Endocrine and metabolic changes associated with periods of spontaneous hypoxia in fetal sheep. Periods of spontaneously occurring hypoxia have been observed in fetal sheep. These were associated with increases in the fetal plasma concentration of glucose, lactate, free fatty acids, ACTH and corticosteroids (for a fetus close to term). These changes are similar to those observed during experimentally induced hypoxia. Hypoxia may be a mechanism by which plasma metabolites and ACTH are increased in fetal sheep under physiological conditions, particularly during labour.


Electron spin resonance study of the synaptosome opiate receptor: kinetics of stereospecific binding of spin labeled morphine. Morphine, spin labeled on the 3- or 6-position has been used as the opiate ligand in a study of the time course of stereospecific opiate binding to intact synaptosomes isolated from non-cerebellar rat brain. The broadening of electron spin resonance lines induced by immobilization of the ligand on binding has been used to determine the concentration of bound opiate. The stereospecificity of the reaction was measured by comparing ligand binding in the presence of thousand-fold molar excesses of dextrorphan or levorphanol. Using both static and flow techniques, the binding process has been continuously monitored at times greater than 4.8 s after mixing spin labeled morphine with synaptosomes. It is shown that for this ligand and receptor preparation, binding takes place primarily during a delayed, abrupt process whose rate and time of onset are temperature dependent and reflect the presence of added opiate agonist or antagonist.


Charge-transfer interactions between metal ions and some uracil derivatives. The interaction between Cu2+ and a few uracil derivatives has been investigated by means of electron spin resonance and optical absorption studies. It could be shown that a charge transfer interaction occurs. Its strength depends upon the electron attracting or releasing properties of the substitutents of the nucleobase.


Changes of cell surface configuration and regulation of cell proliferation. The electric surface charge configuration of 3T3 and SV40-3T3 cells was characterized by determining the product of electrophoretic mobility of the cells times the viscosity of suspension medium. This quantity could be shown to change with temperature and/or treatment with calf serum or trypsin in close correlation with the effects of these agents on characteristics of cell proliferation. The present results, taken together with those of earlier studies on cell-electrophoresis and characterization of lipid constituents of the cells, support the hypothesis of a lateral phase separation in the plasmamembrane as triggering process in stimulation of proliferation of resting normal cells.


Constant negative pressure in the treatment of diaphragmatic paralysis secondary to birth injury. A newborn infant with right Erb palsy and diaphragmatic paralysis was treated with CNP for respiratory failure. There was clinical and arterial blood gas improvement. Several months after treatment, respiratory symptoms had disappeared, but fluoroscopy demonstrated persistence of diaphragmatic paralysis. A review of the literature is included. CNP may allow spontaneous recovery while permitting adequate nutrition.


Platelet interaction with human umbilical cord vascular basement membrane. The basement membrane of the human umbilical vein was studied by electron microscopy with respect to its ultrastructure, susceptibility to digestion by collagenase or trypsin, and reactivity with human platelets. Electron microscopic examination of this vessel showed a continuous reticulated basement membrane which morphologically resembled those of mammalian capillaries and rabbit heart valves. The vascular endothelium was removed by freezing and thawing, thus uncovering the underlying connective tissue. The vessels were sliced into rings which were incubated with collagenase or trypsin. The basement lamella appeared to be susceptible to digestion by either enzyme. Platelet interaction with exposed vascular basement mambrane was studied by rotating frozen-thawed everted and noneverted rings in anticoagulated whole human blood. In heparinized or citrated blood, large aggregates of degranulated platelets adhered to collagenous controls; in contrast, the test rings with exposed basement membrane were partially covered with a monolayer of platelets which appeared to retain discoid or spherical shape and granules. In EDTA-anticoagulated blood, the collagen control rings accumulated a platelet monolayer, whereas little or no adhesion occurred on the basement membrane surface. In this system the basement membrane of the human umbilical vein appears to be a poor platelet reactive surface as compared to collagen.


Isolation and characterization of plasma membrane from rat mesenteric arteries. A method for isolating a plasma membrane-enriched fraction and other subcellular fractions from rat mesenteric arteries by the use of a discontinuous sucrose density gradient is decribed. Electron microscopy showed both plasma membrane and endoplasmic reticulum fractions to be composed of vesicles. 5'-Nucleotidase, alkaline phosphatase, ouabain-sensitive (Na+ + K+)-ATPase and K+-phosphatase, and phosphodiesterase I were concentrated in the plasma membrane fraction. The increase in ATP-dependent calcium uptake in the presence of oxalate was greater in the endoplasmic reticulum than in the plasma membrane fraction. The lack of inhibition of active calcium uptake by azide suggests that the plasma  membrane-enriched fraction was relatively free of mitochondrial contamination. Calcium uptake by the plasma membrane or the endoplasmic reticulum fraction was not enhanced by high-energy compounds other than ATP, and was little affected by 100 mM KCl or NaCl in the Mg++-containing medium. Subcellular fractions isolated by this method will be useful for investigating the biochemistry of small blood vessels of the rat.


Ribosome-lamellae complex in chronic lymphatic leukaemia. The Ribosome Lamellae Complex (RLC) was found in two out of 35 patients with Chronic Lymphatic Leukaemia (CLL). The proportion of lymphocytes bearing RLC was very small (4% in the first case and less than 0.5% in the second case). The significance of this type of inclusion is discussed and the possible relationship to the circular profiles of endoplasmic reticulum is emphasized.


Effect of prostaglandin E1 on neuromuscular transmission in the rat. 1 The effect of prostaglandin E1 on neuromuscular transmission in the phrenic nerve-diaphragm muscle preparation of the rat was studied with intra- and extracellular recording techniques. 2 Prostaglandin E1, in concentrations from 10 nM, induced intermittent failures in the generation of the end-plate potential in response to repeated indirect stimulation. 3 Failures appeared abruptly, the end-plate potential behaving in an all-or-nothing fashion. The effect occurred only at 36-38 degrees C when the nerve was stimulated at 30-80 Hz and was reversible upon washing with drug-free solution. 4 Since miniature end-plate potentials were not affected, such failures must be attributed to a presynaptic action of prostaglandin E1. 5 Extracellular recording suggested that prostaglandin E1 prevented the action potential from reaching the nerve terminal.


The effect of metiamide on acid secretion stimulated by gastrin, acetylcholine and dibutyryl cyclic adenosine 3',5'-monophosphate in the isolated whole stomach of the rat. 1 An isolated stomach preparation from immature rats is described. The lumen of the stomach was perfused and the hydrogen ion activity of the perfusate recorded continuously. 2 The preparation gave dose-dependent responses to gastrin, acetylcholine and dibutyryl cyclic adenosine 3',5'-monophosphate and these responses were readily reversed on washing out the agonist. 3 The acid secretory response to gastrin was inhibited by metiamide at concentrations of 10(-5) M and 3 X 10(-5)M. 4 The acid secretory responses to acetylcholine and dibutyryl cyclic adenosine 3',5'-monophosphate were not inhibited by concentrations of metiamide up to 10(-3) M. 5 These findings are discussed in relation to the role of histamine in the control of gastric acid secretion.


Receptors for 5-hydroxytryptamine and noradrenaline in rabbit isolated ear artery and aorta. 5-Hydroxytryptamine (5-HT) is thought to be implicated in the vascular disturbances of the external carotid artery bed associated with migraine. As part of a study of the pharmacology of some 5-HT antagonists used in the treatment of migraine we have examined the interactions of these drugs with 5-HT and noradrenaline in rabbit isolated ear artery and aortic strip. The results provide new information on the distribution of 5-HT-receptors in these preparations. In the aorta, 5-HT and noradrenaline were of similar potency in producing contractions. Methysergide produced very small contractions and was about 1000 times less potent than the other two agonists. In the ear artery noradrenaline produced monophasic vasoconstrictor responses, whereas 5-HT and methysergide produced prolonged biphasic responses. 5-HT was about 700 times less potent and methysergide about 4500 times less potent than noradrenaline. Methysergide was a better agonist in the ear artery than in the aorta. Biphasic responses to 5-HT and methysergide were also obtained in ear arteries from reserpine-treated rabbits indicating that neither agonist was acting by releasing endogenous noradrenaline. Pizotifen, cyproheptadine and phentolamine had no agonistic actions in either the aorta or ear artery. In the aorta methysergide, pizotifen and cyproheptadine were potent antagonists of 5-HT and much weaker antagonists of noradrenaline. Phentolamine possessed the opposite profile of selectivity. These results show that there are distinct receptors for 5-HT and noradrenaline in rabbit aorta. In the ear artery the pA2 values for each of the four antagonists were virtually identical against 5-HT and noradrenaline and similar to those obtained on alpha-adrenoceptors in the aorta. We conclude that 5-HT and noradrenaline act directly at alpha-receptors to produce vasoconstriction in the ear artery and that this preparation does not contain specific 5-HT receptors. This insight into the distribution of 5-HT receptors and alpha-receptors allows interpretation of the various actions of methysergide. In the aorta, methysergide was a potent antagonist at 5-HT-receptors and a weak partial agonist at alpha-receptors. In the ear artery, methysergide was a partial agonist at alpha-receptors; it was only a weak antagonist of 5-HT because this preparation does not contain specific 5-HT-receptors. The cross-reactivity demonstrated throughout these experiments indicates that 5-HT-receptors and alpha-receptors, although distinct entities, have features in common. These results are discussed in relation to the mode of action of methysergide, pizotifen and cyproheptadine in the treatment of migraine.


Asthma deaths in Cardiff 1963-74: 53 deaths in hospital. In a study of factors associated with death from bronchial asthma in hospital 53 patients were investigated. Typically the fatal attack persisted for several days before admission to hospital and normally occurred in patients with a long history of asthma. The patient or doctor often underestimated the severity of the attack. On admission most patients were severely ill, and over a third died within 24 hours. Peak flow rate and blood gases were rarely measured. Corticosteroid treatment was often underused, and patients rarely received assisted ventilation before death. Infection played a part in 14 deaths, five of them associated with assisted ventilation. Admitting asthmatics to a special respiratory ward with facilities for standardised assessment and treatment and introducing a self-admission service may help to prevent some of these deaths.


