Activity and subcellular distribution of protein kinase dependent on adenosine 3':5'-monophosphate in liver from normal and adrenalectomized rats.

We have examined whether glucocorticoids control the activity and (or) the subcellular distribution of protein kinase dependent on cyclic AMP (adenosine 3':5'-monophosphate), since they influence cyclic-AMP-dependent responses to other hormones. Protein kinase activity was determined in rat liver homogenates and subcellular fractions, nuclear, large granular, microsomal and supernatant obtained by differential sedimentation in 0.25 M sucrose. 63% of the tissue protein kinase activity detected in absence of cyclic AMP reside in the particulate fractions. Upon addition of exogenous cyclic AMP, protein kinase activity is stimulated 1.8, 1.2, 1.2 and 4.5-fold in nuclear, large granular, microsomal and supernatant fractions, respectively. Under these conditions, 66% of tissue activity are found in the supernatant fraction. The activity sensitive to exogenous cyclic AMP resolves into a major (84%) cytosoluble and a minor (16%) nucleomicrosomal component. The latter activity resists elution with isotonic saline and is increased in the presence of Triton X-100. Three groups of rats were studied: control and adrenalectomized with or without cortisol treatment. In whole liver homogenates, both protein kinase activity detected in absence of exogenous cyclic AMP and sensitivity of the enzyme to cyclic AMP were comparable in all groups. Moreover, the distribution patterns of proteins kinase activity amoung the fractions were essentially the same in all groups of animals, whether or not particles had been treated with Triton X-100. Finally, in cell-free experiments, glucocorticoids alone or in combination with their intracellular receptor did not modify protein kinase activity of rat liver. Thus the results reported do not support the possibility that glucocorticoids influence cyclic AMP-dependent protein kinase in rat liver. Yet, this study provides data, not available before, on subcellular distribution of this enzyme in rat liver.


Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase, from human erythrocytes. Evidence for a common active site.

Bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities responsible for 2,3-bisphosphoglycerate metabolsim in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur J. Biochem. 50, 581-593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate. The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities. Both 2,3-bisphosphoglycerate and 1,3-bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations. Inactivation by trinitrophenylation was an apparent first-order reaction. The dissociation constant of the enzyme - 2,3-bisphosphoglycerate complex was determined by analyzing the first-order reaction on the assumption that the protective effect of 2,3-bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3-bisphosphoglycerate in the enzyme reactions, which indicated that 2,3-bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2-phosphoglycerate at high concentrations interacts with the 2,3-bisphosphoglycerate binding site. These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3-bisphosphoglycerate metabolism in human red cells.


Subunit structure and multifunctional properties of yeast phosphoglyceromutase.

1. A new and efficient method for preparation of pure phosphoglyceromutase from baker's yeast (Saccharomyces cerevisiae) is described. Proteolytic alterations of the enzyme during extraction can be minimized by grinding the dried yeast with aluminium oxide at low temperature. 2. Yeast phosphoglyceromutase contains four highly similar, probably idential subunits of molecular weight 28000, a conclusion based on the following observations. Polyacrylamide gel electrophoresis containing dodecylsulphate or urea gives a single band, indicating that the enzyme is composed of four subunits similar in their molecular weight and net charge. Cyanogen bromide cleavage and tryptic digestion of the enzyme yield the number of peptides expected for identical subunites from the amino acid composition analysis. 3. The purified phosphoglyceromutase preparation has bisphosphoglyceromutase activity synthesizing 2,3-bisphosphoglycerate from 1,3-bisphosphoglycerate and 3-phosphoglycerate. It has been reported that yeast phosphoglyceromutase catalyzes the hydrolysis of 2,3-bisphosphoglycerate at the same active site which catalyzes the phosphoglyceromutase reaction [Sasaki, R. et al (1971) Biochim. Biophys, Acta,  227, 584-594, 595-607]. Immunological studies and chemical modification experiments indicate that bisphosphoglyceromutase activity also is due to the phosphoglyceromutase protein and involves amino groups which have been shown to be essential for the other two activities.


Developmental changes in prolyl hydroxylase activity and protein in chick embryo.

Changes in prolyl hydroxylase activity and immunoreactive protein were studied in various chick embryo tissues during the embryonic development. Both the enzyme activity and the amoung of immunoreactive protein increased till the 16th day of development and declined thereafter in all tissues studied. Comparison of the enzyme activity to the content of the total immuno-reactive protein indicated that there are distinct differences in the degree of enzyme activity between different chick embryo tissues, and in the same tissue between different stages of embryonic development. The highest relative enzyme activities were found in cartilage and skin, in which about 60% of the enzyme was active on the 16th day of development and only 20-30% was active on the 20th day of development; the lowest values were observed in spleen and large vessels, in which below 10% of the enzyme protein was in the active form on the 20th day of development Gel filtration studies demonstrated that in cartilage of 16-day-old chick embryos about 60% of the total immunoreactive enzyme in the tissue was present in the form of active prolylhydroxylase tetramer, whereas on the 20th day of development only 30% of the enzyme protein in cartilage was in the tetramer form. By contrast, in large vessels of the 16-day-old chick embryos, essentially all the enzyme was in the form of prolyl hydroxylase monomers.


Intermittent cerebral symptoms in type V hyperlipoproteinemia.

A case with central nervous symptoms in type V hyperlipoproteinemia was described. The 39-year-old male patient suffered from abdominal pains after fatty meals since childhood. After the age of 31 he developed attacks of cephaleas. Headaches were associated with vertigo, paresis and paresthesia of the limbs and loss of consciousness in some instances. During antilipemic treatment combined with a diet of restricted fat and carbohydrate content the patient became free of complaints. Authors emphasize the impairment of cerebral circulation and tissue hypoxia in the development of cerebral disturbances in hyperlipoproteinemia. The mechanism of the development of hypoxia is discussed.


Long-term ACTH and corticosteroid therapy in two siblings with polyneuropathy due to acute intermittent porphyria.

The influence of long-term corticosteroid and ACTH therapy in two sisters was followed up. The periods of observation were 4 and 2 years. The high levels of corticosteroids obviously reversed the fatal progress of the disease. The relapses in the elder sister occurred during longer periods of discontinued corticosteroid therapy. In the younger the therapy was continuous. There was no relapse except for a short one appearing after the tooth extraction and discontinuation of the therapy for 10 days. Some correlation between neurological signs and symptoms and porphobilinogen androporphyrin levels were found. The question arises how it is possible to influence the genetically determined disease by corticosteroids or ACTH.


Turnover of plasma cholesteryl esters and its relationship to other parameters of lipid metabolism in man.

Studies on plasma cholesteryl esters were made in 32 normal and hyperlipidaemic subjects in an attempt to elucidate further the metabolic relationships between cholesterol and other plasma lipids and lipoproteins. Fractional and net turnover rates of plasma cholesteryl esters were measured using two independent (in vivo and in vitro) methods, both of which determined the prevailing activity of plasma lecithin: cholesterol acyltransferase in the ambiance of the patients own plasma. The results obtained by the two methods were in excellent agreement. The turnover rates of plasma cholesteryl esters in hypertriglyceridaemic patients were significantly greater than in patients with only hypercholesterolaemia and in those with normal plasma lipids. In agreement with these observations, the esterified to free cholesterol ratios in hypertriglyceridaemic patients were significantly greater than in those with normal plasma triglycerides. Patients who had only hypercholesterolaemia had normal esterified to free cholesterol ratios. The rates of esterification of plasma free cholesterol correlated well with triglyceride concentrations as well as with the net turnover of plasma triglycerides and also with the turnover or synthesis of endogenous cholesterol. Since the turnover of plasma triglycerides in most patients with modest to moderate hypertriglyceridaemia reflects the turnover of their plasma very low density lipoproteins, it is suggested that it is the turnover of these lipoproteins which correlates with the hepatic synthesis of cholesterol and with the esterification of plasma cholesteryl esters.


Electron microscopic characterization of lipoproteins from patients with familial type III hyperlipoproteinaemia.

The lipoproteins of very low density, low density and intermediate density were isolated by zonal ultracentrifugation from the plasma of subjects with familial type III hyperlipoproteinaemia and studied by electron microscopy. Each of these lipoproteins exhibited a spherical shape when free-standing. The very low density, low density and intermediate density lipoproteins ranged in diameter from 31 - 80, 17 - 23, .and 21 - 35 nm, respectively. These results are consistent with our previous findings that the intermediate density lipoprotein particles characteristic of plasma from type III subjects exhibit properties which are intermediate between those of very low density and low density lipoproteins.


The antagonism of nicotine-induced cardiovascular responses by DMAE and DEO analogs.

A series of quaternary ammonium compounds cinsisting of 4, 4'-bis-[N-(2, 2-diethoxyethyl)-N, N-dimethylammonioacetyl]-biphenyl dibromide (DMAE) and 4, 4'-bis-[N, N-di(2-ethoxyethyl)-N-methylammonioacetyl]-biphenyl dibromide (DEO) analogs was investigated for selective nicotine antagonism. Each series of compounds contained the monophenyl (MPh.DMAE, MPh.DEO); biphenyl (DMAE, DEO); terphenyl (TPh,DMAE, TPh.DEO); half-molecule (1/2DMAE, 1/2DEO); p-phenyl half-molecule (p-Ph.-1/2DMAE, p-Ph.-1/2DEO) and biphenyl half-molecule (p-BPh.-1/2DMAE, p-BPh.-1/2DEO) analogs. Studies were conducted on isolated spontaneously beating guinea pig atria and anesthetized dogs. Relative potencies of the DMAE and DEO series to antagonize the nicotine (20 mug/ml) induced positive chronotropic effect of the guinea pig atria were determined as follows: DMAE (1.0); TPh.DMAE (0.82); P-Ph.1/2DMAE (0.59); DEO (0.54); MPh.DMAE (0.27); TPh.DEO (0.26); MPh.DEO (0.18); p-Ph.-1/2DEO (0.16); 1/2DMAE (0.03); 1/2DEO (0.02); p-BPh.-1/2DMAE (less than 0.01); p-BPh.-1/2DEO (less than 0.01) and C-6 (0.85). The I50 to antagonize nicotine induced responses by DMAE was 0.13 muM (0.10 mu/ml). Several of the above analogs were studied in the dog and their ability to antagonize nicotine (100 mug/kg) induced positive chronotropic effects were compared with their ability to inhibit transmission through the stellate or the superior cervical ganglia. The I50 doses of the drugs antagonizing nicotine, impairing superior cervical ganglionic transmission and the corresponding fold shifts in the dose--response curves follow: DMAE (120 mug/kg, 3.0 mg/kg, 25); TPh.DMAE (40 mug/kg, 1.10 mg/kg, 30), DEO (45 mug/kg, 1.30 mg/kg, 25) and C-6 (140 mug/kg, 0.42 mg/kg, 0.42 mg/kg, 3.0). These findings are suggestive of the hypothesis that receptors normally activated by endogenously released ACh in the stellate or superior cervical ganglia of the dog may be dissociated from those receptors activated by nicotine which results in an increase in blood pressure or heart rate. Clinical implications are that TPh.DMAE as well as other DMAE analogs may pose less of a problem with hypotension than hexamethonium as a nicotine antagonist.


