used in therapy or for the prevention of metastasis may produce their observed effects by a combination of factors including enzyme inhibition.


Use of exercise challenge to investigate possible tolerance to beta-adrenoceptor stimulation in asthma. The effect of prolonged salbutamol administration on beta-adrenoceptor function in asthma has been examined. Six adult patients received salbutamol tablets (16 mg daily) for between 4 and 20 weeks and six adolescents received salbutamol aerosol (800 microgram daily) for 2--5 weeks. Before and after the treatment period the acute bronchodilator response to inhaled salbutamol and the ability of inhaled salbutamol to protect against exercise-induced asthma were examined. Lymphocyte beta-adrenoceptor function was also measured in the patients on tablet therapy. Inhaled salbutamol was less effective in protecting against exercise-induced asthma at the end of the treatment period in the patients who had received tablet therapy, but otherwise there was no significant change in beta-receptor function of either airways or lymphocytes. This apparent loss of efficacy of inhaled salbutamol in the prevention of exercise-induced asthma in some subjects, even when its acute bronchodilator effect is preserved, might reflect differences in the susceptibility of different beta-adrenoceptors to desensitization after prolonged stimulation: its clinical importance remains uncertain.


Effects of amino acid analogues on protein synthesis and degradation in isolated cells. 1. Naturally-occurring and synthetic analogues of phenylalanine, tyrosine, histidine, arginine, proline, tryptophan and the sulphur amino acids have beeen tested in rat reticulocytes and in the Reuber H35 hepatoma for effects on protein synthesis and protein degradation and on the heat lability of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) in the hepatoma cells. The experiments were designed to test whether the analogues could be incorporated into mammalian proteins and whether the resultant proteins would be degraded at an accelerated rate. 2. Several analogues, including thiazolylanine, triazolalanine and selenocystine both stimulated protein synthesis and produced labile protein in reticulocytes. Other analogues, such as dihydroxyphenylalanine, thioproline and pipecolic acid accelerated protein breakdown but probably indirectly via an inhibition of protein synthesis. Azetidine-2-carboxylic acid had the largest effect on protein breakdown in reticulocytes. 3. Labile protein was produced in hepatoma cells incubated in the presence of azetidine-2-carboxylic acid, canavanine, indospicine, triazolalanine, 2-, 3- and 4-fluorophenylalanine. These same analogues, together with 3,4-dehydroproline, beta-2-thienylalanine, dihydroxyphenylalanine, histidinol, 5- and 6-fluorotryptophan, selenocystine and selenomethionine produced heat-labile phosphoenolpyruvate carboxykinase. Enzyme induced in the presence of selenomethionine or indospicine showed the largest increases in heat lability, and for these analogues equimolar concentrations of methionine and arginine respectively were needed to nullify the enzyme abnormality. 4. The toxicity of the same naturally-occurring analogues has been discussed in terms of their ability to be incorporated into cell proteins.


Role of hydrophobicity in the binding of coenzymes. Appendix. Translational and rotational contribution to the free energy of dissociation. We calculate the loss of surface area accessible to solvent associated with coenzyme binding in Clostridium flavodoxin, in dogfish lactate dehydrogenase, and in lobster glyceraldehyde-3-phosphate dehydrogenase. The coenzymes are nearly buried in the complexes and lose on the order of 600 A2, while the proteins lose a similar amount of accessible surface area. Some of the loss can be attributed to conformation changes in the protein, at least in the case of lactate dehydrogenase, where we show that the apoenzyme has a larger accessible surface area than the holoenzyme. Using known correlations with the hydrophobic contribution to the free energy, we demonstrate that hydrophobicity is the major source of stabilization free energy in FMN binding to flavodoxin and in NAD binding to the two dehydrogenases: it contributes 25 to 30 kcal/mol to the free energy of dissociation, more than required in order to compensate for the loss of six degrees of translational/rotational freedom by the coenzyme.


A kinetic study of cyclic adenosine 3':5'-monophosphate binding and mode of activation of protein kinase from Drosophila melanogaster embryos. Cyclic AMP-dependent protein kinase and its regulatory subunit were isolated from Drosophila melanogaster embryos. The profiles of cyclic AMP binding by these proteins were significantly different. In order to explain such a difference and to find the mode of enzyme activation by cyclic AMP, a kinetic study of cyclic AMP binding was carried out. First, the association rate constant k1 and dissociation rate constant k-1 in the cyclic AMP-regulatory subunit interaction at 0 degrees C were estimated to be 2.3 X 10(6)M-1s-1 and 1.1 X 10(-3)s-1, respectively. Secondly, the three possible modes of enzyme activation by cyclic AMP were mathematically considered and could be described by a unique formula: r=APt + BQt (A + B=1) in which the parameters A, B, P, and Q are equivalent to rate constants in the sense that the rate constants are simply expressed by these parameters. Thirdly, the values of the parameters and subsequently the values of rate constants involved in the possible mechanisms were evaluated using a curve-fitting technique and compared with experimental observation. It was then found that the following mechanism was the only one which fitted the experimental observations. Namely, RC + L k3 equilibrium k-3 LRC k4 equilibrium k-4 RL + C where R, C, and L represent the regulatory and catalytic subunits and cyclic AMP as a ligand. Thus, our results indicate that in the presence of cyclic AMP the active enzyme (C) is released from a ternary intermediate which is the primary product of the cyclic AMP-holoenzyme interaction. The estimated values of the rate constants are: k3=3.5 X 10(6)M-1s-1;k-3=7.3 X 10(-1)s-1;and k4=3.8 X 10(-2)s. These estimates indicate that the reaction LRC leads to RL + C is relatively slow and limits the rate of the overall reaction. By comparing k-3 and k4, it is apparent that a large part of newly formed ternary intermediate reverts to the holoenzyme.


Characterization of soluble uterine cyclic nucleotide phosphodiesterase. Soluble cyclic nucleotide phosphodiesterase of rat uterus displays distinct structural and regulatory properties. Like phosphodiesterases from many mammalian sources the soluble uterine enzyme system exhibits nonlinear Lineweaver--Burk kinetics with cyclic adenosine 3':5'-monophosphate (cAMP) as substrate (apparent Kms congruent to 3 and 20 micron) and linear kinetics with cyclic guanosine 3':5'-monophosphate (cGMP) as substrate (apparent Km congruent to 3 micron). Unlike most other mammalian phosphodiesterases, however, numerous separation procedures reveal only a single form of uterine phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP. A single form of the enzyme is observed upon sucrose gradient centrifugation (7.9 S), agarose gel filtration, and DEAE-cellulose chromatography at either pH 8.0 OR 6.0. Heat denaturation (50 degrees C) of soluble uterine phosphodiesterase causes the loss of both cAMP and cGMP hydrolytic activities at the same rate. Isoelectric focusing reveals major (pI = 5.2) and minor forms (pI = 5.8) of phosphodiesterase which both catalyze the hydrolysis of the two cyclic nucleotide substrates. In vivo administration of estradiol produces identical decreases in the activities of cAMP and cGMP phosphodiesterase. These results raise the possibility that the uterus contains a single form of soluble phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP.


Cross-linking of ubiquinone cytochrome c reductase (complex III) with periodate-cleavable bifunctional reagents. Two novel cross-linkers, disuccinimidyl tartarate (DST) and N,N'-bis(3-succinimidyloxycarbonylpropyl)tartaramide (SPT), have been synthesized. These reagents span 6 and 18 A, respectively, between functional groups and contain a vic-glycol bond which can be cleaved with periodate under mild reaction conditions. Both DST and SPT have been used to examine the near-neighbor relationships of polypeptides in ubiquinone cytochrome c reductase (complex III) from beef heart mitochondria. Among the cross-linked products resolved were pairs containing I + II, II + VI, I + V, and VI + VII. Polypeptides III and IV, a cytochrome b aproprotein, and the cytochrome c1 hemoprotein, respectively, were also resolved in several cross-linked products.