Studies on the pharmacology of neurones in the nucleus accumbens of the rat. A study was made of the effects of iontophoretically applied drugs on single neurones in the nucleus accumgens and caudate nucleus of rats anaesthetized with urethane. Neurones in the caudate nucleus were inhibited by dopamine, dibutyryl cyclic AMP, ADTN and ergometrine. Acetylcholine and homocysteic acid caused excitation of striatal neurones. In the nucleus accumbens neurones were inhibited by dopamine, ADTN, ergometrine, dibutyryl cyclic AMP, glycine and gamma-aminobutyric acid. The responses of glycine and gamma-aminobutyric acid were antagonised by strychnine and picrotoxin, respectively. Acetylcholine and homocysteic acid caused excitation of neurones in the nucleus acumbens; the effects of acetylcholine were blocked by atropine. The results are consistent with the suggestion that dopamine is an inhibitory transmitter in the nucleus accumbens and in the caudate nucleus and support the  hypothesis that the effects of dopamine are mediated by cyclic AMP. The locomotor stimulants ADTN and ergometrine mimicked the inhibitory actions of dopamine in both the striatum and in the nucleus accumbens. These results support the suggestion that dopamine receptors in the nucleus accumbens are involved in the actions of locomotor stimulant drugs.


Interactions of nigrostriate synaptic transmission, iontophoretic O-methylated phenethylamines, dopamine, apomorphine and acetylcholine. Recordings were made from, and drugs applied to, neurons in the caudate nucleus of unanesthetized cats, using multibarrel micropipette electrodes. The substantia nigra was stimulated by sterotactically placed electrodes. Three O-methylated derivatives of dopamine, meta-methoxyphenethylamine (m-MPEA), para-methosy-phenethylamine (p-MPEA) and 3,4-demethoxyphenethylamine (DIMPEA), inhibited most, excited a few, and had no detectable effect on a substantial number of the cells upon which they were tested. A statistically significant correlation was found between the effects of dopamine (DA) and the three O-methylated derivatives on the same populations of cells. Iontophoretic release of the O-methylated derivatives could not prevent the actions of DA, nor could it block synaptically mediated effects of the nigrostriate pathway. It is concluded that the three O-methylated products are partial agonists of DA. The findings are difficult to reconcile with the suggestion that the experimental parkinsonian-like symptoms caused by O-methylated phenethylamines are the consequence of blockade of dopaminergic synapses. No correlation, negative or positive, was found between the effects of DA and of acetylcholine (ACh). The findings do not support the theory that balanced sets of antagonistic synapses, one dopaminergic, the other cholinervic, operate upon individual neurons in the caudate nucleus. Apomorphine and dopamine were shown to have similar effects on a substantial number of neurons, even though the onset and offset of the effect of apomorphine were slower than those of DA. This observation agrees with the suggestion that some of the central effects of apomorphine are due to an action at dopaminoceptive receptor sites.


Nervous system antigen-5, an antigenic cell surface component of neuroectodermal origin. The antigenic cell surface component NS-5 (nervous system antigen-5) is recognized by antiserum raised in C3H.SW/Sn mice against cerebellum of 4-day-old C57BL/6J mice. When analyzed in the cytotoxicity test the antiserum detects a cell surface antigen or set of antigens present not only an cerebellum but also other parts of the central nervous system, including retina, as well as on mature spermatozoa and to a lesser degree on kidney. All other non-neural tissues tested, liver, splee, thymocytes, muscle, testis, adrenal gland and epidermis do not express detectable amounts of the antigen. Among seven murine tumors of the nervous system, medulloepithelioma shows high levels of NS-5 expression, whereas neuroblastoma Cl300, glioma G26, glioblastome, ependymoblastoma, ependymoblastoma EPA and glioblastoma G26l do not carry detectable NS-5. All mouse strains tested (C57BL/6J, C3H.SW/Sn, C3H/HeDiSn, A/J, AKR/J, BALB/cJ and DBA/2) express similar levels of NS-5. The antigen is demonstrable not only on postnatal day 4 neural tissue, but also in lower amounts on adult nervous system. On embryonic day 9, the earliest stage tested, and at all subsequent stages during embryonic development, NS-K is already present in brain and spinal cord, but not in gut.


Autonomic nerves, mast cells, and amine receptors in human brain vessels. A histochemical and pharmacological study. The studies were performed on operation material from 17- to 63-year-old patients and on fetuses at 19-23 weeks gestational age. Formaldehyde histofluorescence showed the presence of numerous perivascular adrenergic nerves around pial and intracerebral vessels, the carotid system being better supplied than the vertebral system. Cholinergic nerves, visualized by the cholinesterase technique, followed the adrenergic fibers in the plexus formations of the pial arterial system. Histamine-containing mast cells, often with a perivascular distribution, were located with the o-phthaldiadehyde method. Transmural electrical stimulation of the perivascular nerves contracted isolated pieces of pial arteries in a frequency-dependent manner, and the response was inhibited by the adrenergic nerve blocking agent, guanethidine. On the basis of the relative potency of various amines and related compounds in producing a motor response of isolated pial arteries, and the mode of inhibition caused by specific antagonists, various amine receptors could be demonstrated: adrenergic alpha-receptors (mediating contraction) and beta-receptors (dilation), cholinergic muscarinic receptors (dilation) and histamine H2-receptors (mediating dilation). Thus, the amine mechanisms demonstrated in human brain vessels appear to be principally the same of those shown in more extensive studies on laboratory animals.


Modulation in the sensitivity of noradrenergic receptors in the CNS studied by the responsiveness of the cyclic AMP system. Various characteristics of the altered responsiveness to noradrenaline (NA) of the cyclic Amp (cAMP) generating system of cortical brain slices have been studied in rat after chronic reserpine treatment and in mice after chronic D-amphetamine treatment. Supersensitivity of the cAMP system to NA following reserpine treatment and subsensitivity after D-amphetamine treatment exhibit many common features. Firstly, in both cases the modified responsiveness to NA occurs rapidly after the beginning of the treatment (one day for reserpine and 5 h after amphetamine) and recovers slowly after the end of the treatment. Additionally, the altered states of sensitivity of the cAMP system to NA are the result of a modification of the maximal response rather than of an altered affinity of the system to NA. Since, following D-amphetamine treatment the change of sensitivity is still apparent in the presence of a potent phosphodiesterase inhibitor, the involvement of a gross alteration of phosphodiesterase activity is unlikely. In this case too, while the response to adenosine is slightly decreased, the responses to dopamine and serotonin of the cAMP system observed in brain slices following D-amphetamine treatment is not detected in a cell-free preparation. Thus, it appears that drug treatments, modifying synaptic transmission in opposite directions, lead also to changes in opposite directions of the responsiveness of the cAMP system to NA. The hypothesis that the mechanisms underlying such phenomena are related to a modified number of functional noradrenergic receptors is discussed.


Ultrastructural comparisons of neurons of supraoptic and circularis nuclei in normal and dehydrated rats. A quantitative ultrastructural investigation was undertaken to compare the nucleus circularis (NC) and supraoptic nucleus (SON) of the rat both under normal and water-deprived conditions. NC was found to have dramatically more of its cells and membrane surface involved in direct soma-somatic contact than the SON. Water deprivation, even for one day, brought about a significant increase in both percentage of cells and membrane surface in contact in both nuclei, apparently by the retraction of fine glial processes from between the somata. The normal NC was made up of only one ultrastructurally identifiable cell type. The normal NC had no cells showing expanded endoplasmic reticulum, although these were seen following 5 days (but not 1 day) of water deprivation. The normal SON did have 4.4% of its cells showing expanded endoplasmic reticulum. This percentage significantly increased following water deprivation. The vesicle population per area of cytoplasm was very similar between the two normal nuclei. One day of water deprivation brought about a significant increase in less than 800 A vesicles in NC but not the SON. Five days of water deprivation resulted in a significant decrease in the lysosomal population per unit area in both nuclei. Vesicle changes have been discussed in relation to the volume changes in the cells.


Feeding and body temperature in the rat: diencephalic localization of changes produced by excess calcium ions. In the unanesthetized rat, Ca++ ions in solutions ranging from 2.6 to 112.0 mM in excess of the normal level in CSF were applied at different sites in the brain and by three separate procedures, Colonic temperature was monitored and in certain experiments, the amount of food pellets and water consumed was measured simultaneously following the administration of excess Ca++ ions. An infusion into the lateral cerebral ventricle of excess calcium in a volume of 5.0 mul produced a concentration-dependent hypothermia, This fall in temperature was not attenuated by a prior intraventricular infusion of mecamylamine and often enhanced by atropine. Depending on the site, a microinjection of excess Ca++ ions in a volume of 0.5 to 1.0 mul directly into the hypothalamus produced hypothermia or feeding. The sites of maximum sensitivity at which excess calcium caused a decline in temperature were clustered in the caudal hypothalamus, whereas those at which calcium elicited feeding were distributed widely in caudo-lateral, medial and rostral hypothalamic areas. Push-pull perfusions at a rate of 20 to 25 mul per min for 10 to 20 min at homologous sites caused similar responses but the cation concentration required to evoke feeding or hypothermia was significantly less than that of either microinjection or intraventricular infusion. These findings demonstrate species continuity in the rat concerning anatomical localization of the postulated set-point mechanism for body temperature. Several different pathways in the feeding system are affected by an alteration in the hypothalamic level of calcium.


Sleep cycles in cats during chronic electrical stimulation of the area postrema and the anterior raphe. Sleep-waking profiles were obtained from 130 7 hr stimulation-EEG recording sessions in a series of cats bearing chronically implanted stimulating electrodes in the regions of the area postrema and anterior raphe nuclei. The results indicated that: (a) during electrical stimulation of the region of the area postrema with 0.5 or 10 Hz at 1 and 2 mA there were significant increases in the occurrence of the deeper aspects of slow-wave sleep and in REM sleep. These elevations were significant in comparison to nonstimulation baselines and to sleep profiles obtained during stimulation of points located dorsal and anterior to the area postrema. (b) Stimulation of the medial reticular formation including the anterior raphe using the same parameters employed for the area postrema did not alter the occurrence of any stage of sleep. These findings indicate that the region of the area postrema may be more involved in the generation of sleep than the anterior raphe nuclei.


[Action of the collagenase on the morphogenesis of the duck embryonic urophygial gland cultured in vitro]. Collagenase prevents in vitro the uropygial invaginations differentiation and the ectodermal glandular buds development. The basal lamina and the extracellular material disappear. These data suggest that collagen is essential to preen gland morphogenesis.


