The regulation of glutamate metabolism in Candida utilis. Evidence for two interconvertible forms of NAD-dependent glutamate dehydrogenase. An earlier observation from this laboratory (J. Gen. Microbiol. 64, 423--427) that NAD-dependent glutamate dehydrogenase activity is modulated by rapid inactivation has been extended to show that this mechanism is completely reversible. Changes in properties of the enzyme accompany inactivation and two different forms active (a) and inactive (b) of the enzyme with distinctive properties have been isolated. Incubation of the inactive enzyme with magnesium in vitro produced a rapid increase of activity; this was accompanied by a change in the properties of the enzyme to those of the a form. This control mechanism of enzyme interconversion appears widespread among yeasts. Its probable role in modulating glutamate synthesis and degration is discussed.


Development of Escherichia coli virus T1: repression of host gene expression. Host protein synthesis, measured either as amino acid incorporation into proteins or as enzyme synthesis, is inhibited rapidly after infection Escherichia coli with T1. Analysis of this inhibition, using a technique which distinguishes between translation and transcription, revealed that translation of host mRNA is specifically blocked. Comparison of the time course of T1-induced host repression with inhibition by the drugs rifampicin, nitrofurantoin and chloramphenicol showed that T1 affects the initiation step of host translation. Intact membranes are apparently essential for host repression, suggesting a membrane-mediated process. Concomitant viral protein synthesis is not required. The membrane-altering principle is a constituent of the viral particle.


Mechanism of activation of the protein kinase I from rabbit skeletal muscle. The high-affinity ATP site of the holoenzyme. The high-affinity binding site for ATP of the holoenzyme of cAMP-dependent protein kinase (type I) from rabbit skeletal muscle has been investigated. Binding affinity of a series of ATP derivatives substituted at different sites in the molecule was determined by competition with tritiated ATP. The results were compared with data available from cAMP derivatives with the same substituents, in order to analyse the electronic and steric features of these two sites on the protein kinase. The comparison revealed significant differences of the effect of substituents towards the two sites. In particular the N6-derivatives of ATP and substituents affecting the gamma-phosphate indicate that the high-affinity ATP site of the protein kinase has similar properties as those found for phosphotransferase sites. The present results are consistent with the supposition that the high-affinity site for ATP on the holoenzyme is congruent with the phosphotransferase site of the catalytic subunit. Upon combination of catalytic and regulatory subunits this site would be transformed into a high-affinity site for ATP with simultaneous blocking of the phosphotransferase activity.


Effect of borate on the catalytic activities of muscle glyceraldehyde-3-phosphate dehydrogenase. The effect of borate on glyceraldehyde-3-phosphate dehydrogenase from human, pig and rabbit muscle was studied. At lower concentration of borate only the dehydrogenase activity is inhibited, reversibly and competitively against NAD. At concentration of borate above 6 mM the plots of 1/v versus borate concentration become nonlinear and the inhibition is extended to the esterase and acetylphosphatase activities. In certain conditions a time-dependent inactivation and reactivation was observed. The direct interaction between borate (if present at concentration of at least 6 mM) and glyceraldehyde-3-phosphate dehydrogenase is postulated, the possible site of the reaction being the histidine residue(s). The esterase activity of the human muscle enzyme and the effect of borate on it are different from the other mammalian enzymes.


Case report: Liver scintigraphy in the follow-up of a patient on cytotoxic therapy. A 63-year-old male patient with a small cell bronchial carcinoma on cytotoxic therapy was followed up with the use of liver scintigraphy. In the various scintigrams the remission as well as the late reappearance of the liver metastasis was demonstrated. The abnormalities in the liver scintigrams correlated with the biochemical changes throughout the course.


Effect of exercise and of prolonged oral administration of propranolol on haemodynamic variables, plasma renin concentration, plasma aldosterone and c-AMP. The effect of submaximal exercise upon haemodynamic and biochemical variables was investigated in healthy male subjects, aged 17-27 years, before and at the end of 2 weeks treatment with propranolol (40 mg p.o., q.i.d.). Propranolol reduced the resting blood pressure in normal subjects significantly. This effect was due to reduction of cardiac output and of systemic vascular resistance. No effect of propranolol on BP was seen during maximal exercise, since a reduced cardiac output was accompanied by an increased peripheral resistance. The reduction of cardiac output during exercise can be compensated in part by an increase in stroke volume. The sympathetic activity induced by physical exercise in normotensives increased plasma renin concentration (PRC) and plasma aldosterone (PA), and suppressed urinary excretion of c-AMP. PRC returned to basal levels after 45 min. No increase of PRC was observed after exercise in subjects treated with propranolol. Yet the increase of PA was not completely suppressed. No direct relation was demonstrated between PRC and the haemodynamic variables before or during the administration of propranolol.


Evaulation of the effect of fosazepam (a new benzodiazepine), nitrazepam and placebo on sleep patterns in normal subjects. The nocturnal sleep of eight healthy young volunteers was studied after placebo, Fosazepam 100 mg and Nitrazepam 10 mg. Overall results were very similar after the two drugs. During drug periods there was less of sleep Stages I, III + IV and REM, compensated by an increase in Stage II. The decrease in SWS (III + IV) persisted during the withdrawal periods. There was also an increase in Stage I during Fosazepam withdrawal, whereas the percentage of other sleep stages returned to normal. Sleep quality was reported to be slightly better and morning drowsiness, as well as hangover effects, were said to be less pronounced after Fosazepam than after Nitrazepam.


Smoking and catecholamine and c-AMP concentrations in the coronary circulation of man and the effect of oxprenolol. Changes in catecholamine, c-AMP and lactate concentrations in the coronary circulation of man, during smoking, were studied in 12 patients. The heart rate increase from 63 +/- 2 beats/min (control) to 74 +/- 3 (smoking)(P less than 0.01), falling to 70 +/- 2 (10 min after smoking) (0.05 greater than P greater than 0.01), whilst coronary sinus c-AMP concentrations rose from 11 +/- 0.7 nmol/l (smoking) to 11.9 +/- 0.8 nmol/l (after smoking) (0.05 greater than P greater than 0.01; one tailed 't' test). There was no significant change in blood pressure, catecholamine or lactate concentrations. The study was repeated in eight of the patients following intravenous oxprenolol. Coronary sinus catecholamine concentrations increased from 4.1 +/- 0.7 nmol/l (control) to 5.5 +/- 1.1 nmol/l (after smoking) (0.05 greater than P greater than 0.01; one tailed 't' test), but heart rate and c-AMP concentrations remained unchanged, confirming that smoking-induced tachycardia is a result of a beta-adrenergic mechanism, at least part of which is due to a release of cardiac catecholamines. Arterial lactate concentrations increased only following oxprenolol from 0.74 +/- 0.07 mmol/l (control) to 0.83 +/- 0.09 mmol/l (smoking).


Central and peripheral alpha-adrenergic effects of some imidazolidines. The central hypotensive activity of clonidine and of 12 structurally related analogues was determined following intravenous administration to pentobarbital-anaesthetized, normotensive rats. This sympathoinhibitory action was characterized by means of a pC30, calculated from dose-response curves. The centrally mediated depressor activity (pC30) was correlated with the peripherally induced pressor activity of the compounds (pC100) reported on previously and which had been established after intravenous application to pithed rats. In these correlations, the quantitative ability of the substances to penetrate into the central nervous system had been taken into account. The octanol/buffer (pH = 7.4) partition coefficients were used as a measure of lipophilic behaviour. A positive correlation was found between the central hypotensive activity and the linear combination of peripheral alpha-sympathomimetic activity and lipophilicity.


Properties of the peripheral opiate receptors in the cat nictitating membrane. The noradrenaline overflow and the contractile response elicited by nerve stimulation of the muscle were inhibited by 1 micron morphine in the cat nictitating membrane. This concentration of morphine did not modify the response of the muscle to exogenous noradrenaline. The inhibitory effect of morphine was increased by low Na+ (50 mM), whereas the capacity of naloxone as antagonist to morphine was higher with 150 mM than with 50 mM Na+. These results suggest that the peripheral opiate receptors which interact with noradrenergic neurotransmission could show a sodium allosteric transformation similar to that described for the brain opiate receptor. The effect of morphine was enhanced by manganese ion in the presence of normal Na+. The responses of the cat nictitating membrane to nerve stimulation were not altered in the presence of the protein modifying reagent DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)] but the effect of morphine on the adrenergic neurotransmission was diminished by DTNB with 150 mM Na+. It is postulated that the affinity of the ligands for presynaptic receptors which regulate adrenergic neurotransmission might be modified during the physiological changes in ion concentration which accompany nerve depolarization.


Antagonism of pentobarbital-induced hormonal changes by TRH in rats. In adult male rats, injection of TRH into a lateral ventricle of the brain 5 min prior to pentobarbital (PB) administration caused a significant dose-related inhibition of prolactin (PRL) release, in doses ranging from 500 to 5 ng. Among 8 TRH analogues devoid of thyrotropin-releasing activity, 6 were found to significantly suppress PB-induced PRL secretion at an intraventricular dose level of 10 microgram, and the 3 most effective in this respect were also able to counteract growth hormone (GH) release elicited by PB. The derivative [1,3'-DCM2]TRH was still potent enough to block PB-induced PRL secretion at an intraventricular dosage of 50 ng. The peptide ACTH 4--10 was ineffective, whereas another ACTH derivative H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH (Org 2766) reduced PRL release. TRH did not affect the increase of plasma PRL induced by acute stress. alpha-Methyl-p-tyrosine (alpha-MT) failed to influence the inhibiting effect of TRH on GH secretion but significantly reduced that on PRL release. p-Chlorophenylalanine (PCPA) completely blocked the antagonistic effect of TRH on all PB-induced hormonal changes, suggesting that serotoninergic mechanisms may be involved in the extra-pituitary effect of TRH.


The effect of verapamil on the action of parathyroid hormone on embryonic bone in vitro. Short term in vitro experiments showed that, added alone, verapamil inhibited both glycolysis and Ca uptake in embryonic chicken and rat bone cells. Added together with PTH, verapamil (0.02 MM) enhanced cAMP production, had no effect on lactate production, but significantly inhibited citrate, calcium and phosphate release from embryonic rat and mouse calvaria incubated under hypocalcemic conditions. The depression by verapamil of PTH-stimulated demineralization was confirmed histologically. It is concluded that in addition to cAMP, Ca plays a key role in the action of PTH on bone.


