Cytochemical detection of nucleoside diphosphatase activity in the central nervous system of the rat. The cytochemical localization of nucleoside diphosphatase and thiamine pyrophosphatase occurs within the "mature face" of the Golgi apparatus and over the neurilemma in neurons of the cerebellum, the cerebral cortex and the brain stem. The hydrolytic reaction product of the brain enzyme differs from that of the liver in that it is not found in the endoplasmic reticulum or nuclear envelope. Hydrolysis of IDP, UDP or GDP is not greater than that of ADP or CDP in brain homogenates, in contrast to that found in the liver. The NDPase activity of brain homogenates is optimal at pH 7.2, stimulated by heavy metals and inhibited by uranyl nitrate. Thick section cytochemistry suggests that the reaction product is restricted to a network of polygonally shaped compartments. NDPase activity on the neurilemma may reflect the role of this enzyme in the synthesis of glycoproteins involved in neuronal surface recognition.


Evaluation of myocardial uptake of 99mtechnetium pyrophosphate in clinical exercise-induced ventricular ischemia. Although acute infarction of the myocardium is known to accumulate 99mtechnetium pyrophosphate, it is not entirely clear that ischemia alone without necrosis does not result in abnormal uptake of 99mtechnetium pyrophosphate. The present study investigates whether transient myocardial ischemia is associated with localization of 99mtechnetium pyrophosphate by evaluating images obtained with the scintillation camera at rest and after exercise in 15 patients with unequivocal myocardial ischemia. All patients had angina pectoris, multivessel coronary artery stenoses by selective arteriographic studies, and electrocardiographic ischemic responses on treadmill exercise. Eleven of the 15 patients also underwent radionuclide imaging with 81rubidium at rest and after exercise; the results demonstrated scintigraphic ischemia. The scintiscans with 99mtechnetium pyrophosphate revealed no evidence of increased myocardial radioactivity after exercise compared to rest in 14 of the 15 patients. In contrast, myocardial activity was observed with 99mtechnetium pyrophosphate after treadmill exertion in the remaining patient, in whom a small subendocardial infarction appeared to have occurred with the exercise. It is concluded from these results that transient myocardial ischemia does not cause localization of 99mtechnetium pyrophosphate. These findings support the specificity of abnormal localization of 99mtechnetium pyrophosphate for acute myocardial infarction.


Dipyridamole, an inhibitor of mengovirus replication in FL and L cells. Dipyridamole showed an antiviral activity aganinst mengovirus in FL cells using the agar diffusion plaque inhibition test, plaque reduction test, tube titration test, and virus yield test after one replication cycle. With the last two tests mentioned above the inhibitory action was also confirmed in L cells. In consequence of the known transport inhibition of uridine into the cell in presence of dipyridamole only a very small incorporation of 3H-uridine into acid-insoluble material could be demonstrated. Applying the method of prelabelling of FL cells at 16 degrees C for 1 h with subsequent addition of dipyridamole the drug failed to show an effect on cellular RNA synthesis per se in uninfected cells whereas the viral RNA synthesis in mengo-virus-infected L cells was completely depressed.


Sigma effects of nalorphine in the chronic spinal dog. The effects of graded doses of nalorphine and morphine were studied in nondependent chronic spinal dogs. Morphine and low doses of nalorphine produced behavioral changes characterized by indifference, whereas the largest dose of nalorphine produced canine delirium indistinguishable from that produced by SKF-10, 047 or cyclazocine. Nalorphine depressed the flexor reflex; however, a plateau was observed. The data suggest that nalorphine is a partial agonist of the kappa type and a sigma agonist in addition to being a competitive antagonist at the mu receptor, and further, that the dysphoric and hallucinogenic effects of nalorphine-like drugs are due to their sigma activity.


Ultradian rhythms in the perception of two apparent motions. Fourteen young adults were tested  on the spiral after-effect every 5 min and on the beta movement every 20 min for 8 continuous hrs. Time series analysis revealed significant 100-min rhythms in the perception of the two illusions, which also appeared to be sychronized across the experimental periods.


[Changes in the concentration of biogenic monoamines in different brain regions under the influence of centrophenoxine (experiments on rats)]. The authors administered 50 mg/body weight of centrophenoxine in white rats i.p. for five susseccive days. The content of serotonine in the cerebral cortex, brain stem and cerebellum increased statistically significant in the rats, injected with centrophenoxine. The levels of noradrenaline and dopamine in the brain stem were raised also significantly. Centrophenoxine did not alter the content of cyclic AMP and the activity of phosphodiesterase in the cerebral cortex and brain stem. These data gave foundation to the authors to suggest that in the mechanism of action of centrophenoxine a significant role is given to the changes, induced by this preparation in the content of biogenic amines in the brain stem.


Control of bioactive corticotropin release from the neuro-intermediate lobe of the rat pituitary in vitro. The purpose of this study was to identify agents capable of regulating the release of biologically active ACTH from the isolated neuro-intermediate lobe of the rat pituitary. Agents found to be potent secretagogues included acetylcholine (100 mug/ml), hypothalamic stalk-median eminence extract (0.33 eq), arginine antidiuretic hormone (100 mU/ml) and serotonin (100 mug/ml). Lower doses of arginine antidiuretic hormone (5.5 mU/ml) and serotonin (2 mug/ml) were ineffective. Dopamine 2 and 5 mug/ml) inhibited the release of biologically active ACTH whereas norepinephrine (5 mug/ml) did not. Dexamethasone (0.25 mug/ml) did not alter the basal or stimulated release of ACTH from the isolated neuro-intermediate lobe in contrast to its effect on ACTH release from the isolated anterior pituitary. Similarly, the tripeptide, prolyl-leucyl-glycinamide, which has been reported by some to inhibit MSH release, had no effect on either the basal or stimulated release of ACTH. The data suggest that regulation of ACTH release from the neuro-intermediate lobe in vivo may involve both stimulatory (acetylcholine) and inhibitory (dopamine) inputs.


Control of bovine adrenal cortical cell proliferation by fibroblast growth factor. Lack of effect of epidermal growth factor. Primary functional bovine adrenal cortical cell cultures have been developed to study the factors controlling adrenal cell growth. Cells were prepared by the collagenase technique and maintained in F-12 medium containing fetal calf serum and horse serum. Cells contained abundant lipid as demonstrated by staining with Oil Red O and showed strongly positive staining for delta5,3beta-hydroxysteroid dehydrogenase. ACTH inhibited DNA synthesis and stimulated steriodogenesis in these cells. Fibroblast growth factor (FGF) was shown to be a potent stimulator of the growth of normal bovine adrenal cortical cells maintained in tissue culture. The minimal effective dose of FGF was 1 ng/ml with maximal effects being observed at 100 ng/ml. The effect of FGF was dependent on the serum concentration. Inclusion of FGF in F-12 medium containing serum permitted cloning of functional bovine adrenal cortical cells from cultures seeded at low density (4 cells/cm2). ACTH inhibited the mitogenic effects of FGF. In addition to its mitogenic action, FGF is a migratory factor for bovine adrenal cortical cells. Though ACTH inhibited the mitogenic effects of FGF, it did not block the migratory activity. Epidermal growth factor did not affect the growth of either normal bovine adrenal or functional mouse adrenal tumor cells (Y-1) in tissue culture. FGF is the first direct mitogen identified for adrenal cortical cells; ACTH opposes this mitogenic action and functions directly as a differentiate function signal.