Power spectral analysis of EEG activity obtained from cortical and subcortical sites during the vigilance states of the cat. There is considerable evidence that the raphé system and the region of the nucleus tractus solitarious (NTS), including the area postrema, play significant roles in slow-wave sleep mechanisms and in EEG synchronization. Studies of the interactions between these systems and the neocortex are much needed. If neuronal activity in these lower brainstem regions regulates the degree of cortical synchrony then a high degree of correspondence between the EEG of the area postrema or raphé complex with that of the cortex might be expected. In order to quantitate the reequency characteristics of the EEG obtained from these subcortical sites (nucleus raphé dorsalis, area postrema, as well as anatomical controls adjacent to these regions) during the different vigilance states (waking, slow-wave sleep, REM sleep) in the cat, power spectral analyses techniques were employed. Comparison of these subcortical spectral characteristic with those obtained from cortical (frontal and occipital) sites during the same vigilance state, show that the spectral measures elicited from the region of the area postrema closely correspond to that of the cortex, particularly during slow-wave sleep. On the other hand, the EEG of the anterior portion of the raphé region, although exhibiting a substantial low frequency component during slow-wave sleep in comparison to wakefulness does not show a statistically significant shift to low frequencies such as occurs in the area postrema or the cortex. These results suggest that the increases in the low frequency content of the cortical EEG sites during slow-wave sleep results from synchronizing inputs from the area postrema to a greater extent than from the raphé complex.


[Ultrastructural aspects of cells with 3 beta-HSDH (delta-5-betahydroxysteroid dehydrogenase) activity in female ducks. I. Female Peking duck (Anas platyrhynchos)]. The cells making up the thick internal theca of the follicle of the Peking duck and which contain an active 3 beta HSDH enzyme have a developed smooth reticulum, tubular crested mitochondria, a considerable load in slightly osmiophilic lipid inclusions; all of these characteristics are those of steroid cells. Various physiological states of these cells juxtapose themselves in different proportions according to the season.


Low level potentiation of the brain stem laryngeal reflex. Trains of electrical stimuli were applied to the superior laryngeal nerve (SLN) innervating the upper respiratory tract while evoked potentials were recorded from the recurrent nerve (RN) which innervates the intrinsic laryngeal muscles. Responses of this brain stem reflex (central delay 3.5-5.0 msec) were compared to the post-tetanic potentiation (PTP) of the polysynaptic ventral root response (L6-S1) with trains of stimuli applied to the dorsal root of adult cats. Stimulation of the SLN with a train of pulses varying from 10-45/sec evokes an initial depression of the polysynaptic potential from the RN followed by a low but sustained increase in the amplitude of the integrated signal (1.2-1.6x) lasting 45-130 msec and lacking the early maximum amplitude as seen in the PTP of the polysynaptic spinal reflex. Both the early and late components of the superior laryngeal-recurrent nerve reflex (SLN-RN) demonstrate potentiation whereas the shortest latency components of the spinal reflex account for most of the PTP. Systemic administration of strychnine sulfate (dosage: 0.03-0.45 mg/kg) does not alter the normalized PTP of the SLN-RN reflex despite enhancement of both early and late components of the reflex following control stimuli at 1/sec.


[Ultrastructural aspects of cells with 3 beta-HSDH (delta 5-3 betahydroxysteroid dehydrogenase) activity in female ducks. II. Female Barbary ducks (Cairina moschata)]. The cells making up the thin internal theca of the follicle of the Barbary duck and which contain are 3 beta HSDH enzyme show all of the ultrastructural characteristics of steroid cells: a well-developed smooth reticulum, tubular cristae mitochondria, a considerable load in lipid inclusionologous cells of the Peking duck since they are constantly more dense and their cristae are bigger and less numerous.


[Cytochemical aspects of human polynuclear neutrophils under the in vitro and in vivo action of TRH]. A TRH fixation in vitro occurs at the level of the human polynuclear neutrophil, which suggests putative membrane receptor (s) for the hypothalamic hormone. After TRH administration, enzyme modifications (myeloperoxydase, alkaline phosphatase) and metabolism changes (PAS, Sudan black) happen in vivo within the neutrophil, showing a functional activation of that blood cell. Two processes may be considered to explain those data: 1) the TRH is directly fixed, as observed in vitro; 2) a mediate hormone (TSH, T4, T3) is released capable of binding the granulocyte membrane.


The effect of calcium oncardiac phosphorylase activation, contractile force and cyclic AMP in euthyroid and hyperthyroid rat hearts. Calcium chloride injected into isolated perfused rat hearts produced a positive inotropic effect and increased the levels of phosphorylase a (EC 2.4.1.1). The increase in enzyme activity lagged behind the inotropic effect. Pretreatment of animals with thyroid hormone enhanced the ability of noradrenaline to activate phosphorylase but did not affect the inotropic or phosphorylase activating effect of calcium. Thyroid hormone pretreatment did enhance the chronotropic effect of calcium. Calcium did not affect the cardiac levels of cyclic AMP. It is concluded that calcium can activate phosphorylase by a mechanism other than cyclic AMP and that the enhancement of adrenergic amine-induced phosphorylase activation by thyroid hormone is not a calcium mediated event.


The release and characterization of some periplasm-located enzymes of Pseudomona aeruginosa. Pseudomonas aeruginosa (ATCC 9027) releases four periplasm-located enzymes, i.e., ribonuclease (EC 3.1.4.22; EC 3.1.4.23), alkaline phosphatase (EC 3.1.3.1), cyclic-2', 3'-phosphodiesterase (EC 3.1.4.d), and 5'-nucleotidase (EC 3.1.3.5) into the medium during growth. Ribonuclease and alkaline phosphatase are classed as enzymes which are readily extracted by osmotic shock and spheroplast formation whereas cyclic-2',3'-phosphodiesterase and 5'-nucleotidase are classed as enzymes which are not readily extracted by these procedures. In view of the relative ease of extraction of the former enzymes it is suggested that the lattter enzymes, cyclic-2',3'-phosphodiesterase and 5'-nucleotidase, are bound and located in the periplasm in a manner different to ribonuclease and alkaline phosphatase.


Comparison of vesicular stomatitis virus defective interfering particle synthesis in chick embryo and L cells. A comparison of the ability of vesicular stomatitis virus (VSV) to generate and replicate defective interfering (DI) particles in primary chick embryo (CE) and mouse L cells was investigated as a means of analyzing host control over DI-particle synthesis and interfering capacity. Serial undiluted passage of VSV in CE and L cells indicate that VSV-DI particles are generated and (or) replicate with greater efficiency in CE than in L cells. When DI particles accumulate in L cells, they are able to interfere with infectious particle replication. The DI particles from CE cells interfered to the same extent with infectious particle replication in both CE and L cells. L cells, therefore, are not considered 'low-interference' hosts in which DI particles are produced and do not interfere with infectious virus replication, but rather hosts which restrict the production of DI particles.


Long-term cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain (D) of Rous sarcoma virus. Attempts have been made to keep in vitro, for extended periods of time, cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D. Roller cultures of transformed chick cells kept in serum-deficient medium can be maintained without subcultivation for up to 6 months. The confluent cultures continuously release viruses and viable tumor cells into the medium. The released cells can be plated and have characteristics of growth and morphology which are relatively stable with time until the culture degenerates. Cells released at later stages of the culture produced substantially more viruses than those released earlier, suggesting that cell selection or differentiation occurs during long-term cultivation in low serum concentration. Long-term cultures of untransformed chick embryo fibroblasts can also be maintained in the same way. The release of viable cells by these confluent cultures, however, is negligible.


Lecithinase-negative variants of Clostridium perfringens; the identity of C. plagarum with C. perfringens. Identity of Clostridium plagarum (Prévot) com. nov. (1938) with C. perfringens was demonstrated in the tests for the cultural and biochemical properties, DNA-DNA homology, susceptibility to C. perfringens-specific lysin, complementary synthesis of C. perfringens theta-toxin between C. plagarum and theta-toxin-negative mutants of C. perfringens, as well as the tests for gas-chromatographic patterns.


Lactate dehydrogenase from autotrophic and heterotrophic cells of the marine diatom Cylindrotheca fusiformis Reimann & Lewin. Cultures of Cylindrotheca furisormis grown either autotrohpically or heterotrophically on lactate contained significant amounts of NAD-dependent L(+)-lactate dehydrogenase (EC 1.1.1.27). Polyacylamide gel electrophoresis of crude enzyme extracts revealed a single band which was indistinguishable between autotrohpic and heterotrohpic cells. The Km for lactate of partially purified preparations was lower under heterotrophic conditions. The specific activity in crude extracts was higher under autotrophic than heterotrophic conditions; it dropped precipitously when autotrophic cells were transferred to the dark, increasing again only in the presence of lactate. These and related observations suggest that this enzyme has at most only a minor role in the assimilation of lactate during heterotrophic growth on lactate.


Leakage of periplasmic enzymes from lipopolysaccharide-defective mutants of Salmonella typhimurium. Mutants of Salmonella typhimurium with defects in the heptose region of the lipopolysaccharide (LPS) molecule (heptose-deficient, chemotype Re) leak periplasmic enzymes (acid phosphatase (EC 3.1.3.2), cyclic phosphodiesterase, ribonuclease I (EC 3.1.4.22), and phosphoglucose isomerase (EC 5.3.1.9) (PGI is at least partially periplasmic in E. coli and S. typhimurium; see below)) and do not leak an internal enzyme (glucose-6-phosphate dehydrogenase) into the growth medium. The extent of this leakage is markedly increased at higher temperature (42 degrees C). Leakage of periplasmic enzymes from the strains lacking units distal to heptose I in the LPS molecule (chemotype Rd2) occurs only at 42 degrees C, and not at 30 or 37 degrees C. The extent of leakage of these enzymes from smooth strain and mutants of other LPS chemotypes (Rc, Rd1) is not significant, and is not influenced by growth temperatures. The kinetics of leakage of periplasmic enzymes after shift to 42 degrees C in nutrient broth reveal an accelerated release into the medium from heptose-deficient strains of cyclic phosphodiesterase and ribonuclease I after 30 min at 42 degrees C, and phosphoglucose isomerase after 60 min at 42 degrees C; at 30 degrees C the rate of release of cyclic phosphodiesterase and ribonuclease I is relatively slower. After 60 min at 42 degrees C in nutrient broth, growth of these strains has either slowed down or stopped. In L-broth, which permits the growth of the heptose-deficient strain (SA1377) at 42 degrees C, leakage of cyclic phosphodiesterase and phosphoglucose isomerase occurs, whereas there is no detectable leakage of these enzymes from the isogenic smooth strain (SA1355). Thus, leakage of the periplasmic enzymes from the heptose-deficient strain occurs with or without growth. Mg2+ (0.75 mM), sodium chloride (50 mM), and sucrose (100 mM) in nutrient broth at 42 degrees C prevent the leakage of these enzymes. The shedding of LPS from the heptose-deficient as well as the smooth strains is enhanced by high temperature (42 degrees C), whereas considerable leakage of protein occurs only in the heptose-deficient strain at 42 degrees C and not in the smooth strain. The smooth and heptose-deficient strains are equally sensitive to osmotic shock although a significant proportion of acid phosphatase and cyclic phosphodiesterase activities from the heptose-deficient cells grown at 42 degrees C comes off in the Tris-NaCl wash step suggesting a rather loose attachment of these enzymes onto the cell surface.