Effect of papaverine on cyclic nucleotide levels in the isolated rat aorta. An increase in cAMP  but no significant modification in cGMP content could be demonstrated in rat aorta strips after applying papaverine in concentrations which reduced contractile responses. Accumulation of cAMP was induced in noradrenaline-stimulated on K+-depolarized strips, under omission of external Ca2+. Thus the elevation of cAMP level preceded the reduction of contraction subsequently elicited by readdition of Ca2+. The effects could not be dissociated under the experimental conditions used here.


Isolation of Epstein-Barr virus from cultured cells of a patient with Hodgkin's disease. The suspension lymphoblastoid cell line named ChSL-1 was established from the tumor node of a patient with Hodgkin's disease. The cells of this culture produce a virus with morphological and physicochemical properties of herpes viruses. Immunologic study of the isolated virus demonstrated great similarity with the Epstein-Barr virus (EBV).


Effect of microwave radiation on cells treated with membrane-injuring substances. WISH cells grown in vitro were pretreated with subcytotoxic concentrations of digitonin, cortisol and purified bacterial toxins -- staphylococcal beta-haemolysin or Clostridium perfringens alpha-toxin and irradiated with 3 GHz electromagnetic wave (microwaves) at the field power densities 5 or 40 mW/cm2. At 40 mW/cm2 increase in temperature of the culture medium of about 2-3 degrees C was noted, while at 5 mW/cm2 no detectable increase in temperature was found. Control and pretreated WISH cells after irradiation in the microwave field were used for evaluation of their viability, incorporation of tritiated thymidine, glycine and uridine and level of intracellular cyclic AMP. Irradiation with microwaves resulted in lowering of thymidine and glycine incorporation along with changes in the intracellular amount of cAMP (decrease in cells exposed to 5 mW/cm2 and increase in those exposed to 40 mW/cm2). Under both conditions viability of the cultures was normal. Pretreatment of cells with digitonin or purified bacterial toxins followed by irradiation with microwaves resulted in enhancement of the cytotoxic effect with lowering of cell viability, especially after exposition to power density of 40 mW/cm2. Cortisol led to decrease in 3H-glycine and 3H-uridine incorporation into WISH cells, but did not influence the reaction of the cells to microwave radiation. In view of the results presented it may be concluded that substances injuring cell membranes sensitize cell cultures to electromagnetic radiation of the microwave range and may enhance the specific (non-thermal) effect of microwaves.


Electron microscope study of thallium-induced alterations in the oligodendrocytes of the rat area postrema. Thallotoxicosis following accidental ingestion of thallium in rodenticides and in cases of homicidal and suicidal ingestion are well documented in the literature. The mechanism of action of thallium, in particular the pathogenesis of its neurotoxic manifestations, is not entirely clear. This is the first electron microscopic investigation dealing with the effect of thallium on the oligodendrocytes of the area postrema. The area postrema is devoid of a blood-brain barrier, indicating that the hemoneural transport of thallous ions may be quite substantial in this region. Rats weighing 155-175 g were given intraperitoneal injections of 5 mg/kg thallous acetate for 7 days (35 mg/kg total dose). Following perfusion-fixation, ultrathin sections of area postrema were examined with an electron microscope. The oligodendrocytes exhibited pleomorphic vacuolated dense bodies in their perikaryon. The findings were also suggestive of the fact that either the multiplication of oligodendrocytes or their aggregation to form "giant cell" was operative.


Dissolution of silicic acid from dusts of kaolin, mica and talc and its relation to their hemolytic activity. -- an in vitro study. Hemolysis induced by native kaolin dust was found to run parallel to the amount of silicic acid dissolved from it. Native mica and talc dusts were hemolytic only to a small extent and the silicic acid dissolution from these dusts was also smaller in comparison to that of native kaolin. The proportionality between hemolysis and the amount of dissolved silicic acid was not consistently observed in the case of acid, alkali and water treated kaolin, mica and talc dusts.


Interaction of molybdate with copper(II)-histidine. Molybdate and copper(II)-histidine form an insoluble complex of empirical formula Cu2(His)3(MoO4)2(H2O)2ESR-spectroscopy indicated that the complex had tetragonal symmetry. IR-spectroscopy showed the presence of a carboxylate anion and suggested that the molybdate ion formed an ammonium-type salt with the nitrogens of the imidazole. The complex did not form following dissociation of the protonated imidazole (above a pH of approximately 6).


Alterations in the phospholipid composition of Nocardia polychromogenes during growth. The major phospholipid classes of Nocardia polychromogenes were quantitated at different stages of the growth cycle. Significant differences were observed both in the total lipid phosphorus per g (dry weight) of cells, and in the relative percentages of individual phospholipids. The total amount of lipid-phosphorus increased throughout the growth cycle. Cardiolipin and phosphoinositides contents increased with significant decrease in phosphatidyl ethanolamine and unknown phospholipids.


Cholesterol synthesis in freshly isolated human leukocytes. Cholesterol synthesis has been studied in human leukocytes shortly after the isolation from healthy subjects. Not delipidated human serum reduced the cholesterol synthesis when added to the incubation medium. A similar effect was obtained when the leukocytes were incubated in the presence of physiological concentrations of low density lipoproteins.


Lead potentiation of endotoxin lethality in rats: lack of effect of kininase inhibition. Although lead and SQ20881 are potent in vitro inhibitors of kininase II activity, SQ20881 does not alter the sensitivity of rats to endotoxin. These results indicate that marked changes in plasma kininase activity do not contribute to endotoxin morbidity and that kininase inhibition is not the mechanism whereby lead ions sensitize rats to endotoxin.


Corticotropin and nonshivering thermogenesis. Chronic treatment with corticotropin led to reduced calorigenic effect of norepinephrine in cold acclimatized rats, but potentiated its effect in controls. This inhibitory effect was not due to the observed decrease in corticosterone plasma level, as it was shown by metopirone administration. It is concluded that corticotropin could have a competitive action on receptor sites mediating the calorigenic effect of norepinephrine in nonshivering thermogenesis.


A cholinergic modulator.

A cholinergic modulator. Soluble proteins obtained from presynaptic cholinergic vesicles have been tested regarding their effects to modify postsynaptic spike generation. The results suggest that these proteins (or derivatives, incl. glycopeptides) may act as modulators in increasing the effectiveness and duration of postsynaptic spike generation. They may partake in generation of homosynaptic (posttetanic) potentiation.


Facilitation of synaptic transmission by diazepam in a perfused frog cerebellum. The action of diazepam on the parallel fibres-Purkinje cell synapse was studied in a perfused frog cerebellum. Diazepam facilitates the excitatory input to Purkinje cells and hence increases the inhibition produced by Purkinje cells activity.


Cyclic AMP in neuroblastoma, ganglioneuroma and sympathetic ganglia. Tissue content of cyclic AMP was as much as 10 times greater in ganglioneuroma than in neuroblastoma. This high cyclic AMP in ganglioneuroma was not significantly different from that of sympathetic ganglia.


Effects of various hormones and adrenalectomy on the levels of amylase in rat pancreas and parotid gland. Dexamethasone, adrenocorticotropic hormone and thyroxine increased the amylase activities in both the pancreas and the parotid gland of infant rats. After adrenalectomy, the amylase activities of the pancreas and parotid gland were about half the control levels, suggesting that both glucocorticoid and thyroxine are involved in maintaining the amylase activities in these organs.


[Research on substances with antiviral activity. VII. Activity and lipophilic of property N-alkyl-2-chloro-3-formylindole thiosemicarbazone]. Thiosemicarbazones of N-alkyl-2-chloroindole-3-carboxaldehydes were synthesized and investigated for antiviral activity against RNA virus (parainfluenza type 3, HA-I/CR-8 stock) and DNA virus (vaccinia virus, HID stock). The effect of lipophilicity on the activity against vaccinia virus was considered. Relative lipophilicities were measured by a reversed phase thin-layer chromatographic technique. The most active compound N-isopropyl-2-chloroindole-3-carboxaldehyde thiosemicarbazone appeared comparable for its activity to methisazone.


[Chemical and pharmacologic study on pyran derivatives. IX. Synthesis of 2-dialkylaminochromones]. Under suitable conditions the reaction of phenol and N,N-dialkylethoxycarbonylacetamides, in the presence of phosphorus oxychloride, resulted in the formation of 2-dialkylaminochromones. In a similar manner, variously substituted phenols afforded 2-dialkylaminochromones with substituents in different positions of the benzene ring. Pharmacological screening of all these compounds showed that they markedly affect the CNS, activity being mainly excitatory. Decreased activity was shown by compounds with particular substituents in the positions 6,7,8 among which two products [(V e) - K 12440, (V s) - K 12420] have weak but clear anticonvulsant effect.


[Electrolyte and water content in the kidney cortical sections of rats exposed to catecholamine action]. Functional significance of alpha and beta-adrenoreceptors and also possible involvement of the cyclic 3', 5'-AMP in the catecholamines regulation mechanism of the sodium and potassium ions transport in renal cortex slices of rats were studied. Epinephrine did not change the intracellular sodium and water content, but increased that of potassium in the cells. Phentolamine-a blocking agent of the alpha-adrenoreceptors--did not change the reaction of slices to epinephrine, while inderal--a beta-adrenoblocking agent-prevented accumulation of potassium by the cells occurring under the action of epinephrine. Isopropyl-nore-pinephrine reproduced the action of epinephrine on the electrolytes composition of the renal tissue. It is suggested that accumulation of postassium by the cells of the rats' renal cortex slices is consequent upon excitation of the beta-adrenoreceptors.


[Preparation and pharmacological activity of N6, 02-dipivaloyl-cyclic AMP]. The synthesis of N6,O2'-dipivaloyl-cyclic AMP is described with some pharmacological tests made in comparison with the dibutyryl homolog. The two compounds proved equally active in increasing glycemia in the rabbit whereas the dipivaloyl derivative is less effective in counteracting contractions induced by imidazole in the isolated guinea-pig trachea.


[Antidiuretic activity of dithiocarbamic acid derivatives]. Chemically pure dithiocarbamates, thiuramsulphides and ethylene-bis-dithiocarbamates display a well-marked antidiuretic action. They materially reduce diuresis, natri- and kaliuresis in rats (1/5 LD(50) per 1 kg into the stomach) and in dogs (sodium N, N-diethyl-dithiocarbamate-into renal artery). Without changing the tubular reabsorption DEDTC inhibits the glomerular filtration of the dogs' kidneys. Furosemide (lasix) prevents the development of the an antidiuletic anidiuretic effect and reduction of the sodium and potassium excretion provoked in rats with DEDTC, zinc N, N', N"-tetraethylthiuram-disulphide and ethylene-bis-dithiocarbamate. Furosemide is recommended for the treatment of acute poisonings with dithiocarbaminic acid derivatives.