Lipid-protein associations in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides. Lipid-protein interactions were examined in chromatophores isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides using lipid spin-labels. The chromatophores contain fluid bilayer and a significant amount of lipid immobilized by membrane proteins. For a typical preparation of cells grown under 600 ft-c illumination, 59% of the spin-labeled fatty acids were bound. Essentially the entire length of the 18-carbon fatty acid chain was immobilized, judging from results obtained with the spin-label at the 7, 12, and 16 positions. The amount immobilized varies directly with the bacteriochlorophyll content of the chromatophore material, suggesting that a significant fraction of the lipid spin-labels is immoblized on the hydrophobic surfaces of the chlorophyll-binding proteins. Changing the lipid spin-label head group from a negatively charged carboxyl group to a positively charged quarternary amine greatly decreased the amount of immobilized lipid. The changes in immobilized lipid with light level and polar head group suggest that the anntenna bacteriochlorophyll-binding proteins preferentially associate with negatively charged lipids.


Mode of coupling between the beta-adrenergic receptor and adenylate cyclase in turkey erythrocytes. The mode of coupling of the beta-adrenergic receptor to the enzyme adenylate cyclase in turkey erythrocyte membranes was analyzed in detail. A number of experimental techniques have been used: (1) measurement of the kinetics of cyclase activation to its permanetly active state in the presence of guanylyl imidodiphosphate, as a function of hormone concentrations; (2) measurement of antagonist and agoinst binding to the beta-adrenergic receptor prior and subsequent to the enzyme activation by hormone and guanylyl imidodiphosphate. On the bases of these two approaches, all the models of receptor to enzyme coupling which involve an equilibrium between the enzyme and the receptor can be rejected. The binding and the kinetic data, however, can be fitted by two diametrically opposed models of receptor to enzyme coupling: (a) the precouped enzyme-receptor model where activation of the enzyme occurs, according to the following scheme: formula (see text) where H is the hormone, RE is the precoupled respetor-enzyme complex, k1 and k2 are the rate constants describing hormone binding, and k is the rate constant characterizing the formation of HRE' from the intermediate HRE. According to this model, the activated complex is composed of all of the interacting species. (b) The other model is the collision coupling mechanism: formula (see test) wheere KH is the horome-receptor dissociation constant, k1 is the bimolecular rate constant governing the formation of HRE, and k3 the rate constant governing the activation of the enzyme. In this case the intermediate never accumulates and constitutes only a small fraction of the total receptor and adenylate cyclase concentrations. In order to establish which of the two mechanisms governs the mode of adenylate cyclase activation by its receptor, a diagnostic experiment was performed: Progressive inactivation of the beta receptor by a specific affinity label was found to cause a decrease in the maximal binding capacity of the receptor and a proportional decrease in the rate of activation, but no change in the maximum level of activity was attained. Progressive inactivation of the enzyme by p-hydroxymercuribenzoate was found not to change the rate of activation nor the capacity of the receptor to bind hormone. Only the maximal level of activation was found to be decreased. These results are not compatible with the precoupled model of receptor and cyclase nor with floating receptor models in which an intermediate of hormone, receptor, and cyclase is in equilibrium with its reactants. The data strongly suggest that the collision coupling is the mode of coupling between the beta receptor and cyclase coupling in turkey erythrocyte membranes.


Nucleic acid interaction with VERO cells. A temperature barrier in the interaction pattern. The interaction of VERO cell monolayers with spin (nitroxide)-(labeled polynucleotides (1(N)n) was examined by electron spin resonance (ESR) spectroscopy at various temperatures. Nitroxide labels covalently linked to (A)n, (dUfl)n, (U)n and (A)n . (U)n were used to monitor the interaction. The VERO cells were grown on small quartz plates with a cell viability of 95% or better and then used directly for the ESR studies. The ESR results indicated that the interaction between VERO cells and spin-labeled nucleic acids is temperature dependent. No temperature dependence was found when VERO cells were in contact with nitroxide radicals which were free in solution or covalently bound to Sepharose 4B. The temperature dependence established with nitroxide-labeled nucleic acids indicates that a temperature barrier must exist between 20 and 26 degrees C for the interaction between nucleic acids and VERO cells; namely, at 26 degrees C or above spin-labeled nucleic acids interact significantly with a VERO cell surface; whereas, at 20 degrees C the ESR signal reports no interaction. It is concluded that a temperature-dependent phase transition of membrane components or cell surface products active at 26 degrees C or above play a key role in the nucleic acid cell surface interaction process.


In vitro translation of nematode cuticular collagens. Phenanthroline treatment of growing cultures of the free-living nematode Panagrellus silusiae was used to lower the degree of hydroxylation of nascent collagen chains at the polysomal level. Under these conditions, the bound pentasome-hexasome fraction provided substrate for prolyl hydroxylase. When this polysomal fraction was subsequently tested in a cell-free wheat germ system, collagenase-susceptible translation products were observed after sodium dodecyl sulfate-acrylamide gel electrophoresis. The electrophoretic mobilities of each of these four major collagen products were similar to four collagens that are isolated from intact cuticles. In addition, purified polysomal RNA that adhered to unmodified cellulose directed the synthesis of four pepsin-resistant polypeptides that had molecular weights that coincided with four pepsin-resistant collagens that can be purified from the cuticle of this species. Thus, the polysomal site of the messenger RNAs for the cuticular collagens of P. silusiae was located. Although precursor forms of the cuticular collagens were not produced in the cell-free system, the question whether additional amino acid segments occur on the primary translational products of the cuticular collagens in vivo remains open.


Phosphatidate phosphatase: activity and properties in fetal and adult rat lung. The purpose of this work is to compare the properties of phosphatidate phosphatase (L-alpha-phosphatidate phosphohydrolase, EC 3.1.3.4) in fetal and adult rat lung and to establish the developmental profile of activity measured under optimal conditions. The maximal pH of 6.0--7.0 and the inhibition by fluoride, Ca2+ and detergents were simialr for both adult and fetal. Phosphatidate phosphohydrolase activity was located in both mitochondria and microsomes. The localizations of marker enzymes indicated that the activity in these subfractions was not a result of cross contaminations. Very low activity was detected in the supernatant fraction and no Mg2+ requirement was demonstrable. The activity in the particulate fraction was about 50% of the adult from 18 day gestation until birth. Following birth, the activity rapidly increased to adult levels. Dipalmitoyl, dioleoyl and diacyl glycerol 3-phosphates are all utilized well as substrates. 1,2-dipalmitoyl-sn-glycerol 3-phosphate was hydrolyzed  faster under maximal conditions. The velocity-substrate curves tended to be sigmoidal, particularly when 1,2-dipalmitoyl-sn-glycerol 3-phosphate was the substrate. Estimated apparent Km values of 0.02--0.03 mM were obtained for fetal and adult preparations.


Cholesteryl ester exchange protein in human plasma isolation and characterization. A protein catalyzing the exchange of cholesteryl esters among the lipoproteins was found in human plasma. A rapid method for assaying this activity was developed based on the transfer of radioactive cholesteryl esters from low density lipoprotein with MnCl2 in the presence of phosphate. Fractionation of plasma through a combination of ammonium sulfate precipitation, ultracentrifugation at p = 1.25, and chromatography on Phenyl-Sepharose, CM-cellulose, and concanavalin A-Sepharose, yielded a preparation purified 3500-fold compared to the starting plasma. The exchange protein was found to be a glycoprotein with an isoelectric point of 5 and apparent molecular weight of 80 000. On the basis of these properties and its immunological characteristics the exchange protein was judged to be distinct from any of the known apolipoproteins. This protein could also be separated from plasma phosphatidylcholine cholesterol acyl-transferase on DEAE-cellulose. The exchange protein did not appear to influence cholesterol esterification in lipoproteins by phosphatidylcholine cholesterol acyl-transferase, and the latter had no effect on the transfer of low density lipoprotein cholesteryl esters to high density lipoprotein. The exchange protein did not esterify cholesterol or hydrolyze cholesteryl esters in lipoproteins.