Comparison of four eluents in the recovery of indigenous viruses from raw sludge. The efficiency of 3% casein hydrolysate (CH), 3% lactalbumin hydrolysate (LH), 3% beef extract (BE), and 10% fetal calf serum (FCS) was compared for the recovery of viruses from raw sludge. CH and LH proved to be inefficient and were eliminated from the study after initial testing. In tests with 20 different samples of raw sludge, beef extract eluted virus in 15 (75%) and FCS revealed virus in 19 (95%) of the samples using BS-C-1 cells. That different eluents were not eluting different viruses from the same sample was shown by the serologic and electron-microscopic examination of 43% (18/42) of the isolates. The identified viruses included members of the entero- (coxsackie B, and polio) and reo-virus groups.


A comparison of the prophylactic and therapeutic effects of poly I:C and endotoxin in mice infected with Mengo virus. We have compared the protective effect in AKR mice of poly I:C and bacterial endotoxins against lethal doses of Mengo virus. Administered intravenously or intraperitoneally, both interferon inducers protected mice to about the same extent from virus challenges of 2-3 LD50's. Endotoxin, however, was unable to protect the mice effectively against higher challenge doses of virus. Evidence is presented that the level of protection afforded by both inducers is related to the level of circulating interferon produced. We have also shown that a single intravenous dose of poly I:C results in the appearance of two distinct bursts of interferon activity, with maxima at about 2 h and 9 h post injection. Endotoxin, on the other hand, produced only one peak of activity, at 2 h post injection.


Polymorph-mediated antibody-dependent cytoxicity--modulation of activity by drugs and immune interferon. Bovine polymorphonuclear leukocytes (PMN) mediated antibody-dependent cell cytotoxicity (ADCC) against erythrocyte and herpes virus-infected target cells. The extent of cytotoxicity was not affected by drugs that inhibited DNA, RNA, or protein synthesis. The effect did not occur in the absence of divalent cations, was suppressed by pretreatment of PMN with silica and cytochalasin B, and was subject to the bidirectional control by cyclic nucleotides; drugs decreasing cyclic AMP or elevating cyclic GMP levels enhanced ADCC. The ADCC phenomena was also enhanced by supernates containing immune interferon activity from antigen-stimulated-immune lymphocyte-macrophage cultures. The possibility that immune interferon(s) might be causing the elevation of ADCC and the relevance of this observation in terms of the part interferon might play in modulating recovery from herpes virus infections was discussed.


Indirect microhemagglutination test for varicella--zoster antibody determination. An indirect microhemagglutination assay (IHA) was devised because of a need to provide an alternative test to complement fixation (CF) for varicella-zoster (V-Z) antibody determination. Human erythrocytes were sequentially treated with 2% glutaraldehyde, 0.04% tannic acid, and 2% pyruvic aldehyde then exposed to sonicated V-Z infected cells. This same tanning procedure was suitable for herpes simplex and Epstein-Barr virus antigen attachment but unsatisfactory for several non-herpes-group viruses. V-Z antibody titres determined by IHA were generally 2 to 6 times higher than CF titres. Cross-reaction with herpes simplex antibody was minimal.


Temperature-sensitive mutants of vesicular stomatitis virus: viral RNA synthesis in cells infected with mutants belonging to complementation group I. The RNA polymerase in cells infected with three group I mutants of vesicular stomatitis virus has been examined. Mouse L cells were incubated at the permissive temperature (30 degrees C) for a few hours after infection to allow the development of secondary transcription. The temperature dependence of the secondary transcription system was determined from the incorporation of labelled uridine, in the presence of cycloheximide, at 30 and at 38 degrees C, the later temperature being non-permissive for viral replication. In cells infected with mutants W14, W28, and G11 at a low multiplicity (20 PFU/cells) secondary transcriptase activity was markedly temperature-sensitive after 3 and 5 h of infection at 30 degrees C. At a high multiplicity of infection (1000 PFU/cell) cells infected with W28 showed considerable RNA synthesis at 38 degrees C after 3 h at 30 degrees C. RNA synthesis was also observed in W28-infected cells in which protein synthesis was allowed to continue after the shift from 30 to 38 degrees C. In the latter two cases the RNA synthesized contained 12-18S species but little or no 30S mRNA.


Pathways of glucose catabolism in Mycobacterium smegmatis. Glucose metabolism in Mycobacterium smegmatis was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Glucose is oxidized in this organism mainly through the Embden-Meyerhof-Parnas pathway, irrespective of the carbon source used for growth. The pentose phosphate pathway plays only a minor role and its extent depends on the carbon source used for growth. Enzymes of glycolytic and oxidative pathways were detected in cells grown on glucose, glycerol, or pyruvate but enzymes of the Entner-Duodroff pathway could be detected only in glucose-grown cells. Labeled acetate is utilized by cells cultured on glucose, glycerol, and pyruvate. In all cases more of C1 of acetate was converted to CO2 while incorporation into cellular constituents was maximum from C2 of acetate.


Anomalous aggregation of Clostridium perfringens enterotoxin under dissociating conditions. Polyacrylamide gel electrophoresis of highly purified Clostridium perfringens enterotoxin revealed electrophoretic microheterogeneity of the enterotoxin, apparently because of slight charge differences in the peptides. Detergent gel electrophoresis showed that purified enterotoxin formed high molecular weight aggregates in the presence of both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide. No conditions capable of inhibiting this phenomenon were found. Although a molecular weight of 35 000 daltons has been reported in the literature, the experimentally determined molecular weight values in the presence of detergents corresponded to multiples of a theoretical subunit molecular weight of 17 500 daltons. Binding studies performed by equilibrium dialysis and ultracentrifugation methods revealed that the enterotoxin bound very small amounts of SDS per gram of protein. The evidence presented indicates possible detergent induced structural alterations of the protein.


Liver transplantation in a patient with cholangiocarcinoma and ulcerative colitis. A 39 year-old patient with cholangiocarcinoma and pre-existing ulcerative colitis was successfully treated by orthotopic liver transplantation. He was given low doses of prednisone and azathioprine and survived for more than 9 months, dying with tumour metastases, thrombosis of the inferior vena cava and an intra-abdominal abscess. At autopsy the homograft showed little evidence of rejection. Preoperatively the patient had septicemia. Removal of his liver was difficult. The discrepancy between donor and recipient in size of blood vessels and the presence of two hepatic arteries in the donor caused problems during the vascular anastomoses. During the operation cardiac arrest occurred. Postoperatively there were several medical and surgical problems, including intraperitoneal and gastrointestinal hemorrhage, paralysis of the right dome of the diaphragm, sinus bradycardia, massive diuresis, peroneal nerve palsy, and one major and three minor episodes of rejection, which were reversed by giving pulse doses of methylprednisolone intravenously.


Primary hyperlipoproteinemia in childhood and adolescence: identification and treatment of persons at risk for premature atherosclerosis. Determination of serum cholesterol values in three populations of children and adolescents, totalling 4013 subjects aged 1 month to 20 years, revealed 16 cases of primary hyperbetalipoproteinemia (overall frequency, 1:251) and led to the detection of the disorder in 12 asymptomatic siblings. The upper limit of normal for serum cholesterol concentration was approximately 200 mg/dl at all ages studied. Dietary treatment was instituted in patients whose serum cholesterol value exceeded this limit and in whom a primary lipid defect was confirmed; the serum cholesterol value decreased in all patients who adhered to the diet. However, since the potential hazards and long-term results of dietary treatment, with or without drug therapy, in growing children are not known, such treatment should be reserved for affected children with a family history of premature atherosclerosis, and follow-up is essential.


Cushing's disease in a child with lymphosarcoma and acute leukemia. A 6-year-old girl with lymphosarcoma developed Cushing's syndrome. Suppression-stimulation studies verified adrenal hyperfunction secondary to bilateral adrenocortical hyperplasia, and the course suggested the possibility of "ectopic ACTH" production by the neoplasm as the etiology.


The treatment of Wilms' tumor: Results of the national Wilms' tumor study. The National Wilms' Tumor Study, initiated in 1969, tested competing treatment strategems for patients with tumors ranging from Group (Gp) I (tumors confined to the kidney and totally removed) to Gp IV (remote metastases present at diagnosis). Three hundred and fifty-nine of 606 registered patients were randomized in the trial. Gp I patients under 2 years of age fared well whether postoperative radiation therapy (RT) was or was not added to 15 months' maintenance actinomycin D (AMD). Their prognosis was better than that for older cohorts similarly treated, in whom the difference in relapse rates between treatment groups were suggestive of an RT effect. Combined AMD and vincristine (VCR) gave better results than either agent alone in patients with more advanced tumors (Gps II and III) still confined to the abdomen, all of whom received postoperative RT as well. Preoperative VCR given Gp IV patients in addition to postoperative RT, AMD, and VCR did not improve results. The frequency of mesoblastic nephroma (1%), of bilateral tumors (5%), and of incorrect preoperative diagnosis of Wilms' tumor (5%), the toxicities of the various regimens, and other ancillary data are presented and discussed.


Preoperative versus postoperative radiotherapy, single versus multiple courses of actinomycin D, in the treatment of Wilms' tumor. Preliminary results of a controlled clinical trial conducted by the International Society of Paediatric Oncology (S.I.O.P.). The preliminary results of a controlled clinical trial organized by the S.I.O.P. of radiotherapy and chemotherapy in patients with nephroblastoma are presented. Forty-two centers have participated. Between September 1971 and October 1974, 398 patients were registered; 195 were eligible for the trial and were randomized. The remaining 203 patients were excluded from the trial, but were followed in the same way as the patients in the trial. The results were evaluated in terms of recurrence-free survival rate and survival rate. Results in patients who received preoperative and postoperative radiotherapy (group A, 73 patients) were compared with the results in patients who recieved only postoperative radiotherapy (group B, 64 patients). The tumor ruptured at surgery in three patients of group A, and in 20 patients of group B, a difference that is statistically significant. No significant difference in survival and recurrence-free survival between groups A and B is observed at present. Results in patients treated with a single course of actinomycin D (group I, 80 patients) were compared with the results in patients treated with multiple courses (group II, 80 patients). At present, no significant difference is found between the two groups.