[Effect of combinations of apressin, obsidan, diprazin, adenosine, NAD and nicotinamide on the resistance of rats to hypoxia and on carbohydrate metabolic indices]. As evidenced from experiments on rats, a combined application of apressin with obsidan and diprazine, and also of adenozine with nicotine-amidadenine-dinucleotide (NAD), as well as of adeozine with nicotine amide potentiates the protective effect of these substances in hypobaric hypoxia, increases the resistance of the animals to cerebral ischemia, brings down the excess lactate level and raises the redoz potential of the system lactic-acid-pyruvic acid in the brain of rats exposed to the effects of rarefied atmosphere.


[Role of the medial hypothalamus in regulating the pituitary-adrenocortical system]. In unrestrained rabbits, inhibition of ACTH release as determined by changes in plasma levels of corticosteroids, followed electrolytic lesion of the dorso-and ventromedia nuclei of the hypothalamus. Electric stimulation of the dorsal midbrain tegmentum had activated ACTH prior to lesion; after the lesion of the dorso-and ventromedial nuclei the stimulation produced no response. The dorso-and ventromedial nuclei of the hypothalamus are essential for basal and induced secretion of the ACTH.


Isolation of mutant adrenocortical tumor cells resistant to cyclic nucleotides. A somatic cell genetic approach was used to study the role of cyclic nucleotides in adrenal steroidogenesis. 8-Bromoadenosine 3',5'-monophosphate (8BrcAMP) stimulated steroidogenesis (K'd=0.1 mM) in cultured mouse adrenocortical tumor cells (Clone Y1). In addition, 8BrcAMP inhibited Y1 cell growth and caused Y1 cell monolayers to assume a rounded morphology. As a consequence, 8BrcAMP (at concentrations greater than or equal to 0.4 mM) reduced the relative plating efficiency of Y1 cells to less than 10(-5). Y1 cells were mutagenized with ethyl methanesulfonate (300 microgram/ml) and grown in the presence of 0.4 mM 8BrcAMP. A surviving colony (8BrcAMPr-1) was shown to be resistant to growth inhibition (relative plating efficiency at 1.0 mM 8BrcAMP=50 percent)) and to morphological changes induced by 8BrcAMP. 8BrcAMPr-1 cells had diminished steroidogenic responses to cyclic nucleotides and to ACTH (less than or equal to 33 percent of maximum). In 8BrcAMP(R)-1 cells, adenylate cyclase activity remained responsive to ACTH, and cyclic AMP phosphodiesterase activity was not increased. These data suggest that 8BrcAMPr-1 cells are defective at a point common to cyclic AMP action on growth, morphology and steroidogenesis. The associated decrease in responsiveness of the steroidogenic pathway to ACTH suggests that ACTH-regulated steroidogenesis is via a cyclic nucleotide-mediated mechanism.


Discontinuity of thyroid gland response to hormonal stimulation: effect of TSH and cAMP on iodide organification. The action of TSH on the process of iodide organification was studied in rat thyroid glands under different experimental in vitro incubation conditions. The effects on glands of both short and prolonged exposure to TSH were evaluated using two different procedures: continuous and pulse labelling of thyroids with radioactive iodide. It was demonstrated that during prolonged contact with thyroid cells, TSH stimulates iodide organification periodically. This periodic effect is cyclic and is composed of a stimulatory and an inhibitory phase. Each cycle lasts 30-45 min, and several cycles follow one another in a regular manner. Furthermore, it has been shown that the periodic effect of THS is due to an intrinsic property of the thyroid cell to respond in a cyclic manner to hormonal stimulation. TSH stimulated the accumulation of organic iodide only when introduced at a precise phase of the cycle. The same type of discontinuous thyroid cell response was obtained when TSH was replaced by its intracellular mediator, cAMP. This indicates that the initiation of the cyclic response to hormonal stimulation is localized at the steps after that of cAMP formation. It seems, therefore, that this cyclic response of thyroid glands is not related to the recently observed phenomenon of desensitization. This phenomenon, characterized by the development of resistance in many target organs and cells toward their respective hormonal stimulators, is due to modifications in steps preceding those of cAMP formation. The discontinuity of thyroid gland response to both TSH and cAMP described in this work seems to be a new phenomenon, whose physiological significance and possible molecular mechanisms are discussed.


Studies on the synergistic effect of androgen on the stimulation of progestin secretion by FSH in cultured rat granulosa cells: a search for the mechanism of action. Cultures of granulosa cells from immature hypophysectomized DES-treated rats were unable to maintain progestin production of more than 48 h in medium without hormone supplementation or in the presence of FSH only. Production of progestin (20alpha-dihydroprogesterone, as measured by radioimmunoassay) remained unimpaired in the presence of androstenedione (Ad) and was markedly increased in the presence of both Ad and FSH. The combined treatment with FSH and Ad during the first 48 h of culture brought about persistent changes in the cultured cells, since progestin accumulation did not decline upon subsequent removal of these hormones during days 3 and 4 of culture. Dibutyryl cyclic AMP (DBC) was able to mimic the changes in steroidogenic capability induced by the combined action of FSH and Ad. The extent of [125I]-FSH binding, FSH-stimulable cAMP accumulation and cyclic 3',5'-nucleotide phosphodiesterase activity were not affected by addition of Ad to the culture medium. Ad synergized with DBC in the stimulation of progestin accumulation in granulosa cell cultures. It is suggested that androgen acts at a step in the regulation of progestin biosynthesis distal to cAMP production.


Biological and immunological properties of tritiated salmon calcitonin. Tritiated salmon calcitonin was prepared by methylation of the free amino groups using tritiated sodium borohydride as precursor. Specific radioactivity was measured in competitive inhibition studies with specific anticalcitonin antibodies or tubular membranes as binding sites for calcitonin. The value observed, approx. 4 Ci/mmol, corresponded to methylation of one third of the available N-H bonds. Tritiated calcitonin prepared in this way retained full biological activity as assessed in vitro by stimulation of adenylate cyclase and in vivo by rat bioassay. Tritiated calcitonin specifically bound to isolated renal cells and nonspecific binding did not exceed 10% of total binding. Equilibrium was obtained after 15 min incubation. The hormone-receptor complex could be dissociated in the presence of an excess of unlabelled calcitonin. This data shows that tritiated calcitonin can be used in metabolic and receptor studies.


Steroid and FSH action on LH receptors and LH-sensitive testicular responsiveness during sexual maturation of the rat. This study examines the effect of FSH, testosterone and estradiol on testicular LH receptors and in vitro testicular responsiveness to LH in immature rats under various conditions. FSH treatment of 15-day-old immature rats significantly increased the number of LH receptors but did not alter testicular responsiveness. FSH treatment of hypophysectomized immature rats increased the number of LH receptors and markedly increased testicular responsiveness. Treatment of hypophysectomized rats with testosterone proprionate for 4 days, followed by a 5-day treatment with FSH, enhanced the effect of FSH on the number of LH receptors but did not increase the effect of FSH on testicular responsiveness. In contrast, treatment with estradiol for 4 days before FSH treatment had no effect on the FSH-induced increase in LH receptors but completely inhibited the FSH-induced increase in testicular responsiveness. These observations suggest that during male sexual maturation (1) regulation of LH receptors is distinct from regulation of testicular responsiveness to LH, (2) estradiol may be a factor in the regulation of testicular responsiveness to LH, and (3) testosterone may enhance the FSH-induced increase of LH receptors.


Hormone receptors: VI. On the nature of the binding of glucagon and insulin to human circulating mononuclear leukocytes. Several characteristics of the binding of insulin and glucagon to human circulating mononuclear leukocytes have been studied. Functional analysis (latex bead ingestion) revealed that cell mixtures, as prepared according to Boyum and used generally in studies of insulin resistance in humans, consist of 20-29% phagocytic monocytes, with the remainder being lymphocytes. Partial separation of monocytes from lymphocytes on columns of Sephadex G-10, followed by correlation of insulin binding with cell type, confirms that the monocyte is the binding species. Insulin influenced neither glucose uptake nor the further conversion of glucose to lipids and CO2 by the leukocytes. The transport of alpha-aminoisobutyrate, a nonmetabolizable amino acid, into these cells was also unaffected by insulin. Monocyte/lymphocyte mixtures specifically bound glucagon and prostaglandin E1. At physiological concentrations of these hormones, steady states were reached in 15 min and 45 min, respectively. In contrast to the 8-10-fold increases in cellular cyclic AMP produced by prostaglandins, the effect of glucagon was very small but apparently real. Under appropriate preincubation conditions, sodium azide and iodoacetamide inhibited phagocytosis and insulin binding in parallel. The binding of glucagon was unaffected by these agents. Although both antimycin A and actinomycin D inhibited phagocytosis of the monocytes, only the former inhibited insulin binding; there was only a slight effect on glucagon binding. We would conclude that the binding of insulin to human circulating monocytes, although reflective of insulin resistance in diabetes mellitus and obesity, may not be to traditional receptors. In contrast, the binding of glucagon to lymphocyte/monocyte mixtures may be to function-linked receptors.


Increased spontaneous mitotic segregation in MMS-sensitive mutants of Saccharomyces cerevisiae. Methyl methanesulfonate (MMS)-sensitive mutants of Saccharomyces cerevisiae belonging to four different complementation groups, when homozygous, increase the rate of spontaneous mitotic segregation to canavanine resistance from heterozygous sensitive (canr/+) diploids by 13-to 170-fold. The mms8-1 mutant is MMS and X-ray sensitive and increases the rate of spontaneous mitotic segregation 170-fold. The mms9-1 and mms13-1 mutants are sensitive to X rays and UV, respectively, in addition of MMS, and increase the rate of spontaneous mitotic segregation by 13-fold and 85-fold, respectively. The mutant mms21-1 is sensitive to MMS, X rays and UV and increases the rate of spontaneous mitotic segregation 23-fold.


Spontaneous and ethyl methanesulfonate-induced mutations controlling viability in Drosophila melanogaster. I. Recessive lethal mutations. The efficiency of the adult feeding method for EMS treatment in Drosophila melanogaster was studied by measuring the frequency of induced recessive lethals on the second chromosome. The treatment was most effective when mature spermatozoa or spermatids were treated and was much less effective on earlier stages. The number of mutations induced was proportional to the concentration except at the highest doses. The recessive lethal rate was estimated to be about 0.012 per second chromosome per 10(-4) M. In addition, about 0.004-0.005 recessive lethals per 10(-4) M were found in a later generation in chromosomes that had not shown the lethal effect in the previous generation. When the experiments are done in a consistent manner and gametes treated as mature sperm or spermatids are sampled, the results are highly reproducible. However, modifications of the procedure, such as starvation before EMS treatment, can considerably alter the effectiveness of the mutagen.