A spectrophotometric method for determination of sphingomyelinase. A colored derivative of sphingomyelin was synthesized and used as substrate for several sphingomyelinases. The compound is N-omega-trinitrophenyl-aminolaurylsphingosylphosphorylcholine. The rate of hydrolysis of this substrate was compared to that of bovine brain sphingomyelin, labelled with tritium in the choline moiety. The following enzyme preparations were used: homogenate-less debris of brain, assayed at pH 5.0 or 7.4; a solubilized preparation derived from rat brain lysosomes, assayed at pH 5.0 and a purified enzyme of Staphylococcus aureus. With all preparations, the rates of hydrolysis of the yellow derivative were very similar to those of the brain sphingomyelin. Extracts of skin fibroblasts of normal and Niemann-Pick patients as well as amniotic cells were also used. Again, the rates of hydrolysis of the yellow derivative practically equalled those using brain sphingomyelin.


[ESR study of the superslow diffusion of lysozyme macroradicals in an aqueous matrix]. Using kinetic data on recombination macroradicals in freezed water solutions of lyzozyme, superslow molecular motions of biopolymer have been studied. Recombination of macroradicals is observed at 160--240 K and it has a stepwise character. The kinetic data are described quantitatively by the model, in which the set of processes with different rate constants is considered (the so-called model of polychromatic kinetics). It has been shown that polychromatic character of the process is conditioned by energy activation distribution, which is changed from Emin = 8+/-1 kcal/mol to Emax = 13 +/- 1 kcal/mol with "rectangular" distribution function f(E) approximately equal to: formula: (see text). Preexponential factor K0 = 2 . 10 (-9)cm3.s-1. The recombination-kinetic method used permits to estimate frequencies (inverse correlation time) translation mobility macroradicals of protein up to the frequency order 10(-2)s-1 (or velocities of motions approximately 1 A/s).


[Study of the diffusion of water absorbed on proteins by the ESR methods using a new magnetic field impulse gradient program]. Modification of the program of impulse gradient removing anomalous suppression of the echo at small time intervals between gradient impulses is presented. The results are given of measuring the diffusion of water adsorbed on bovine serum albumin. An efficient diffusion coefficient for 20 degrees C is evaluated as 2.4.10(-6) cm2 sec at the activation energy in the temperature range 5 divided by 40 degrees C for 0.42 g H2O/g of protein and 20 divided by 50 degrees C for 0.22 g H2O/g of protein equal to 6.5 kcal/mol.


[Study of electron transport in the photosynthetic systems of higher plants by the ESR method. VI. Kinetics of photo-induced P700 redox transitions in a system of continuous and flash illumination of different duration]. The kinetics of P700 redox-transients in leaves and isolated been and spinach chloroplasts was studied by the ESR method. P700 oxidation after a short flash (less than 10 microseconds) of white light was not resolved. P700 oxidation may be resolved only after "long" (more than 500 microseconds) flash of red light (700 or 720 nm) with a slow reduction of P700+ in the dark. In case of 500 microseconds flash exciting both photosystems (600 nm) P700 response was not observed. Fast P700+ reduction was observed if the short pulse of white light was given simultaneously with a continuous background 707 nm light. We can conclude from these data that P700+ accepts an electron from its primary donor within 10--500 microseconds.


[Analysis of the ESR spectra of irradiated liver and hepatoma specimens]. The analysis of ESR low temperature spectra of in vitro gamma-irradiated (1 Mrad) liver, hepatoma 22-a and tumor host liver has been carried out. On the ground of differences in thermostability and relaxation parameters of paramagnetic centres it has been shown, that there is a large amount of different paramagnetic centres in irradiated normal and tumor tissues. The ESR spectra parameters and properties of observed paramagnetic centres are presented. The main result is the detection of a specific "tumor signal" (asymmetrical singlet deltaH = 6, Oe, g = 2.005) and peroxide radical ROO. signal in tumor and tumor host liver.


Binding of platinum to human transferrin. A complex of platinum and human transferrin has been formed by appropriately combining apotransferrin (metal free protein) and potassiumchloroplatinate (K2PtCl4). Atomic absorption spectroscopy indicated that both primary bind sites on the protein participated in the complex. Electron paramagnetic resonance (EPR) examination showed that the bound platinum was not paramagnetic, and thus it is highly probable that the Pt ion is in the +2 oxidation state. The results suggest a possible mechanism for physiological distribution of third-transition-series metals.


The ontogenetic evolution of acidic phosphoprotein phosphatase activity in the lymphatic tissue and the liver of the rat. We have shown that an acidic phosphoprotein phosphatase (APP-ase) has a different pattern of postnatal maturation in the spleen, thymus and liver of rats and mice. The APP-ase activity increases during the first eight months of postnatal life in the spleen of rats (when it attains an 8--10 times higher value than at birth) and up to the sixth month of life in the spleen of mice. It increases considerably during the first two weeks of postnatal life in the thymus of rats and mice; in the liver of rats it reaches maximum activity before birth, but continues to increase up to the sixth month of postnatal life in the liver of mice. The results show also that the APP-ase from the spleen, thymus and liver of rats is equally active in dephosphorylating ATP and phenyl phosphate during the whole life span of rats, but not in relation to beta-glycerol phosphate. After analyzing its substrate specificity, its pH dependence in relation to different substrates, its kinetic properties, as well as its behaviour towards ascorbic acid and different inhibitors (sodium tungstate and sodium molybdate, L-tartrate, L-phenylalanine and L-cysteine) we have come to the conclusion that the rat spleen APP-ase is different from "nonspecific" acid and alkaline phosphatases and very similar to the EC 3.1.3.16 acid phosphoprotein phosphatase.


[Participation of free radicals in ATP synthesis]. To study ESR absorption of mitochondrial ATPase a special flow system was developed allowing to maintain surviving conditions for mitochondria. It has been shown that ESR free radical signal of mitochondria observed at room temperature (g-factor 2.00 and halfwidth of about 15 Gs) depends on their metabolic state. An increase of free radical content during mitochondrial energization can be associated with operation of ATPsynthetase. It is supposed that free radicals take part in the reaction of ADP phosphorylation and that a molecule of ADP itself bound in the active centre of ATPase can become a free radical to facilitate the process of inorganic phosphate incorporation.


[EPR study of redox titration of Chromatium minutissimum chromatophores]. Redox titration of chromatophores through the region +300 +570 mV at room temperature results in generation of two dark ESR signals, a well-known reaction center signal and a new narrow line. This line is characterized by deltaH pp = 4.2 Oe, g-factor about that of RC and a Lorentzian shape. It is insensitive to illumination. The determination of an RC signal midpoint potential is affected by the presence of the narrow line. Taking into account the superposition of the two ESR signals it was measured to be +(486+/-+/-18)mV.


[The role of copper atoms in the cytochrome oxidase reaction]. It was found that cytochrome oxidase from bovine cardiac muscle possesses marked superoxide dismutase activity. Superoxide dismutase activity is inhibited by cyanide and azide or by alkaline or thermal treatments. This activity is also suppressed by chelating agents, e.g. bathocuproin. The data obtained indicate that superoxide dismutase activity of cytochrome oxidase is due to the copper atoms of the enzyme. The experiments on the copper-containing subunit support this conclusion. Possible physiological significance of superoxide dismutase activity of cytochrome oxidase is discussed.


[Some properties of glial membrane Na, K-ATPase]. Na,K-ATPase activity in glial membranes is rather low that in the nerve ending membranes, but is characterized by the same kind of Na+/K+-dependence. Glial Na,K-ATPase is insensitive to acetylcholine (ACh), 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) while norepinephrine activates Na,K-ATPase at low concentrations and inhibits it at high concentrations. Participation of Na,K-ATPase in the regulatory mechanisms of the neuron-neuroglia relations is discussed.