Effect of a cholinergic stimulant on interaction of isoprenaline with beta-adrenoceptors.

The maximum contraction induced in taenia from the guninea pig caecum by a cholinergic stimulant, butyltrimethylammonium (BuTMA) was relaxed by papaverine (10(-4) M) but not by l-isoprenaline (5 X 10(-7) M). The concentrations used relaxed the taenia completely in the absence of BuTMA. BuTMA (10(-4) M) decreased the increase of cyclic AMP caused by isoprenaline but not that due to papaverine. Futhermore, photoinactivation of the beta-adrenoceptors by l-isoprenaline was not observed on the taenia contracted by BuTMA (10(-4) M). These results suggest that l-isoprenaline does nto interact with the beta-adrenoceptors in the taenie contracted by the high concentration of BuTMA.


Effects of neuroleptic agents on cyclic GMP in rat cerebral cortex.

Cyclic guanosine 3' ,5'-monophosphate(cyclic GMP) levels were increased in incubated tissue slices from rat cerebral cortex in response to added cholinomimetic agents (carbachol and choline chloride) and neuroleptic compounds (chlorpromazine, 8-hydroxychlorpromazine, 7-hydroxychlorpromazine methiodide, haloperidol, thioridazine, chlorpromazine sulfoxide and promethazine). Calcium ion was required for this effect. Moreover, selected agents namely, 7,8-dihydroxychlorpromazine, 7,8-dimethoxychlorpromazine, chlorpromazine and clozapine prevented the rise in cyclic GMP induced by carbachol.


Somatostatin and ACTH are peptides with partial antagonist-like selectivity for opiate receptors.

The peptides, Met-enkephalin and somatostatin were analysed for competitive affinity to opiate agonist receptor sites in rat brain. Met-enkephalin, like morphinomimetic compounds, being equal to marked selectivity for agonist sites while somatostatin and ACTH showed much less selectivity, being equal to partial antagonists like nalorphine. These results are compared with those observed for these peptides in vivo. It is suggested that somatostatin and acth act as partial agonist--antagonist on opiate receptors in the cns.


Effect of some phosphodiesterase inhibitors on central dopamine mechanisms.

The effect of five phosphodiesterase (PDE) inhibitors (papaverine, IBMX, theophyllamine, dipyridamol and M & B 22,948) was studied on adenylate cyclase and on cyclic nucleotide phosphodiesterase activities in extracts of rat caudate nucleus. For comparison the effect on DA turnover and on turning behaviour in rats with unilateral lesions of the nigro-neostriatal DA nerurons was studied. Cyclic AMP PDE was inhibited by papaverine, dipyridamol, IBMX, M & B 22,948 and theophyllamine in that order of potency. Cylcic GMP PDE was inhibited by IBMX, papaverine, M & B 22,948 and theophyllamine, but not by dipyridamol. Basal adenylate cyclase washigher if assayed in the presence of papaverine or dipyridamol than if theophyllamine or IBMX was present. The degree of stimulation caused by DA was not significantly influenced by the PDE inhibitors. Papaverine and dipyridamol enhanced DA disappearance in the caudate nucleus and the tuberculum accumbens, but not in the median eminence. Caffeine had no significant effect. Papaverine (1-28 mg/kg) had no signigicant effect on L-dopa (5 mg/kg)-induced turning, and actually inhibited turning induced by the combination of L-dopa (10 mg/kg) and atropine (5 mg/kg). The other four PDE inhibitors all potentiated L-dopa-induced turning. Theophyllamine (20 mg/kg) and IBMX (5 mg/kg) even caused turning when given alone. The data are compatible with the opinion that PDE inhibition leads to an enhanced effect of DA in the caudate nucleus. However, the results also demonstrate that several of the PDE inhibitors have effects on central DA mechanisms that are difficult to explain solely on the basis of PED inhibition.


The potentiating effects of 4-aminopyridine on adrenergic transmission in the rabbit vas deferens.

The effects of 4-aminipyridine on adrenergic transmission in rabbit vas deferens were investigated. 10(-5)-10(-3) M 4-aminopyridine markedly potentiated the sustained, secondary contractile response to transmural stimulation and significantly increased the amount of (+/-)-|3H]-metaraminol released at 5 Hz, but had little effect on responses to exogenous (-)-noradrenaline. 10(-5)-10(-4) M 4-aminopyridine did not significantly alter the accumulation or efflux of (-)-[3H]-noradrenaline in rabbit heart. It is suggested that 4-aminopyridine potentiated responses of rabbit vas deferens to transmural stimulation mainly by increasing transmitter release, possibly as a result of prolongation of the action potential. Responses to transmural stimulation were also potentiated by by 2- and 3-aminopyridine and by 3,4-diaminopyridine but not by pyridine, aniline, 4-dimethylaminopyridine, 4-aminoethylpyridine or 2-aminopyrimidine.


Effects of ethanol on the cyclic AMP system of the dog gastric mucosa.

The effect of ethanol on the cyclic AMP system of the dog fundic mucosa was studied in vitro. The gastric mucosal content of cyclic AMP was increased by 2.5% ethanol, whereas 10 and 20% ethanol decreased the mucosal content of cyclic AMP. The activity of adenylate cyclase was increased by 2.5 and 5% ethanol, whereas 10% ethanol did not significantly affect it. The activity of cyclic AMP phosphodiesterase was inhibited by ethanol in a competitive manner. The increase in the gastric mucosal content of cyclic AMP, induced by low concentrations of ethanol, is apparently due to the stimulation of adenylate cyclase and inhibition of phosphodiesterase. Changes in the phosphodiesterase or adenylate cyclase activites do not explain the decrease of the mucosal content of cyclic AMP by higher concentrations of ethanol. The mechanism of the decrease is discussed.


Brain dopamine receptors and sleep in the rat: effects of stimulation and blockade.

A high dose of apomorphine, a stimulator of brain dopamine receptors, caused a reduction in total sleep, intermediate sleep and a delayed appearance of paradoxical sleep. With a lower dose, a small and not significant trend toward an increase of paradoxical sleep was observed. Spiroperidol, considered as a specific blocker of dopamine receptors, produced a dose-dependent increase of total sleep and a decrease of paradoxical sleep. Chlorpromazine induced a different effect, that is a clear enhancement of paradoxical sleep. Taken together, these results indicate that an activation of dopamine systems in the brain is partly involved not only in behavioral activation, but also in cortical activation of waking and paradoxical sleep. The effect of chlorpromazine on paradoxical sleep cannot be attributed to the antidopaminergic properties of this drug.


Quantitative studies of intracellular postsynaptic potentials in the lateral geniculate nucleus of the cat with respect to optic tract stimulus response latencies.

LGN cells were intracellularly recorded with glass micropipettes. Electrical stimuli of different amplitude and frequency were applied to the optic tract close to the optic chiasm. The cells were classified according to stimulus response latencies of action potentials as belonging to class I (1.0-16 msec) Or class II (1.7-3.0 MSEC). Class I EPSPs had shorter latencies (1.0-1.5 msec), durations (4-12 msec), rise times to peak (0.5-1.4 msec), and decay times (3.0-8.5 msec); the synaptic transmission time was on the average 0.41 msec. Class II EPSPs (1.6-2.6 msec latency) had longer durations (10-30 msec), rise times (1.6-3.7 msec), and decay times (9.0-25 msec); the synaptic transmission time was on the average 0.67 msec. With repetitive stimulation the EPSPs of latency class I revealed almost no stimulus frequency dependence between 1 and 120 HZ, while class 22 EPSPs decrease in amplitude between 30 and 70% with increasing frequency. Comparable temporal summation of excitation occurred in cells of both latency classes. Negative serial correlation coefficients of first order were found for consecutive EPSP amplitudes of all cells recorded for sufficient periods of time. The IPSPs were subdivided into two groups according to their optic tract response latency. Group 1 IPSPs had shorter latencies (2.0-2.6 msec), durations (15-50 msec), and times from the onset to maximal hyperpolarization (2.4-4.2 msec) than group 2 IPSPs (3.0-4.8 msec latency, 40-100 msec duration, 2.7-7.5 msec time from onset to extremum). The group 2 IPSPs decreased in amplitude by about 90% when the stimulus frequency was increased form 1 to 50 HZ, while the group 1 IPSPs displayed a comparable decrease in the frequency range between 50 and 120 HZ. Effective temporal summation was found in group 2 IPSPs in the frequency range below 70 HZ, and in group 1 IPSPs at stimulus frequencies between 70 and 120 HZ. The EPSP peak latencies and the latencies to the minimum of IPSPs proved to be invariant with respect to PSP amplitude and stimulus fre quency in individual cells. The latencies to the extrema of EPSPs and IPSPs as well as the amplitude values were symmetrically distributed.


Influence of saccadic eye movements on geniculostriate excitability in normal monkeys.

Using permanently implanted electrodes in squirrel monkeys and macaques, transmission through the lateral geniculate nucleus (LGN) was assayed from the amplitude of potentials evoked in optic radiation by and electrical pulse applied to optic tract. Averaging of either individually or machine selected potentials, elicited at 0.3, 1.0, 20 or 50 HZ, in all cases showed a decrease in transmission ranging from 5-60% in the period after saccadic eye movements made ad libitum. The suppression was greater in a patterned visual environment than in diffuse illumination, which in turn was greater than that occurring following saccades in the dark. Demonstration of the effect in darkness always required data averaging and never exceeded 20%. The effect was consistently greater in the magnocellular than parvocellular component. Suppresion was often abruptly terminated and replaced by a facilitation of 5-15% about 100 msec after saccade detection. Comparable effects were observed for excitability of striate cortex tested by a stimulus pulse applied to optic radiation. In addition, sharply demarcated potentials inherently arising in LGN and striate cortex were found in association with saccades made even in total darkness. Neglecting a possible but dubious contribution from eye muscle proprioceptors, the experiments establish the existence of a centrally originating modulation of visual processing at both LGN and striate cortex in ralation to saccadic eye movement in primates. This modulation may partially underlie the phenomenon of "saccadic suppression" and hasten the acquistion of a meaningful visualsample immediately following an ocular saccade. It remains uncertain as to how it may relate to similar or greater effects accompanying changes in alertness, or to fluctuations of unknown origin occurring sometimes semirhythmically at 0.05-0.03 HZ (Fig 7).