Spontaneous and ethyl methanesulfonate-induced mutations controlling viability in Drosophila melanogaster. II. Homozygous effect of polygenic mutations. Polygenic mutations affecting viability were accumulated on the second chromosome of Drosophila melanogaster by treating flies with EMS in successive generations. The treated chromosomes were later made homozygous and tested for their effects on viability by comparison of the frequency of such homozygotes with that of other genotypes in the same culture. The treated wild-type chromosomes were kept heterozygous in Pm/+ males by mating individual males in successive generations to Cy/Pm females. The number of generations of accumulation was 1 to 30 generations, depending on the concentration of EMS. A similar experiment for spontaneous polygenic mutations was also conducted by accumulating mutations for 40 generations. The lower limit of the spontaneous mutation rate of viability polygenes is estimated to be 0.06 per second chromosome per generation, which is about 12 times as high as the spontaneous recessive lethal mutation rate, 0.005. EMS-induced polygenic mutations increase linearly with the number of treated generations and with the concentration of EMS. The minimum mutation rate of viability polygenes is about 0.017 per 10(-4)m, which is only slightly larger than the lethal rate of 0.013 per 10(-4) m. The maximum estimate of the viability reduction of a single mutant is about 6 to 10 percent of the normal viability. The data are consistent with a constant average effect per mutant at all concentrations, but this is about three times as high as that for spontaneous mutants. It is obvious that one can obtain only a lower limit for the mutation rate, since some mutants may have effects so near to zero that they cannot be detected. The possibility of measuring something other than the lower limit is discussed. The ratio of the load due to detrimental mutants to that caused by lethals, the D/L ratio, is about 0.2 to 0.3 for EMS-induced mutants, as compared to about 0.5 for spontaneous mutants. This is to be expected if EMS treatment produces a large fraction of small deletions and other chromosome rearrangements which are more likely to be lethal.


Spontaneous and ethyl methanesulfonate-induced mutations controlling viability in Drosophila melanogaster. III. Heterozygous effect of polygenic mutations. Spontaneous and EMS-induced mutations were accumulated for several generations on the second chromosome of Drosophila melanogaster by keeping this chromosome heterozygous under conditions of minimal natural selection. This article reports studies of heterozygous effects of these mutants.--Both lethal and mildly deleterious mutants have a deleterious heterozygous effect. There was no discernible difference between heterozygotes in which all the mutants were on one chromosome and those where the mutants were distributed over both homologs; thus the coupling-repulsion effect of MUKAI and YAMAZAKI (1964, 1968) is not confirmed. The spontaneous polygenic mutants have a dominance of 0.4 to 0.5, and the same value is found at very low EMS doses. However, the value at higher EMS doses is only about half as high. Since the low doses have a large fraction of spontaneous mutants, the dominance of EMS mutants is less, in the range 0.1 to 0.3, but still larger than for lethals.


[Antidotes in acute poisonings].

[Antidotes in acute poisonings]. Inhalation of dexamethazone (Auxiloson) prohibits a toxic edema of the lungs after poisoning with irritants. Poisoning with methanol is to be treated with ethanol trisbuffer and tetrahydrofolic acid (Folsan). "Solvents" should be "bound" in paraffinum liquidum in order to prohibit their absorption. Poisoning with organophosphates is to be treated with high doses of atropine and with the reactivator obidoxime (Toxogonin). Cyanide poisoningis are to be treated with 4-dimethylamino-phenol (DMAP) and sodium thiosulfate. In methemoglobin-poisoning the reduction of ferrihemoglobin is accelerated when toluidine-blue is applied intravenously.


Insulin release from collagenase-isolated islets of rat pancreas in the presence of cyclic AMP and somatostatin. In order to study the role of cyclic AMP in the inhibition by somatostatin of glucose-induced insulin release, the effect of somatostatin on the potentiation by dibutyryl-cyclic AMP (db-cAMP) of insulin release from isolated pancreatic islets of rats was examined. Isolated islets were obtained from the rat pancreas by the collagenase method. Ten islets were incubated for periods of 30 min in Krebs-Ringer bicarbonate buffer containg albumin and glucose 2.0 mg/ml in the presence or absence of somatostatin (1 microgram/ml or 100 ng/ml) and/or db-cAMP 1 mM. Glucose-induced insulin release was reduced by somatostatin in concentrations of 1 microgram/ml. Somatostatin in a concentration of 100 ng/ml significantly abolished the potentiation by db-cAMP of insulin release (p less than 0;01), in spite of exerting no inhibition of glucose-induced insulin release. However, in the presence of theophylline 5 mM, somatostatin 100 ng/ml did not show that inhibitory effect on the potentiated insulin release.


Glycogen metabolism and cyclic AMP levels in isolated islets of lean and genetically obese mice. The levels of glycogen and cyclic AMP, incorporation of glucose into glycogen and activities of glycogen synthetase and phosphorylase were determined in pancreatic islets isolated from genetically obese mice and their lean litter-mates. Islets from obese mice had elevated glycogen levels, increased phosphorylase activity and an increased amount of glycogen synthetase in the physiologically more effective I-form, indicating an increased turnover of glycogen. There was no significant difference in cyclic AMP levels between islets of lean and obese mice, but inhibition of phosphodiesterase or stimulation of adenyl cyclase increased cyclic AMP levels more in obese than in lean mouse islets, indicating a more rapid turnover of cyclic AMP in the former. It is suggested that cyclic AMP stimulated phosphorolytic breakdown of glycogen may be one of the mechanisms responsible for the increased insulin secretory response to glucose observed in islets from genetically obese mice.


Effects of hypothalamic extract and corticosterone on incorporation of 14C-phenylalanine into newly synthesized ACTH-like protein in vitro. The purpose of the present experiments was firstly, to examine the efficacy of the oxycellulose-Amberlite CG-50 extraction procedure for the isolation of rat pituitary ACTH and secondly, to study the effects of both hypothalamic extract and corticosterone on the synthesis of rat pituitary ACTH. Briefly, 'cold' rat pituitary ACTH was monitored by bioassay in each step of the isolation procedure as a function of corticosterone stimulation in incubated adrenal slices. Labeled ACTH was isolated from rat pituitaries previously incubated in vitro and obtained from adrenalectomized animals and cortisone acetate treated animals to provide further data to substantiate that the labeled isolated protein contained ACTH. Cold ACTH was detected in two purified fractions (oxycellulose and Amberlite CG-50). Adrenalectomy stimulated and cortisone acetate suppressed the incorporation of 14C-phenylalaine into the ACTH-like protein in these two fractions. A continuously increasing uptake of the label into ACTH-like protein in the isolated fractions occurred with time providing evidence that de novo synthesis had taken place. Corticosterone inhibited ACTH synthesis at both 10(-4) and 10(-6) M, whereas, hypothalamic stalk median eminence (HSME) caused a significant, but not dose-related, increase in ACTH synthesis with 3/5, 1, and 2 HSME equivalents added.


On the molecular weight of mitochondrially synthesized subunits of rat liver cytochrome oxidase. The subunit composition of cytochrome c oxidase from rat liver mitochondria was studied by dodecylsulfate polyacrylamide gel electrophoresis. The apparent molecular weight of the seven subunits are in reasonable agreement with published data on cytochrome c oxidase subunits from other sources. Two additional subunits were found if the electrophoresis was performed with 8m urea, due to splitting of the smallest subunit. Performic acid oxidation of the isolated subunits I and II increased the apparent molecular weights from 38000 to 48000 and from 24500 to 29000, respectively, accompained by a normalization of the anomalous behaviour of subunit I in the Ferguson plot. It is suggested that performic acid, by splitting extremely inaccessible disulfide bridges, mediates full complexing of the subunits by dodecylsulfate, thus permitting the determination of the real molecular weights by dodecylsulfate polyacrylamide gel electrophoresis.


Inactivation of pyruvate kinase type M2 from chicken liver by phosphorylation, catalyzed by a cAMP-independent protein kinase. A cAMP-independent protein kinase from chicken liver phosphorylated and inactivated pyruvate kinase type M2 from the same tissue. Complete inactivation was reached when 4 mol of phosphate were incorporated/mol of tetrameric pyruvate kinase. The protein kinase bound with high affinity to pyruvate kinase type M2 (Km value for pyruvate kinase = 6 X 10(-10)M; it phosphorylated phosvitin and casein but not histones, ATP and GTP were substrates. The differences between the properties of this protein kinase in the interconversion of pyruvate kinase and that described previously are discussed.


Irreversible inhibition of hypoxanthine phosphoribosyltransferase. Further studies on the specificity of periodate-oxidized GMP. Inactivation of hypoxanthine phosphoribosyltransferase caused by periodate-oxidized GMP is irreversible, even under the conditions of polyacrylamide gel electrophoresis and during affinity chromatography on GMP-Sepharose. Partial binding of the inhibitor to the enzyme protein can be demonstrated on dodecyl sulfate gel electrophoresis: The substrate, phosphoribosyl diphosphate in the presence of Mg2, and the product GMP protect the enzyme against inactivation. Periodate-oxidized GMP, AMP and oxidized purine nucleosides do not influence ribosephosphate pyrophosphokinase, 5'-nucleotidase, purine-nucleoside phosphorylase and guanylate kinase. A variety of other purine nucleosides and nucleotides, tested in their periodateoxidized form, do not lead to a compound comparable or superior to oxidized GMP in its effect on hypoxanthine phosphoribosyltransferase. In an erythrocyte system it is clearly demonstrated that oxidized GMP cannot act across an intact cell membrane.


Structure of a cytochrome b-c 1 complex from Saccharomyces cerevisiae YF. 1) An isolation and purification procedure is reported for an active cytochrome b-c1 complex from Saccharomyces cerevisiae. The complex acts as an antimycin A-sensitive duroquinone-cytochrome c reductase and contains cytochromes b and c1 at a concentration of 8 nmol/mg protein and non-heme iron at a concentration of 15 nmol/mg protein. 2) Difference spectra at room temperature and at 70 degrees K show that the preparation is free from contamination with cytochromes c or aa3. Assays of enzyme activity indicate the absence of any of the other catalytic functions normally associated with the mitochondrial respiratory chain. 3) On dissociation and separation on sodium dodecylsulfate-polyacrylamide gels the complex gives rise to seven bands corresponding to subunit polypeptide molecular weights of 43 000, 40 000, 32 000, 24 000, 22 000, 20 000 and 18 000. These appear in a regular stoichiometry of 1:1:3:1:1:1:1.