A superfusion system technique for the study of the sites of action of glucocorticoids in the rat hypothalamus-pituitary-adrenal system in vitro. I. Pituitary cell superfusion. Rat anterior pituitary cells were placed in a superfusion column and were stimulated by rat hypothalamic extract. The rate of ACTH secretion was monitored by collecting fractions eluting from the column every 2 min. Dispersed rat adrenocortical cells were used to determine the amounts of ACTH in the superfusate. ACTH appeared in the medium 6-12 sec after stimulation of the cells. A linear log dose-response relationship existed between 1/8 to 1 rat hypothalamus extract and the amounts of ACTH released. Repeated pulses of CRF stimulation of the same column of pituitary cells resulted in repeated, identical peaks of ACTH. Constant stimulation caused a plateau in hormone release that lasted as long as the stimulus was present. The stimulated, but not the basal ACTH release, could be inhibited by superfusion with corticosterone in vitro (0.2 mug/ml), or by treating the pituitary donor animal in vivo with corticosterone (5 mg/100g BW), cortisol or dexamethasone. Adrenalectomy of the donor animal caused increases in the basal and the stimulated ACTH release.


Cortisol secretion and clearance in the rhesus monkey. Cortisol secretory patterns were studied in two chair-adapted rhesus monkeys by simultaneous measurement of plasma concentration and specific activity of cortisol after an iv bolus of 14C-labeled hormone. The results indicated that fluctuating plasma cortisol concentrations are the result of episodic secretion by the adrenal cortex. Specific activity changes during these spontaneous secretory bursts indicated occasional submaximal activity. In addition, cortisol secretory rates calculated during basal (20.4 mug/min) and ACTH-stimulated (28.4mug/min) conditions in a total of seven monkeys were significantly different (P less than 0.05), further demonstrating that spontaneous secretory bursts were usually sub-maximal. From plasma samples collected at 10 min intervals, a cortisol distribution t1/2 of 6 min and a clearance t1/2 of 66 min were found. The apparent volume of distribution for this hormone was 4.8 liters, a value far in excess of extracellular fluid volume estimates. The circadian pattern of plasma cortisol in these monkeys resembled that reported for man, but monkeys had twice as many episodic bursts and over twice the mean cortisol levels as man. However, the 24 h production rate was 10.5 mg, a value within the range of human production.


The mechanism of damping of the serum thyroxine and triidothyronine levels caused by increasing thyrotropin dosage in mice. The time course of the effect of bovine TSH (bTSH) on serum concentrations of thyroxine (T4) and triiodothyronine (T3) was measured in the normal mouse. The basal, unstimulated levels were 3.2+/-1.1 mug/100 ml T4 and 104+/-25 ng/100 ml T3 (mean+/-SD). With doses of bTSH from 0.5 to 100 mU the peak levels of the thyroid hormones were only 2.6 and 1.8 times the basal level for T4 and T3, respectively. With increasing doses of bTSH there was a proportional prolongation of the increased serum levels of thyroid hormones, i.e., about 2 h for 0.5mU to 12 h for 100 mU TSH. The integrated response with time was linearly related to the log dose. This would suggest a control mechanism which prevents excessive concentration of thyroid hormones in the serum. This pattern of response to TSH differs somewhat from that obtained by following radioiodine release in the McKenzie type bio-assay. To avoid the problems of changing blood concentrations of thyroid hormones and TSH, the release of T4 and T3 from the mouse thyroid was measured in vitro. The secretion increased with bTSH concentrations in the range of 0.02-0.8 mU/ml for T4 and 0.02-0.4 mU/ml for T3. The maximal response was 8.8+/-0.5 ng T4/3h/thyroid and 3.6+/-0.3 ng T3/3h/thyroid as against the basal secretion of 2.4+/-0.2ng T4 and 0.8+/-0.1 ng T3 (mean+/-SEM). Further in crease in bTSH concentration was associated with a decreased rate of thyroid hormone release. Thyroidal cAMP accumulation was enhanced with increasing bTSH concentration, even when there was a decrease in secretion. The dichotomy in the dose-response pattern between the two parameters indicated that the effect of high TSH concentrations on the release was induced at a step beyond cAMP accumulation. This was corroborated by the similar pattern of release induced by increasing concentration of DBcAMP. These findings indicate the existence of an intrathyroidal autoregulatory mechanism which prevents excess increase of thyroid hormone levels in the blood.


Functional clones of pituitary cells derived from Rathke's pouch epithelium of fetal rats. Sixty-five clonal strains fo anterior pituitary cells have been established from Rathke's pouch epithelium of fetal rats. Radioimmunoassay of the conditioned media has shown that some clones produce only prolactin, some produce prolactin and growth hormone and others produce prolactin, growth hormone and ACTH. The clonal cells have retained a normal diploid karyotype for extended periods of time in culture. The results indicate that differentiation into somatotrophs, coricotrophs and prolactin cells can occur in vitro from a single progenitor cell.


Adenylyl cyclase and cyclic nucleotide phosphodiesterases in GH-Strains of rat pituitary cells. The properties of adenylyl cyclase and cyclic nucleotide phosphodiesterases from GH-strains of rat pituitary tumor cells have been investigated. Adenylyl cyclase was inhibited by calcium ion and stimulated by fluoride ion, 5'-guanylylimidodiphosphate and by prior treatment of intact releasing hormone (TRH), which stimulates prolactin releasing hormone (TRH), which stimulates prolactin release and synthesis in GH-cells, did not cause a significant stimulation of adenylyl cyclase activity under a wide variety of assay conditions; under the same conditions, [3H]TRH bound to a previously characterized membrane receptor. GH-cells contain phosphodiesterase activity catalyzing the hydrolysis of cAMP which gives nonliner Lineweaver-Burk plots with apparent Km's for cAMP of 1.5 muM and 4mM. TRH did not affect the activity of cyclic nucleotide phosphodiesterase at high or low cAMP concentrations when added to broken cell preparations. Treatment of intact cells with TRH caused no changes in the total adenylyl cyclase and cyclic nucleotide phosphodiesterase activites within the first 2 h of incubation, when stimulation of prolactin release occurs, but did lead to slight decrease in adenylyl cyclase and the apparent low Km phosphodiesterase after 72 h of treatment.


The influence of propranolol on the thyrotropin receptor. Propranolol, a beta adrenergic blocking drug, is known to inhibit the thyrotropin (TSH) stimulation of adenosine-3',5'-monophosphate (cyclic AMP production in thyroid membranes but the mechansim of this inhibitory action is known. We have therefore investigated the influence of propranolol on the binding of 125I-labelled TSH to human thyroid membranes. Both d- and l-propranolol were found to enhance the binding of 125I-labelled TSH to thyroid membranes. The amount of label bound increased from about 30% in the absence of propranolol to about 60% in the presence of 3.3 x 10(-3)M propranolol. Scatchard analysis of the binding data indicated that propranolol increased the association constant of the thyrotropin-thyrotropin receptor interaction. Practolol, lithium carbonate, methimazole, and somatostatin had no effect on thyrotropin binding. This effect of propranolol appeared to be due to a direct reversible action of propranolol on the thyroid membranes and could be attributed to the membrane-disrupting properties of the drug rather than its beta-blocking activity. The increased TSH receptor occupancy which resulted from the increased association constant of the TSH-thyroid membrane interaction corresponded with a decrease in TSH-stimulated cyclic AMP formation. These data could indicate that propranolol reduced the efficiency of the receptor-adenylyl cyclase coupling system.