Noradrenaline-refractoriness in cultured glioma cells.

Rat glioma cells in culture (subclone C6-SK71) respond to 0.1 mM noradrenaline by a 100 fold elevation of the intracellular cyclic AMP concentration, and by a subsequent change in morphology. The glioma cells extend multiple processes to resemble the morphology of normal astrocytes in brain tissue. Evidence is presented that the responses are mediated by way of a beta-adrenergic receptor. Both the biochemical and the morphological responses to noradrenaline are visible within minutes, but the cells revert to the untreated condition within 8 hours. Addition of fresh noradrenaline does not alter the sequence of events. A refractory period in which neither effect could be evoked by a subsequent exposure to noradrenaline was observed to last about 50 hours and was not correlated with a change in the rate of cell growth.


Measurement of current spread from microelectrodes when stimulating within the nervous system.

Tungsten stimulating microelectrodes have been tested in monopolar, bipolar and concentric configurations for the extent to which unwanted current spread occurred. Current spread from monopolar electrodes in close conformity with the predictions of the inverse square law, both in vitro in vivo. The bipolar and concentric configurations, tested only in vitro, had current-spread characteristics which did not follow the inverse square law so closely. The bipolar configuration gave little reduction in spread, compared wpith a monopolar electrode, but the concentric configuration did reduce the extent of stimulus spread. The extent of spread depends greatly upon a number of experimental variables, including the dimensions of the microelectrode tip. For studies requiring precise localization with low stimulus currents, it is advisable to determine the inverse square law constant for each experimental situation. For more generalized stimulation purposes within the mammalian C.N.S., employing monopolar microelectrodes of moderate tip size, brief pulses of 10 muA can be expected to stimulate myelinated axons with about 0.15 mm of the tip, and 100 muA WILL Stimulate with a radius of about 0.5 mm.


Cyclic AMP during development and ageing of the insect Ceratitis capitata.

Cyclic AMP was determined during the development of the diptera Ceratitis capitata. The concentration of the nucleotide reaches a peak at apolysis with a sharp decline in the pharate adult stage. A gradual increase takes place through the longevity of adult stage reaching a maximum plateau at the end of life.


Tele-encephalic versus cerebellar control upon ponto-geniculo-occipital waves during paradoxical sleep in the cat.

Studying the effects that removal of the cerebellum and the frontal lobes had upon the phasic activities (PGO waves) of paradoxical sleep in the cat, it is shown that in this phase of the sleep-wakefulness cycle the cerebellum exerts an inhibitory action upon the amplitude of the GPO, while the frontal lobes influences the pattern of their discharges.


The effect of combined chronic hypoxia and N-ethyl-N-nitrosourea on the carotid bodies of rats.

We measured the carotid body volume of rats treated with chronic hypoxia alone and chronic hypoxia together with a single neonatal injection of N-ethyl-N-nitrosourea (10 mg/kg). All the animals so treated showed enlargement of their carotid bodies, but no carotid body chemodectomas occurred.


Antagonism of prostaglandin-induced cyclic AMP accumulation in the rat anterior pituitary in vitro by somatostatin analogues.

The PGE2-induced cyclic AMP accumulation in the rat anterior pituitary in vitro is inhibited by [desamino1]-[desamino1] [descarboxy14]- and [D-Lys4]-somatostatin similarly to somatostatin, while the [descarboxy14]-somatostatin exhibits reduced activity; [D-Lys9]-somatostatin is ineffective at a higher concentration.


Isolation of a protease from the cell-free medium of in vitro cultured mammary carcinoma.

Trypsin-like protease with wide spectrum of enzymatic activities have been isolated from cell-free medium from in vivo cultured human mammary carcinoma cells, and from peripheral blood lymphocytes of patients with mammary carcinoma cultured in presence of cell-membrane carcinoma-associated glycoprotein.


Failure to SV40-transformed lizard cells to induce tumors in autogeneic or allogeneic hosts.

Lizard cells from the tails of geckos were readily morphologically and antigenically transformed in vitro by SV40 virus. Neither autografts of these cells nor allografts of SV40 transformed gecko embryo cells produced tumors in animals under observation for 1 to 3 years.


On the mineralocorticoid and hypertensogenic properties of 16beta-hydroxy-dehydroepiandrosterone.

Dosages of either 1 or 2 mg daily of 16beta-hydroxy-dehydroepiandrosterone, given to mononephrectomized, salt-loaded female rats, had no detectable effect upon saline consumption, blood pressure, kallikrein excretion or heart and kidney weight. Its alleged mineralocorticoid properties, as judged by these criteria, were not demonstrable.


Effect of progesterone on the in vivo binding of estrogens by uterine cells.

Progesterone selectively inhibits estradiol uptake by the nuclei of the luminal epithelial cells but not by other uterine cells. This inhibition in estrogen binding parallels the inhibition by progesterone of some estrogenic responses in the luminal epithelial cells only.


Timing of single daily meal influences relations among human circadian rhythms in urinary cyclic AMP and hemic glucagon, insulin and iron.

Relations among circadian rhythms in serum iron, glucagon and insulin and urinary cyclic AMP excretion differ drastically when diurnally active, nocturnally resting human adults consume all daily food for one week as breakfast only and for another week as dinner only - a finding of interest to diverse fields, e.g., for optimizing certain kinds of therapy or for a better utilization of calories.


Rapid purification of lactate dehydrogenase X from mouse testes by two steps of affinity chromatography on oxamate-sepharose.

At concentrations of 200 muM NADH and 0.5 M NaCl LDH-X is separated from the other LDH isozymes of mouse testes on oxamate-sepharose. In a second step LDH-X is bound to the same matrix at lower NADH and NaCl concentrations and the pure enzyme can subsequently be eluted.


Intermediary metabolism of adipose tissue.

Metabolism of ruminant adipocytes involves the synthesis and mobilization of lipids. Rates of lipid synthesis from the uptake of preformed fatty acids (via lipoprotein lipase) and de novo synthesis of fatty acids are related to the energy balance. Acetate is the major carbon source for fatty acid synthesis with NADPH originating from the pentose cycle and the isocitrate cycle. Ruminant adipose tissue lacks the ability to utilize for lipogenesis those substrates that generate mitochondrial acetyl CoA because of an absence of ATP citrate-lyase and NADP-malate dehydrogenase. Lipid mobilization in ruminant adipocytes is apparently regulated via cAMP levels and a summary of the compounds investigated for lipolytic responses is presented. The control of lipid synthesis and mobilization is interrelated in ruminant adipose tissue. The coordinated manner in which these two functions are regulated is examined with regard to adipocyte responses to insulin and epinephrine. In both lipid synthesis and lipid mobilization, ruminant adipocytes are uniquely different from nonruminant adipose tissue. The physiological significance and possible basis for these species differences in adipose metabolism are discussed.


The specificity of gonadotropin binding by the human corpus luteum.

The specificity of gonadotropin binding was studied in fresh and frozen human corpora lutea. Ovine, bovine, and porcine luteinizing hormone (LH) competed with 125I-labeled human LH (125I-hLH) and 125I-labeled human chorionic gonadotropin (125I-hCG) for binding to tissue receptors in homogenates of human corpora lutea frozen for 3 to 12 months. In contrast, oLH, bLH, and pLH competed minimally for 125I-hLH and 125I-hCG binding sites in homogenates of fresh human corpora lutea. Ovine follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) did not compete in homogenates of fresh or frozen tissue. Competition of oLH and hCG for 125I-hCG binding sites at several dose levels in a homogenate of a fresh corpus luteum was studied. One hundred micrograms of oLH and ten nanograms of hCG gave an equivalent competition--a 10,000-fold difference in competitive potency. Only hCG competed with 125I-hCG for binding when the competition of oLH, bLH, pLH, oFSH, oTSH, hCG and hCG subunits, and hCG were compared at the 10-mug level in a homogenate of fresh human corpus luteum. The binding of 125I-labeled homologous human hormones by the corpus luteum was examined in a limited fashion. 125I-Prolactin did not bind to preparations of fresh stroma from a patient with polycystic ovaries nor did it bind to three separate preparations of fresh corpora luteum which did bind 125I-hCG. 125I-hTSH did not show significant binding to a fresh human corpus luteum preparation which did bind 125I-hCG. These studies indicate that the gonadotropin receptor of the fresh human corpus luteum possesses a unique species specificity and illustrate the importance of working with human corpora lutea in their most native state.


[The effect of metallic ions on steroid hormone receptors in the rabbit uterus (author's transl)].

The copper-IUD has been proved to be more effective for contraception than the popular IUD. To understand this mechanism of action, the effects of metallic ions on estrogen and progesterone receptors were examined in the cytosol of estrogen primed with female rabbits. The 274,200 X G supernatant of the uterine homogenate was used as the cytosol. The cytosol and 3H-progesterone (3.12 X 10(-9)M) or 3H-estradiol-17 beta (3.46 X 10(-9)M), with or without various metallic ions of different concentrations had been incubated for 2 hours. Bindings of steroids were estimated by the dextran coated charcoal assay (0.001% dextran and 0.1% Norite A) and were evaluated by 5 approximately 20% sucrose linear gradient centrifugation 1) Effect of various metallic ions on steroid hormone-receptor binding (determined by dextran coated charcoal assay. Fig. 1) : Thesteroid hormone-receptor interactions were markedly inhibited by Cu++, Fe++, and Zn++ ions and moderately by Mn++, but K+ and Ca++ increased of slightly affected the binding at concentrations between 10(-2)M and 10(-4)M. There were some differences between estrogen and progesterone receptors in their sensitivities to various metallic ions. 2) The effect of copper ion on the binding of steroid to the receptor : The dextran coated charcoal assay (Fig. 2) demonstrated that the inhibitory effect of Cu++ appeared at 10(-6)M and the steroidhormone-receptor bound decreased down to 10% at 10(-2)M Cu++. The estrogen receptor was less affected by copper than the progesterone receptor. It was demonstrated by 5 approximately 20% sucrose linear gradient centrifugation (Fig 3) that estrogen and progesterone receptors, which both sedimented at 8 S, were changed to more sedimenting forms in the presence of 10(-4)M Cu++, and were dissociated to 6.5 S form with moderate loss of steriod hormone binding affinity in 10(-2)M Cu++. The kinetic study (Fig 4), determined by dextran coated charcoal, showed that Cu++ was a competitive inhibitor against steroid hormone receptor bindings with Ki in equilibrium 2.7 X 10(-5)M to estrogen receptor (Kd in equilibrium 1.4 X 10(-9)M), and with Ki in equilibrium 5.1 X 10(-6M to progesterone receptor (Kd in equilibrium 8.1 X 10(-10)M). These results indicate the inhibiting factor of copper is the direct interference at the steriod binding site of the receptor, resulting in the increase in the effectiveness of the IUD. While progesterone receptor is more affected by copper ion than estrogen receptor, it is suggested that estrogen receptor survives longer than progesterone receptor and thus the biolgoical effect of copper seems to be somewhat estrogenic.