Electron microscopy of a feminizing Leydig cell tumor of the testis. The ultrastructural characteristics of a feminizing interstitial (Leydig) cell tumor of the testis were compared with those of normal Leydig cells and with the findings described in 10 published cases of Leydig cell tumor. The neoplastic Leydig cells superficially resembled normal Leydig cells. Similarities included abundant smooth endoplasmic reticulum, lipid, and microbodies. Contrastingly, Reinke crystalloids and paracrystalline inclusions were absent and lipochrome pigment and lysosomes very rare. The nuclei were large and contained enlarged, often multiple, nucleoli. The nuclear membranes tended to be irregular and undulating. Cytoplasmic membranous whorls and myelin figures were conspicuous. Fairly homogeneous fibrous septa were evident between single and grouped tumor cells. Despite several individual variations, there is a general resemblance between the neoplastic Leydig cells in this patient and those previously reported. No distinguishing ultrastructural characteristics were discerned between feminizing and virilizing Leydig cell tumors.


Malignant mixed tumor of the parotid gland presenting as a scalp nodule: a case report. Malignant mixed tumors of the parotid gland are rare. Metastasizing lesions are even more rare. To the best of our knowledge, this is the first case reported in the English literature in which the scalp nodule was the primary manifestation of the malignant nature of the parotid tumor.


Interaction between cyclic nucleotides and herpes simplex viruses: productive infection. Infection of human fibroblasts and HEp-2 cells with herpes simplex virus type 1 (HSV-1) produced a decrease in the intracellular levels of cyclic adenosine 5'- monophosphate (cAMP) and a concomitant increase in the cyclic guanosine 5'- monophosphate (cGMP) levels. In both cell cultures, changes in cyclic nucleotide levels were first observed at 6 h after viral inoculation and were maximal at 12 h. In human fibroblasts, the addition of theophylline, dibutyryl cAMP, or papaverine (cAMP-enhancing compounds) decreased significantly the yield of HSV-1, whereas the addition of insulin or dibutyryl cGMP (cGMP-enhancing compounds) increased the viral yield. In HEp-2 cells, only theophylline decreased the yield of HSV-1, and the cGMP-enhancing compounds had no apparent effect. Cyclic nucleotide enhancing compounds exhibited their effect only if added to either cell culture within the first 3 h after inoculation with HSV-1.


New test for endotoxin potency based upon histamine sensitization in mice. The results of a test of endotoxic potency based upon the development of histamine hypersensitivity in mice were compared with the results obtained by testing the same materials for pyrogenicity in rabbits and lethality for chicken embryos (CELD50). The results of the histamine hypersensitization test (HHT) correlated well with those of the other two tests. The sensitivity of the HHT was about the same as that of the CELD50 assay. The HHT may provide a relatively inexpensive, fast, and reliable assay method for endotoxin laboratories that do not have the facilities for the more elaborate assays.


Immunological and chemical characterization of the extracellular antigens from Corynebacterium vaginale. Supernatants from 72-h peptone-starch-dextrose broth cultures of Corynebacterium vaginale contained significant quantities of three extracellular, soluble antigens (ESA). The ESA were concentrated and partially purified by dialysis followed by ammonium sulfate and ethanol precipitation. Diethylaminoethylcellulose columns were used to isolate two of the three ESA. The ESA were shown to be similar to antigens found on whole C. vaginale cells. Absorption studies indicated that the cell antigens are located at, or near, the surface. On the bases of heat stability, resistance to protease treatment, concanavalin A binding activity, and susceptibility to periodate, it appears that all three ESA are polysaccharide or glycoprotein in nature.


Effect of interferon, elevated temperature, and cell type on replication of acute hemorrhagic conjunctivitis viruses. Five strains of enterovirus type 70 (E 70) and four of coxsackievirus type A 24 (CA 24) were studied for their sensitivity to interferon (IF), ability to induce IF, replication at various temperatures, and adaptability to human and mouse cell cultures. We found that isolates ranged from 0.01 to 16 times as sensitive to fibroblast IF as vesicular stomatitis virus, depending upon the cell type used and the multiplicity of infection. Most of the isolates induced no detectable IF; however, when induction occurred the titers were relatively low (5 to 300 U). Only E 70 virus isolates were adaptable to growth in L-cells. Replication of all viruses was inhibited approximately 90% at 37 to 39 degrees C depending upon the cell type. These results and the accessibility of the eye to application of IF and/or heat suggests the possibility of their use for treatment. The adaptation of certain E 70 viruses to mouse L-cells opens the possibility of development of a mouse model infection.


Mitogenic response of mouse spleen cells and gelation of limulus lysate by lipopolysaccharide of Yersinia pestis and evidence for neutralization of lipopolysaccharide by polymyxin B. Lipopolysaccharide (LPS) extracted with phenol and water from Yersinia pestis was compared with LPS of Escherichia coli for stimulation of deoxyribonucleic acid synthesis in mouse spleen cells (lymphocyte mitogenesis), gelation of limulus lysate, pyrogenicity in the rabbit, and susceptibility to inhibition of these activities by polymyxin B sulfate (PBS). LPS of Y. pestis stimulated deoxyribonucleic acid synthesis in mouse spleen cell cultures over the same quantitative range as LPS of E. coli. In the limulus tests and rabbit pyrogenicity studies, the LPS of Y. pestis was active but about 10 times less potent than E. coli LPS on a weight basis. PBS in concentrations from 1 to 10 microgram/ml diminished the rate of deoxyribonucleic acid synthesis in spleen cell cultures stimulated by LPS of both Y. pestis and E. coli. Addition of PBS to LPS of both Y. pestis and E. coli in a ratio of 100 parts of PBS to 1 part of LPS by weight increased by 10-fold the concentration of LPS required to produce gelation of limulus lysate and inhibited significantly pyrogenic responses in rabbits. These results demonstrating similarities of LPS of Y. pestis and E. coli may suggest that the pathogenesis of plague is similar to that of other gram-negative bacterial infections.


Experimental infection of human leukocytes with parainfluenza 1 (6/94) virus. Parainfluenza 1 (6/94) virus replicated in both unstimulated and phytohemagglutinin (PHA)-stimulated human peripheral blood leukocytes (PBL). After exposure of PBL to 6/94 virus at a multiplicity of infection of 1, the presence of viral antigen was demonstrated by immunofluorescence in the cytoplasm of less than 1% of unstimulated PBL and 1 to 5% of macrophages. Small amounts (less than 50 mean egg infective doses per ml in most instances) of cell-free virus were present in 18 of 30 (60%) cell cultures tested from 3 to 8 days postinfection. Cell-free virus peaked 6 days postinfection. Virus replication was enhanced in PHA-stimulated cells. Approximately 1 to 10% of PHA-stimulated PBL contained viral antigen as evidenced by immunofluorescence, and cell-free virus was present in 19 of 25 (76%) of the cell cultures tested from 3 to 8 days postinfection. Paramyxovirus nucleocapsids and tubular aggregates were seen in the cytoplasm of approximately 5% of PHA-stimulated PBL and were visualized only in lymphocytes. No other unusual intracytoplasmic or intranuclear structures were seen.


Detection of hepatitis A antigen by immunofluorescence. Hepatitis A antigen (HA Ag) was demonstrated by immunofluorescence (IF) in liver biopsies from chimpanzees with experimental hepatitis A virus infection. Blocking experiments with paired sera from patients with hepatitis types A, B, or non-A, non-B, as well as with purified HA Ag, showed that the fluorescence was specific for HA Ag. HA Ag could be demonstrated only in biopsies from chimpanzees inoculated with hepatitis A virus. In two of four chimpanzees biopsied weekly, HA Ag could be detected by IF before stool shedding of HA Ag, elevation in serum alanine aminotransferase (SGPT), or histopathological evidence of liver disease was seen. The HA Ag was detected for 4 to 5 weeks; the last IF-positive biopsy was obtained after SGPT activity had returned to normal. In the two other chimpanzees, HA Ag could be detected only in the biopsy taken at the time of SGPT elevation. In the early IF-positive biopsies, HA Ag was diffusely distributed in the cytoplasm of many cells, but it later accumulated in a focal distribution in the cytoplasm of a few of the hepatocytes and Kupffer cells. This cytoplasmic distribution agrees with previous electron microscopic data.


Physical changes in the epsilon prototoxin molecule of Clostridium perfringens during enzymatic activation. Enzymatic activation of Clostridium perfringens epsilon prototoxin removed a small basic part of the molecule, causing a slight change in molecular weight (32,700 to 31,200) and a large change in isoelectric point (from pH 8.02 into fractions of 5.36 and 5.74).


Immunological relatedness of papovaviruses of the simian virus 40-polyoma subgroup. Viral antigens in permissive cells infected with JC virus, K virus, and SA12 virus were reactive in immunofluorescence tests to antisera against sodium dodecyl sulfate-disrupted simian virus 40 capsids and polyoma VP1. The major capsid polypeptides of all papovaviruses of the simian virus 40-polyoma subgroup are immunologically related.


Isolation of infectious EB virus from the epithelial tumour cells of nasopharyngeal carcinoma. Evidence of herpesvirus replication has been found by light and electron microscopy in the malignant epithelial cells of two out of six nasopharyngeal carcinomas (NPC) examined directly after growth in nude mice to eliminate non-malignant infiltrating cells. The agent has been identified as EB virus by immunofluorescence tests for EB virus capsid antigen, and has been shown to be biologically active by its ability to infect and transform foetal cord blood lymphocytes. Lymphoblastoid cell lines which express the EB virus nuclear antigen have been established from the transformed foetal lymphocytes, and thus carry the first isolate of the virus from the actual epithelial tumour cells of NPC, in a form suitable for further investigation. The results are discussed in terms of the relationship of EB virus to NPC epithelial cells.


Complement-dependent cytotoxic antibodies in the course of cervical carcinoma. Complement-dependent cytotoxic antibodies to three cervical carcinoma cell lines (Me-180, SW-732 and HeLa) and to Herpes simplex virus type 2 (HSV-2)-infected cells were determined in a long-term study of women with cervical carcinoma. Cytolysis of surface antigens differed significantly between the cervical carcinoma cell lines and HSV-2. Regression of the tumor during treatment was accompanied by decreasing cytolysis in the cervical carcinoma lines, while tumor bearers and patients who became severely ill had high or increasing cytotoxic antibodies, The opposite was noted for the antibody response to HSV-2 infected cells: patients with less advanced cancer had significantly higher cytolytic activity than those who were severely ill or had advanced cancer; long-term survivors demonstrated high, stable lysis of HSV-2 infected cells. As a control of the cervical carcinoma cell lines, cytolytic activity was tested also on a lung carcinoma cell line, A-549. No significant differences were found between the patients with cervical cancer and the control women. Tumor bearers and patients treated for advanced cervical cancer showed a slight but non-significant increase in cytolytic activity on the A-549 line.