Pulmonary changes induced by amphophilic drugs. Administration of amphophilic drugs to experimental animals causes formation of myeloid bodies in many cell types, accumulation of foamy macrophages in pulmonary alveoli, and pulmonary alveolar proteinosis. These changes are the result of an interaction between the drugs and phospholipids which leads to an alteration in physicochemical properties of the phospholipids. Impairment of the digestion of altered pulmonary secretions in phagosomes of macrophages results in accumulation of foam cells in pulmonary alveoli. Impairment of the metabolism of altered phospholipids removed by autophagy induces an accumulation of myeloid bodies. In summary, administration of amphophilic compounds causes a drug-induced lysosomal disease or generalized phospholipidosis.


Electron spin resonance (ESR) study of cigarette smoke by use of spin trapping techniques. The technique of spin trapping has been applied to the gas phase of cigarette smoke to identify and quantify the radicals present. It was found that radicals could be trapped only if the smoke was filtered. Three spin traps were used: N-tert-butyl-alpha-phenyl nitrone (PBN). 5,5-dimethyl-delta1-pyrroline-1-oxide (DMPO) and alpha-[3,5-di-tert-butyl-4-hydroxyphenyl)-N-tert-butyl nitrone (OHPBN). From the electron spin resonance (ESR) splitting constants of the radicals produced by the reaction of smoke radicals with the spin traps and also from the effec of varying the path length between the cigarette and the spin trap solution, it is concluded that three types of signals are observed. Type I signals indicate the presence of oxygenated radicals which appear to be a mixture of alkoxy radicals (RO) and aroyloxy (ArCO2-) radicals. Our data do not allow conclusions about the nature of the R or Ar groups in these two oxy radicals; however, considerations based on lifetimes suggest that the R group probably is tertiary. Type II and III signals are not typical spectra of spin adducts. Instead, we believe they result from reaction of smoke (and probably radicals in smoke) with the PBN spin trap and indicate that smoke has the ability to effect one-electron oxidations. Only type I signals are observed with DMPO and OHPBN. A quantitative study shows that 4 x 10(14) spins/puff are present in the smoke, in contrast with the result of a recent study which used a very different method for determining the radical content of smoke. A discussion of the nature of the radicals in smoke and some tentative conclusions are presented.


Purification and control of bovine adrenal cortical cholesterol ester hydrolase and evidence for the activation of the enzyme by a phosphorylation. A procedure for the purification of cholesterol ester hydrolase from bovine adrenal cortical 105000 x g supernatant is described. Preincubation of a crude enzyme extract with [gamma-32P]ATP followed by purification resulted in the isolation of a phosphorylated preparation of cholesterol ester hydrolase. The phosphorylated cholesterol ester hydrolase appeared to be composed of 4 subunits, each having a molecular weight of 41000 +/- 280, only one of which may be phosphorylated. Preincubation of the crude enzyme preparation with [alpha-32P]ATP followed by purification did not produce a phosphorylated preparation of cholesterol ester hydrolase. Cyclic-AMP-dependent protein kinase, cyclic AMP, ATP and magnesium ions were required for activation of purified cholesterol ester hydrolase in vitro and the time course of activation closely paralleled the time course of phosphorylation of the enzyme. The addition of ATP, cyclic AMP and magnesium ions to the bovine adrenal cortical 105000 x g supernatant produced a 2.5-fold stimulation in cholesterol ester hydrolase activity. This stimulation was abolished if protein kinase inhibitor was added prior to the addition of ATP cyclic AMP and magensium ions. The addition of magnesium ions or calcium ions to a crude preparation of cholesterol ester hydrolase was found to inhibit activity; however the same additions made to a purified preparation of cholesterol ester hydrolase were not inhibitory. The decrease in cholesterol ester hydrolase activity on incubation with magnesium ion was accompanied by a loss of 32P radioactivity from the protein. Preincubation of a crude preparation of cholesterol ester hydrolase with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic-AMP-dependent protein kinase. Deactivation of cholesterol ester hydrolase is accomplished by dephosphorylation catalysed by a phosphoprotein phosphatase, dependent on magnesium or calcium ions.


Adenosine 3':5'-monophosphate metabolism and turnover in dog thyroid slices. The metabolism and turnover of adenosine 3':5'-monophosphate (cyclic AMP) have been studied in intact thyroid cells incubated in vitro. Thyroid slices have been stimulated by 1 mU thyrotropin/ml, then washed with buffer, or with buffer containing thyrotropin antibody, or trypsin so as to cut off the stimulation. The decline of cyclic AMP levels has been followed and the time required to decrease this level to half of the initial value estimated. Computer simulation taking into account the penetration of trypsin in the slices, the kinetics of thyrotropin inactivation and the relation between thyrotropin concentration and cyclic AMP concentration at the steady state has made it possible to estimate the true cellular half-life of cyclic AMP in the stimulated cell to 1 min 50 s. The method provides an experimental approach to the demonstration in intact cells of effective on cyclic AMP disappearance. The methodology of the calculation of half-life and turnover from such data is discussed.


Terminal nucleotide sequences of 17-S ribosomal RNA and its immediate precursor 18-S RNA in yeast. The 5' and 3'-terminal nucleotide sequences of 17-S rRNA and its immediate precursor 18-S RNA from the yeast Saccharomyces carlsbergensis have been analysed. Identification of the terminal oligonucleotides, as present in Ti ribonuclease digests, was performed by diagonal procedures. The major (molar yield 0.9) 5'-terminal oligonucleotide (molar yield 0.15) with the overall composition pU (U2,C2)G was observed. 18-S precursor RNA was found to contain the same 5'-terminal sequences as 17-S rRNA. However, the 3'-terminal sequences of the two types of RNA appeared to be different. The 17-S rRNA yields the oligonucleotide A-U-C-A-U-U-AOH while at least half of the 18-S RNA molecules contain the sequence U-U-U-C-A-A-U-AOH. In addition 18-S RNA yields several minor 3'-terminal oligonucleotides which appear to be structurally related to the major 3'-terminal sequence. These results demonstrate that the extra nucleotides in 18-S RNA relative to 17-S RNA are located exclusively at the 3'-terminus of the 18-S RNA molecule. The possibility that the 3'-terminal nucleotide sequence of 18-S RNA plays a role in the maturation process is discussed.