[Polypeptide synthetase activity of chromatin from eukaryotic cells]. It is shown that the polypeptide synthetase activity (PS-activity) of chromatin from rat liver is increased 9--21 hrs after partial hepatectomy. Among 9 amino acids studied alanine, methionine, lysine, tyrosine and arginine are not incorporated into the system in question. The highest rate of polymerization is observed in case of glutamic acid. The rate of glutamine, asparagine and glycine incorporation is 7--8 times slower. The PS-activity of chromatin is enhanced by chromatin preincubation with NAD (but not with its analogs). The activation is prevented by thymidine and nicotinamide. Storage of chromatin for 18 hrs at 2--4 degrees C results in a complete loss of PS-activity. Ability of "old" chromatin to incorporate of amino acids may be restored by its preincubation with NAD. Storage of chromatin in the presence of 5 mM cAMP does not decrease the PS-activity. It is assumed that in the system described poly-ADP ribose is an energy source for amino acid activation.


[The role of histidine residues of formate dehydrogenase from Bacterium sp. 1]. The reaction between formate dehydrogenase from Bacterium sp. 1 and diethylpyrocarbonate results in the enzyme inactivation. 4 histidine residues can be blocked per subunit by this reagent. The enzyme activity correlates with the disappearance of free histidines. The process of enzyme inactivation is biphasic and obeys pseudo-first-order kinetics. NAD and NADH slow down the rate of inactivation, but do not protect histidine residues against modification. Formate does not protect the enzyme. The modification of 80% of histidines increases the Km value for both substrates 3-fold. The general conformation of enzyme in the course of modification is preserved. The modification of histidines markedly decreases the reactivity of an essential SH-group of formate dehydrogenase against the Ellman reagent.


[Factors regulating gluconeogenesis in chick embryo liver]. The ratio NAD+/NADH in cytoplasm and mitochondria of chicken embryo liver does not change up to the stage of hatching. After the hatching this ratio decreases 2-fold in both cytoplasm and mitochondria. The hatching is also accompanied by the decrease of total and mitochondrial contents of oxaloacetate and of oxaloacetate/malate ratio, the activity of citrate synthase and the ratio acetyl-CoA/CoA being unchanged.


Angiotensin-I-converting enzyme activity in term and premature infants. Cord blood angiotensin-I-converting enzyme (ACE) activity was examined in 21 term and 21 premature infants. Values were significantly higher in premature infants than in term infants. Cord blood ACE activity was found to have a significant negative correlation with birth weight and gestational age. ACE activity measured from peripheral blood during the first 24 h of life was higher in premature than term infants but similar to levels in maternal and adult controls. It was not possible to predict those premature infants who would develop respiratory distress syndrome on the basis of cord blood ACE activity. However, ACE may serve as a marker for maturation of the pulmonary vascular endothelial cell.


[Mechanisms of inhibition of synaptic transmission in the sympathetic ganglia of rats with alloxan diabetes]. In experiments on the isolated superior cervical sympathetic ganglia of rats with alloxan diabetes rhythmic stimulation of preganglionic nerves was effected; summation presynaptic spikes and EPSPs of ganglionic neurons were registered. In rats with moderately severe alloxan diabetes progressive depression of rhythmic ganglion potentials was connected with suppression of the mediator emission to the impulse due to rapid exhaustion of its operational fraction. Rats with severe diabetes displayed also postsynaptic suppression of the ganglionic neurons. Dynamic characteristics of the transmitter turnover assessed on the basis of consideration of the successive patterns of posttetanic potentiation showed insignificant changes in the mediator output and a significant (by 38%) suppression of the mediator reserve per sec in comparison with control.


The role of prostaglandins in the study of intestinal water and electrolyte transport in man. The objective of this paper is to assess current knowledge on the pharmacologic and presumed physiologic effects of prostaglandins (PGs) on water and ion transport in the human intestine, from data in the literature and results of authors' studies. PGs, when administered intraluminally or intravenously, induce a profuse net secretion of water and ions into the jejunum or ileum; there is evidence that this effect is unrelated to changes in motility but due to a direct interaction of PGs with mucosal transport processes. Relations of PGs to cholera toxin, gastrointestinal hormones and adenylate cyclase-cyclic AMP system are reviewed. We propose to consider PGs as one of the most convenient model for studying intestinal secretion in vivo in man, in view of the rapid onset, magnitude, reversibility and reproducibility of their striking effect on mucosal transport.


[Electron paramagnetic resonance study of the products of the reaction between nitrogen oxides and several organic compounds]. A method of electron paramagnetic resonance was applied to the study of a possibility of paramagnetic centres formation in the nitrogen oxide reaction with saturated and unsaturated fatty acids and aromatic amino acids. Several paramagnetic centres are formed in interaction of NO2 with oleic acid at 20 degrees C. Saturated fatty acids formed no paramagnetic centres in the reaction with NO2. NO formed no paramagnetic centres not only with butyric, palmitic, and stearic acids, but also with oleic acid. The capacity of NO2 to form paramagentic centres with tyrosine in its saturated solution was revealed. In  case of interaction between NO and NO2 with saturated phenylalanine and tryptophane solutions no formation of paramagnetic centres was observed. It is suggested that unsaturated fatty acids and tyrosine remnants of the membrane lipoprotein complexes could serve as peculiar targets with which NO2 interacted, and that formation of paramagnetic centres in the NO2 reaction with the mentioned substances was the primary act of the membrane damage.


[Increase in the antiviral resistance of cells after treating them with messenger RNA for antiviral protein]. The authors studied antiviral resistance of murine cells L-929 following single introduction into them of homologous and heterologous informative RNA preparations for antiviral protein (i-RNA-AVP). As shown, in using homologous i-RVA-AVP preparations suppression of the virus production constituted 90--93%, and was stably traced in cell passage in the course of 1 1/2 months (observation period). Following cell contact with heterologous i-RNA-AVP preparations suppression the first 6 passages of the virus production constituted about 90%, rising by the 16th passage to 99.9%. The data obtained pointed to the possibility of stable increase of cell antiviral resistance with the aid of i-RNA-AVP, this could serve as a new effective method of nonspecific cell protection from the viruses.


Induction of hyperlipemia by carbohydrate-rich fractions isolated from crude papain. A hyperlipemia was obtained in rabbits injected intravenously with two carbohydrate-rich fractions isolated from crude papain dialyzate. The fractions have an approximative molecular weight of 15,000 and 5,000 daltons and contains 35% and 15% of sugars respectively. The hyperlipemia appears 20 hours after the injection and consists in a hypertriglyceridaemia as suggested by the enrichment of serum in very light lipoproteins of density about 1.006. Concomittantly a decrease of the post-heparin lipase activity (PHLA) of the plasma was observed. After a few days the hyperlipemia disappears and the PHLA returns to a normal value. It is suggested that the hypertriglyceridaemia can be due to an inhibitory effect of the injected fractions on the removal action of the lipases which are released into the blood by an heparin injection.


[Reaction between dexamethasone-receptor complexes and isolated nuclei from Zajdela hepatoma and rat liver cells]. Temperature-activation of the hormone-receptor complex (HRC) was shown to be necessary to ensure its translocation from cytoplasm to nucleus both in the rat liver and hepatoma. Hepatoma nuclei bind 20 times less HRC derived from homologous hepatoma cytosol (0.15 pmol/mg DNA), but twice as much (5.6 pmol/mg DNA) of HRC from heterologous liver cytosol, as compared with the binding of HRC from normal liver cytosol by liver nuclei (3 pmol/mg DNA), Ka of HRC with the acceptor sites in hepatoma and liver nuclei were found to be practically of the same order of magnitude. The above findings suggest an inhibition of cytosol-nucleus translocation of HRC from the cytosol of hepatoma cells as a possible cause of the nonresponsiveness of the latter to the hormone.


[Mechanism of the inhibitory effect of BCG vaccine on specific antitumor immunity in Syrian hamsters]. The duration of inhibiting influence of BCG vaccine on specific antitumour immunity and possibility of SV40-induced resistance restoration in Syrian hamsters after the vaccination was studied. Antitumor immunity in animals immunized with SV40 virus and then inoculated with BCG was demonstrated to be abrogated one year after the vaccination. Reinoculation of the subjected to combined immunization animals with SV40 virus induced specific antitumour resistance in them again. The data presented may be interpreted as an argument favouring the cell-mediated nature of the phenomenon described.