Distribution of adenosine triphosphatase in infiltrating ductal carcinoma and non-neoplastic breast. The histochemical reaction for adenosine triphosphatase (ATPase) has previously been used to differentiate myoepithelial from epithelial cells in the breast and to investigate the possible contribution of myoepithelial cells to mammary carcinoma. Discrepancies in published reports prompted this study of ATPase in non-neoplastic breast and infiltrating ductal carcinoma. ATPase was localized mainly on myoepithelial cells of normal breast and was identified with significant frequency on epithelial cells in hyperplastic ducts. Infiltrating ductal carcinomas usually displayed a variable reactivity. In one instance, malignant cells demonstrating mucin production were found to be ATPase-positive. An infiltrating ductal carcinoma of the papillary type with apocrine features was also strongly ATPase-reactive. It is concluded that ATPase is not an exclusive marker of myoepithelial cells and, therefore, data resulting from the use of this enzyme to study the role of the myoepithelium in mammary carcinoma must be interpreted with caution.


Primary liver cell carcinoma in the presence or absence of hepatitis B antigen. The clinical course and pathological patterns of a group of 13 patients with both primary liver cell carcinoma and Hepatitis B surface antigen (HBsAg) are described and contrasted with those of 43 patients with primary liver cell carcinoma but without HBsAg. HBsAg-positive carcinoma patients demonstrated a higher incidence of splenomegaly, transudative ascites, and the presence of alpha-fetoprotein, although none of these reached statistical significance. Serum bilirubin was significantly higher in patients with HBsAg. HBsAg-positive carcinoma patients most frequently originated from countries where the presence of HBsAg is high in the general population. Survival time from the diagnosis of primary liver cell carcinoma was shorter in patients with HBsAg.


Effect of prostaglandins on adenylate cyclase activities in membranes from liver and transplantable hepatomas. The effects of prostaglandins on adenylate cyclase activity have been examined in membranes purified from normal rat liver and from a series of Morris hepatomas. Prostaglandin E1 gave the greatest stimulation (up to two-fold) in all membranes. However, prostaglandins A1, A2, and F2alpha, although stimulatory in liver and four tumor membranes, were inhibitory of adenylate cyclase activity in membranes from two of the fast-growing tumors. Arrhenius plots yielded broken line curves (at 20 degrees C) for the basal activity of all enzymes. Addition of various prostaglandins caused shifts in the broken line curves and/or produced nonbroken (straight) line curves for the liver and many of the hepatoma adenylate cyclases.


A relative deficiency of cytochrome P-450 and aryl hydrocarbon [benzo(a)pyrene] hydroxylase in hyperplastic nodules induced by 2-acetylaminofluorene in rat liver. The concentrations of cytochrome P-450 and the activities of aryl hydrocarbon [benzo(a)pyrene] hydroxylase (AHH) and reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase were measured in early (gray-white) and remodeled (brown) hyperplastic nodules induced in the livers of rats with 2-acetylaminofluorene and were compared to the values in control livers and in the liver surrounding the nodules. Cytochrome P-450 content of early (14 weeks) hyperplastic nodules is 30% of the activity of untreated control livers and 48% of the activity of the surrounding liver. AHH activity of the  early nodules is 10% of the control activity and 33% of the activity in the surrounding nonnodular liver. Nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity in the microsomes of early nodules is 76% of the control activity and 78% of the activity in the surrounding liver. In the late remodeled nodules, (22 and 25 weeks), the cytochrome P-450 content is 40% of that of controls and AHH activity is 15% of the control activity. In primary hepatomas induced by 2-acetylaminofluorene, cytochrome P-450 content is 21% of that of controls, AHH activity is 11% of the activity of controls, and reductase is 50% of the control activity. These results, indicating a relative nodule deficiency in some of the cellular components believed to be important in the activation of hepatocarcinogens and hepatotoxins, offer one possible explanation for the relative resistance to carcinogen cytotoxicity of hyperplastic liver nodules.


Virus-infected avian cell lines established in vitro. Four virus-infected avian cell lines have been established in culture. Two of these lines, infected with BAI strain A virus, liberate only small quantities of virus in the culture fluid. The cells retain the ability to induce myeloblastic leukemia when inoculated i.v. into 1- to 2-day-old chicks, but do so less efficiently than freshly obtained myeloblasts. These cells do not appear to be transplantable, since the disease produced is characterized by the presence of myeloblasts that liberate large quantities of virus. The other two cell lines, infected with the MC29 strain of avian leukosis virus, liberate normal levels of infectious virus in the culture fluid. When these cells are inoculated into the wing web of 1- to 2-day-old chicks, tumors develop at the site of inoculation which are detectable as early as 4 to 7 days after challenge. Chromosome studies demonstrate that the four cell lines have karyotypes typical of Gallus domesticus. The myeloblastic cell lines (D.U. 11157 and D.U. 1765) show a reduction in the number of microchromosomes. These cell lines have been carried in continuous culture for various lengths of time, can be frozen, are easily recovered in viable form, and appear to be capable of indefinite growth.


Prolactin receptors in 7,12-dimethylbenz(a) anthracene-induced mammary tumors following endocrine ablation. We have determined the effect of ovariectomy and hypophysectomy on prolactin receptors in 7,12-dimethylbenz(a)anthracene-induced mammary tumors. Growing tumors from intact rats show a wide range in the number of prolactin receptors. Ovariectomy causes a slight (approximately 30%) decrease in receptors regardless of whether tumors regress or continue to grow, while the affinity of the receptor for prolactin remains unchanged. Hypophysectomy, which causes a prompt 10-fold decrease in prolactin receptors in rat liver, causes only a slight reduction in prolactin receptors in tumors from these same animals. We conclude that autonomous and ovariectomy responsive 7,12-dimethylbenz(a)anthracene-induced mammary tumors cannot be distinguished on the basis of prolactin receptor sites and that endocrine regulation of prolactin receptors is distinctly different in normal liver and neoplastic mammary tissue.


Cyclic adenosine 3':5'-monophosphate phosphodiesterase activity in normal and chronic lymphocytic leukemia lymphocytes. The specific activity of cyclic adenosine 3':5'-monophosphate phosphodiesterase was measured in lymphocytes isolated from the blood of normal subjects, from patients with chronic lymphocytic leukemia, and from tonsil tissue. The mean specific activity of cyclic adenosine 3':5'-monophosphate phosphodiesterase in the lymphocytes from patients with untreated chronic lymphocytic leukemia was lower than that in lymphocytes from the blood of normal subjects or from tonsils. Cyclic adenosine 3':5'-monophosphate phosphodiesterase levels did not correlate with differences in B- and T-cell lymphocyte subpopulations or with peripheral blood lymphocyte counts.


Histogenesis and Morphology of periosteal sarcomas induced by FBJ virus in NIH Swiss mice. FBJ virus was injected i.p. into 145 neonatal NIH Swiss [N:NIH(s)] mice. Eighty mice developed a total of 110 neoplasms by 5 months of age. The mean latent period of the tumors was 71 days (26 to 145) postinjection. The frequency of occurrence of neoplasms at different sites was: diaphragm, 45%; ribs, 14%; vertebrae, 14%; femora, 9%; pelvic bones, 5%; tibiae, 4%; sternebrae, 3%; and inguinal area, 7%. The neoplasms were characterized histologically by elongated or rounded cells associated with an abundant connective tissue stroma. Occasional areas of bone formation and apparent osteoid metaplasia were seen. Bone tumors appeared to arise from periosteal cells, to grow by expansion, and to invade locally, but they failed to metastasize. Neoplasms of the diaphragm originated in the central aponeurosis and appeared histologically similar to bone neoplasms. Histochemical studies demonstrated abundant alkaline phosphatase in tumor cells, and ultrastructural observations revealed subcellular characteristics of osteoblasts and chondroblasts. Tumors were readily transplantable and had histopathological characteristics similar to those of the primary viral-induced tumors. The results of this study indicate that the FBJ virus induces in NIH Swiss mice a unique type of chondroosseous neoplasm derived from periosteal cells which has a resemblance to human juxtacortical (parosteal) osteosarcoma.


Virus-dependent cytostatic activity to mammary tumor cells of lymphocytes from normal mice. A cytostasis assay has been used to study the natural immunity of mice to murine mammary tumor virus (MTV). Spleen cells from adults of all strains tested were found to be cytostatic to a variety of MTV-positive mammary tumor cell lines. Newborn spleen cells were unreactive in the same cytostasis assay. The degree of reactivity to the target cells was greater in spleen cell preparations from low MTV expressors than from syngeneic, high MTV expressors. The cytostasis was specific, since MTV antigens prepared from gradient-purified whole MT virions significantly blocked the reaction. In addition, spleen cells were totally nonreactive to MTV-negative cell lines. Other types of lymphoid cells, such as lymph node cells as well as peritoneal macrophages, were highly cytostatic under similar conditions. Spleen cells from nude athymic donors were not cytostatic. Since depletion of splenic T-lymphocytes by use of anti-theta serum also did not significantly affect cytostasis, it was concluded that T-cells were required for initiation of immunity to MTV but that the effector cells were not theta positive. Blocking factors were found to exist in the sera of mammary tumor-bearing animals that prevented cytostasis by reactive spleen cells.


Mode of action of the bioreductive alkylating agent, 2,3-bis(chloromethyl)-1,4-naphthoquinone. The bioreductive alkylating agent, 2,3-bis(chloromethyl)-1,4-napthoquinone (CMNQ), has been shown to inhibit the growth of Sarcoma 180 ascites cells in vivo. Evidence for the reductive activation of this agent via the mitochondrial respiratory chain was provided by CMNQ-induced oxidation of reduced nicotinamide adenine dinucleotide; the interaction was shown to be on the substrate side of the site of rotenone inhibition. Consistent with the concept that reduction of CMNQ to a hydroquinone results in the generation of an alkylating species (i.e., a quinone methide) was the finding that radioactivity from [14C]CMNQ present in Sarcoma 180 ascites cells was associated with DNA, RNA, and protein for a period of up to 72 hr after exposure to tumor-bearing animals to this agent. Inhibition of the incorporation of [3H]thymidine, [3H]uridine, and [14C]leucine into DNA, RNA, and protein, respectively, of Sarcoma 180 ascites cells was produced by this agent, with DNA biosynthesis being the most susceptible. The inhibitory effect of CMNQ on the formation of DNA was, at least in part, the result of a prevention of the conversion of thymidine to its nucleotide forms. This action was due to (a) a drug-induced decrease in intracellular levels of adenosine 5'-triphosphate, presumably resulting from uncoupling of oxidative phosphorylation by CMNQ; and (b) a partial loss of thymidine kinase activity in Sarcoma 180 cells, which did not appear to be due to direct inhibition of the enzyme by the drug. Although the primary event produced by CMNQ at the mitochondrial level appeared to be release of respiratory control, other effects of mitochondrial metabolism occurred. These included inhibition of reduced nicotinamide adenine dinucleotide and succinoxidase activities, as previously demonstrated, and mitochondrial swelling, which suggested interaction of CMNQ with the inner mitochondrial membrane. These findings indicate a variety of biochemical lesions are associated with the antineoplastic activity of CMNQ and demonstrate a relationship between the effects of this drug on mitochondrial respiratory control and DNA biosynthesis.