2'-Deoxy-2'-azidocytidine, a new inhibitor of DNA replication in mammalian cells. Cell growth is reversibly inhibited by the nucleoside analogue, 2'-deoxy-2'-azidocytidine and the inhibition is a result of interference with DNA replication. The 5'-diphosphate of the analogue was earlier shown to specifically inactivate the enzyme ribonucleotide reductase in vitro. However, measurements of the pools of deoxyribonucleoside triphosphates in cells incubated in azidocytidine showed only minor changes which appeared to result from and not to be the cause of the inhibition of DNA replication. The DNA synthesized in polyoma-infected cells after incubation in azidocytidine showed a sedimentation pattern quite different from that seen after inhibition of DNA synthesis with arabinosyl cytosine or hydroxyurea. Experiments with nuclei isolated from azidocytidine-inhibited, polyoma-infected cells indicated (a) that the number of replicating molecules is decreased during the inhibition and (b) that upon incubation of the nuclei there is a rapid synthesis of DNA occurring in a new class of DNA molecules which are at a very early state of replication. Neither the 5'-triphosphate of azidocytidine nor the nucleoside itself inhibit DNA synthesis in vitro in isolated nuclei from polyoma-infected cells and at present the nature of the DNA-synthesis-inhibiting compound acting in the cells after addition of azidocytidine is unknown. Taken together the results suggest that azidocytidine inhibits DNA synthesis at an early stage, possible by blocking the initiation of DNA synthesis at the origin or by interfering with the elongation of newly initiated DNA molecules.


The synthesis of macromolecular 3-hydroxyproline by attaching and confluent cultures of human fibroblasts. The synthesis of collagen has been studied during the attachment of freshly trypsinized human fibroblasts to culture vessels by measurement of the incorporation of radioactive proline into macromolecular hydroxyproline. Collagenous protein(s) was found to be a component of a substrate-attached material ('microexudate carpet') synthesized rapidly during cell attachment in the absence of serum. The ratio of 3-hydroxyproline/4-hydroxyproline in the collagenous proteins synthesized during cell attachment was found to be 4-5 fold higher than that of normal type I collagen. The synthesis of 3-hydroxyproline by confluent cultures was diminished by serum deprivation, and was shown to require higher concentrations of ascorbate than the synthesis of the 4-hydroxy isomer.


Purification of bovine adrenal-cortex plasma-membrane vesicles containing a highly corticotropin-sensitive adenylate-cyclase system and angiotensin-II-binding sites. The purpose of this experimental investigation was to provide a purified plasma membrane fraction containing a highly hormone-responsive adenylate cyclase system. Bovine adrenal cortex was homogenised and a washed pellet (450 000 X g - min) was fractionated by zonal centrifugation in a sucrose and dextran gradient. Adenylate cyclase activity was purified up to 60-fold to a specific activity of 55, 340 and 210 pmol of adenosine 3':5'-monophosphate (cyclic AMP) produced/minute per mg of protein at 38 degrees C for the basal, adrenocorticotrophin and fluoride-activated states, respectively. The time course of the adenylate cyclase activity is linear. The concentration necessary for half-maximal stimulation by adrenocorticotrophin-(1-24)-tetracosipeptide is 0.5 muM. The high hormone-responsiveness of the membrane preparation allows one to demonstrate activation of adenylate cyclase by very weakly agonistic adrenocorticotrophin fragments. The F- activated state can be detergent-dispersed by Lubrol and shows a Km (ATP) different from that of either the basal or adrenocorticotrophin-stimulated state. Other marked enzymes such as 5'-nucleotidase, glucose-6-phosphatase and cytochrome oxidase were followed during purification. The plasma membrane fraction shows rather homogeneous, relatively large vesicles (mean diameter 0.5 mum). It contains high-affinity binding sites for angiotensin II (about 2 pmol per mg protein) with an apparent association constant of 2 X 10(7) (1/mol) at 12 degrees C. The yield, 20 mg of membrane protein per preparation, may make it a tool in either affinity-labelling studies with the peptide hormones mentioned or the starting point for solubilisation and purification of adenylate cyclase.


Purification and properties of a periplasmic protein related to sn-glycerol-3-phosphate transport in Escherichia coli. Protein GLPT, a periplasmic protein previously recognized as closely related to the active transport of sn-glycerol-3-phosphate in Escherichia coli was isolated by the cold osmotic shock procedure. It was purified by Sephadex chromatography and isoelectric focussing. The purified protein does not exhibit any detectable binding activity toward sn-glycerol-3-phosphate. It has no activity as a glycerol phosphatase nor as a glycerol kinase. Polyacrylamide gel electrophoresis in the presence of dodecylsulfate of the protein subsequent to treatment in urea, boiling in dodecylsulfate and crosslinking indicates that it occurs as an oligomeric protein composed of four identical subunits of 40 000 molecular weight. Membrane vesicles of wild-type strains that contain protein GLPT in whole cells loose it during vesicle preparation. However, they still exhibit high transport activity toward sn-glycerol-3-phosphate. Membrane vesicles prepared from glp T mutants that may or may not contain protein GLPT do not transport sn-glycerol-3-phospahte. We conclude from these results that protein GLPT does not participate in the energy-dependent active transport through the cytoplasmic membrane but could be involved in facilitating the diffusion of sn-glycerol-3-phosphate through the outer layers of E. coli.


The roles of c-type cytochromes in algal photosynthesis. Extraction from algae of a cytochrome similar to higher plant cytochrome f. A membrane-bound cytochrome resembling higher plant cytochrome f in many respects has been extracted from the algae Chlamydomonas. Euglena and Anacystis, and partially purified. The spectra of the cytochromes from Chlamydomonas and Euglena are virtually identical to that of parsley cytochrome f, with alpha-band maxima near 554 nm, very asymmetrical beta-bands, and gamma-band maxima at 421 nm. The cytochrome from Anacystis had alpha and gamma-bands both shifted to slightly longer wavelengths. The redox potential of the cytochrome from Chlamydomonas was determined as +350 mV, and its minimum molecular weight in sodium dodecyl sulphate as 31 000. The cytochrome from Euglena showed a rate of reaction with higher plant plastocyanin at least 100 times that of the soluble Euglena cytochrome c-552, and was unaffected by Euglena cytochrome c-552 antiserum. A very fast rate of electron transfer occurred between this cytochrome purified from Euglena and cytochrome c-552. The roles of the membrane-bound and soluble c-type cytochromes in algal photosynthesis are discussed, and it is recommended that the name cytochrome f should be reserved for the membrane-bound cytochrome (to emphasize its affinity with higher plant cytochrome f), while the soluble one should be named by its alpha-band (c-552, c-553, etc.) to make clear its distinctness from higher plant cytochrome f and homology with mitochondrial cytochrome c.


The binding of carbon monoxide to cytochrome c oxidase. The effect of CO on the optical absorbance spectrum of partially reduced cytochrome c oxidase  has been studied. The changes at 432 and 590 nm suggest that the cytochrome alpha2/3+ - CO compound is formed preferentially and that concomitantly a second electron is taken up by the enzyme. From the CO-induced changes at 830 nm it is concluded that in the partially reduced enzyme addition of CO causes reoxidation of the copper component of cytochrome c oxidase. Addition of CO to partially reduced enzyme (2 electrons per 4 metal ions) also brings about a decrease in the intensities of electron paramagnetic resonance signals of high-spin heme iron near g = 6 and of the low-spin heme at g = 2.6. Concomitantly both the low-spin heme a signal at g = 3 and the copper signal at g = 2 increase in intensity. These results demonstrate that formation of the reduced diamagnetic cytochrome a3 - CO compound is accompanied by reoxidation of both the copper component detectable by electron paramagnetic resonance and possibly also by cytochrome a.