[Effects of low salt plus upright posture, angiotensin II, ACTH, and potassium upon plasma renin activity, aldosterone, and cortisol (author's transl)].

In order to study the control system of plasma aldosterone in human, we examined the effects of low salt plus upright posture, angiotensin II, ACTH and potassium upon plasma renin activity, aldosterone and cortisol in five subjects who were supposed to be normal. All of the procedures, low salt diet with below 3 g of salt and 2 hr-upright posture, 0.25 mg of Cortrosyn, angiotensin infusion to increase 20mmHG of diastolic pressure for an hour, and 30 mEq of potassium infusion stimulated plasma aldosterone significantly. Furthermore, in each subject the degrees of response to each of these stimulations were almost same. In an old woman aged 68, responses to all of stimulations were significantly lower than those in other subjects. Plasma cortisol was significantly stimulated by ACTH, but slightly reduced by potassium infusion. From these results, it is certain that plasma aldosteron levels are easily affected by a small amount of changes in angiotensin, ACTH, potassium and sodium. However, responses of aldosterone to these changes seem to be decreased in old subjects.


[Studies on the responsiveness of human adrenocortical tumors to ACTH the clinical and experimental observations (author's transl)].

Six cases of Cushing's syndrome with adrenocortical tumors which showed a variety of responsiveness to ACTH were investigated in relation to their clinical pictures and laboratory findings. Abnormal responses to ACTH in tumors was analyzed by in vitro experiments with surgically obtained tumor tissues, and the ACTH responsive mechanism of the tumors was discussed. An 8 hour intravenous ACTH infusion test showed that three of these patients were ACTH responsive, and the other three unresponsive. Histological observation of the tumors revealed that ACTH responsive tumors were adenomas and that ACTH unresponsive tumors were "black adenomas" in two and a carcinoma in one. To investigate possible factors which might account for these differences in ACTH responsiveness, tumor specimens of each one of the responsive and unresponsive adenomas, and a carcinoma were subjected to in vitro studies. When incubated with ACTH or cyclic AMP, tissue sections of a responsive adenoma enhanced cortisol secretion, while that of a black adenoma failed to show any change. Steroidogenesis by carcinoma sections were significantly suppressed in the presence of ACTH or cyclic AMP. Cycloheximide abolished a stimulatory effect of ACTH and cyclic AMP on steroidogenesis in a responsive adenoma without affecting its basal secretion of cortisol. Steroidogenesis by unresponsive tumors (an adenoma and a carcinoma) were decreased by cycloheximide. Since the conversion of cholesterol to pregnenolone, the rate limiting step in steroidogenesis, takes place in adrenal mitochondria, the effect of cyclic AMP on pregnenolone formation from 14C-cholesterol by mitochondrial fractions of these tumors was examined. Cyclic AMP stimulated pregnenolone formation by mitochondrial fraction of an ACTH responsive adenoma, while with that of an unresponsive adenoma pregnenolone formation was not affected. Pregnenolone formation by cancer mitochondria was significantly suppressed by cyclic AMP. These results suggest that the unresponsiveness to ACTH of these tumors might be explained by the ineffectiveness of cyclic AMP to stimulate pregnenolone formation by tumor mitochondria, and that the steroidogenic pathway in unresponsive tumors are in an enhanced state even without cyclic AMP. It should be mentioned that all unresponsive adenomas gave a characteristic appearance of a "black adenoma". Histologically, tumors were composed of compact cells with abundant lipofuscin granules. The possible relationship between the ACTH responsiveness of adrenocortical tumors and some clinical pictures caused by them was also noticed. ACTH unresponsive adenomas resulted in shorter duration, severer conditions of the disease and higher 17-ketosteroid excretion than responsive adenomas. The growth of unresponsive tumors seemed faster than that of responsive ones.


[Effect of cyclic AMP and Ca++ on steroidogenesis by rat adrenal mitochondrial fraction. Studies on the mechanism of ACTH action (I) (author's transl)].

Despite the accumulation of a number of studies, the mechanism of action of ACTH remains to be clarified. Although it is now clear that cyclic AMP acts as a intracellular mediator of ACTH action, the mechanism of its action on the stimulation of steroidogenesis is not known. The present studies were carried out to test the hypothesis that cyclic AMP might act directly on adrenal mitochondrial fraction to stimulate the metabolism of cholesterol to pregnenolone and progesterone, and to determine whether Ca++ might modulate the action of cyclic AMP. Adrenal mitochondria were obtained from male Sprague-Dawley rats pretreated with dexamethasone. Steroidogenesis by the mitochondrial fraction from cholesterol-4-14C (0.2-0.25 muCi, 3.6-4.5 mmumole/sample) were measured in a system containing 20 mM tris-HCl buffer (pH 7.4), 11.5 mM NaCl, 15.4 mM KCl, 70 mM sucrose, 10 mM sodium succinate and mitochondrial fraction (0.16-0.22 mg protein/sample). Incubations were performed at 37 degrees C, with shaking, in the presence or absence of cyclic AMP, cyclic GMP and cycloheximide. After incubation, the medium was extracted with chloroform, and the extracts were analyzed by thin-layer chromatography. And the radioactivity of the separated steroids was measured. The products from cholesterol-4-14C were mainly pregnenolone and progesterone, and the other products were almost negligible. Cyclic AMP effected the formation of pregnenolone and progesterone by mitochondria. Cyclic AMP exerted its effect even at low concentrations (5 X 10(-6) approximately 5 X 10(-5)M), which was presumably near the intracellular level. On the other hand, cyclic GMP (5 X 10(-5) M) failed to enhance steroidogenesis. The effect of Ca++ on the action of various concentrations (5 X 10(-6) approximately 3 X 10(-3) M) of cyclic AMP was also clearly demonstrated. Addition of Ca++ (1 mM) to the incubation medium intensified the stimulatory effect of cyclic AMP in each concentration. And in the presence of Ca++, the most effective level of cyclic AMP was shifted from 5 X 10(-4) approximately 3 X 10(-3)M to the lower concentration (5 X 10(-5)M). In addition, cyclic AMP action was modified by the changes in the concentration of Ca++ in the medium. At concentration of 10(-6) M of Ca++, steroid formation of mitochondria was maximally activated by cyclic AMP. These observations suggest that cyclic AMP enhances steroidogenesis by acting directly on adrenal mitochondria to stimulate pregnenolone and progesterone formation from cholesterol, and that Ca++ plays a significant role in its action.


Characterization of two forms of cyclic 3', 5'-adenosine monophosphate-dependent protein kinase in rat testicular interstitial cells.

The adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-dependent protein phosphokinase of rat interstitial cells was characterized by ion-exchange chromatography and sucrose density gradient centrifugation. The 0.2 M NaCl fraction from DEAE-Sephadex showed a small 2.9-S peak of basal enzyme activity, and a large 6.5-S peak of cyclic AMP-dependent protein kinase activity; fractions eluted from DEAE-Sephadex with 0.3-0.5 M NaCl contained a major 3.8-S peak of cyclic AMP-dependent enzyme activity. Activation of protein kinase in cell extracts by cyclic AMP, and in intact interstitial cells by trophic hormone, caused a major shift of enzyme activity to the 2.9-S cyclic AMP-dependent form which was eluted from DEAE-Sephadex by 0.2 M NaCl. These results are consistent with the presence of two distinct protein kinase holoenzymes, with a common 2.9-S catalytic subunit. During hormonal activation of protein kinase in dispersed interstitial cells by 10-10 M human chorionic gonadotropin (hCG), conversion to the 2.9-S catalytic subunit was observed between 2 and 30 min of incubation. Protein kinase activity was correlated with cyclic AMP production, and full enzyme activation occurred at the time of maximum intracellular cyclic AMP concentration. The presence of two forms of cyclic AMP-dependent protein kinase in the Leydig cell provides a potential mechanism whereby progressive occupancy of gonadotropin receptors could evoke a series of discrete target cell responses.


Biphasic effect of testosterone propionate on Sertoli cell secretory function.

When various doses of testosterone propionate (10 to 10,000 mug/day) were given to 21-day-old rats for 10 days a biphasic effect was seen both on testis weight and production of androgen-binding protein (ABP). At low doses (10 to 100 mug testosterone propionate/day) there was a reduction in testis weight as well as ABP content in the epididymis. At higher doses of testosterone propionate, there was a stimulation of both testicular weight and ABP production in spite of suppressed serum FSH and LH levels. These effects of testosterone propionate on Sertoli cell secretory function strongly suggest that the Sertoli cell is a target cell for androgen.


The effects of cholera toxin on the adrenal weight in hypophysectomized rats.

The effects of cholera on adrenal weight in hypophysectomized rats were investigated, in an attempt to demonstrate an ACTH-like, adrenal trophic effect of the toxin. The results suggested that the toxin probably exerts is ACTH-like action on the adrenal via adenylate cyclase. Cholera toxin was also shown to have a thermolytic action, similar to that of ACTH, probably due to stimulation of adrenal glucocorticoid secretion.


Partial purification of the activated hepatic glucocorticoid - receptor complex.

A rapid procedure for the purification of the hepatic glucocorticoid receptor has been developed which exploits the observation that "activation" of this complex enables it to bind to anionic substances such as DNA and phosphocellulose. The procedure consists of two phosphocellulose columns operated in sequence. The first column removes from unfractionated cytosol all basic proteins which adhere to the immobilized phosphate residues; the steroid - receptor complex elutes in the flow-through of this first column. This complex is then thermally activated and applied to a second phosphocellulose column where it is retained, washed, and eluted by a salt gradient. This simple procedure is capable of purifying the steroid - receptor complex over 1000-fold.


Apparent multiple forms of cyclic AMP phosphodiesterase from rat erythrocytes.