[Specific variability in the number of cells with X-chromatin in short-term buccal epithelium cultures from women following exposure to hormones at different phases in their individual diurnal biorhythm]. Changes in the number of cells with X- and K-chromatin were studied in the buccal epithelium of 4 women in vivo for 29 h, every 2--4 h, and similarly in cultures (3 h) with and without any hormone: ACTH, insulin, thyroxin. The biorhythm of the cells with X-chromatin (established by Kanyuka in 1973) both in vivo and ie vitro within the range of from 44 to 340 0/00 per 24 h was confirmed; individual variability of the mean heterochromatin index value (X-HClm) was revealed in vitro with and without any hormones. Cell incubation with hormones at the time of minimal X-HCl in vivo led to increase in the number of cells with X-chromatin within 3 h from 50 to 240 0/00, whereas incubation at the time of maximal X-HCl in vivo--to reduction from 190 to 70 0/00. It is suggested that the specific response to the hormones is preceded by the nonspecific tissue response, determined by its condition at the time of action (by the individual X-HCl biorhythm phase) and realized in the systemic reconstruction of the number of cells at various mitotic cycle phases. Stereotypic reaction was expressed in the changes in character of the nuclear chromatin sondensation; these processes were reversible.


Polyoma virus, as a model for viral skin carcinogenesis. Polyoma virus is presented as the simplest model of transformation by a DNA virus. The three possible ways of virus/cell interaction are recalled: lytic infection, abortive infection and malignant transformation. Nuclear and surface antigenic modifications are also recalled as well as their possible correlation with the transformation mechanism. Lastly, in "in vivo" systems, we have tried to schematize the interactions of host and virus-induced tumour.


The human papillomaviruses.

The human papillomaviruses. Recent biochemical and serological studies have shown the existence of at least four distinct types of human papillomaviruses (HPVs) causing benign skin lesions. These viruses show hardly no antigenic relationships; their DNAs differ by their sensitivity to restriction endonucleases, and show little, if any, sequence homology, as detected by molecular hybridization using complementary RNAs transcribed in vitro. Data on the pathogenicity of HPVs are still incomplete but indicate that some types of benign skin lesions (plantar warts, common warts, flat warts) may be preferentially associated with some types of HPV. Most interesting is that epidermodysplasia verruciformis has been found associated with two types of virus, and that malignant conversion of some lesions has been observed in all the patients infected with one of them. This suggests that at least a HPV may have a higher oncogenic potential, as do rabbit (Shope) papillomavirus and bovine alimentary tract papillomavirus. Much remains to be known on human papilloma-viruses and further studies may lead to the characterization of additional types of HPVs, especially in genital condylomata acuminata and laryngeal papillomas whose malignant conversion, although rare, may be observed. Progress in this field has been and remains hampered by the lack of cell culture systems allowing replication of these highly host and tissue specific viruses, and by the widely variable virus content of the different human lesions known to be associated with a papillomavirus. Further studies are warranted by the possible role of these widespread and epitheliotropic viruses in the origin of some carcinomas in man.


Heterogeneity of human papilloma viruses. Two types of human papilloma viruses (HPV-1 and HPV-4) were characterized, which show no relationship as far as the following criteria are concerned: the pattern of restriction enzyme fragments of their DNA, DNA-cRNA hybridization, and complement fixation with specific rabbit antisera. Furthermore, the molecular weights of the major proteins differed significantly. Both types of viruses could be isolated from plantar warts and from verrucae vulgares. This paper gives the molecular weights of the major proteins of a third type of HPV from skin warts, which reacts neither with HPV-1 nor with HPV-4 rabbit antiserum.


Epidermodysplasia verruciformis. An autosomal recessive disease characterized by viral warts and skin cancer. A model for viral oncogenesis. EV is an autosomal recessive disease which usually begins in infancy or childhood, with an average age of onset of 9. Flat warts are most common, but pityriasis-like warts occur in approximately 75% of patients. Warts are disseminated and chronic. Cancer may develop as early as age 13; the average age of onset is 31. About 1/3 rd. of patients develop cancer of the skin usually multiple and in light-exposed areas. It may take only two years from wart to cancer, or many more years. The cancer is carcinoma-in-situ of the Bowenoid type or squamous cell carcinoma. Two deaths have occured by local invasion and only one metastasis has been reported. One patient died of Burkitt's lymphoma and another was reported to have disseminated reticulosis. 8% of the patients are mentally retarded. Humoral immunity is normal; cell mediated immunity is depressed in most patients. The gene defect in EV has not yet been uncovered.


Immunological studies in epidermodysplasia verruciformis. Immunofluorescence and cell mediated immunity studies have been performed in 14 cases of epidermodysplasia verruciformis (EV), 3 of those abortive or regressing in members of the families of the patients with EV. Two different types of human papillomavirus (HPV)--HPV3 and HPV4--have been found in cases of EV. HPV3 was detected also in flat warts without features of EV. There was no cross-reactivity between these two viruses, neither with HPV1 responsible for plantar warts nor with HPV2 inducing common warts. There was a relationship between the type of HPV and the clinical picture of EV as well as the malignant transformation, namely HPV4 has been found to be more oncogenic. Cell mediated immunity (CMI) seems to be an important factor because it was depressed in a vast majority of active cases and preserved in regressing and abortive cases (in the members of the families of EV patients). However, low CMI has been found in EV cases infected with HPV3 and in persistent flat warts also due to HPV3, which did not undergo malignant transformation. In contrast, in a case of EV due to HPV4 a malignant transformation occured in spite of still preserved, although lowered CMI. Various human papillomaviruses seem to differ in their oncogenic potential. HPV1 responsible for plantar warts, and HPV2 for common warts have no evident oncogenic potential, HPV3 inducing both EV and flat warts has a low oncogenicity, whereas HPV4 inducing some cases of EV seems more oncogenic.


Transformation of warts to malignancy in alimentary carcinoma in cattle. There is a high incidence of alimentary carcinoma in beef cattle in the highland areas of Britain. Evidence is presented to shown that this is associated with the presence of bracken fern on high incidence farms and that many of the carcinomas arise in pre-existing virus-induced papillomas. Papillomas are much commoner and occur in greater numbers in animals of all ages on the cancer farms as compared to lowland cattle. Papillomas are found in all ages of cattle; carcinomas have only been seen in cows over 6 years of age. The virus has been isolated, visualized and purified; it produces papillomas when inoculated into the alimentary tract and skin.


Interactions of Shope papilloma virus with some other DNA viruses. The interactions of Shope papilloma virus (SPV) with primate and rabbit cells in tissue culture have been investigated. The rabbit cell cultures were derived from normal epidermis, from SPV-infected epidermis, from SPV-induced papillomas, and from an SPV-associated carcinoma. None of these cell cultures, whether infected in vitro with SPV or derived from tissues infected in vivo, ever produced infectious SPV or even detectable viral antigens. Some other DNA viruses behaved differently in cells which had been in previous contact with SPV either in vitro or in vivo. Adenovirus type 5 multiplies better in human cells infected 24 h previously with SPV than in the untreated controls. The production of infectious virions of either herpes simplex virus or Shope fibroma virus is reduced in cells derived from SPV-induced papillomas or carcinoma, due, apparently, to a defect in viral maturation. Of the rabbit cells, only those derived from in vivo infected tissue, or those previously infected in vitro with SPV, could be transformed by SV40. The rabbit cell lines derived from papillomas or carcinoma differed from their counterparts derived either from normal epidermis or from tissue infected 24 hrs before biopsy, in their karyotype, and in their ability to grow in soft agar. Similar karyotypic alterations were induced in cells derived from healthy epidermis by infection in vitro with SPV.