Evidence for the identity of nuclear and cytoplasmic adenosine-3':5'-monophosphate-dependent protein kinase from porcine ovaries and nuclear translocation of the cytoplasmic enzyme. Protein phosphokinase activity from a 0.5 M NaCl extract of purified porcine ovary nuclei has been resolved by Sephadex G-200 gel filtration into three forms of kinase, protein kinase I and III, both independent of adenosine 3':5'-monophosphate (cyclic AMP), and cyclic-AMP-dependent protein kinase II. Cyclic AMP-binding activity was associated with protein kinase II but not with protein kinases I and III. Protein kinases I, II, and III exhibited different cyclic nucleotide dependency and substrate specificity. Protein kinase II was inhibited by a heat-stable protein from rabbit skeletal muscle, whereas protein kinases I and III were not inhibited. According to previously established criteria [Traugh, J.A., Ashby, C.D. and Walsh, D.A. (1974) nuclear protein kinase II can be classified as cyclic-AMP-dependent protein kinase consisting of regulatory and catalytic subunits. Nuclear protein kinases I and III are cyclic-AMP-independent enzymes. Evidence for the identity of nuclear cyclic-AMP-dependent protein kinase II with cytosol (105 000 X g supernatant fraction) cyclic-AMP-dependent protein kinase was obtained in several ways. Nuclear and cytosol cyclic-AMP-dependent protein kinases exhibited identical elution characteristics on DEAE-cellulose and Sephadex G-200 indicating that both kinases are of similar molecular size and possess similar ionic charge. Both kinases exhibited an identical Km for ATP of 8 muM, showed similar substrate specificity, and revealed similar antigenic properties. Cyclic-AMP-dependent protein kinase II was also identified in nuclei isolated in nonaqueous media, eliminating the possibility that the cyclic-AMP-dependent protein kinase activity identified in nuclei isolated in aqueous media may have arisen as the result of cytoplasmic contamination. After incubation of neonatal porcine ovaries which lack nuclear cyclic-AMP-dependent protein kinase with 0.1 muM 8-p-chlorophenylthio cyclic AMP, considerable cyclic-AMP-dependent protein kinase II activity was identified in nuclei isolated in nonaqueous media. From these data it is concluded that the nuclear cyclic-AMP-dependent protein kinase II is related to or identical with the ovary cytoplasmic cyclic-AMP-dependent protein kinase, supporting the concept that nuclear cyclic-AMP-dependent protein kinase is of cytoplasmic origin.


Different types of mitochondria in parenchymal and non-parenchymal rat-liver cells. 1. Intact and pure parenchymal and non-parenchymal cells were isolated from rat liver. The specific activities of several mitochondrial enzymes were determined in both parenchymal and  non-parenchymal cell homogenates to characterize the mitochondria in these liver cell types. 2. In general the activities of mitochondrial enzymes were lower in non-parenchymal liver cells than in parenchymal cells. The specific activity of pyruvate carboxylase in non-parenchymal cells expressed as the percentage of that in parenchymal cells was onlu 2% for glutamate dehydrogenase 4.3% and for cytochrome c oxidase 79.4%. Monoamine oxidase, as an exception, has an equal specific activity in both cell types. 3. The activity ratio of pyruvate carboxylase at 10 mM pyruvate over 0.1 mM pyruvate is 3.35 for parenchymal cells and 1.50 for non-parenchymal cells. This indicates that non-parenchymal liver cells only contain the high affinity form of pyruvate carboxylase in contrast to parenchymal cells. 4. The ratio of glycerol-3-phosphate cytochrome c reductase over succinate cytochrome c reductase activity differs from parenchymal (0.01) and non-parenchymal cells (0.10). This might indicate that the glycerol-3-phosphate shuttle, which is important for the transport of reduction equivalents for cytosol to mitochondria is relatively more active in non-parenchymal cells than in parenchymal cells. 5. The activity pattern of mitochondrial enzymes in parenchymal and non-parenchymal cell homogenates indicates that these cell types contain different types of mitochondria. The presence of these different cell types in liver will therefore contribute to the heterogeneity of isolated rat liver mitochondria in which the mitochondria from non-parenchymal cells might be considered as "non-gluconeogenic".


Effect of hyperoxide radicals on bovine-erythrocyte membrane. 1. Bovine erythrocytes exposed to the action of an enzymic source of hyperoxide radicals (hypoxanthine + xanthine oxidase) exhibited hemolysis, which was prevented by the presence of hyperoxide dismutase. 2. Exposing bovine erythrocyte membranes to the source of hyperoxide radicals resulted in a decrease of (Mg2+ + Na+ + K+)ATPase activity which could be partially prevented by addition of hyperoxide dismutase. 3. The damage observed to erythrocyte membranes under the conditions applied is ascribed to toh formed in the Haber and Weiss reaction since a protection by OH scavengers was also observed.


Cyclic nucleotide phosphodiesterases from rat anterior pituitary. Characterization of multiple forms and regulation by protein activator and Ca+. Phosphodiesterase activities for adenosine and guanosine 3':5'-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl2 to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphadiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1% Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to Ca2+ and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N'-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 10500 X g supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 muM substrate being 20 muM and 24 muM for cyclic AMP and 7 muM and 10 muM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent Km values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction II enzyme was stimulated 6--7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+.


2-Deoxy-D-galactose metabolism in ascites hepatoma cells results in phosphate trapping and glycolysis inhibition. The metabolism of 2-deoxy-D-galactose has been studied in AS-30D rat ascites hepatoma cells in suspension. Using 2-deoxy-D-(1-14C)galactose and an alkaline ethanol deproteinization procedure, the quantitatively identified metabolites included 2-deoxy-D-galactose 1-phosphate comprising 99.3%, and UDP-2-deoxy-D-galactose and UDP-2-deoxy-D-glucose, together amounting to 0.4% of the total metabolites. After incubation for 5 h in the presence of 2-deoxy-D-galactose (1 mmo1/1), the content of 2-deoxy-D-galactose 1-phosphate reached 35 mmo1x(kg cells)-1. The rate of phosphorylation of 2-deoxy-D-galactose was rapid during the first 30 min and decreased to approximately 20% of this rate during the subsequent hours. The rapid trapping of Pi in the form of 2-deoxy-D-galactose 1-phosphate resulted in a depression of free intracellular Pi in spite of a concomitant increase in net 32Pi uptake from the medium and a decrease of ATP and other 5'-nucleotides. The rates of glucose utilization and lactate production were depressed by more than 80% in the presence of 2-deoxy-D-galactose (1 mmo1/1). Interruption of Pi trapping by removal of 2-deoxy-D-galactose from the medium reversed the depressions of Pi and ATP and resulted in a rapid but incomplete relief of glycolysis inhibition. Crossover analysis of glycolytic intermediates indicated an inhibition at the 6-phosphofructokinase step. The depression of glucose utilization may be mediated by the increased level of glucose 6-phosphate, a potent inhibitor of hexokinase. An additional inhibitory effect of a metabolite of 2-deoxy-D-galactose at the 6-phosphofructokinase step was indicated by crossover analysis after reversal of Pi and ATP depressions in the presence of a high intracellular content of 2-deoxy-D-glactose 1-phosphate. The quantitative analysis of the metabolites of 2-deoxy-D-galactose demonstrated the predominance of the monophosphate and the negligible formation of UPD derivatives of this sugar analog in AS-30D hepatoma cells. This provides a system for the investigation of a galactose analog as a phosphate-trapping agent in the virtual absence of uridylate trapping.