Bio-Gel A-5m chromatography has been used to separate apparent multiple forms of cyclic nucleotide phosphodiesterase from rat erythrocytes. Cyclic AMP phosphodiesterase was resolved by gel filtration into three peaks of activity with apparent molecular weights of about 300,000, 225,000 and 100,000, while cyclic GMP phosphodiesterase activity in gel column fractions was too low to permit meaningful estimates of its molecular weight. All three of the separated peaks of cyclic AMP phosphodiesterase activity displayed anomalous kinetic behaviour suggestive of negative cooperativity. The possibility that multiple phosphodiesterase activities could arise from in vitro alterations of a single enzyme was investigated. Similar changes in gel filtration profiles resulted when erythrocyte extracts were treated with trypsin or ammonium sulfate or were incubated at 37 degrees C. After these treatments, a large proportion of the enzyme activity occurred in low (ca. 100,000) molecular weight regions. The low molecular weight phosphodiesterase activities from untreated, incubated, and trypsin-treated extracts possessed similar properties. All were inhibited by methylxanthines, had pH optima of approximately 8.0, and similar kinetic properties and requirements for divalent cations. These observations raise the possibility that preparative procedures or limited proteolysis occurring during preparation and handling of extracts can contribute to the apparent multiplicity of enzyme forms seen after gel filtration of phosphodiesterase from rat erythrocytes and perhaps other cell types.


Cyclic nucleotide phosphodiesterases, insulin and thyroid hormones.

The in vitro effects of insulin on different phosphodiesterase activities present in rat epididymal fat cells from normal and hypothyroid rats have been studied. Evidence is presented that insulin increases the maximum velocity of a particulate, low Km, cyclic adenosine-3', 5'-monophosphate (cyclic AMP) phosphodiesterase in both types of cells, this effect being more clearly evident with the fat cells from hypothyroid animals; combination of insulin and thyroidectomy resulted in a 400% stimulation with 10-10 - 10-9 M insulin. A clear and significant effect was apparent at 10-11 M insulin. However, the dose-response curve was biphasic, since stimulation by insulin was suppressed for doses of hormone higher 10-8 - 10-7 M. Moreover, insulin effects were very fast, since clear stimulation was observed after only 2 min of incubation; the maximal increase was obtained after 10 min. Insulin did not significantly affect the soluble cyclic AMP phosphodiesterase activity in normal cells, thus confirming results obtained by others. However, the soluble cyclic AMP phosphodiesterase activity was clearly stimulated by insulin when the fat cells were prepared from hypothyroid rats. Maximal stimulation was obtained with 10-9 M insulin; the response was again very fast. Soluble cyclic GMP phosphodiesterase activity was also increased additively by hypothyroidism and insulin, maximal stimulation being obtained with 10-9 M insulin. With this dose of insulin the additive effects of thyroidectomy and insulin produced a 5-fold stimulation. The effect of insulin on the soluble cyclic GMP phosphodiesterase was very fast (2-5 min). With both soluble cyclic nucleotide phosphodiesterase activities, insulin increased the maximal velocity but not apparent Km of the enzyme. Thus, hypothyroidism and insulin produced additive effects suggesting a different mechanism of action of these two hormonal situations on the degradation of the intracellular pools of cyclic AMP and cyclic GMP.


Insect prothoracic glands: a role for cyclic AMP in the stimulation of alpha-ecdysone secretion.

The prothoracic glands of the tobacco hornworm, Manduca sexta, were studied to determine if cyclic AMP is involved in the regulation of alpha-ecdysone secretion. Culturing glands in the presence of the phosphodiesterase inhibitors, aminophylline and 1-methyl-3-isobutylxanthine, caused a greater than 2-fold stimulation of ecdysone secretion while cyclic AMP alone was ineffective. Based on a dose-response analysis, 1-methyl-3-isobutylxanthine was 200 times more potent than aminophylline. Measurements of endogenous prothoracic gland cyclic AMP during the fifth larval instar demonstrated that dramatically increased levels preceded the increase in in vitro ecdysone-secretory ability. The data suggest that cyclic AMP may act as a second messenger in the stimulation of prothoracic gland alpha-ecdysone secretion by the prothoracicotropic brain hormone.


Stabilization and characterization of the dexamethasone-binding proteins in rat liver cytosol.

Conditions were worked out for maximal stabilization of dexamethasone binding activity of rat liver cytosol in the absence of the protective steroid ligand. Important stabilization factors are ionic strength, thiol-protecting agents, glycerol and pH. Maximal stability of the cytosol is observed in a buffer consisting of 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 25 mM beta-mercaptoethanol and 20% glycerol. Chromatography of cytosol on DEAE-cellulose revealed the existence of three dexamethasone receptors, binder DE-1, present in the flow-through fraction and binders DE-2 and DE-3, eluting from the column with salt concentrations of 100 and 190 mM, respectively. Binders DE-2 and DE-3 are not adsorbed on phosphocellulose at pH 7.5, whereas binder DE-1 is. All three receptors are retained to varying degrees on DNA-cellulose columns: binder DE-1 is eluted with salt concentrations of 270 mM, whereas binders DE-2 and DE-3 are eluted between 180 and 200 mM NaCl. The dexamethasone receptors also bind natural glucocorticoids, but to varying degrees, the highest binding being observed to binder DE-2. The receptors obtained after chromatography on DEAE-cellulose, but not on phosphocellulose, cannot be to an appreciable extent charged with dexamethasone.


Cholinergic regulation of cyclic nucleotide levels, amylase release, and K+ efflux from rat parotid glands.

Carbachol increased amylase release and K+ efflux from rat parotid tissue slices. The amount of amylase released was small compared to that released by isoproterenol. The effect of carbachol on amylase release and K+ efflux was a direct effect. This conclusion was based on the finding that the stimulatory effects of carbachol were blocked only by atropine and not by propanolol or phentolamine. In addition to the above effects, carbachol also caused a rapid increase in the parotid guanosine-3', 5' cyclic monophosphate (cGMP) levels without a discernable effect on adenosine-3',5' cyclic monophosphate (cAMP) levels. The increase in cGMP level caused by carbachol was blocked by atropine and not by phentolamine. The stimulatory effect of carbachol on amylase release was not additive with that of isoproterenol or dibutyryl cAMP. Although carbachol had no effect on basal cAMP levels it did inhibit increases in cAMP caused by isoproterenol. Similarly isoproterenol inhibited increased in parotid cGMP levels caused by carbachol. Unlike the apparent nonadditivity between the effects of isoproterenol and carbachol on amylase release and cAMP and cGMP accumulation, the effects on K+ efflux were additive. The possibility of a role for cGMP in mediating the effects of cholinergic agonists on K+ efflux was lessened by our observations that 1-methyl-3-isobutylxanthine enhanced the effect of limiting concentrations of carbachol on cGMP accumulation while not enhancing the effects of carbachol on K+ efflux.


Non-specific immunity after local immunization.

The most important advantage of local immunization is the non-specific effect. The active protection test in mice shows an immunity against oral infection with virulent Salmonella typhimurium bacteria after oral immunization with heterologous inactivated enterobacteria. We observed the same non-specific protection in a viral model. After oral immunization with inactivated S. typhimurium bacteria, the mice are protected against oral infection with a virulent parapoliomyelitis virus. In the phagocytosis test after local immunization a non-specific effect is also demonstrated. The rate of phagocytosis of macrophages against streptococci rises significantly after oral immunization or after inhalation with heat-killed S. typhimurium bacteria.


Characteristics of the response of human adrenocortical cells to ACTH.

The effects of adrenocorticotrophic hormone (ACTH) on human adrenocortical steroidogenesis were studied in adrenocortical cells which had been isolated from normal and hyperplastic glands by a technique combining tyrpsin digestion and mechanical dispersion, and incubated in the presence of ACTH or dibutyryl cyclic AMP (dbcAMP). The response was measured in terms of cyclic AMP, cortisol, corticosterone, 11-deoxycortisol and cortisone production. A classical sigmoid curve, calculated by non-linear, least square method, related the increase in cAMP production or in steroidogenesis to the log dose of ACTH. For the normal adrenocortical cells, the estimated concentration of ACTH inducing a half-maximal response was approximated 2h0 pg ACTH 1-24/ml for steroidogenesis, against 437 pg/ml for cAMP production. The estimated Vmax (per 107 cells/ml, on average) was 27 pmol cAMP/2 and for steroidogenesis (in ng/2 h): 188 for cortisol, 106 for corticosterone, 37 for 11-deoxycortisol, and 32 for cortisone, dbcAMP (1.0 mM) stimulated steroidogenesis to a comparable extent. The cells from a hyperplastic adrenal gland exhibited a steroidogenic response to ACTH and dbcAMP which was 2-3 times greater than the response of a similar number of normal adrenocortical cells. Calculated per pmol cAMP generated, the ACTH-stimulated cortisol production by cells from hyperplastic gland was also increased with respect to normal cell response. These data suggest a prolonged effect of ACTH on cortisol biosynthetic pathway beyond the membrane step of cAMP generation.


Similarity of responses of cultured Sertoli cells to cholera toxin and FSH.

The effects of cholera toxin on the responses of cultured Sertoli cells were compared with those elicited by follicle-stimulating hormone (FSH), and N6O2'-dibutyryl-3',5'-cyclic AMP (bu2cAMP). Addition of FSH or cholera toxin increased cAMP levels. Subsequently there was greater rates of conversion of testosterone to 17beta-estradiol, formation of androgen-binding protein (ABP), and incorporation of [3H]thymidine into DNA by Sertoli cells prepared from testes of immature rats and cultured in the presence of either FSH or cholera toxin. Addition of bu2-cAMP also resulted in enhanced rates of formation of ABP, synthesis of 17beta-estradiol and synthesis of DNA. Cholera toxin and bu2-cAMP elicited changes in morphology of cultured Sertoli cells indistinguishable from those following FSH addition. It is concluded that elevated intracellular cAMP levels can duplicate known actions of FSH on cultured Sertoli cells, but the possible obligatory role of cAMP in mediating FSH actions remains to be evaluated.


Oral immunization against pox. Studies on fowl pox as a model.

Oral vaccination against fowl pox is both effective and harmless. A suitable vaccine strain is the attenuated HP-1 strain in the 200th to 400th tissue culture passage. For optimal immunization virus of the 200th to 270th passage level should be applied twice at an interval of 3 to 4 weeks. Vaccination dose should contain 10(7.0)TCID50.  Chickens may be effectively immunized already at 5 days. Immunity is against both homologous and heterologous virus and proves equally resistant to oral, cutaneous and intravenous challenge. Protection of mucous membranes of the upper respiratory and digestive tract forms faster than after cutaneous immunization. Compared to conventional fowl pox vaccination, drinking water vaccination facilitates the technicalities of vaccination; there are immunological advantages and risks of complications are avoided. It can therefore be recommended as a model for vaccination against smallpox in man.


Hormonal control of cyclic AMP turnover in isolated fat cells.