The effects of release and depletion of endogenous noradrenaline on the transmission of impulses in the mouse vas deferens. 1 The effects of endogenous noradrenaline released by tyramine and the influence of depletion of the tissue noradrenaline with reserpine and/or alpha-methyl-p-tyrosine on the twitch responses of the field-stimulated mouse vas deferens have been studied.2 Tyramine (10-40 muM) inhibited the twitch responses to field stimulation and failed to produce a contraction. The inhibition decreased as the rate of stimulation increased.3 The inhibition produced by tyramine was antagonized by cocaine (10 muM) and by yohimbine (10 nM), which indicated that it was produced by released noradrenaline acting on presynaptic alpha-adrenoceptors.4 Depletion of the tissue noradrenaline by 39% by blockade of the synthesis of noradrenaline with alpha-methyl-p-tyrosine, was without effect on the twitch response but it reduced the inhibitory effect of tyramine.5 Depletion of the tissue noradrenaline by 96.5% with reserpine alone and by 99.4%, with a combination of reserpine and alpha-methyl-p-tyrosine, reduced the twitch responses by approximately 66% and virtually abolished the inhibition produced by tyramine. It also increased the rate of decline of the responses when the tissue was continuously stimulated. The remaining twitch was not antagonized by phenoxybenzamine (15 muM).6 Residual twitches were bigger in tissues depleted by 99.4% than in those depleted by only 96.5%. This difference was eliminated in the presence of yohimbine (128 nM).7 It is concluded that inhibition of the twitch responses by tyramine is produced by stimulation of presynaptic alpha-adrenoceptors and that the twitch response is associated with stimulation of the sympathetic neurone, but that it is not mediated by postsynaptic alpha-adrenoceptors.


The role of beta1- and beta2-adrenoceptors in the inhibition of gastric acid secretion in the dog. 1. Characterization of the beta-adrenoceptors mediating inhibition of gastric acid secretion in the conscious Heidenhain pouch dog has been investigated by determination of the effects of propranolol, (+)-propranolol, practolol and H35/25 on salbutamol and isoprenaline-induced inhibition of gastric acid secretion. 2. The gastric antisecretory effect of salbutamol was significantly blocked by propranolol and H35/25 but not by practolol or (+)-propranolol. The effect of isoprenaline was significantly blocked by propranolol and practolol but not by H35/25 or (+)-propranolol. 3. It is concluded that both beta1- and beta2-adrenoceptors can mediate inhibition of pentagastrin-induced gastric secretion in conscious dogs with a Heidenhain pouch. Salbutamol exerts its antisecretory effect through beta2-adrenoceptors, whereas isoprenaline mediates its effects primarily through beta1-adrenoceptors. 4. The results are discussed with regard to the sub-classification of beta-adrenoceptors and to the possible role of adrenoceptors in the physiological control of gastric secretion. 5. In this study it is concluded that the tachycardia induced by isoprenaline or salbutamol is mediated primarily through reflexes activated by beta2-adrenoceptor mediated vasodilatation.


Central actions of benzodiazepines: general introduction. After a brief review of the characteristic somatic and psychotropic effects of benzodiazepines evidence is presented which supports a specific facilitatory action of these drugs on GABA ergic synapses within the mammalian central nervous system. Benzodiazepines enhance presynaptic inhibition in the spinal cord and dorsal column nuclei as well as postsynaptic inhibition in dorsal column nuclei, hippocampus, hypothalamus, cerebral cortex, cerebellar cortex, which are all examples of recurrent and collateral inhibition mediated by GABA ergic intrinsic neurones. In addition, the compounds also enhance the inhibitory effect of GABA ergic long projection neurones in the substantia nigra and the lateral vestibular nucleus of Deiters. Several problems remain to be solved, such as the exact site at which benzodiazepines initiate their action (pre-synaptically at GABA ergic nerve endings or postsynaptically at the target cells) and the possible existence of endogenous ligands for the benzodiazepine receptor. Some suspected implications which studies on benzodiazepine binding sites could have for a deeper understanding of the mode of action of these drugs are discussed.


Molecular mechanisms mediating the action of diazepam on GABA receptors. Two types of crude synaptic membranes have been prepared which differ in their kinetic properties to bind 3H-GABA in a Na+-free medium. One type (B) has one type of receptor (affinity or KD about 0.2 micrometer), the other type (A) has two populations of receptors which have a high (0.16 micrometer) and a low ((.02 micrometer) KD for GABA binding. This difference is due to the presence of a selective endogenous inhibitor of the high affinity receptor for GABA, a thermostable protein of about 15,000 molecular weight. Inhibitor action is counteracted by diazepam (7 X 10(-7) M), competitively. Clonazepam is more active but chlordiazepoxide is less active than diazepam. Two enantiomers of a benzodiazepine were studied. One of them is endowed with anxiolytic activity and nullifies the action of the endogenous inhibitor on high affinity GABA binding, the other is devoid of anxiolytic activity and is inactive against the inhibitor. The endogenous protein inhibitor also competitively blocks the high affinity binding of 3H-diazepam to Type A membranes. It is concluded that the endogenous inhibitor and diazepam act on the same receptor; and suggested that the endogenous inhibitor may be the natural ligand for the brain receptors that bind benzodiazepines with high affinity.


The benzodiazepine receptor in normal and pathological human brain. Benzodiazepines bind with high affinity to a specific benzodiazepine receptor, which occurs exclusively in the central nervous system. The affinity of various benzodiazepines to the receptor closely parallels their pharmacological and therapeutic potency. Binding to the receptor is stereospecific. The receptor is mainly localized in the synaptic membrane fraction and has its highest density in cortical areas of the brain. In Huntington's chorea a decrease in benzodiazepine receptor binding is found in caudate nucleus and putamen, which, at least in putamen, is due to a loss of benzodiazepine receptors apparently located on GABA neurones, which degenerate in Huntington's chorea. The loss of benzodiazepine receptors might explain why the ameliorative effects of benzodiazepines in the early stages of the disease are not sustained in the later stages.


Excitability increase of neurons in olfactory cortex slices of the guinea pig after penicillin administration. The mechanism underlying the effects of penicillin on slices of the olfactory cortex of the guinea pig was examined. In a previous report it was shown that penicillin increases the amplitude of the presynaptic action potential, the population EPSP and, more strongly, the population responses of the postsynaptic cells. Moreover, the postsynaptic population responses increased in number and suggested strong repetitive firing. These results were confirmed in the present study. Analysis of stimulus-response relationships suggested that the enhancement of the postsynaptic response was due to an increase in excitability of the postsynaptic neurons by penicillin. The amplitude changes of the presynaptic action potential and the EPSP were probably largely, if not completely, due to an increase in resistance of the bathing fluid. It was found that the changes in population responses paralleled to a large extent changes in cell discharge. In addition, penicillin was found to induce spontaneous firing of the postsynaptic cells. The changes in cell discharge were consistent with an increase in excitability of the postsynaptic cells.


Some actions of catechol on synaptic transmission in the isolated spinal cord of the frog. The isolated frog spinal cord was used to investigate the synaptic effects of the convulsant agent catechol. Addition of the compound to the superfusate consistently enhanced orthodromic reflex activity recorded from ventral roots and augmented primary afferent depolarization. Concomitantly catechol altered the polarization changes produced in ventral and dorsal roots by putative neurotransmitter amino acids when these compounds were applied in Mg2+-containing Ringer. Catechol reduced the hyperpolarizations induced in motoneurons by the neutral amino acids, GABA, beta-alanine, taurine and glycine, but did not affect the depolarizations produced by the dicarboxylic amino acids, L-glutamate and L-aspartate. In contrast, catechol increased the dorsal root depolarizations elicited by both neutral and dicarboxylic amino acids and also the depolarizations produced by elevated potassium concentrations. Catechol did not bring about significant changes in the passive electrical properties of motoneurons or dorsal root fibers. In addition, it did not alter either the high affinity uptake or the depolarization-evoked release of tritiated GABA, glycine, L-glutamate and L-aspartate. It appears that the postsynaptic actions of catechol explain its ability to enhance spinal reflexes.