The mechanism of 5-fluorouridine toxicity in Novikoff hepatoma cells. The mechanism of 5-fluorouridine (FUrd) cytotoxicity in Novikoff hepatoma cells appears to vary under different experimental conditions. Continuous exposure to 1 X 10(-7) M FUrd results in a simple thymineless death that can be prevented by the addition of 1 X 10(-4) M thymidine to the culture medium. In contrast, 1 X 10(-4) M thymidine does not prevent the growth inhibition caused by a 1-hr exposure to 1 X 10(-5) M FUrd, despite the fact that it does prevent the inhibition of DNA synthesis. Although it has no effect on the inhibition of DNA synthesis, 1 X 10(-4) M uridine prevents the growth inhibition caused by a 1-hr exposure to 1 X 10(-5) M FUrd. Since 1 X 10(-4) M uridine, but not 1 X 10(-4) M thymidine, prevents the inhibition of ribosomal RNA maturation caused by a 1-hr exposure to 1 X 10(-5) M FUrd, it seems likely that the effects of the analog on RNA metabolism contribute significantly to the cytotoxic activity of the analog in this specific experimental situation.


Cellular hypersensitivity of gp55 of RIII-murine mammary tumor virus and gp55-like protein of human breast cancers. Previous studies suggested that immunogenic breast cancer tissues contained a component(s) that is antigenically similar to some component of murine mammary tumor virus (MuMTV) and resembles the glycoprotein, M.W. 55,000 (gp55), of RIII-MuMTV in molecular weight and charge density. This investigation measured in vitro cellular hypersensitivity responses of breast cancer patients to RIII mouse milk, purified RIII-gp55, C3H-MuMTV, autologous and homologous breast cancer tissues, gp50 of A-MuMTV, and preparations of Rauscher leukemia virus and Mason-Pfizer monkey virus. Particular attention was paid to cross-reactivity between gp55 and the other targets. The data indicate that responsiveness to C3H-MuMTV and RIII milk are linearly correlated with responsiveness to gp55. A preferential relationship was demonstrable between responses to gp55 and to those breast cancer tissues containing a gp55-like protein component (S-p50). The critical role of a gp55-like protein as the antigen responded to by breast cancer patients' in leukocytes was also suggested by the ability of anti-gp55 antiserum to decrease leukocyte responsiveness to RIII-gp55, C3H-MuMTV, and breast cancer tissues. In vitro cellular hypersensitivity against RIII-gp55 was preferentially found in prognostically favorable cases with immunogenic lesions. Further studies are needed to test the possibility that gp55 might be of value in the immunodiagnosis of early breast cancer, the monitoring of prognostically significant cellular hypersensitivity, and the induction of such hypersensitivity (immunoprophylaxis).


The metabolism-dependent maintenance of cell volume and ultrastructure in slices of Morris hepatoma 3924A. The changes in water and electrolyte content of slices of Morris hepatoma 3924A induced by various conditions of incubation have been compared with the ultrastructural appearance of the tissue. Incubation at 1 degrees led to an increase of water, Na+, and Cl- content and to a loss of K+. There was simultaneous increase in size of the cells and intercellular spaces, loss of junctional complexes, increase in the number of microvilli, and fragmentation and dilation of the cytocavitary network. Subsequent incubation at 38 degrees in oxygenated medium led to a substantial reversal of all of these changes of composition and structure, which was well advanced within 10 min and largely complete by 60 min. The presence of 20 mM glucose in the medium somewhat enhanced the degree of recovery. A reduction of cell volume and intercellular spaces was evident both from the electron microscopic observations and measurements of the volume of inulin distribution. The presence of ouabain inhibited the net accumulation of K+ and much of the Na+ extrusion, but permitted about 50% of the net extrusion of water (accompanied by Na+ and Cl-) and had little effect on the ultrastructural recovery. The presence of glucose increased the resistance of volume and structural recovery of ouabain without releasing the inhibition of K+ accumulation. A marked feature of the recovering tissues was the Golgi apparatus, which assumed an appearance suggestive of increased activity when water extrusion was active. In slices using only endogenous substrate, cyanide and (to a lesser extent) oligomycin greatly inhibited the recovery of volume and structure. The presence of glucose permitted some recovery in the presence of cyanide. The control of cell volume in hepatoma 3924A appears to involve two separate components of water transport, one of which is sensitive, and one insensitive to ouabain. The ouabain-insensitive component appears to be especially related to the recovery of cell ultrastructure after incubation at 1 degrees, to be more sensitive to paucity of adenosine 5'-triphosphate, and to proceed by secretion of water, Na+, and Cl- into vesicles that fuse with the Golgi apparatus. This mechanism may be related to that for bile secretion in normal liver. The ouabain-sensitive component of water transport is a function of the mechanism for the coupled transport of Na+ and K+.


Interaction of Na+ and K+ transport with aerobic energy metabolism in slices of Morris hepatoma 3924A. Addition of increasing concentrations of glucose to slices of Morris hepatoma 3924A greatly stimulated aerobic lactate production and reduced respiration by 20%. Neither the adenine nucleotide content of the slices nor the calculated rate of adenosine 5'-triphosphate synthesis was altered. Ouabain reduced the rate of O2 uptake (by 20 to 25%) and of aerobic lactate production (by 25 to 50%) without affecting adenine nucleotide contents. The reduction by ouabain of the calculated rate of adenosine 5'-triphosphate synthesis was similar whether the slices were utilizing only endogenous substrate or exogenous glucose also. Raising the medium K+ concentration (and correspondingly reducing Na+) partially overcame the inhibition of ion transport by ouabain and partially restored the rates of respiration and aerobic lactate production toward control levels. Electron microscopic observations of mitochondria within the slices incubated under different conditions showed variations in configuration between "orthodox," "condensed" and degenerating forms. Slices preincubated at 1 degrees showed mitochondria in the condensed form: they were restored to the orthodox configuration during incubation at 38 degrees in oxygenated medium. Oligomycin and glucose enhanced the transition, but ouabain reduced the number of mitochondria undergoing the change. The results suggest that in hepatoma 3924A utilization of adenosine 5'-triphosphate by ion transport exerts a simultaneous control of both respiration and aerobic glycolysis, which is presumably mediated by alterations in the availability of adenosine 5-diphosphate. The mitochondria undergo conformational transitions under conditions likely to affect local availability of adenosine 5'-diphosphate within cell compartments, but the transitions are not all externally added adenosine diphosphate on isolated mitochondria.


In vitro differentiation of teratomas and the distribution of creatine phosphokinase and plasminogen activator in teratocarcinoma-derived cells. Mouse teratocarcinoma cells from embryoid bodies were cultured in vitro to permit their differentiation into a number of cell types. Two enzyme activities, creatine phosphokinase (CPK) and the protease plasminogen activator, were studied to follow the developmental sequence of events in these embryoid body-derived cell cultures. CPK activity increased with time in culture, indicating the appearance of new cell types with brain- or muscle-specific enzyme activities. Plasminogen activator was detectable in extracts of embryoid bodies. This protease activity first increased and then decreased to a low level as the embryoid bodies in culture developed into differentiated cell types. These cell cultures also showed a decreased potential for tumor formation in syngeneic mice as a function of time in culture. This decrease in tumorigenic potential was correlated with the appearance of differentiated cells in vitro. Simian virus 40 (SV40) was used to infect and transform cells derived from embryoid bodies in culture. This was done to permit the establishment of cloned teratocarcinoma-derived cell lines. Twenty-nine distinct cloned permanent cell lines (called SVTER) containing the SV40-specific tumor antigen were obtained. None of these cell lines was capable of producing tumors in syngeneic mice. An analysis of the levels of creatine phosphokinase and plasminogen activator in these SVTER cell lines indicated that : (a) some cell lines had high CPK activity and little or no plasminogen activator activity, (b) some cell lines contained high levels of plasminogen activator activity with little or no CPK activity, and (c) some cell lines contained neither of these enzyme activities. No example of a cell line with high levels of both enzyme activities was observed, indicating that these two enzymes may participate in mutually exclusive developmental pathways. The SVTER cell lines may therefore be useful in reconstructing these developmental pathways in vitro.


Genes responsible for transformation by avian RNA tumor viruses. Avian sarcoma viruses probably contain 4 genes. One of these, src, controls the process of transformation of the infected cell and has been located near the 3' end of the viral genome. The remaining 3 genes direct the synthesis of virion proteins: env (envelope proteins), pol (polymerase), and gag (internal proteins). They are probably in the order gag-poly-env-src. A scheme for the replication of these viruses is proposed, and several possible mechanisms for their origin and variability are discussed.


Homochromatographic and immunological analysis of controls of nucleolar gene function. To compare regulation of nucleolar function of tumors and other tissues, it was necessary to develop assays of the fidelity of ribosomal DNA readouts. For this purpose, homochromatography analyses of complete T1 ribonuclease digestion products of the in vivo labeled 45 S preribosomal RNA were compared with those of 18S and of 28 S ribosomal RNA. Homochromatography analysis of the in vitro readout product of isolated nucleoli showed the presence of many large marker nucleotides of the in vivo 45 S preribosomal RNA. Moreover, no other large oligonucleotides were detected. The in vitro readout product of nucleolar chromatin had the same T1 ribonuclease digestion products, including the large marker of oligonucleotides. However, the in vitro readout product of nucleolar DNA contained no large marker T1 ribonuclease oligonucleotides. These results indicate that the fidelity of nucleolar readouts is controlled by regulatory proteins of the nucleolar chromatin. Differences were found in nucleolar proteins of normal rat liver and Novikoff hepatoma by immunological analyses. The possibility exists that differences in readout rates of tumor and other nucleoli are related to the protein difference detected by these immunological studies.