Radiochemical purity of various 99mTc-labelled bone-scanning agents. The radiochemical purity of preparations for 99mTc-bone-scanning was tested using thin-layer chromatography and electrophoresis. The investigation included ten commercial kits, comprising four types of phosphorus compounds, i.e. sodium polyphosphate, sodium pyrophosphate, disodium  diphosphonate, and sodium monofluorophosphate. Out-spoken differences in the content of free pertechnetate were found between the different kits. The contents in preparations from the same kit were fairly constant, but in some cases a change could be referred to the age of the 99mTc-generator or the time of analysis. No significant difference was found between preparations made from eluate with and without oxidizer. A 99mTc-tin-colloid was sporadically present in the preparations.


Adrenocorticotrophic hormone during the first day of life. The plasma concentration of ACTH (by radioimmunoassay) was measured in 56 healthy parturients and their newborns. Umbilical cord and maternal venous blood were collected immediately after delivery. In addition one venous blood sample was taken from 50 newborns at the age of 15, 30, 60 min, 2, 6, 12, or 24 h. The maternal and cord plasma ACTH levels were higher than the levels in healthy nonpregnant women. There are no differences between the mean maternal (226 +/- 146 pg/ml) and cord (226 +/- 147 pg/ml) values of ACTH. The high ACTH levels of cord plasma remain unchanged for 30 min, decrease significantly during the 1-6 h after birth, because of the elimination process of the circulated foetal ACTH, and increase over the next 12-24 h and slightly thereafter indicating an initiation of neonatal pituitary ACTH secretion. The plasma ACTH level in the mothers with membranes ruptured for 1-5 h was significantly (P less than 0.05) higher compared with that in the mothers with membranes ruptured for less than 1 h. However, the duration of ruptured membranes appeared to have no effect on the cord blood ACTH level.


Variations among individual mice in binding of growth hormone and insulin to membranes from animals of different ages. Binding of 125I-labeled insulin and growth hormone to membranes from liver and hearts of C57BL/6J mice was measured and the data analyzed by the graphical method of Scatchard. Animals were studied at ages 2, 10, 20, and 31 months; preparations from each animal were analyzed individually. There was no significant and progressive age-related difference in either dissociation constant or binding capacity for insulin in heart and liver, or for growth hormone in liver. Variations among individual animals were large; in some cases, the standard deviation exceeded the mean for a particular age group. We conclude that there are no large age related differences in binding of these anabolic hormones to the target tissues studied, and that any small changes would be masked by the large variations among individual animals.


Morphine analgesia in mice of different ages. The analgesic response to 5 mg per kg morphine sulfate was examined in male C57BL/6J mice aged from 2 months to 28 months by the tail-flick assay. The baseline latency of old mice was slightly lower than that of young mice. There was a smaller increase in latency after morphine in old mice than in young mice. Decreased analgesic response in old mice was accompanied by a slower rate of uptake of labeled morphine into the blood after i.p. injection and a longer half-life of the drug. There was no difference between young and old mice in the number of narcotic receptors in the brain.


Cholinergic transmission in subcortical and cortical visual centers of rats: no evidence for the involvement of primary optic system. The effect of unilateral enucleation, ablation of the visual cortex or coagulation of the lateral geniculate nucleus (LGN) upon the activity of choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) in different structures of the visual system of albino rats was studied. The localization and extent of the degeneration pattern were followed up by histological silver degeneration methods. Afferents from the retina project mainly contralaterally to the dorsal and ventral LGN, the pretectal region and the superior colliculus. Afferent fibres from the dorsal LGN enter the visual cortex in area 17 only. Neurons of this area project back ipsilaterally to the LGN and the superior colliculus (SC). No significant decrease in the activity of the cholinergic marker enzyme choline acetyltransferase could be observed under any of the experimental conditions; there was rather a tendency to increased activity in the subcortical centres. AChE as a less specific marker also exhibited no gross changes in activity in the lesioned animals. The results add more direct proof to pharmacological and physiological evidence that ACh is not involved in the synaptic transmission of the direct optic projections in rats, either at the subcortical or at the cortical level.


Use of 3H and 14C doubly labeled glucose and amino acids in the study of hormonal regulation of gluconeogenesis in rats. Double isotope procedures (3H and 14C) were used in vivo to investigate a) slow long-term gluconeogenic actions of adrenal glucocorticoids, and b) rapid stimulation of gluconeogenesis by glucagon. [U-14C,6-3H]Glucose was administered to normal and adrenalectomized rats. No effect was observed on the [6-3H]glucose half-life suggesting the dicarboxylic acid shuttle is unaffected by adrenalectomy; the Cori cycle is also not influenced. Loads of [14C]aspartate, [14C]glutamate, or [14C]alanine were given to normal and adrenalectomized rats. Simultaneously, in vivo transaminase activity was studied by measuring the appearance of 3H2O in body water after administration of [2-3H]aspartate, [2-3H]glutamate, or [2-3H]alanine, Adrenalectomy has no influence on the incorporation of glutamate or aspartate into glucose or on their in vivo transaminases. Diminution of incorporation of [14C]alanine into glucose and alanine transaminase activities occurs only when rats are given unphysiological loads. These studies support the contention that glucocorticoid rate-limiting actions occur in extrahepatic tissues to produce an increased flow of glucose precursors to the liver. [U-14C,3-3H]Glucose was used to investigate the effect of glucagon on the hepatic fructose-6-phosphate (F-6-P) cycle. Glucagon administration resulted in a rapid drop in the 3H/14C ratio of circulating glucose, suggesting an increase in F-6-P recycling caused by activation of FDPase with little or no decrease in phosphofructokinase. Such a change would direct substrate flux toward gluconeogenesis.


Integrated control of hepatic glucose metabolism. The rates of storage and release of carbohydrate by the liver are determined by the plasma concentrations of several blood-borne signals; most important are the concentrations of glucose, and of the hormones insulin and glucagon. To understand the complex control relationships of these three signals as they affect the liver, their individual dynamic influences have been determined experimentally, and the findings have been integrated by means of a computer simulation of the pathways of hepatic glycogen metabolism. The simulation studies have led to specific hypotheses about the biochemical effects of glucose and insulin on the liver. The simulation studies have also led to the conclusion that glucose exerts a rapid moment-to-moment influence on the rate of uptake of glucose by the liver. Insulin, however, by exerting a slower influence on the sensitivity of the liver to glucose, is very effective in "optimizing" the amount of glycogen which the liver stores during food intake. Thus, integrated experimental and simulation studies can lead to a view of a physiological regulating system which does not emerge from either approach used alone.-


Reversible inhibition of the motility of human spermatozoa by cytochalasin B. Cytochalasin B inhibits the motility and metabolism of washed human spermatozoa at low concentrations (20 to 200 mum). Spermatozoal motility (primarily the frequency of flagellar contraction) declines slowly after addition of the antibiotic but is not abolished even after treatment for several hours. The addition of caffeine or dibutyrylcyclic adenosine 3' :5'-monophosphate to washed sperm suspensions increases the percentage of motile cells, the frequency of flagellar contraction, and the rate of glycolysis. These effects are blocked by cytochalasin B. However, cytochalasin B-treated spermatozoa regain their responsiveness to these agents when the antibiotic is washed out of supporting media. These effects are discussed in terms of an interaction of cytochalasin B with the sperm plasma membrane.