F-wave and cervical somatosensory response conduction from the seventh cervical spinous process to cortex in multiple sclerosis. Transit (conduction) times from the wrist to the seventh cervical spinous process (C7) were measured by using the F-wave response (TA) and the cervical somatosensory response (SEPC). The mean values obtained in 25 controls were 10.1 +/- 0.9 s-3 and 10.9 +/- 1.0 s-1 respectively. The 0.8 +/- 1.0 s-3 difference between the two methods represents central delay of SEPC, possibly at the level of the dorsal root ganglion. The mean onset latency of the cortical somatosensory response (SEPA) evoked by median nerve stimulation in the same subjects was 15.5 +/- 1.0 s-3. Transit time from C7 to the cortex (TB) given by either SEPA - TA or SEPA - SEPC - 0.8, where 0.8 is the necessary corrective factor for central delay of SEPC, WAS 5.3 +/- 1.0 S-3. These techniques were applied to 10 patients suspected of having multiple sclerosis, but without clinical evidence of involvement above C7. Six of the 10 cases showed prolongation of TB. In 4 of these, this could only be calculated by the F-wave method since SEPC was absent. It is concluded that transit times derived from either the F-wave or SEPC are equally valid and interchangeable. The absence of one response allows for its replacement by the other.


Mucin embolism to cerebral arteries: a fatal complication of carcinoma of the breast. The case is reported of a woman with a mucin producing lobular carcinoma of the breast with metastases to many bone sites, whose terminal neurological illness was the result, not of cerebral metastases, but of cerebral infarcts. These were caused by emboli of mucin and emulsified fat, originating in bone metastases. The pathogenesis of this embolism is compared with that of traumatic fat embolism. Attention is drawn to this process because emboli of this type have never been reported and because this distant nonmetastatic effect of carcinoma may have been overlooked in other cases. It is suggested that this mechanism should be considered in the diagnosis of otherwise obscure cerebral infarction.


Human colon tumors: enzymic and histological characteristics. In samples of colonic adenocarcinomas, the mean activities of thymidine kinase, glucose-6-phosphate dehydrogenase, phosphoserine phosphatase and pyrroline-5-carboxylate reductase were several fold higher than those of nonneoplastic colon. The presence of considerable, cold labile pyrroline-5-carboxylate reductase activity provided an additional criterion for distinguishing tumors from the control tissue. Deviations from the pattern of enzymes in normal colon were much more pronounced in the five moderately well-differentiated than in the single well-differentiated adenocarcinoma.


Ultrastructural observations in pleomorphic liposarcoma. An ultrastructural study of pleomorphic liposarcoma with an unusual clinical presentation is described. A 69-year-old Caucasian female presented with recurrent superficial phlebitis of left leg, which responded only partially to conventional therapy. Following investigations, a mass was excised. Diagnosis of pleomorphic liposarcoma was made on microscopic examination. Numerous lipid droplets in the cytoplasm and often within intranuclear extensions of cytoplasm were seen on oil red O stain of frozen sections. Ultrastructural features are quite distinctive. The cells varied from small to often large pleomorphic cells with numerous lipid spaces without limiting membrane, large pleomorphic nuclei, and an abundance of cytoplasmic organelles. Centrioles were very prominent, in close proximity to nuclei, and collections of autophagocytic inclusions. Intercellular collagen was immature and scant. These features are compared to ultrastructural features of malignant fibrous histiocytoma, pleomorphic rhabdomyosarcoma, and cardiac myxoma, indicating that ultrastructural features are distinctive and help differentiate similar soft tissue tumors.


Trace element concentrations in renal cell carcinoma. Cadmium, zinc, copper levels and zinc-copper, zinc-bromine, iorn-zinc, iron-copper and iron-bromine ratios are measured in neoplastic and normal kidney samples from humans by the particle induced X-ray emission analysis (PIXE) technique. It is found that cadmium which is normally present in the tubular cells of kidney is not detectable in tumor samples. It is also observed that the zinc-copper ratios in all neoplastic kidney tissues are decreased, but this observation cannot be extended to other element ratios.


Retroperitoneal fibrohistiocytic tumors in children: report of five cases. Five rare retroperitoneal fibrohistiocytic tumors of children are described mainly in reference to the pathological aspects. A more detailed description is given of a retroperitoneal xanthogranuloma, including an ultrastructural study. This case is particularly interesting for its rapid transformation into a fibroxanthosarcoma. The other cases include two fibroxanthosarcomas, a storiform fibrous xanthoma, and a malignant histiocytoma. The most difficult points in the recognition and in the differential diagnosis of these tumors are discussed, and emphasis is placed on some interesting pathological features. Prognosis of such tumors appears difficult to assess from our series since two patients died in the immediate postoperative period, one died one and a half years after the discovery of the tumor, and two are alive and well, but with a short follow-up.


Treatment of transplanted murine tumors with an oncolytic virus and cyclophosphamide. Effective treatment of transplanted murine mammary tumors with an oncolytic virus and cyclophosphamide is reported. The oncolytic virus was cultured initially from a murine ascites tumor that had undergone "spontaneous" regression. This virus used alone causes rapid lysis of ascites tumors, but successful treatment of solid tumors requires the addition of cyclophosphamide. The virus must be injected directly into the tumor. The virus causes no detectable disease in mice even when administered in conjunction with cyclophosphamide. Fluorescent antibody techniques revealed that all A/S stock mice have antibody against cells infected with this virus.


Growth of a human breast cancer cell line in serum-free hormone-supplemented medium. ZR-75-1, a human breast cancer cell line, has been grown in hormone-supplemented medium without serum. The factors required for optimal growth include 17beta-estradiol, insulin, transferrin, dexamethasone, and L-triiodothyronine. If estradiol, insulin, or L-triiodothyronine is omitted, cells cease division within 7 days, but viability is retained for at least 14 days. Omission of transferrin leads to cell death within 7 days. The cells have been continuously maintained in this environment without morphological alteration or cessation of growth for more than 5 months. Addition of the anti-estrogen, Tamoxifen (10(-6) M), inhibited cells below the growth rate seen when estradiol was omitted from the medium, even when Tamoxifen was added 4 days and two medium changes after the removal of estradiol from the medium, thus suggesting an action of Tamoxifen which may be independent of competition with estradiol. The availability of a human breast cancer cell line that can be propagated in hormone-supplemented medium without serum should aid in the study of the mechanisms by which hormones effect cell growth.


A new in vitro cell line established from human large cell variant of oat cell lung cancer. A new tissue culture cell line, SHP-77, has been established from explant cultures of a primary human lung cancer. Although the latter exhibited histological features of the so-called large or polygonal cell undifferentiated variant, neurosecretory granules were detected ultrastructurally in cells of the original tumor, culture line, and neoplasms developing after transplantation of the latter in nude mice. These features attest to the identity of the original tumor as a large-cell variant of oat cell cancer. In addition, electron microscopy revealed the presence of gland formation and intracytoplasmic lamellar bodies in the cells of SHP-77. This information indicates the potential for varied differentiation and morphological expression of so-called undifferentiated lung cancer cells and is pertinent to nosological considerations concerning human lung cancer. The cell line has maintained its morphological, karyotypic, chromosomal, and growth characteristics after transplantation to nude mice. Because of this stability, SHP-77 appears to represent a propitious cell line for in vitro and in vivo biological and therapeutic studies of this type of lung cancer.