Fatty acid oxidation, substrate shuttles, and activity of the citric acid cycle in hepatocellular carcinomas of varying differentiation. Fatty acid oxidation, reconstituted substrate shuttles, and the activity of the citric acid cycle were studied in mitochondria isolated from Becker transplantable hepatocellular carcinoma H-252 AND Host livers, and the results were compared with those obtained with Morris hepatomas 7288CTC and 5123C. Whereas the activities of the malate-aspartate and the alpha-glycerophosphate shuttles were only slightly lower than those of host livers, the activity of the fatty acid shuttle was much lower in H-252 mitochondria. Oxygen uptake and CO2 production associated with the oxidation of fatty acids was much lower in tumors H-252 and 7288CTC, compared with host livers, whereas tumor 5123C mitochondria show a high capacity to oxidize fatty acids. Ketogenesis and beta-hydroxybutyrate dehydrogenase activity were also lower in tumor H-252 mitochondria. However, neither oxygen uptake associated with the oxidation of other respiratory substrates nor CO2 production from succinate or malate was strikingly elevated in these tumors. These factors suggest that the respiratory phosphorylation chain and activity of the citric acid cycle are fully functional in tumors H-252 and 7288CTC. The defects responsbile for the lower rates of fatty acid oxidation in these tumors probably involves the beta-oxidation pathway, as well as the activation of fatty acids. The impairment of fatty acid oxidation may explain the lower activity of the reconstituted fatty acid shuttle for transporting reducing equivalents into H-252 mitochondria. The different properties with regard to fatty acid oxidation in Morris hepatoma 5123C, compared with those in Becker H-252- AND Morris hepatoma 7288CTC, may reflect the different extent of differentiation in these tumors, the former being a slow-growing, well-differentiated tumor, whereas the latter represent tumors that are less differentiated and of more rapid growth rate.


The limiting effect of transfer RNA's on the rate of protein synthesis in cell extracts of rapidly growing tumors. Using crude cell extracts from rapidly growing animal and human tumors, we found that (a) the addition of homologous transfer RNA (tRNA) to these extracts stimulated polypeptide synthesis two-to threefold, while addition of heterologous tRNA did not have a similar effect; (b) addition of homologous as well as heterologous ribosomal RNA was also stimulatory; and (c) both stimulatory effects were additive. The possibility that the effect of homologous tRNA could be mediated by contaminating material (such as the "translational control" RNA) seems to be rulted out by experiments with highly purified tRNA preparations, which did not contain even traces of 18 S, 7 S, 5 S, and smaller than 4S RNA's. Control experiments showed that no loss of tRNA occurred either during preparation of the cell extracts or under the conditions of in vitro protein synthesis. The results obtained suggest possible occurrence of a deficiency in specific isoaccepting tRNA's in rapidly growing solid tumors.


Influence of vaccination with A/PR 8/34 (HO-N1) influenza virus on the oncogenic activity of polyoma virus in newborn Wistar rats. Vaccination with the A/PR 8/34 (HO N1) strain of influenza virus can decrease significantly the oncogenic activity of polyomavirus in newborn Wistar rats. This effect was regularly observed when animals were vaccinated with 24 hr after birth, whereas vaccinations performed on Days 3 and 10 failed to influence the oncogenic potential of polyoma virus. In 3 series of experiments, influenza virus vaccination resulted in sigificantly decreased tumor rates, and, in a 4th series, in reduced tumor growth only. Heat inactivation of influenza virus failed to abolish its influence on the oncogenicity of polyoma virus. In most of the experiments, the antibody response to influenza virus was enhanced by inoculation of animals with polyoma virus. In contrast, polyoma virus antibody titers were, in some experiments, decreased, and, in others, not influenced by vaccination with influenza virus.


Analysis of the transfer RNA population of mouse mammary glands infected with a latent mammary tumor virus. Mammary gland transfer RNA's  (tRNA'S) of CEH mice infected with mammary tumor virus were analyzed in the preneoplastic state and compared to tRNAs of virus-free C3Hf mice and another uninfected strain, C57BL/6, which is completely resistant to cancer. This quantitative study was based on the ability of each tRNA to fix its corresponding amino acid. The amount of each of the 17 tRNA's tested was identical for the three mammary glands. In addition, tRNA populations during lactation correlated with the amino acids incorporated into the lactoproteins synthesized, which indicates adapation of the tRNA's to protein biosynthesis. Qualitative chromatographic studies on reverse phase capillary columns Type 5 of 10 aminoacyl-tRNA's did not reveal any difference in the isoacceptor elution profiles. This shows that no new isoaccepting tRNA is associated with the mammary tumor virus at that stage, and that no viral modification of a host tRNA has occurred.


Biosynthesis of albumin via a precursor protein in Morris hepatoma 5123tc. The mechanism of albumin biosynthesis was studied in Morris hepatoma 5123tc in vivo and in hepatoma cell suspensions obtained by solubilizing the intercellular matrix with collagenase and hyaluronidase. In the in vivo experiments, L-[-14C]leucine was injected i.v. into rats bearing hepatomas in the muscles of both hind legs. After 14 min, tumors were removed and homogenized. A protein fraction quantitatively precipitable with antialbumin was isolated from the homogenate by acetone fractionation and precipitation with antiserum against serum albumin. This protein fraction was not homogeneous. With the use of 3 consecutive chromatographies on diethylaminoethyl cellulose, a very highly radioactive albumin-like protein could be separated from a large amount of only slightly radioactive albumin. In hepatoma cell suspensions incubated with L-[1-14C]leucine followed by a chase with excess nonradioactive L-leucine, radioactivity was incorporated first into the albumin-like protein and transferred thereafter into albumin, suggesting that albumin was synthesized via the albuminlike protein as precursor. In vivo, 1.8% of newly synthesized hepatoma protein was albumin or its precursor, compared with 1.2% in cell suspensions.


Prognostically significant protein components of human breast cancer tissues. Cryostat sections of clinicopathologically characterized breast cancer tissues were eluted with phosphate-buffered 0.9% sodium chloride solution, pH 7.2. The proteins were then characterized by polyacrylamide gel electrophoresis with  and without prior treatment with sodium dodecyl sulfate. Approximately 65% of the brease cancer tissue eluates contained a prominent protein fraction with a molecular weight of 47,000 to 55,000 (p50). No such component was found in 15 of 17 eluates of benign breast tissue. Charge density studies disclosed that the p50 component included three populations of proteins that could be characterized according to the migration relative to gp55 derived from  RIII murine mammary tumor virus, namely, fast (F-p50), intermediate (I-p50), and slow (S-p50). Prognostically favorable pathological characteristics, i.e., stage, nuclear grade, and lymphoreticuloendothelial responses, were proportionately most frequently found among S-p50 bbreast cancers and were least frequently found among F-p50 breast cancers. It appears that the S-p50 component acts in vivo as a prognostically significant immunogen. Further knowledge of the relationship between protein characteristics and clinicopathological features of human breast cancers would contribute to our understanding of mammary carcinogenesis and biological behavior.


Loss or persistence of the differentiated state of simian virus 40-induced hamster tumor cells before and after serial passage in culture. The transformed cells that arise from among the hamster epithelial and mesenchymal cells exposed to SV40 in vitro are, as a rule, fibroblastoid and pleomorphic rather than epithelioid. Moreover, the neoplasms that these transformed cells induce in the allogeneic host are spindle cell sarcomas and pleomorphic sarcomas rather than carcinomas. Since this phenomenon may result from cellular dedifferentiation in culture, to the extent that the anaplastic morphology and lack of specialized function can no longer suggest the cell or origin, we investigated the fate of the differentiated state of cells of three types of SV40-induced hamster tumors before and after serial passage in vitro. The tumors evaluated were three reticulum cell sarcomas, three osteogenic sarcomas, and two lymphosarcomas of B-cell origin. Our data demonstrate that reticulum cell sarcoma cells lose their morphological differentiation soon after the original tumors are dissociated into cell suspensions but preserve their phagocytic activity throughout their in vitro passage. Osteogenic sarcoma cells lose their differentiated phenotype and their capacity to form osteoid during but not before their serial passage in culture. Lymphosarcoma cells preserve their lymphoid morphology and their ability to produce immunoglobulin even after many in vitro passages. These results indicate that, in many types of SV40-induced tumors, neoplastic cell dedifferentiation, following serial passage in culture, is responsible to a great extent for the emergence of new cell phenotypes lacking in morphological and functional features characteristic of the cells originally transformed by SV40.


A cyclic adenosine 3':5'-monophosphate-mediated effect of cholera toxin on high-molecular-weight glycoprotein species of malignant cells. A comparison of the Pronase-sensitive glycosylated species detectable under permissive and nonpermissive conditions by normal rat kidney cells transformed by a temperature-sensitive derivative of Rous sarcoma virus reveals relative decreased labeling of high-molecular-weight glycosylated species under conditions that allow the expression of transformation, in medium supplemented either with 0.5% calf serum or with human alpha2-macroglobulin, 100 mug/ml. Exposure of the cultures to cholera toxin or dibutyryl cyclic adenosine 3':5'-monophosphate leads to a relative increase in the glycosylation of the high-molecular weight species in both wild-type transformed and temperature-sensitive cells exposed to conditions that allow the expression of transformation.


Detection of the major glycoproteins of Friend leukemia virus (gp71) and the murine mammary tumor virus (gp52) on the surface of mouse cells. Specific rabbit antisera to the major glycoproteins of Friend leukemia virus (gp71) and the mouse mammary tumor virus (gp52) were utilized to study the surfaces of C3H-, DBA-, BALB/c-, and C57BL-transformed and normal cells by immunoelectron microscopy. Antiserum to gp71 showed reactivity with all of the mouse cells tested regardless of strain, virus production, or state of transformation. In cells producing murine leukemia virus, budding viruses and other areas of the cell surface were consistently labeled with gp71 antiserum. A gp52-like antigen was likewise detected on both cell surfaces and virions of C3H and DBA cells producing the mammary tumor virus. Budding virions with surface spikes but with crescent-shaped nucleoids in C3H/HeJ cells were labeled specifically with gp52 antiserum. The antigen localized with anti-gp52 serum was detected in low concentration on the surface of nonvirus-producing cultures of a C57BL/6 sarcoma induced by the Schmidt-Ruppin D strain of Rous avian sarcoma virus (SRD-2), a BALB/c bone marrow culture (JLS-V9), and a normal BALB/c fibroblast culture (BALB/cF). Other cell cultures transformed by either C-type virus or methylcholanthrene failed to demonstrate gp52 antigen. Both gp52- and gp71-like antigens were found to be expressed simultaneously in C3H/HeJ, C3H-MT, DBA-MT, SRD-2 (transformed) and BALB/cF, JLS-V9, and C3H-1 (normal) cultures. Expression of gp52 antigen in the absence of gp71 was not detected in any of the cultures examined. These findings demonstrate the ubiquitous expression of gp71 in a wide variety of normal and transformed mouse cells while gp52 tends to be expressed predominantly in cells from mice with high mammary tumor incidence (C3H) and DBA), but only to a minor extent in cells from low mammary tumor incidence strains (BALB/c and C57BL).