Lymphocyte subpopulations and mitogenic responses in nasopharyngeal carcinoma, prior to and after radiotherapy. Eighteen patients with nasopharyngeal carcinoma (NPC) were compared to matched controls, before or after cobalt therapy, for the ability of their peripheral blood lymphocytes to: (1) form E and EAC rosettes and (2) mount a proliferative response with PHA, Con A and ALG. A slight decrease in the percentage of E rosettes and a moderate hyporesponsiveness to PHA and Con A were observed before treatment. The statistical significance of these alterations was borderline. Within the group of treated patients a much greater depression, including the response to ALG, was found, although a few long-term survivors responded to mitogens as well as the controls. These findings stress the difficulty of interpreting the results of a longitudinal study of cell-mediated immunity, specific or non-specific, in cancer patients. Finally, by comparing the proliferative response to the three mitogens before and after radiotherapy, it is suggested that their differential effect on these responses might be used in man, as it was in mice, to delineate lymphocyte subpopulations.


Glycopeptides from epithelial cell mutants: temperature sensitive for the transformation phenotype. Fucose-labelled glycopeptides obtained from the cell surfaces of normal and transformed epithelial cells were compared by co-chromatography on Sephadex G-50. The material obtained from epithelial cells transformed in vitro or from hepatoma cells in culture elutes earlier than the fucose-containing glycopeptides obtained from normal rat epithelial cells. A mutant (TS 223) of a transformed epithelial cell that is temperature-sensitive for maintenance of the transformed phenotype, varies in its Sephadex G-50 profile of cell surface glycopeptides when grown at the permissive (36 degrees C) or the non-permissive temperature (40 degrees C). When grown and labelled at 36 degrees C the gel filtration profile of the glycopeptides resembles that of transformed cells. At 40 degrees C there is an enrichment of later eluting glycopeptides. These differences are more striking in confluent-phase cultures than in log-phase culture. The differences are reversible following upward or downward shifts in growth temperature although there appears to be a lag of at least 6 h before the alteration can be demonstrated by these procedures.


Virus expression in different tissues of normal and tumor-bearing mice inoculated with a murine leukemia virus. Evolution of virus expression in different lymphoid organs as well as in solid syngeneic tumors of mice inoculated with an MuLV was studied with the aid of in vitro XC co-culture technique. When normal adult mice of strain XLII were inoculated intraperitoneally with a cultured Rauscher virus (RC), the virus could be detected, 10 days after inoculation, only in bone marrow in small amounts and thereafter no virus could be found in any of the organs tested, including bone marrow, spleen, thymus, lymph node and kidney. However, when age- and sex-matched parallel mice bearing syngeneic subcutaneous non-viral tumors were inoculated similarly with the RC virus, the virus could be detected abundantly not only in bone marrow and spleen but also in tumors during the first 3 weeks and even 6 weeks after virus inoculation. Transitional decrease or disappearance of the virus was observed around the 25th-31st day in organs and tumors of the inoculated mice. When the tumor mass was removed from these mice by surgery, the virus disappeared rapidly and definitely from all the organs tested. The virus recovered from in vitro explanted and cultured tumors, taken from mice inoculated with the virus, induced typical lymphoid leukemia in BALB/c mice inoculated as newborns. However, from certain aspects (hypertrophy of the thymus and lymph nodes), this virus was different from the original RC virus.


Immunologic identification of fetal calf serum-derived proteins on the surfaces of cultured transformed and untransformed rat cells. The antigens of rat embryo fibroblasts (REF) and of rat Rous sarcoma cells (derived by in vivo passage of oncogenically transformed REF) were studied using the technique of non-ionic detergent solubilization of radiolabelled cells. Solubilized antigens were complexed with rat immune IgG, and precipitation of the complexes was accomplished with rabbit anti-rat IgG. The precipitated radiolabelled antigens were then dissolved in sodium dodecyl sulfate and separated by polyacrylamide gel electrophoresis. This investigation disclosed the existence of cell surface antigenic proteins which are derived from the fetal calf serum (FCS) used in the cell-culture medium. These FCS-dependent antigens include at least three molecular species of approximate molecular weights 95,000, 80,000 and 98,000 daltons. They are probably derived from simple adsorption of FCS proteins to the cell surface, although more complex interactions are possible. One of these proteins (95,000 daltons) is of particular interest. It tenaciously adheres to the cell surface so that a trace amount remains even after subculture in the absence of FCS. Rat Rous sarcomas which are morphologically highly transformed appear to bind very little or none of this protein to their surfaces, whereas untransformed rat embryo fibroblasts bind large quantities. A rat Rous sarcoma line which is intermediate in morphological transformation binds an intermediate amount of this antigen. These findings invite speculation that the interaction of certain serum components with the cell surface may be related to plasma membrane properties which distinguish untransformed and transformed cells.


Induction of tumor resistance with BCG-associated tumor antigen. Soluble material enriched in tumor-associated antigen was prepared by affinity chromatography from a KCl extract of the chemically-induced D-23 rat hepatoma. Microgram quantities of the above material bound spontaneously to living BCG when the two were incubated briefly in vitro. When injected into normal syngeneic rats, the BCG-associated tumor antigen induced a measure of resistance against challenge with D-23 tumor cells. Peritoneal exudate cells (PEC) obtained from such actively immunized subjects were able to suppress the growth of D-23 tumor cells at a test site in muscle. In contrast, immunization with either BCG alone, tumor protein alone, or tumor protein admixed with BCG in circumstances designed to impede association of the protein, failed to provoke the formation of tumor suppressor PEC. The results encourage of the belief that binding of tumor antigen to BCG favors the induction of a cell-mediated tumor suppressive response.


A review of treatment and rehabilitation legislation regarding alcohol abusers and alcoholics in the United States: 1920-1971. A review of various legislative and judicial decisions in the area of alcohol abuse and alcoholism is presented. With heightened awareness of alcoholism as our nation's number one drug problem, the federal government has developed a national institute to coordinate multimodal prevention, treatment, and rehabilitation programs. It is noted that several historical cases promoted the courts and investigating committees to recommend to our states that the crime of public drunkenness be removed. The recently developed Uniform Alcoholism Intoxication Treatment Act is discussed along with some of the intervention techniques being employed to deal with drinking drivers.


The effects of various kinds of sparsely-ionizing radiation on total cell RNA and preribosomal nuclear RNA of Novikoff hepatoma ascites cells after in vivo labelling. The effects of 300 kV X-rays, 60Co gamma-rays, 43 MV X-rays and 43 MeV electrons on total cell RNA and 45 S pre-r RNA were investigated in Novikoff Hepatoma ascites cells. Six hours after local irradiation of the animals with 600 rad, the tumour cells were labelled in vivo for 30 and 60 min. The specific activities of high-molecular-weight RNA were influenced differently. This may be due to divergent effects of the applied radiation qualities at the level of transcription and transformation (processing). In addition, these radiation effects are mediated by the adrenal glands in a rather complex manner. Therefore, the results presented in this study support the suggestion that an in vivo labelling system is unsuitable for the evaluation of quantitative radiation effects on RNA.


A C5'-centred deoxyribose radical in single crystals of 5-chloro-and 5-bromodeoxyuridine X-irradiated at low temperatures. X-irradiation at 77 K and subsequent warming to 150 K of single crystals of 5-chloro-and 5-bromodeoxyuridine produces a radical located at position C5' of the deoxyribose moiety. The radical exhibits identical spectral properties in both crystal systems. It is characterized by interaction of the unpaired electron with an alpha-proton(-17-0, -8-7, -33-7G), a beta-proton (18-6, 21-3, 17-5 G) as well as an OH-proton (4-8, 7-9, 12-8 G). The principal values of the g-tensor are 2-0034, 2-0049 and 2-0042. The spectral parameters are discussed in relation to those of the same or similar radicals in other nucleosides (-tides).


In vitro determination of the ability of drugs to bind to adrenergic receptors. Alpha- and beta-adrenergic receptors were studied by measuring the binding of 3H-dihydroergocryptine and 3H-dihydroalprenolol, respectively, to membranes prepared from homogenized rabbit iris--ciliary bodies. The binding of 3H-dihydroergocryptine appears to be specific for alpha-adrenergic receptors, since adrenergic agents displace this radioligand with the following order of potency: phentolamine greater than epinephrine greater than or equal to norepinephrine greater than or equal to isoproterenol = propranolol. The binding of 3H-dihydroalprenolol appears to be specific for beta-adrenergic receptors, since adrenergic agents displace this radioligand with the following order of potency: propranolol greater than or equal to isoproterenol greater than or equal to epinephrine greater than norepinephrine greater than or equal to phentolamine. Clonidine and dopamine bind to the alpha-adrenergic receptor but have little activity at the beta-adrenergic receptor. Timolol, d-isoproterenol, and dipivalyl epinephrine bind to the beta-adrenergic receptor but have little activity at the alpha-adrenergic receptor. The results demonstrate that in vitro binding assays for alpha- and beta-adrenergic receptors are useful for studying the mechanism of drug action.


Human cytomegalovirions and dense bodies: glycopeptide analysis and mechanism of cell rounding and polykaryocytosis. Human cytomegalovirions and dense bodies labeled with [14C]glucosamine were analyzed by means of sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and autoradiography. The same glycopeptide composition was found in both cytomegalovirions and dense bodies. These consisted of at least 11 glycopeptides which ranged in molecular weight from 46,500 to over 170,000 daltons. Addition of purified human cytomegalovirus (CMV) at high multiplicity to confluent monolayers of human fibroblasts produced cell rounding and polykaryocytes containing 3 to 150 nuclei. The cell rounding was induced by the cytomegalovirions, but not by the cytomegalo dense bodies. Inhibition of protein synthesis, but not of DNA synthesis, prevented this effect, suggesting that cell rounding is protein mediated. In contrast, the formation of polykaryocytes by CMV was not affected by inhibitors of protein synthesis. UV irradiation of the virus, which abolishes infectivity, does not affect its fusion properties. CMV dense bodies, which contain very little or no DNA, also produce cell fusion although this effect is less pronounced than with virions. CMV-induced polykaryocytosis therefore appears to be a direct result of the interaction of cells with the input viral particles, a phenomenon usually referred to as early polykaryocytosis.