A differential interaction of daunomycin, adriamycin, and N-trifluoroacetyladriamycin 14-valerate with mouse peritoneal macrophages. The interaction of three anthracycline drugs, daunomycin, Adriamycin, and N-trifluoroacetyladriamycin 14-valerate, with mouse peritoneal macrophages was explored. As assessed by drug-specific cytofluorescence, Adriamycin and daunomycin accumulated slowly within macrophages, first staining the nucleus and then the cytoplasmic inclusions that were induced by the drug treatment. N-Trifluoroacetyladriamycin 14-valerate distributed rapidly into the cells, was excluded from the nucleus, and induced numerous cytoplasmic inclusions. Electron microscopy revealed that the cytoplasmic inclusions were vacuoles containing some amorphous material and not the classical autophagic vacuoles containing organelles and membrane lamellae. All the drugs induced cell shrinkage with time and brought about cell death within 24 hr. Loss of cell function and viability was dose and time dependent; i.e., a 6-hr incubation with daunomycin or Adriamycin, 2.5 microgram/ml, brought about a 50% reduction in the phagocytic capacity of the treated macrophages. The damaging potency towards macrophages (daunomycin greater than Adriamycin greater than N-trifluoroacetyladriamycin 14-valerate) is in the reverse order to the in vivo therapeutic efficiency.


5-Methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid (folinic acid), and folic acid requirements of normal and Rous sarcoma virus-infected chicken fibroblasts. Normal and Rous sarcoma virus-infected chicken fibroblasts proliferate maximally in a culture medium containing a physiological (10 ng/ml) concentration of 5-methyltetrahydrofolic acid or folinic acid (5-formyltetrahydrofolic acid), while their maximal proliferation requires a hyperphysiological (1000 ng/ml) concentration of folic acid. The normal and Rous-infected fibroblasts do not differ in their requirements for 5-methyltetrahydrofolate, folinic acid, or folic acid.


Role of exogenous female hormones in altering the risk of benign and malignant neoplasms in humans. The epidemiological and clinical evidence for various forms of exogenous estrogens altering the risk of neoplasms of the female genital system, breast, and liver are reviewed and evaluated. It is virtually certain that in utero exposure to diethylstilbestrol can cause clear cell adenocarcinomas of the vagina and cervix. There is strong evidence that various estrogens given for treatment of menopausal symptoms can cause endometrial carcinoma and that sequential oral contraceptives probably also do so. Oral contraceptives very probably reduce the risk of both cystic disease and fibroadenoma of the breast and increase the risk of liver cell adenomas. Studies to date do not provide consistent and convincing evidence that any form of exogenous estrogen alters the risk of cancers of the breast or ovary or that oral contraceptives alter the risk of cervical neoplasia or focal nodular hyperplasia of the liver, although recent reports suggest that continued vigilance is warranted. Specific topics requiring further epidemiological investigation are suggested.


Hormonal induction of mammary tumor viruses and its implications for carcinogenesis. The purpose of this study was to evaluate the possibility that hormones and mouse mammary tumor virus (MuMTV) are cocarcinogenic for mammary epithelium. Results of our investigations in vivo and in vitro suggest: (a) Hormones promote mammary carcinogenesis in BALB/c females whether MuMTV is germinally (BALB/c) or horizontally (BALB/cfC3H) transmitted; the rate of carcinogenesis in BALB/cfC3H females is substantially faster than it is in BALB/c, but the final mammary carcinoma incidence is approximately the same. The rate-limiting step in malignant transformation in BALB/c, which infectious MuMTV overcomes, is in premalignant transformation from normal. (b) One characteristic of horizontal MuMTV transmission in BALB/c that is not observed in germinal transmission is integration of new MuMTV sequences in mammary cell DNA. Integration is mammary cell specific and constant at 2 to 3 copies/cell from tumor to tumor. (c) MuMTV expression is changed in mammary epithelial cells during hormonal carcinogenesis. The nature of the change is qualitatively similar in both BALB/c and BALB/cfC3H. Expression of envelope glycopeptides (glycoprotein with a molecular weight of 52,000) is induced, which correlates with amplification of MuMTV RNA sequence content. Quantitative differences exist in induced levels in BALB/c and BALB/cfC3H. (d) MuMTV RNA and a glycoprotein with a molecular weight of 52,000 were not inducible with dexamethasone in normal mammary epithelial cells in culture. These structural components were induced in both premalignant (BALB/cfC3H) and malignant (BALB/c and BALB/cfC3H) cells. MuMTV RNA was induced by dexamethasone in normal cells pretreated with 5-iodo-2'-deoxyuridine. (e) Both premalignant and malignant cells have altered (vis-à-vis normal) surfaces, discernible by differences in reactivity with concanavalin A in hemadsorption assays. Indirect evidence suggests that the alteration includes membrane incorporation of MuMTV-related determinants of a glycoprotein with a molecular weight of 52,000. (f) Malignant cells exhibit enhanced sensitivity to insulin for reinitiation of DNA synthesis and mitosis in contact-inhibited homotypical monolayers. These findings have been organized into a hormonal cocarcinogenesis hypothesis in which expression of germinally transmitted MuMTV genes is the proximal cause of neoplastic transformation.


Relationships of the structure and function of the interferon receptor to hormone receptors and establishment of the antiviral state. This report describes similarities between the structure and function of the interferon receptor and receptors for glycoprotein hormones and several bacterial toxins. Specifically, it describes several common molecular and mechanistic elements, including: (a) the presence of a glycoprotein as well as a ganglioside component in the receptor; (b) changes in membrane structure as a consequence of interferon action; (c) interferon-induced intracellular cyclic adenosine 3':5'-monophosphate changes; and (d) alterations in the flux of certain ions across the membrane. Since interferon has an antiviral effect, these results define a relationship between hormonal perturbation of cellular events and the ability of an agent to prevent or suppress viral infections of cells. Further definition of these relationships should be important to our understanding of the oncogenic state, of hormonal effects on the oncogenic state, and of other human diseases in which hormonal perturbations of non-target tissues or cross-reactivity of receptors could be pathogenic.


Investigation of hormone-receptor interactions by means of fluorescence labeling. Fluorescent-labeled hormones can be used to study hormone-receptor interactions by means of fluorescence polarization, visualization by fluorescence microscopy, or separation methods, e.g., dextran-coated charcoal. Subcellular fragments, single cells, and tissue preparations are amenable to study; in this work rat uterine cytosol was used unless otherwise noted. Estrone labeled with fluorescein at position 17 gives 50% inhibition in the radiometric dextran-coated charcoal assay at 8.3 X 10(-7) M as compared to 3.4 and 3.5 X 10(-8) M for diethylstilbestrol and estradiol, respectively. Scatchard plots from fluorescence polarization are hyperbolic and consistent with two classes of binding sites having association constants 5.6 X 10(10) and 6.4 X 10(7) M-1. Binding by high-affinity sites, which were present at about 3 times the concentraion of "specific" sites (radiometric dextran-coated charcoal assay), was abrogated by estradiol or diethylstilbestrol. Kinetic measurements showed that binding sites that can be blocked by excess estradiol or diethylstilbestrol are those that are both slowly associating and slowly dissociating. Staining of tissues by estrone labeled with fluorescein at position 17 as seen in the fluorescence microscope showed specificity. In normal rat uterus only epithelial cells were stained. In one human infiltrating ductal carcinoma only the malignant ductoid elements stained, while in another there was essentially no staining.


Regulation of S49 lymphoma cell growth by cyclic adenosine 3':5'-monophosphate. S49 lymphoma tissue culture cells arrest in the G1 phase of the cell cycle when treated with agents that elevate endogenous cyclic adenosine 3':5'-monophosphate (cAMP), such as cholera toxin or exogenously added active congeners of cAMP such as N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP). This phenomenon requires that cells contain the appropriate receptors: Mutant cells deficient in adenylyl cyclase fail to arrest in response to cholera toxin, and another mutant that lacks cAMP-dependent protein kinase does not respond to cholera toxin or to Bt2cAMP. The size distribution of cell populations treated with Bt2cAMP changes in a manner that reflects only the perturbation of cell cycle distribution. Arrested G1 cells in particular have the same volume as the G1 cells of an exponentially growing population. When G1 cells that have been arrested by Bt2cAMP are grown in fresh medium free of Bt2cAMP, they begin to reenter S phase after a delay of about 6 hr and do so with pseudo-first-order kinetics, with a half-life of 5 hr. These and other properties previously described suggest that cAMP regulates S49 cell growth by physiologically significant rather than artifactual mechanisms.