Protein phosphotransferase activities and cyclic nucleotide action in proliferating lymphocytes. Cyclic nucleotide levels, protein phosphotransferase activities, and cyclic nucleotide-binding proteins have been determined and partially characterized in the mouse lymphosarcoma P1798. This system is used as a model to understand the function of these activities in a rapidly proliferating cell. Adenosine 3':5'-monophosphate (cAMP) concentrations are 5-fold higher in the lymphosarcoma cells than in thymocytes. In both the thymocytes and malignant tissue, cAMP concentrations are increased by physiological concentrations of epinephrine and prostaglandin. The guanosine 3':5'-monophosphate (cGMP) level in the lymphosarcoma is 0.1 pmole/10(6) cells and is not modified by acetylcholine, prostaglandin F2alpha, or concanavalin A. Four protein phosphotransferase activities have been identified in the lymphosarcoma. These are the cAMP-dependent protein kinase type I and II isozymes and a "histone kinase" and a "phosvitin kinase"; neither of the latter two is regulated by cyclic nucleotides. Characterization of these enzymes was based on fractionation by DE 52 chromatography, substrate specificity, interaction with the protein inhibitor of cAMP-dependent protein kinases, and sucrose gradient sedimentation rates. Both the cAMP-dependent protein phosphotransferase activity and the phosvitin phosphotransferase activity are 2-to 4-fold elevated in the lymphosarcoma cells in comparison to thymocytes. cAMP binding is associated with both the type I and II isozymes and with a fraction tentatively designated as the regulatory subunit of these enzymes. cGMP also binds to this later fraction and to the partially purified fraction containing the type IcAMP-dependent enzyme. The histone phosphotransferase activity of this fraction is also stimulated by cGMP, but studies of the number of binding sites and of absorption to cAMP and cGMP affinity resins indicated that this fraction contains more than one species of cyclic nucleotide-binding protein.


Alteration of mitochondrial function and lipid composition in Morris 7777 hepatoma. Mitonchondria isolated from the Morris hepatoma 7777 demonstrated a markedly different phospholipid composition from those of control mitochondria, both with respect to the amounts of the various types present and the fatty acid composition. The level of polyunsaturated fatty acids in the mitochondrial phospholipids was lowered, wheras there was an increase in the level of monounsaturated fatty acids. Moreover, the usual distribution of saturated fatty acids at position 1 and polyunsaturated fatty acids at position 2 does not exist in hepatoma phospholipids; a high percentage of monounsaturated fatty acids was found at both positions. The cardiolipin content was lower in hepatoma mitochondria (3.7%) than in livers of animals with hepatomas (5.2%). There was, however, some compensation in the amount of acidic phospholipids in these mitochondria due to an increase in phosphatidylserine (4.9% versus 1.3%). The force-area curves of the hepatoma phospholipids spread on a monomolecular film demonstrated a smaller area per  molecule than those from liver mitochondria. The zeta potential of liposomes of the hepatoma phospholipids (-45) was less than those of control mitochondria (-81), as determined by microelectrophoresis. The calcium-stimulated phospholipase A activity of the hepatoma mitochondria appeared to be more readily expressed than the same activity in liver organelles. The maximal activity was lower, however, than that noted in liver mitochondria. Furthermore, by following the incorporation of [3H]ethanolamine into mitochondria phospholipids, it was established that the conversion of glycerophosphorylethanolamine to glycerophosphorylcholine was increased in the hepatoma. These observations suggest dramatic changes in phospholipid metabolism in the hepatoma, at the level of both the endoplasmic reticulum and the mitochondrion. Accompanying the changes in phospholipid compositon and metabolism were alterations in mitochondrial energy-linked processes. The hepatoma mitochondria demonstrated lower respiratory control ratios even when isolated in an isotonic solution containing 1mM ethylenediaminetetraacetate and bovine serum albumin (0.5 mg/ml). This was due to increased state 4 respiration.


Susceptibility of xeroderma pigmentosum cells to transformation by murine and feline sarcoma viruses. Among various strains of skin fibroblasts tested, two strains derived from xeroderma pigmentosum (XP) patients (ages 19 and 25) with neurological complications and two strains obtained from heterozygotes (ages 54 and 18) showed relatively higher susceptibility than normal age-matched controls to transformation by feline sarcoma virus (FSV). Only one strain from a normal individual also showed a high susceptibility. Generally, there was a parallelism in susceptibilities to FSV and Kirsten murine sarcoma virus (KiMsv). However, cells from normal individuals of 46 years or older exhibited high ratios of FSV:KiMSV titers which were due to their lower susceptibility to KiMSV. Cells from two XP patients (ages 25 and 22) and a heterozygote (age 18), who were in a younger age group, manifested such a differential susceptibility to FSV and KiMSV. There was a correlation between the relative sensitivity of XP cells to the cytotoxic effect of 4-nitroquinoline 1-oxide and killing effect of UV light. Pretreatment of fibroblasts from three XP patients by a subtoxic dose of 4-nitroquinoline 1-oxide 24 hr before viral infection facilitated transformation by KiMSV and FSV, whereas no such effect was observed with three normal cells strains similarly treated.


Prolactin receptors and androgen-induced regression of 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma. Prolactin reverses the inhibitory effects of pharmacological doses of androgen on 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth (Quadri, S.K., Kledzik, G.S., and Meites, J.J. Natl. Cancer Inst., 52: 875-878,1974). To determine whether this effect is due to an alteration in the ability of the tumor cell to bind prolactin, we have quantitated prolactin receptors in androgen-responsive and nonresponsive tumors. Prolactin receptors were measured with 125I-labeled ovine prolactin in a subcellular fraction which reproducibly contained 60 to 80% of the total receptor present in tumor homogenates. Prolactin binding was reversible, reached a steady state in 9 hr, and was completed by excess unlabeled prolactin. Prolactin bound to its receptor with a Kd of approximately 1 X 10(-10) M. Growing tumors were biopsied, and rats bearing regrown tumors were given injections of 4 mg testosterone propionate twice a week. Prolactin receptors were reduced in most of the tumors, which regressed after testosterone treatment by an average of 63% compared to the pretreatment biopsy specimens. Nonresponsive tumors and vehicle-injected controls showed no signifcant alterations in receptor content. This reduction of prolactin receptors is probably insufficient to account for androgen-induced mammary tumor regression.


Antigenically active nonhistone chromatin proteins in cancer cells. Two-dimensional polyacrylamide gel electrophoresis shows that in nuclei of Novikoff hepatoma ascites cells there are approximately 75 proteins in the chromatin fraction soluble in 3 M NaCl:7 M urea. Dialysis of this fraction to an ionic strength of 0.15 produces a soluble fraction and a precipitate. The proteins in the soluble fraction have been reported to be active in gene control. Antibodies to the soluble fraction distribute diffusely throughout the nucleus, and antibodies to the precipitate localized primarily in the nucleolus and the nuclear ribonucleoprotein network. The nucleolar proteins differ from the extranucleolar proteins in antigenicity and labeling patterns. The development of methods for isolation, purification, and identification of nuclear proteins provided the opportunity for analysis of chromatin antigens in tumor cells. Utilizing two-dimensional preparative polyacrylamide gel techniques as well as conventional procedures, several nuclear proteins have been isolated in electrophoretically homogeneous states including protein A-24, a histone-like nonhistone protein; C-14, a protein that stimulates nucleolar RNA polymerase; and a chromatin antigen soluble in 3 M NaCl:7 M urea that remains soluble after dialysis to 0.15 M NaCl to precipitate the histones and the DNA. This antigen has been found in the chromatin of both the Novikoff hepatoma and the Walker 256 carcinosarcoma but not in the chromatin of either normal or regenerating liver. It is a nonhistone nuclear protein as indicated by its amino acid analysis in which the ratio of the number of acidic to basic amino acids is approximately 1.4. Further studies are in progress on the function and structure of this chromatin protein. As an approach to analysis of relative  rates of synthesis of this antigen and otherproteins, the products of translation of messenger RNA of Novikoff hepatoma and normal liver are being analyzed by autoradiography of two-dimensional electrophoretic gels.


The effect of exposure to dibutyryl cyclic adenylic acid on the membrane antigenicity of SV40 tumor cells. When SV40 tumor cells (F5-1) in culture were treated with dibutyryl cyclic adenylic acid (DbcAMP),marked alterations occurred in their growth and morphology. Additionally, the incorporation or uptake of labeled thymidine, uridine, phenylalanine, and choline were reduced by this treatment. These modifications with DbcAMP exposure produced conditions simulating those of a contact-inhibited state. The immunization of hamsters with X-irradiated tumor cells previously cultured in the presence of DbcAMP indicated that the tumor-specific transplantation antigens and/or fetal antigens were altered during short-term expsoure to DbcAMP. Further examination of membrane antigens of SV40 tumor cells using the isotopic antiglobulin technique demonstrated a significant reduction in the tumor membrane antigen on cells cultured with DbcAMP.


Biological and biochemical properties of soluble tumor-specific transplantation antigen of a simian virus 40-induced neoplasm. We have solubilized by limited papain digestion and partially purified the tumor rejection antigen, tumor-specific transplantation antigens (TSTA), from membranes of a simian virus 40-induced sarcoma. Uniform-sized materials with a molecular weight range of 50,000 have retained their tumor rejection activities through the purification procedures. The simian virus 40 TSTA have been separated from H-2 activity by affinity chromatography on concanavalin A columns and no evidence was found for H-2 antigens in the unbound fraction (I) of concanavalin A containing TSTA activity. A reduced yield from the crude soluble fraction was observed with Fraction I of concanavalin A material and this may indeed represent fragmentation of antigen during papain digestion. These results stand in contrast to purification of histocompatibility antigens (H-2alpha) using the same methods and techniques. Low concentrations of simian virus 40 TSTA crude soluble materials were nervertheless biologically active. A concentration as low as 4 mug protein provided 50% tumor rejection and 0.1 mug protein provided lymphocyte stimulation. Both assays reflected specificity of response.


Alterations in peripheral nerves of rats treated with chlorphentermine or with iprindole. This study deals with the effects of two amphiphilic lipidosis-inducing drugs (chlorphentermine, iprindole) upon the ultrastructure of peripheral nerves of rats. After prolonged drug treatment the preterminal and terminal axoplasm of motor and sensory nerves within skeletal muscles contain numerous abnormal inclusions (osmiophilic conglomerates, autophagic vacuoles, lamellated bodies). By contrast, the axons within large peripheral nerves are little effected. The present observations are tentatively interpreted as resulting from interference with catabolic processes involved in the normal turnover of axoplasmic constituents at the nerve terminal. The exact pathogenesis and the functional significance of these alterations remain to be elucidated.