Role of the Food and Drug Administration concerning chemical contaminants in animal feeds. Chemical contamination of animal feeds and feed ingredients is of concern to the Food and Drug Administration. Contamination by industrial chemicals such as polychlorinated biphenyls and polybrominated biphenyls; heavy metals such as lead, cadmium, and mercury; and pesticides such as dieldrin and chlordane exemplify the problem in feeds and the resulting problem of tissue residues in human foods.


Arrhenoblastoma in a mare.

Arrhenoblastoma in a mare. An ovarian neoplasm measuring 10 by 8 by 6 cm was surgically removed from a 14-year-old Appaloosa mare. For 2 years prior to surgery, the mare had manifested marked behavioral changes, becoming aggressive toward other broodmares. Histologically, the tumor was found to be an arrhenoblastoma. Preoperative endocrinologic findings (high serum testosterone and low serum estradiol concentrations) supported the diagnosis.


Pelvic endometriosis and simian foamy virus infection in a pigtailed macaque. Pelvic endometriosis and simian foamy virus infection occurred in a pigtailed macaque. Diffuse omental, peritoneal, and intestinal implantation of endometrium resulted in massive adhesions between adjacent abdominal and pelvic viscera, with formation of a large mass in the right caudal quadrant of the abdomen. Simian foamy virus type 1 was isolated from ectopic endometrium and from the uterine wall but was considered to be merely epiphenomenal.


Encephalomyocarditis virus infection of captive elephants. Four African elephants at each of 2 widely separated zoologic gardens in Florida died following a fulminating illness. Tissue suspensions obtained from an elephant from each of the zoologic gardens were inoculated into newborn mice, 3- to 4-week-old mice, and buffalo green monkey and baby hamster kidney cell cultures. Encephalitis and myocarditis developed in the mice. The cell cultures were destroyed within 24 to 72 hours, and intracytoplasmic viral inclusions were observed in infected cells by electron microscopy. The viral agent was neutralized by known antiserum to encephalomyocarditis virus.


High pressure liquid chromatographic determination of vitamin D3 in livestock feed supplements. Vitamin D3 is determined in livestock feed supplements by high pressure liquid chromatography (HPLC). Extracts of the samples are quantitated using normal phase chromatography. If interfering co-extractives are present, an aliquot of the extract is injected on the normal phase column, and the fraction corresponding to vitamin D3 is collected. The vitamin fraction is then further cleaned up and separated from interferences by reverse phase chromatography, and quantitated by measuring the ultraviolet absorption at 254 and 280 nm. The method measures actual vitamin D3 content in the presence of pre-vitamin D, tachysterol, isotachysterol, and vitamin A.


Identification of adenosine 3',5'-monophosphate in Mycobacterium smegmatis. Cyclic adenosine 3',5'-monophosphate isolated from Mycobacterium smegmatis cells was identified by thin-layer chromatography, stepwise conversion to adenosine 5'-monophosphate and adenosine, ultraviolet absorption spectrum, phosphate analysis, and detection by two relatively specific radioisotopic methods.


Outer membranes of gram-negative bacteria. XV. Transmembrane diffusion rates in lipoprotein-deficient mutants of Escherichia coli. Permeability of the outer membrane to 6-aminopenicillanic acid was unaltered in an lpo mutant, lacking the Braun lipoprotein, a result suggesting that the lipoproteins by themselves form no or few diffusion pores.


Direct selective procedure for isolating Neurospora mutants defective in nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase. A procedure has been developed for isolating nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (am) mutants of Neurospora. Physiological, genetic, and enzymatic tests show that the new mutants are am alleles. Reconstruction tests and analysis of the new alleles show that the procedure yields a broad spectrum of lesions at the am locus. The isolation of am mutants by this technique appears to be related to the effect of am mutants on the control of the general permease.


Synthesis of ribonucleotides and their participation in ribonucleic acid synthesis by Coxiella burnetii. Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a approximately P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates.


Polyadenylate in the virion RNA of mouse hepatitis virus. Mouse hepatitis (MH) virus was grown in SR-CDF1-DBT, a mouse cell line, and purified by ammonium sulfate precipitation and by density gradient centrifugation. Extraction of RNA from purified virions with 1% SDS and sedimentation analysis of the RNA revealed a major 50S component and two minor components. Treatment of virions with phenol/chloroform also produced the 50S component, although its yield was lower. MH virion RNA can bind to a poly(U)-fiberglass filter, indicating that MH virion RNA contains poly(A). A poly(A)-like fragment was isolated by digestion with ribonuclease A [EC 3.1.4.22] and T1 [EC 3.1.4.8] and by DEAE-Sephadex column chromatography. Analysis of the fragment for base composition showed it to be an adenine-rich material. Its chain length was about 90 nucleotides, as determined by ion-exchange chromatography and gel electrophoresis.


Stimulatory effect of histones on phosphorylation of nuclear phosphoproteins. Effect of histones on phosphorylation of nuclear phosphoproteins was studied using two species of phosphoprotein kinases with different kinetic and catalytic properties; namely, protein kinases A1 and A2 (Takeda, M., Matsumura, S., & Nakaya, Y. (1974) J. Biochem. 75, 743-751). The reaction rate for protein kinase A1 was markedly enhanced when histone or polylysine was added to the reaction mixture. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the basic protein served as a stimulator rather than acted as a substrate in this reaction. In contrast, when protein kinase A2 was employed, the stimulatory action of these basic proteins was less marked than for protein kinase A1. It seems likely that the phosphorylation of nuclear phosphoproteins, particularly the reaction catalyzed by protein kinase A1, may be strongly influenced by histones which are integrated in the chromatin structure.


Essential arginyl residues in yeast phosphoglyceromutase. Inactivation of yeast phosphoglyceromutase (tetramer) with 1,2-cyclohexanedione correlates with the modification of six arginyl residues per mole of the enzyme. Protection experiments using 3-phosphoglycerate suggest that four arginyl residues (one residue per subunit) are involved in the binding of the substrate to the enzyme. The modified enzyme reversibly regained its activity upon incubation with hydroxylamine. The reactivity of lysyl residues which have been shown to be involved in the active site is markedly reduced in the enzyme inactivated with 1,2-cyclohexanedione, indicating that the lysyl and arginyl residues are in close proximity in the active site.


Spin-labeling of adenosine triphosphatase in sarcoplasmic reticulum membrane and change in the state of the spin labels induced by deoxycholate. Fragmented sarcoplasmic reticulum (SR) was reacted with a thiol-directed spin label, N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)maleimide, under various conditions. It was found that ATP inhibited the binding of the label to SR protein in the initial phase of the reaction, but as the incubation time was extended up to 18 h, the amount of label bound to SR protein in the control and ATP-containing samples became almost identical. The Ca2+-dependent ATPase control and ATP-containing samples became almost identical The Ca2+-dependent ATPase (ATP phosphohydrolase [EC 3.6.1.3]) of SR was protected by the presence of ATP during incubation with relatively low concentrations of spin label, irrespective of the total amount of label bound, although with increasing concentration of bound label the ATPase activity decreased. Deoxycholate slightly reduced the rotational freedom of the label bound to SR protein and decreased the initial rate of quenching of protein-bound nitroxide by ascorbate. From an analysis of these results, it was concluded that the binding of deoxycholate to protein decreases the accessibility of ascorbate to the protein-bound label.


Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles. The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.


An ultracentrifugal study of the self-association of canine apolipoprotein A-I in solution. The sedimentation behavior of canine apolipoprotein (apo) A-I in 0.02 M EDTA, pH 8.6, was studied as a function of protein concentration by the techniques of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge. At concentrations of less than 1 g/liter, apo-A-I exhibited a monomodal sedimentation pattern, with apparent sedimentation coefficients which varied from 2.3 to 3.5 S with increasing protein concentrations. Above 1.5 g/liter, apo-A-I had two well resolved peaks with s20,w values of 4.15 S and 5.75 S. The proportion of the 5.75 S component increased with increasing apo-A-I concentrations, with a concomitant decrease of the 4.15 S component. By sedimentation equilibrium ultracentrifugation with both the conventional and meniscus-depletion methods, the apparent weight-average molecular weight of apo-A-I was found to be concentration-dependent. At a protein concentration of 5.25 g/liter, an apparent weight average molecular weight of 138,000 was determined, indicating that molecular species larger than a tetramer (monomer molecular weight = 28,000) were present in solution. When analyzed in terms of a reversible self-associating system, the experimental data could best be described according to a monomer-dimer-tetramer-octamer model, as previously reported from human apo-A-I (Vitello, L. B., and Scanu, A. M. (1975) J. Biol. Chem. 251, 1131-1136). The equilibrium constants were: K2 = 4.5 liters/g, K4 = 470 liters3/g3, and K8 = 41,600 liters7/g7, respectively.


An endonuclease activity of venom phosphodiesterase specific for single-stranded and superhelical DNA. A homogeneous preparation of venom phosphodiesterase from Crotalus adamanteus possesses an intrinsic endonuclease activity, specific for superhelical (form I) and single-stranded DNA. The phosphodiesterase degrades single-stranded T7 DNA by endonucleolytic cleavages. Duplex T7 DNA is hydrolyzed by the liberation of acid-soluble products simultaneously from the 3' and 5' termini but without demonstrable internal scissions in duplex regions. Since venom phosphodiesterase is known to hydrolyze oligonucleotides stepwise from the 3' termini, the cleavage at the 5' end of duplex T7 DNA is ascribed to an endonuclease activity. Form I PM2 DNA is nicked to yield first relaxed circles and then linear DNA which is subsequently hydrolyzed only from the chain termini. The linear duplex DNA intermediates consist of a discrete series of fragments (11 are usually resolved on agarose gels) with initial molecular weights ranging from 6.3 x 10(6) (the intact PM2 DNA size) to approximately 1 x 10(6). The cleavage of the form I molecule must, therefore, occur at a limited number of unique sites. The enzyme also cleaves nonsuperhelical, covalently closed circular PM2 DNA but at a 10(4) times slower rate. Both the endonuclease activity on form I DNA and the known exonuclease activity co-migrate on polyacrtkanude gels, are optimally active at pH 9, are stimulated by small concentrations of Mg2+, and are similarly inactivated by heat, reducing agents, and EDTA.