Control of the levels of cAMP in the early phase after addition of catecholamines and the effect of insulin is discussed under consideration of own findings from experiments with isolated fat cells of the rat. Data on the kinetics of cAMP are interpreted in the light of results from several groups of a rapid activation of phosphodiesterase activity along with the adenylate cyclase system. Comparison of energy metabolism of fat cells with the formation of cAMP under conditions of near-maximal activation of the adenylate cyclase system by isoproterenol shows that about half of the cellular ATP turnover is used for information transfer. Insulin reduces cAMP concentrations in the presence of isoproterenol within one min of incubation when added either together with or after the catecholamine. Experiments with propranolol and the phosphodiesterase inhibitor, methyl isobutylxanthine suggest an effect of insulin on formation and breakdown of cAMP.


Oral immunization against smallpox.

Attenuated vaccinia strain penetrates the mucous membranes of the oral and pharyngeal cavity; local lesions are not produced, even if high doses of attenuated virus are applied. Attenuated vaccinia virus thus offers the possibility of  oral and  nasal immunization against smallpox in man. Several groups of monkeys were immunized by the oral and nasal route. The subsequent challenge with virulent smallpox virus resulted in strongly mitigated clinical symptoms as compared to non-immunized controls. In the human, oral immunization with live attenuated virus was affected by the administration of virus-containing tablets. The procedures caused no untoward effect in primary vaccinees. Subsequent skin testing and conventional cutaneous vaccination resulted in accelerated takes, demonstrating successful oral pre-immunization.


A synergistic effect of androgen on the stimulation of progesterone secretion by FSH in cultured rat granulosa cells.

Granulosa cells from preovulatory follicles (PO) or from the enlarged preantral follicles of hypophysectomized immature diethylstilbestrol-treated (Hx-DES) rats were cultured with various combinations of FSH, androst-4-ene-3,17-dione (Ad), estradiol-17beta and dibutyryl cyclic AMP (dbcAMP). Progestin levels (progesterone and 20alpha-dihydroprogesterone) in the medium after 2 days of culture were assayed by radioimmunoassay. The control levels of the two progestins were lower for Hx-DES than for PO cells. Rat FSH (NIAMD-1-3;0.1 mug/ml) caused a 2-fold rise in progestin accumulation in both PO and Hx-DES cultures, dbcAMP (1 mM) increased progestin accumulation in PO cultures 4-5-fold, and to an even greater extent (10-20 fold) in Hx-DES cultures. Androstenedione (1.0 mug/ml) augmented progestin accumulation (1.5-3-fold), and synergized the steroidogenic action of FSH: in cells from Hx-DES rats, combined treatment with FSH and Ad caused a 5-10-fold increase over the values obtained with FSH alone. Testosterone and 5alpha-dihydrotestosterone, but not estradiol-17beta or estrone, mimicked these effects of Ad, Ad did not synergize the action of dbcAMP on progestin levels in Hx-DES cultures. It is proposed that androgen may play a role in the development of the FSH-responsive mechanism in preantral granulosa cells.


Local immunity in Newcastle disease: some recent experiments.

Following primary exposure by the ocular route to lentogenic Newcastle disease virus (NDV) the lacrymal fluid, saliva and tracheal washes  of three-week old specific pathogen free chickens acquired specific virus-neutralizing activity which considerably exceeded transudation of circulating antibody. All three secretions contain IgA which, at least in saliva, accounted for 85% of its activity, the remanider being due to IgG. Antibody in secretions limited, but did not prevent, reinfection of the trachea when birds were challenged two weeks  later. In contrast to an elevation of circulating antibody titre, challenge induced only a repeated primary response in secreted antibody. Ocular infection induced marked lymphoid and plasma cell activity in the Harderian gland which is a major source of specific antibody in lachrymal and possibly other fluids. Functional ablation of the gland can be effected by occlusion of its draining duct, providing a means for evaluation of its immunological significance.


The proposed international standard for infectious bronchitis vaccine (live).

An H-120 strain of infectious bronchitis virus was prepared in a form suitable to serve as an international standard for  Infectious Bronchitis Vaccine (live). Accelerated degradation studies carried out on this vaccine showed it to be of suitable stability to serve as a standard. Results from a collaborative assay involving ten laboratories demonstrated that the proposed standard reduced the variation in virus titrations both within and between laboratories. This assay also showed that firstly the proposed standard could be used as a reference vaccine for the different safety tests described by the collaborating laboratories, and secondly as a  reference vaccine in the different potency tests.


Studies on vaccination against infectious laryngotracheitis by the drinking water.

The authors, after a brief reference to the wide use in the field of a mild vaccinal strain of infectious laryngotracheitis virus by the drinking water to prevent the disease, describe laboratory experiments to confirm the efficacy of this route of administration compared with the conventional one (ocular). Four groups of 7-week-old chickens were vaccinated as follows: a) ocularly with the normal dose, b)by instillation per os, c) by the drinking water with one dose, d) by the same route with ten doses; a fifth group was left as negative control. To compare the effect of  vaccination by the above different methods of administration the serum-neutralization test was used. The results obtained are finally discussed.


Vaccination by the non-parenteral route of virus disease in the veterinary field.

After a brief reference to the importance of the non-parenteral route of vaccination of domestic animals in general, the author deals, for each animal species separately, with the most important vaccines utilised by this method of administration. On the basis of bibliographical data, he describes the history of this use, discusses the results of the application in the field and draws the relative conclusions.


Comparative tests on safety and potency of IBA vaccines.

During a comparative test on the pathogenicity and immunogenicity of a non-adapted and an embryo-adapted IDA virus, following observations were made: 1) the non-adapted virus remained pathogenic as determined by weight loss and bursal lesions. Clinical signs and mortality did not occur. A change in the virulence did not occur during back passages; 2) the immunosuppressive effect of the non-adapted virus was diminished by maternal antibodies; 3) the embryo-adapted virus produced little pathogenicity as demonstrated by minor weight depressions. The vaccinated birds had no recognizable pathological lesions in the bursae. Immunosuppression with this virus was not observed; 4) the embryo-adapted strain was able to induce neutralizing antibodies in birds with or without maternal antibodies. However, precipitins were not detected; 5) immunization with the embryo-adapted strain after 8 days of age was protective whereas prior to that age it was not confirming the age susceptibility factor reported by Hitchner (1971); 6) no change in virulence occurred during back passages with both test materials.


Infectious bursal disease vaccine: some remarks on production and control.

A vaccine against infectious bursal disease (IBD) is prepared with a strain of IBD virus attenuated in chicken embryos. Safety and potency tests are carried out on embryos and chickens susceptible to IBD virus.


Standard  requirements for vaccines against infectious bursal disease.

A variety of criteria must be considered when examining the suitability of preparations of live infectious bursal disease (IBD) virus for use as vaccines: identity and purity of the vaccine strain, lack of spread, or of tendency to revert  to virulence, lack of immunosuppressive effect, safety and potency. Arguments as to th identity of IBD virus are well documented, and after ensuring that the virus strain is in fact IBD virus, it must be ascertained  that it is free from contaminating agents. If the vaccine strain spreads, lack of reversion to virulence must be determined by several direct chick to chick passages: histological observations of bursae from first and final  passages must be made to ensure that no change has occurred. In order to formulate proposals for standard tests of safety, lack of immunosuppressive effect and potency for IBD vaccines, comparative studies were made of products of different origins. Safety of the vaccines was assessed by observing young chicks after administration and measuring loss in weight gain. The effect on the bursa was judged by weighing and making  microscopic observations at intervals. The immunosuppressive effect of the vaccines  was assessed by comparing the serological and protective responses to subsequent Newcastle disease (ND) vaccination with the response in chicks given no IBD vaccine. Potency of the vaccines was determined by challenge of vaccinated chicks with virulent IBD virus. Various properties were found in the products examined. Some were concluded to be insufficiently attenuated because of the effect on the bird or upon its bursa and the consequent effect on the bird's immune competence. The ability to confer protection was not correlated with the degree of damage caused in the bursa. Vaccines which had no adverse effect on the health of the bird, caused little or no damage to the bursa and conferred adequate protection against challenge were considered to be of a satisfactory standard.


The epizootiology of infectious bursal disease and prevention of it by immunization.

Transport of leftover feed from infectious disease (IBD) farms to susceptible flocks on clean premises is believed to have played an important role in the transmission of this disease to new farms and to new areas. Servicemen, caretakers, contaminated equipment and air were suspected in the spread of the disease, but these modes were not demonstrated. Litter, drinking water and samples of feed from feeders of IBD flocks were found to be infectious when added to rations of susceptible chickens  but were not when added to  drinking water. Infectious bursal agent (IBA) remained viable for at least 6 months in dry litter and in unused dry chicken houses for more than 1 year. IBA was found in circulating blood of infected chickens 36 hours after inoculation, but not after 72 hours. Liver, spleen, thymus, kidney, pancreas and intestines were infectious at 72 hours. Bursa and feces were infectious from 48 hours through  7 days after inoculation. Tests on 4 1/2 to 6-week-old birds from 10-12 farms of each of several broiler complexes in three southeastern states of the U.S. revealed IBA present on a high percentage of farms even though none had used vaccine. Vaccination with modified or unmodified IBA administered to millions of 4-10-day-old chicks gave good protection for the life of broilers and laying birds. Effect of IBD on development of immunity to other diseases will be discussed.


Oral immunization of ducklings with attenuated duck hepatitis virus.

Duck hepatitis, a highly fatal disease of ducklings, must be controlled in successful commercial duck enterprises. Several procedures which have been effective in controlling the disease are 1) vaccination of adult breeders with passive transfer of antibodies through the yolk to the duckling, 2) administration of antisera to ducklings at the onset of signs of disease, and 3) parenteral vaccination of ducklings with attenuated DH virus after the loss of passive immunity. However, in each of these procedures, some losses usually occur and the administration of antiserum or vaccine to 2- to 3-week-old ducklings is costly and involves physical stress. Experimental studies of oral immunization in ducklings from immunized and nonimmunized parents were conducted to determine the feasibility of a convenient control program with little stress to the ducklings. Ducklings immunized with attenuated live duck hepatitis virus by intramuscular method, oral instillation, and through the drinking water were challenged with virulent duck hepatitis virus either intramuscularly or orally. Intramuscular, oral instillation, and drinking water application all provided adequate protection on challenge with virulent virus. Rise in neutralizing antibody was detected following immunization especially in sera of ducklings from nonimmunized parents. On the basis of successful experimental results, over 350,000 ducklings from immunized parents on a commercial farm have been sucessfully vaccinated by the  drinking water method.


Vaccination against avian encephalomyelitis with special reference to the spray method.