[Inhibition of spontaneous lymph vessel contraction evoked by transmural stimulation]. In isolated sections of mesenteric lymphatic vessels, the constrictory responses to transmural electrical stimulation were studied. Inhibition of the spontaneous rhythm occurring primarily at low frequencies was revealed as well as the inhibition following the excitatory effect of high--frequency stimulation. Analysis of the transmural inhibition showed the latter to occur as the result of activation of adrenergic nervous plexus and to be fulfilled by the beta--adrenoreceptors.


[The effects of ACTH, aminoglutehimide and hypophysectomy on the rat adrenal lipids (author's transl)]. The lipid patterns in the adrenal glands of rats with the adrenocortical steroidogenesis stimulated by ACTH or inhibited by hypophysectomy or by aminoglutehimide (AGT) were compared with those in intact rats. The administration of ACTH caused the gradual decrease in the concentration of cholesteryl ester with the preferential decrease in the proportion of arachidonic acid. The deposition of cholesteryl ester was observed in the adrenal glands of both hypophysectomized and AGT treated rats. Hypophysectomy was accompained by the increase in the proportion of 16:0 and 18:0 and the decrease of 18:2 and 20:3, while AGT administration caused the increase in the proportion of 20:4 and the decrease of 22:6 in the cholesteryl fraction, ACTH and AGT treatments or hypophysectomy resulted in minor alterations of the concentrations and fatty acid compositions of triglyceride and phospholipid. The marked differences in the concentration as well as fatty acid compositions of cholesteryl ester under the stimulated or inhibited adrenal steroidogenesis suggest the important role of fatty acids esterified with cholesterol in the steroid hormone synthesis.


[Clinical study of pseudohypoparathyroidism Type I & Type II (author's transl)]. Pseudohypoparathyroidism (PHP) is a disorder characterized by renal and/or skeletal refractoriness to the effect of parathyroid hormone (PTH). In 1973, it was first proposed by Drezner et al that PHP could be divided into two types, e.g., PHP Type I and PHP Type II. PHP Type I is a disorder which fails to show the increase of urinary c-AMP and phosphaturia by the administration of PTH, but PHP Type II is that which responds to the administration of PTH with a marked rise of urinary c-AMP, but no increase of phosphate excretion, In 1974 Rodriguez et al demonstrated a patient with PHP Type II who restored normal renal responsiveness to PTH by calcium administration. Here we present two patients who fitted in the categories of PHP Type I & II, respectively, and restored normal renal responsiveness by the combined Ca-PTH administration or dibutyryl-cAMP infusion. Case I (PHP Type I) was a 31 yr old male with marked hypocalcemia and hyperphosphatemia, who showed neither increase of urinary c-AMP nor phosphate excretion in response to PTH infusion. Case II (PHP Type II), also diagnosed as Sjögren syndrome was a 22 yr old female with relatively mild hypocalcemia and hyperphosphatemia, who showed a marked rise of ordinary c-AMP, but no increase of phosphate excretion by PTH administration. Acute infusion of calcium, followed by PTH administration restored renal responsiveness to PTH in both types, though calcium infusion showed only little effects. The patient of PHP Type I received calcium gluconate and Vitamin D therapy and serum Ca improved, when PTH or DBc-AMP was given with the reappearance of PTH-like action. Thus in PHP Type I, the lack of c-AMP response to PTH, coupled with the ability of infused DBc-AMP to evoke a normal renal response suggests that the metabolic defect in this disorder may exist in hormone receptor-adenyl cyclase complex. Calcium infusion followed by PTH administration probably might evoke calcium influx and c-AMP production, resulting in the reappearance of hormone action. In PHP Type II, as c-AMP generation system is intact, the metabolic defect can be thought to exist in the rather poorly defined process beyond c-AMP generation. The combined Ca-PTH administration also restored normal renal response, probably partly due to the improved calcium environment of renal tubular cells. Though it is difficult to explain why endogenously generated c-AMP had no effect, but DBc-AMP had on reappearance of PTH-like action, it is speculated that DBc-AMP may have much more stronger effects for intracellular receptor site of c-AMP in PHP Type II. Conclusively these results clearly suggest that the nature of the metabolic defects in PHP is not a genetically determined inrreversible disorder, but rather a functional one which can be reversibly restored by special conditioning.


[ACTH secretion and adrenocortical responsiveness in Cushing's syndrome due to adrenocortical hyperplasia (author's transl)]. A radioimmunoassay for plasma ACTH has been developed utilizing highly purified human ACTH (Li) labelled with 125I by the lactoperoxidase method as a tracer and an ACTH antibody produced by immunization of rabbits with ACTH-Z (Organon). The assay is highly specific, reproducible and sensitive to 20 pg of ACTH per ml. Utilizing this technique, endogenous ACTH secretion, adrenal responsiveness to endogenous ACTH and hypothalamic pituitary adrenal feedback mechanisms have been assessed in normal subjects and in patients with Cushing's syndrome due to adrenocortical hyperplasia and nodular cortical hyperplasia. The potential effect of a negative feedback mechanism on the circadian rhythmicity after SU-4885 administration was assessed by initiating SU-4885 either at 9 p.m. or 8 a.m. In normal subjects, the circadian rhythm of ACTH was persistent and independent of a decrease in cortisol. However, in a single case of Cushing's syndrome due to adrenocortical hyperplasia, the circadian rhythm was different from that of normal subjects, possibly influenced by a negative feedback mechanism when SU-4885 was initiated at 8 a.m. In Cushing's syndrome due to adrenocortical and nodular cortical hyperplasia, a significant correlation was observed between the mean plasma ACTH and urinary 17-OHCS values before and after SU-4885 administration (r=0.743, p less than0.01). A significant correlation was also obtained in normal subjects between plasma ACTH and urinary 17-OHCS values (r=0.889, p less than 0.01). However, there was quantitatively more 17-OHCS excreted in the urine for a given plasma ACTH level in patients with Cushing's syndrome than in normal subjects. To assess the relative biological activity of endogenous and exogenously administered ACTH, the ratio of daily 17-OHCS during SU-4885 administration and Cortrosyn-Z administration was expressed as the Cortrosyn Equivalent Quotient (C.E.Q.). The correlation between plasma ACTH and C.E.Q. was similar and significant for normal subjects and patients with Cushing's syndrome (r=0.670, p less than 0.01). These data suggest that there is hyper-responsiveness of the adrenal glands to endogenous ACTH in Cushing's syndrome due to adrenocortical hyperplasia and nodular cortical hyperplasia and that the adrenal hyperactivity is not engendered by a qualitative change in the ACTH release from the pituitary gland. To assess pituitary suppressibility, dexamethasone was administered 40 days or more later following total adrenalectomy in 9 patients with Cushing's syndrome, 6 with adrenocortical hyperplasia and 3 with nodular cortical hyperplasia. One day after discontinuation of substitution therapy, 2 mg of dexamethasone was administered orally followed on successive days by 4 and 8 mg doses. In each instance, dexamethasone was given at midnight and the plasma ACTH concentration was determined at 9:00 a.m. on the day before and after administration of the dexamethasone. A patient with Addison's disease was studied as a control...