Substrate-induced conformational changes in lactate dehydrogenase. Proteolysis of the immobilized enzyme in the presence of specific substrates. We report here a new approach to the study of the conformation of enzymes in the presence of specific substrates. Rabbit muscle lactate dehydrogenase was attached to CL-Sepharose via a cleavable spacer arm (-NH-(CH2)6NHCO(CH2)2SS(CH2)2CO-). The bound lactate dehydrogenase was digested with subtilisin BPN' in the presence of substrates of lactate dehydrogenase. The use of a flow system permits the maintenance of saturating levels of substrates. Proteolysis was followed by loss of activity of the enzyme column. The time course of proteolysis in the presence of either NADH, NAD+, or pyruvate alone did not differ from the control. However, when NADH and pyruvate were present simultaneously, the enzyme became more susceptible to proteolysis. The initial rate of proteolysis was increased by 40%. The abortive ternary complex (lactate dehydrogenase - NAD+ - pyruvate) also showed an increase in susceptibility to proteolysis. These findings clearly show that the productive ternary complex (lactate dehydrogenase - NADH - pyruvate) is conformationally different from the apoenzyme and binary complexes under optimal catalytic conditions.


Conformational relaxations of urea- and guanidine hydrochloride-unfolded ferricytochrome c. Several recent studies of protein the unfolded proteins. In urea- and guanidine HCl-unfolded ferricytochrome c (horse heart), an acid-induced spin state transformation of the heme group has been detected by the heme absorptions, Trp-59 fluorescence, and the intrinsic viscosity of protein. Kinetics of this second conformational transition, by the temperature jump and stopped flow methods, are complex. One rapid reaction (tau1), pH-independent, occurs in a 50-mus range; the second reaction (tau2), in a 1-ms range, depends linearly upon pH and is faster at the alkaline side; a third reaction (tau3), in a 1-s range, shows a sigmoidal transition at pH 5.1 and is faster at the acidic side. The results are consistent with a kinetic scheme which involves protein conformational changes in the transformation of the heme coordination state. The kinetics, along with previous equilibrium studies, indicate that ligand or charge interactions within a protein molecule are not completely prohibited even in strongly denaturing conditions, such as in high concentrations of urea and guanidine HCl. Thus, local structures of peptide chain associated with these interactions can exist in the unfolded protein.


Failure of cultured chick embryo fibroblasts to incorporate collagen into their extracellular matrix when transformed by Rous sarcoma virus. An effect of transformation but not of virus production. Whole chick embryo fibroblasts were infected with the Prague wild type Rous sarcoma virus and with a temperature sensitive mutant of this strain, RSVtsLA24. Normal fibroblasts and fibroblasts infected with the temperature-sensitive mutant and cultured at the nonpermissive temperature-sensitive mutant and cultured at the nonpermissive temperature, secreted procollagen into the medium and incorporated collagen into their extracellular matrix. On the other hand, transformed fibroblasts and fibroblasts infected with the temperature-sensitive mutant and cultured at the permissive temperature, were able to secrete procollagen into the medium, but there was no evidence that they were able to convert procollagen to collagen and incorporate collagen into an extracellular matrix. The inability of the infected cells to incorporate collagen into an extracellular matrix was found to be a result of transformation rather than of virus production in these cells.


Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus. Tunicamycin, an antibiotic which prevents the glycosylation of newly synthesized proteins, inhibits the replication of both vesicular stomatitis virus and Sindbis virus. In tunicamycin-treated infected cells, all of the viral proteins are synthesized but the glycoproteins are devoid of carbohydrate. The nonglycosylated glycoproteins could not be detected on the outside of the plasma membrane by lactoperoxidase labeling, indirect immunofluorescence staining, or chymotrypsin treatment of intact cells, whereas the glycosylated glycoproteins were readily detected by all three methods. These results indicate that the bulk of the nonglycosylated glycoproteins are unable to undergo the normal migration to the cell surface. In contrast to the normal glycosylated viral glycoproteins, the nonglycosylated glycoproteins were insoluble in nonionic detergents such as Triton X-100. The nonglycosylated glycoprotein of vesicular stomatitis virus could be solubilized using a combination of 6 M guanidine hydrochloride and 0.2% Triton X-100, but precipitated when the 6 M guanidine was removed by dialysis. These results suggest that the lack of carbohydrate alters the properties of the glycoproteins, which may explain their impaired mobility through the intracellular membranous system.


Studies of low molecular weight RNA from cells infected with adenovirus 2. III. The sequence of the promoter for VA-RNA I. VA-RNA I is one of the very few RNA species produced in animal cells whose transcriptional initiation site is known precisely. We have analyzed the nucleotide sequence of the DNA preceding the 5' end of VA-RNA I and compared it with known prokaryotic promoters and presumptive eukaryotic promoters.


Mitotic activity of differentiated goldfish erythrophores in culture. Integumental erythrophores on the scales of xanthic goldfishes were dissociated and cultured in a monolayer for about 30 days. The proportion of erythrophores in the culture was about 50% at the beginning of cultivation. Erythrophores in culture were characterized by the aggregate of orange-coloured pigment in the centre of the cell body. With the progress of culture the erythrophores develop a few elongated dendritic processes and came to rest on a monolayer sheet of coexisting non-pigmented cells. The erythrophores responded to adrenocorticotropic hormone (ACTH) by producing reversible pigment dispersion and this response was retained throughout the whole period of culture. Dividing erythrophores were found first within 3 days of cultivation and at all times after that. The number  of the erythrophores doubled within 9 days of cultivation and trebled within 16 days. Thus, evidence was provided that differentiated erythrophores of the goldfish still showed mitotic activity in vitro, in contrast to the generally accepted view that fish chromatophores are kind of terminally differentiated cell.


Adrenocortical function in hyperprolactinemic women. To study the effects of prolactin (PRL) on adrenocortical function in humans, dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), androstenedione (delta) and testosterone (T) were measured in serum obtained from 35 hyperprolactinemic women with galactorrhea and amenorrhea before and after treatment with bromocriptine-induced fall in mean PRL levels from 82 +/- 8 (SE) to 14 +/- 2 ng/ml (n = 39, P less than 0.0005), DHAS fell from 322 +/- 21 to 237 +/- 21 microgram/dl (n = 39); P less than 0.0005), DHA fell from 492 +/- 47 to 378 +/- 30 ng/dl (n = 39; P less than 0.01) while T (n = 16) and delta (n = 13) levels were unchanges (44 +/- 4 vs. 49 +/- 4 ng/dl and 280 +/- 55 vs. 236 +/- 40 ng/dl, respectively). In addition, 4 women were infused iv with 25 microgram synthetic ACTH over 4 h and serial blood samples drawn while hyperprolactinemic, and again 2-4 months later following normalization of PRL levels by bromocriptine. Although pre-infusion levels of DHAS were lower when PRL levels were normalized, no significant differences in responses of circulating DHAS, DHA, T, cortisol and 17-hydroxyprogesterone concentrations were detected between the two infusions. Since DHAS is virtually an exclusive product of the adrenal cortex, and since high PRL levels appear to inhibit ovarian steroid production, the findings suggest that hyperprolactinemia selectively stimulates adrenocortical androgen production.


Anaerobes in human biliary tracts.

Anaerobes in human biliary tracts. During a 2-year period, 1,892 patients underwent biliary tract surgery at the Mayo Clinic. Both aerobic and anaerobic cultures of bile were performed in 371 of these patients. Sixty-nine percent of the cultures were positive, and 41% (117) of these grew anaerobes, although they were present in pure culture only twice. Mixed cultures most commonly contained four different organisms (three aerobes and one anaerobe). Bacteroides fragilis was the single most commonly isolated anaerobe and ranked fourth in terms of overall isolates behind Escherichia coli, group D streptococci, and Klebsiella B. fragilis accounted for 7.0% of the total group D streptococci, and Klebsiella. B. fragilis accounted for 7.0% of the total aerobic and anaerobic isolates and was present in 21% of all positive cultures. Pseudomonas aeruginosa and Clostridium perfringens ranked fifth and sixth, providing 6.5 and 5.9% of all isolates, respectively. This study demonstrates the frequent presence of anaerobes in patients with bactibilia and suggests that they be considered in the formulation of antimicrobial therapy for infections involving human biliary tracts.


Enzyme-linked immunosorbent assay for diagnosis of rotavirus infections in calves. An enzyme-linked immunosorbent assay for the diagnosis of rotavirus infection in calves is described. The assay was more efficient for the detection of rotavirus antigens in calf feces than were electron microscopy and immunoelectroosmorphoresis.


The development and degeneration of Purkinje cells in pcd mutant mice. Purkinje cell degeneration (pcd), an autosomal recessive mutation in the mouse, causes the postnatal death of virtually all cerebellar Purkinje cells during the third and fourth postnatal week. We have compared the postnatal development of normal and pcd mutant Purkinje cells. The early deviations from normal development involve primarily the perikaryonal polysomes and endoplasmic reticulum. Many of the mutant Purkinje cells retain abnormally the basal accumulation of polysomes, a finding which permits the identification of affected animals at postnatal day 15, one week prior to the onset of behavioral abnormalities. In addition, the affected Purkinje cells possess unusual configurations of endoplasmic reticulum with associated electron-dense particles similar to but larger than ribosomes, mature and forming intracisternal A particles and nematosomes. Before the pcd Purkinje cells degenerate they appear to receive all their appropriate synaptic contacts. Some disruption, however, of parallel fiber: Purkinje spine synaptogenesis occurs at late stages of development. Some spines lack presynaptic elements, postsynaptic thickenings are present along the dendritic shafts and parallel fibers appear to make synaptic contacts directly onto the shafts. The spectrum of early morphological changes that has been observed in pcd mutant Purkinje cells is thus far unique to this cerebellar abnormality.


The effects of formocresol on rat sponge implant tissue: a biochemical study. An investigation was conducted on the effect of formocresol treatment of the aqueous extractability and enzymatic susceptibility of excised implant tissue. The treated tissue demonstrated a decreased solubility and a diminished digestibility by trypsin, pepsin and collagenase. However, its reactivity toward hyaluronidase showed little alteration.


A laboratory evaluation of the effect of two surface-wetting treatments on soft denture liners. The wettability of acrylic and silicone soft denture-liners was improved by two methods of surface treatment. They consisted of formation of a thin film of silica from the application of silicon tetrachloride, and by cross-linking the surface molecules and bombarding with hydroxyl free radicals. Softness and water sorption were monitored throughout the study.


Inorganic trimetaphosphatase as a histochemical marker for lysosomes in light and electron microscopy. A new cytochemical method is presented for the light and electron microscopic localization of lysosomes in mineralized and soft tissues. Inorganic trimetaphosphate is used as substrate in a lead chelate incubation medium at pH 3.9. Lysosomes in several tissues are strongly reactive, and reaction product is frequently present in Golgi saccules and GERL. The reaction can be differentiated from acid glycerophosphatase activity, is relatively insensitive to fixation and demineralization procedures, and the reaction is often complete after short incubation times.