Three vaccination methods against avian encephalomyelitis have been tested in different types of housing. The product used was a vaccine improved by Philips-Duphar, having an optimal disseminative and immunogenic  capacity, and a high virus titer per  bird dose. We have tested: 1) administration per os in 2-5% of the flock, 2) administration via drinking water in the whole flock and 3) spray vaccination. Observations by Schneider (1967), that administration in the beak in part of the animals may only be satisfactory under optimal conditions, have herewith been confirmed. With the actual rearing on batteries, the chance of horizontal spreading of the vaccine virus is minimal. The two other methods of administration eliminate this problem. Already 3 weeks after vaccination more than 50% of the animals show a positive serological reaction. Later this level increases quickly to more than 80%, even in batteries. Under widely varying circumstances (concentaration of animals, ventilation, length of water tubes, etc) a dosage response  effect becomes apparent. The practical results obtainable will be dependent on the titer of the vaccine, care while vaccinating and conditions of housing.


Application of the turkey herpesvirus vaccine  by the aerogenic route for the prevention of Marek's disease.

Recently in laboratory and field trials, birds were vaccinated with cell-free HVT vaccine by aerosol spray. In laboratory trials, birds vaccinated by aerosol spray were as well protected against challenge with virulent MD (GA isolate) as those vaccinated by subcutaneous inoculation. Birds were vaccinated with a sprayer (Spraying Systems Company, Wheaton, Illinois) which  emits a particle size in which 90% are less than microns. The chicks were sprayed with 1,000 to 10,000 PFU per bird dose and remained in contact with the aerosol for 30-60 min. Turkey herpesvirus was isolated from the blood or kidney of these birds as early as 7 to 10 days postvaccination. In field trials approximately 300,000 birds have been vaccinated by aerosol spray with between 2,000 to 5, 000 PFU of cell-free HVT vaccine per chick. Chicks were sprayed while they were in the trays of the hatchers which still contained the dust  and down which remained after hatching. Prior to spraying, the incoming and outgoing aid ducts were closed. The chicks were sprayed for 12-15 min with 250-300 ml of diluent, and after completion of spraying the  chicks remained in contact with tha aerosol for an additional 5 to 8 min. In the birds vaccinated by aerosol spray the incidence of MD in the processing plant varied from 0.0% to 23.4%, while the incidence of MD in birds vaccinated by subcutaneous inoculation varied from 0.02 to 23.9%. Overall the incidence of MD in birds vaccinated by aerosol spray was essentially the same as those vaccinated  by subcutaneous inoculation.


Immunity studies in  calves vaccinated with a multivalent live respiratory vaccine composed of I.B.R., parainfluenza 3 and bovine adenovirus type 3.

The persistence of systemic and local antibodies was studied after two intranasal administrations of the vaccine, six weeks apart. Systemic antibodies to I.B.R. and adenovirus 3 evoked by the vaccine were still present 21 weeks following the second dose of the vaccine. Inconslusive results were obtained regarding the persistence of systemic and local PI-3 antibodies because of an intercurrent natureal PI-3 infection occurring during the observation period. Local antibodies to adenovirus type 3 were found in a high percentage of vaccinated animals 21 weeks after the second  dose of the vaccine, whereas local antibodies to I.B.R. remained detectable in 50% of the animals eight weeks after thesecond  dose. The results of a challenge study 21 weeks after revaccination show that the presence of local and systemic antibodies prevent the multiplication of PI-3 and BAV-3 in the upper respiratory tract. Protection against I.B.R. was achieved in the absence of detectable local antibodies.


Further studies on the neonatal ferret model of infection and immunity to and attenuation of human parainfluenza viruses.

Earlier we reported on the susceptibility via the intranasal route of inoculation of newborn ferrets to infection by wild and serially egg passaged parainfluenza type 3 virus. The wild type consistently induced fatal infection whereas with serial egg passage, mortality rate decreased while the degree of infectivity remained the same. This attenuation was accompanied by a change in capacity to induce interferon and decreased growth in human and primate cell cultures. The present work reports, similarly, on the susceptibility of newborn ferrets to wild and serially egg passaged parainfluenza types 1 and 2. Wild type infections caused significantly greater mortality than did egg passaged materials, although infectivity rates were approximately the same. Wild and egg passaged strains produced silent infections in adult ferrets and induced specific circulating hemagglutinin inhibiting antibodies. Newborn offspring of  dams inoculated with wild or egg passaged viruses resisted challenge with wild virus. The apparent attenuation of the wild type virus with serial egg passage viruses resisted challenge with wild virus. The apparent attenuation of the wild type virus with serial egg passage was correlated with alteration in cell culture host range and capacity  to induce interferon. The low and mid level passages of types 1 and 3 were the subject of clinical vaccination trials in man in four age groups: 18-35 years, 14-16 years and 6-12 years. The clinical and serologic results of these studies will be reported  elsewhere.


Intranasal vaccination of cattle against IBR and PI3: field and laboratory observations in dairy, beef, and neonatal calf  populations.

The author first summarizes the development of intranasal vaccination by Nasalgen P and particularly Nasalgen IP, previously reported, and then reviews present knowledge regarding safety and efficacy of Nasalgen IP in feeder calves, dairy animals and neonates. The minimal immunogenic dose is evaluated, as well as the secretory antibody response and the development of interferon. Two studies are reported on the transmission from vaccinates to susceptible non-vaccinates and the evaluation of the potential to revert to a more virulent form. An important part is devoted to the latest studies carried out which confirm that wide acceptance should be given to the intranasal vaccination of cattle.


Transmissible gastroenteritis of swine. Efficacy assay of an intramammary use vaccine.

TGE seronegative pregnant gilts were vaccinated by intramammary inoculations with a chemically inactivated (betapropiolactone) cell culture virus vaccine and their nursing pigs were exposed to virus at three days of age. The pig morbidity and survival rates were compared with those of pigs nursing gilts exposed orally to virulent TGE virus during late gestation and  also with pigs nursing non-vaccinated seronegative sows. The morbidity rates were 100%, 19% and 19%, while the survival rates were 33%, 100% and 17% respectively. The intramammary vaccine used to vaccinate pregnant gilts in this study did not stimulate sufficient antibody response to provide an acceptable level of protection to nursing pigs against experimental challenge with virulent TGE virus. The serum, colostrum and milk TGE neutralizing antibody responses are reported.


Comparative studies on parenteral and intranasal inoculation of an attenuated feline herpes virus.

Using a temperature sensitive mutant of feline herpes virus, comparative studies were carried out regarding efficacy of the intramuscular and the intranasal routes of administration of virus in inducing resistance to feline rhinotracheitis disease. Susceptible adult animals and kittens with and without parental immunity were used. Resistance to disease was measured by serological and challenge methods. Results in terms of rapidity or induced resistance or immunity as well as the degree and duration of immunity are discussed.


Effectiveness of orally administered Bordetella pertussis vaccine in mice.

Oral administration of killed Bordetella pertussis organisms to mice results in increased  resistance to an intracerebral infection with virulent B. pertussis cells. The rate of survival is dependent on the dose of antigen. But besides specific systemic immunity, which is persistent over a long period, also transient non-specific resistance is increased. These effects are evidently induced without penetration of bacterial substances into the circulation.


Preliminary results with an oral application of killed Purtussis bacteria in newborn infants.

Newborn babies have been vaccinated orally on five consecutive days, with 5 droplets of a vaccine containing 10(11) killed Bordetella pertussis. Eight out of twelve subjects showed antibody titres varying between 1:20 and 1:80.


Effects of early experience on the metabolism and production of corticosterone in rats.

Rats were handled for 3 min daily for 3 weeks before or after weaning or were left totally undisturbed. At 80 days of age, animals from each group were selected for corticosterone half-life determination. Adrenal tissue from thr remaining animals was incubated in vitro in the presence or absence of ACTH. Handled and unmanipulated animals did nto differ in half-life or in adrenocortical output. These data do not support the hypothesis that the reduced adrenocortical reactivity that results from manipulating animals is due to either the metabolsim of corticosterone or the response of the adrenal cortex to ACTH.


Individual consistency in behavioral states in neonates.

In this study we identified consistent characteristics of the sleep-wake state of newborn infants. Forty-one normal, full-term infants were observed in a warmer-skin temperature maintained at 36.1 degree C--located in a sound-attenuated room for 1 hr during 2 successive mid-feeding periods. The infant's state was recorded on a check-list every 10 sec using the following categories for sleep and wakefulness: Quiet Sleep A, Quiet Sleep B, Active Sleep Without REM, Active Sleep With REM, Active Sleep With Dense REM, Drowsy, Alert Inactivity, WAKING Activity, Fussing, Crying, and Indefinite State. Immediately following the completion of each observation, the 2 observers made independent judgments of the degree of organization shown by the infant, using a 6-point scale. Over the two 1-hr observations, only Quiet Sleep A, Quiet Sleep B, and Crying showed any consistency. Active Sleep as typically defined showed no consistency whatsoever. However, 2 of the 3 components of Active Sleep were significantly reliability measures for the 2 observations; in sleeping infants all 3 components of active sleep showed high reliability. These results indicate that Active Sleep may be a composite of at least 3 meaningful categories. To look at overall state organization in the neonate, including both sleep and wake states, we considered Active Sleep as 3 separate states and then combined the resulting 11 behavioral states into derived clusters such that each combination of states showed a test-retest reliability above .52. With these 5 state clusters we viewed each infant's state behaviors in terms of a profile depicting the percent of time spent in each state cluster. We found a close association between these profiles and the subjective judgments of sleep-wake state organization made by the observers. The results clearly indicate that a 1-hr observation provides reliable information on individual sleep-wake states in the newborn.


Standardization fo a digestion-filtration method for isolation of pancreatic islets.

Standardization of a technic for isolating large numbers of pancreatic islets is described. This procedure employed collagenase digestion of rat pancreatic tissue in a cylindrical wire screen in order to separate isolated islets from undigested pancreas. From this basic protocol the following conditions were established: (1) the duration of the initial digestion period was found to be optimal at six minutes; (2) three subsequent digestions of one minute each effected maximum islet yield; (3) the optimal initial collagenase concentration was found to be 1,000 U. (Worthington)/ml.; and (4) proper reductions of collagenase concentrations during the three subsequent digestions were found to be 50 per cent of each preceding incubation period. This method, combined with Ficoll gradient separation, yielded a mean of 800 islets per two rat pancreases. The isolated islets appeared morphologically intact, contained 0.36 +/- 0.05 mug. protein/islet, and demonstrated a normal biphasic release of insulin in response to stimulative levels of D-glucose. The present method provides a means for obtaining a large mass of viable islet cell tissue in a short time.


Incorporation of 75Se-selenomethionine into human apoproteins. III. Kinetic behavior of isotopically labeled plasma apoprotein in man.

The metabolism of lipoprotein-apoprotein was examined in four subjects with normal lipid metabolism and in one subject with type II hyperlipemia by means of isotopic tracer methodology. Studies were performed after intravenous injection of a radioactive amino acid precursor for apoprotein synthesis (75Se-selenomethionine), in both the basal state and following the acute injection of intravenous heparin. Computer technics were used to evaluate a series of multicompartmental models, and a general model is proposed that yields optimum fitting of experimental data for serum free amino acid precursor, very-low-density lipoprotein-apoprotein (VLD-apoprotein), and low-density lipoprotein-apoprotein (LDL-apoprotein) in man. The analysis demonstrates that approximately half of the transport of 75Se-apoVLDL from the plasma VLDL pool is converted to 75Se-apoLDL. The acute injection of heparin in two normal subjects results in a two-and-a-half-fold increase in this rate of conversion of 75Se-apoVLDL to 75Se-apoLDL. 75Se-apoLDL is metabolized by rapid transport into a recycling extravascular pool and by irreversible catabolism. The fractional rate of recycling is large relative to the fractional rate of catabolism of apoLDL (3.7:1.0), suggesting extravascular recycling as a potential site of regulation of the plasma concentration of apoLDL. In a patient with type II hyperlipemia, the extravascular recycling pathway is reduced and is not corrected with D-thyroxine therapy. However, this therapy did reduce conversion of apoVLDL to apoLDL in this type II patient. The kinetic data support the validity of the compartmental model in simulating both normal and pathologic apoprotein metabolism and that perturbation of physiology seen with heparin injection and D-thyroxine therapy. These data support a quantitative role of apoVLDL as a precursor of apoLDL and identify an important recycling pathway of apoLDL metabolism in addition to that of catabolism.


Stimulatory and inhibitory effects of cyclic AMP on pancreatic glucagon release from monolayer cultures and the controlling role of calcium.

When glucagon release from monolayer cultures of newborn rat pancreas was measured over four hours in media containing 2.5 mM Ca++, a significant cyclic AMP-related inhibition of release was observed. This was noted whether intracellular cyclic AMP levels were raised by the addition of exogenous cyclic AMP or dibutyryl cyclic AMP, by phosphodiesterase inhibition with theophylline, or by the stimulation of adenylate cyclase with cholera toxin. The inhibition was concentration dependent for cyclic AMP and could not be reproduced by the addition of AMP, ADP or ATP. Adenosine also inhibited glucagon release while ATP was stimulatory. From time course studies it appeared that the inhibitory effects of cyclic AMP and cholera toxin were progressive after two hours of incubation. With cholera toxin an early stimulation of glucagon release was observed. The effects of cyclic AMP and cholera toxin on arginine-stimulated glucagon release were to stimulate further the glucagon release during the first hour of the incubation. Thus, the effects of raising intracellular cyclic AMP levels were biphasic in that both an early stimulation and a late inhibition of glucagon release were observed. In examining the nature of these responses a remarkable controlling role for Ca++ was uncovered: at Ca concentrations of 0.3 mM and lower no effect of cyclic AMP on glucagon release was found. With 1 mM Ca++ in the medium cyclic AMP stimulated glucagon release early (30 min) and thereafter had no further effect. In the presence of 2.5 mM Ca++ cyclic AMP did not stimulate early but did cause the delayed inhibition of release. It is concluded that the effect of cyclic AMP on glucagon release can be either stimulatory or inhibitory depending upon the Ca++ concentration of the medium and the duration of exposure to raised cyclic AMP levels.


[In vitro induction of numerical changes in the karyotype of normal and transformed Djungarian and Chinese hamster cells].

The comparative study of the frequency of colcemid-induced aneuploidy and polyploidy in cultured normal and transformed cells of Djungarian hamster is described. The occurrence of variants with changed chromosome number is much higher in populations of SV40-transformed cell line (4/21) than in normal embryonic cultures. In transformed lines of Djungarian and Chinese hamsters (4/21 and V-79) the frequency of cells with changed chromosome number was found to be dependent on the culture density: the percentage of polyploids was 4-5-fold higher when the number of seeded cells was 2-fold lower. The highest number (18-29%) of hypermodal cells was produced at drug concentrations of 0.02-0.025 mkg/ml. The percengate of polyploids under these conditions reached 10-20. At further increase of colcemid concentrations the proportion of polyploid cells increased. In Djungarian hamster embryonic cell cultures there were single cells with changed chromosome numbers at a concentration of the drug of 0.015-0.1 mkg/ml.


[Mutagenesis under the influence of simian virus 40 (SV40). II. Induction of mutations for resistance to analogs of purine bases in human and Chinese hamster cells].

Induction of gene mutations by SV40 was studied in aneuploid human and Chinese hamster cell lines. It is shown that SV40 penetrates into the cells studied and induces the T-antigen synthesis. The efficiency of the infection in Chinese hamster cells was tested additionally by the ability of infected cells to form colonies in the medium lacking serum growth factor. The maximal number of colony-forming cells was detected 24 hours after the infection. By culturing cells in factor-free medium a Chinese hamster cell subline was isolated, which proved to synthetize T-antigen within 60 days after the virus treatment. This is regarded as an indirect proof of the integration of viral genome into the cell genome. The increased frequency of mutants resistant to 60 mkg/ml of 8-azaquanine (human cells) and to 15 or 30 mkg/ml of 6-mercaptopurine (hamster cells) was observed on the 1-4th days following the infection. The analysis of the results of all experiments taken together revealed that the induction is highly significant according to Wilcoxon (greater than 0.99). The resistance of the isolated clones proved to be stable after the prolonged culturing under non-selective conditions. It is suggested that viral genome integration, gene mutations and chromosome aberrations may have common molecular mechanisms. The role of gene mutations in virus-induced carcinogenesis and tumour progression is discussed.


[Genetics and phenogenetics of the hormonal characteristics of animals. II. Changes in the reactivity of the pituitary-adrenal system and influence of the sex glands in selection of silver-black foxes by behavior].

Seasonal changes in the reactivity of adrenal cortex to ACTH and of the pituitary-adrenal system to psychic stress in intact and castrated domesticated (tame) and non-domesticated (non-tame) silver foxes of both sexes were studied. It is demonstrated that in tame foxes there are differences among the intact and castrated animal in their reaction to ACTH and psychic stress. These differences are maifested especially in autumn (September, October) which is the period of extra-seasonal sexual activity for domesticated animals. This points to the fact that the sex glands exert a great influence on the condition of central regulating mechanisms of the pituitary-adrenal complex and that the selection of silver foxes for tame behaviour changes the functional state of these mechanisms through the change of the functional state of the sex glands.


Evidence for the absorption of bile acids in the proximal small intestine of normo- and hyperlipidaemic subjects.

Bile acid composition was determined in duodenal and jejunal aspirates obtained under fasting conditions in normolipidaemic controls and in patients with the type IIa and type IV lipoprotein patterns. In 17 out of the 22 subjects studied, the duodenal as compared with the jejunal aspirates contained proportionally more CD (chenodeoxycholic acid) than C (cholic acid). The two types of aspirates also differed slightly with regard to the D (deoxycholic acid): CD ratio, which was higher in samples drawn from the jejunum. These findings, compatible with an absorpiton of about 30% of CD in the upper small intestine, were approximately the same in all groups of patients. As evidenced by additional experiments in two subjects, an oral intake of cream does not significantly influence the absorption of CD in the upper small intestine.


[Effect of aldosterone and ACTH administration on fluctuation of 5-HT content of the brain and pentobarbital anesthesia in adrenalectomized rats].

The present study was undertaken to determine whether or not adrenalectomy influences anesthesia and increase in brain 5-HT induced by pentobarbital and if such is the case, whether or not aldosterone or ACTH inhibit the effect of adrenalectomy. Male Wistar rats were adrenalectomized or sham-operated and injected with aldosterone (20mug5hg i.p.), ACTH (5.0 mg/kg i.p.) or the vehicle (1.0 ml/kg i.p.) everyday for 1 week after a postoperative interval of 3 days. These rats ware given a normal diet and 1% saline approximately 5% glucose solution. Animals were injected with pentobatbital (50 mg/kg i.p.) 18 hr after the last injection of each hormone. It was found that adrenalectomy enhanced "pentobarbital-anesthesia", although there was no such effect in the adrenalectomized rats pretreated with each hormone. Adrenalectomy inhibited an increase of 5-HT in the brain after pentobarbital, but the hormone pretreatment to those rats did not antagonize the inhibition of the 5-HT increase.


Clinical application of somatosensory cerebral evoked response for the localization and the level diagnosis of neuronal lesions.

A new SER recording system using the bilateral simultaneous peripheral nerve stimulation on cervical, thoracic and lumbar segments was found to be helpful as a diagnostic aid for the localization and the level diagnosis of sensory neuronal lesions including peripheral nerves. SER abnormalities were observed in 32 (76.2%) of the 42 cases with cerebrovascular disease, in 11 (64.7%) of the 17 cases with spinal cord lesions and 12 (75.0%) of the 16 cases with peripheral nerve lesions. As an interesting finding, marked SER abnormalities corresponding with the level and the distribution of the lesions were observed in cases with spinal cord tumor, myelopathy and polyneuropathy. In a case with recurrent myelitis, lesions of different levels of spinal cord were suggested from the SER changes. The recording system gives also valuable information about chronological modifications of the lesions, and may reveal subclinical neuronal damage in a certain neurological disorder.


An anatomicopathological approach to the study of articular aging.

Bone and joint aging results from the combined action of various metabolic and mechanical factors. Osteoarticular aging produces primary changes in bone (intrinsic rarefaction, but also extrinsic osteogenesis), cartilage, synovial membrane, and tendon. The association of these primary changes can result basically in three anatomicopathological entities: hyperostosis and osteophytes at tendon insertions, senile remodeling, and osteoarthritic remodeling. Some of these entities are asymptomatic. Others form part of nosologic entities such as osteoarthritis and various types of joint pain and stiffness. The standard methods of morbid anatomy provide a link between clinical findings and modern research methods. By separating nosology and morphology they permit a better analysis of the boundaries and transitions between articular "aging" and "disease". With advancing age, osteoarthritis becomes increasingly frequent but not inevitable.


[Diagnosis of breast carcinoma. Value of x-ray findings for the diagnosis of carcinoma in situ of the breast from the clinical point of view].

The x-rays-results of 35 mammae with a carcinoma in situ are presented and compared with those of 2 groups without carcinoma. In contradiction to invasive malignancies the carcinoma in situ do not show pathognomonic signs on x-ray examination. However, suspicion is raised by the appearance of microcalcifications and by striped differences in tissue density. These findings should compell to perform a biopsy, even though the clinical and roentgenologic findings may otherwise suggest a fibrocystic disease. Therefore, provided the discrete signs described in this paper are carefully considered, the technique of mammography may contribute to the pre-operative presumptive diagnosis of carcinoma in situ.