Progressive systemic sclerosis (scleroderma). First case report in a Nigerian. The first case of progressive systemic sclerosis in a Nigerian is described. In addition to the typical features of the disease, the case shows affection of the peripheral nerves, a very rare complication. The latter led to a mistaken diagnosis of leprosy. The reasons for the rarity of this disorder in the indigenous Africans and its differentiating features from leprosy are discussed.


Secretion of immunoreactive insulin and glucagon in hamsters bearing a transplantable insuloma. Glucose, insulin (IRI), pancreatic (IRG) and total (GLI) immunoreactive glucagon were measured in the serum of normal hamsters and of hamsters with an insulin- and glucagon-secreting, transplantable insuloma. The tumor-bearing animals were hypoglycemic, hyperinsulinemic and hyperglucagonemic. The pancreatic islets of tumor-bearing animals secreted less glucagon and insulin in response to arginine or to changes in the glucose concentration of the medium, than did the islets of control hamsters. In addition, the introduction of glucose into the gastro-intestinal tract, which caused a significant rise in the serum GLI concentration of normal hamsters, failed to do so in the tumor-bearing animals. The results suggest that  the high levels of serum glucagon and insulin induced by the tumor, suppressed IRI, IRG and GLI secretion in these animals.


Somatostatin: a review of its effects especially in human beings. A review is given of the early 1976 situation in somatostatin research: the effects on pituitary hormones and the effects outside the pituitary gland, the production of somatostatin outside the hypothalamus, the artificial analogues, the problem of "side effects", and the mode of action. The possible clinical applications of somatostatin and somatostatin analogues are discussed.


Endocrine and cardiovascular consequences of angiotensin converting enzyme inhibition. One of several novel peptidic inhibitors of angiotensin converting enzyme (CEI) has been studied intravenously both in normal male volunteers and severely hypertensive patients without any clinically significant adversity or intolerance. Hypertensive patients experienced a significant yet gradual reduction in resting arterial pressure without hypotension. The addition of a diuretic agent was observed to potentiate this antihypertensive effect. Normal, sodium replete volunteers received this nonapeptide intravenously in doses up to 2-0 mg/kg without any significant cardiovascular effect. Both patients and normal subjects exhibited reversible dose related increases in angiotensin I and renin levels after receiving the peptide. The plasma renin response to tilting was also potentiated by CEI. These findings suggest that intravenous CEI may be of value in the treatment of severely elevated hypertension and as a tool to evaluate vasoconstrictor and volume factors in hypertension.


Properties of detergent-solubilized adenylate cyclase and gonadotropin receptors of testis and ovary. The relationship between solubilized hormone-binding sites and adenylate cyclase was examined in detergent extracts of particulate testis and ovarian fractions. Both basal and fluoride-stimulated activities of the particulate enzyme were markedly increased in the presence of detergents, and about 60% of the enzyme activity was recovered as the soluble form in the 300,000 g supernatant. Enhancement of adenylate cyclase activity was more marked with Lubrol PX and WX than with Triton X-100, and the highest recovery and activation of adenylate cyclase were obtained with 0.5% Lubrol PX. The particulate and solubilized testicular enzymes were more active in the presence of Mn2+, and the detergent-extracted soluble ovarian cyclase showed a small and inconstant response to gonadotropin. Fractionation of Lubrol-solubilized testis and ovarian preparations on Sepharose 6B showed two peaks of free gonadotropin receptors. The binding activity eluted with Kav of 0.32 corresponded to the receptor sites previously characterized in detergent-solubilized gonadal particles, and was coincident with the elution profile of adenylate cyclase activity. An additional peak of binding activity with Kav of 0.56 was not accompanied by detectable adenylate cyclase activity. These observations suggest that the peak of larger molecular size could represent dissociated receptors or binding sites which were not coupled to adenylate cyclase.


The use of dynamic models to study the role of calcium in the oxytocin-induced contractions of the uterus. The question as to whether calcium can be considered to be a mediator of oxytocin-induced myometrial contraction has been investigated. Assuming that the contraction is linearly proportional to the myoplasmic calcium concentration, several possible molecular mechanisms leading to its increase (calcium release from the cell membrane, acceleration of calcium transport from extracellular space by a 'gate' mechanism, release from intracellular organelles, blockade of calcium pumps) were modelled on an analog computer. The oxytocin intervention in the calcium distribution was mimicked by a discontinuous change of the appropriate rate constants. The computed transient simulating the myoplasmic calcium concentration was then compared with an experimental time profile of uterine tension. The result of screening the models shows that oxytocin must act predominantly via release of calcium bound to the cell membrane. A quantitative comparison, however, requires that the kinetics of oxytocin distribution in myometrium also be considered in the model. The problem treated in this paper demonstrates the possibilities and limitations of a screening procedure based upon direct comparison of time profiles of experimental processes with several computed model alternatives.


Prolactin binding in rat testis: specific receptors in interstitial cells. Specific receptors for [125I]hPrl (human prolactin) are present in membrane preparations of rat testis. The receptors are specific for lactogenic hormones (prolactin and human growth hormone) but do not bind gonadotropins. The prolactin receptors are localized exclusively in the interstitial cell tissue, and are not present in membrane preparations from isolated seminiferous tubules. The localization of prolactin receptors interstitial tissue suggests that the effect of prolactin on LH/hCG-stimulated testosterone production is due to a direct effect of prolactin of Leydig cells.


Involvement of intracellular calcium in hormone secretion from pituitary cells. Cells were dissociated from normal rat pituitaries by a combination of mechanical agitation and enzymatic action, seeded into culture flasks and grown in monolayer culture. When such cells were exposed to an extract of the hypothalamic stalk-median eminence area (HSME) a dose-dependent secretion of ACTH was observed. A 2-h exposure to Ca-free media significantly reduced the HSME-stimulated release of ACTH but the measured levels were still greater than the unstimulated controls. When the 45Ca2+ uptake into the cultured cells was measured both control and HSME-stimulated cells yielded identical results (60-80 nmol Ca/mg cell protein). Upon removal of the calcium associated with the surface coat it was found that HSME actually decreased the cellular uptake of calcium. Since variations in uptake  can result from changes in influx or efflux as well as from variations in pool size or turnover times of calcium exchange with intracellular compartments, a series of isotope washout experiments were performed. Neither HSME nor theophyline affected the rate constant of calcium efflux from what is believed to be the cytosol pool to the extracellular media. Both agents, however, prompted a shift of intracellular calcium into a more tightly bound compartment. The data suggest that the calcium required for pituitary hormone secretion is derived primarily from an intracellular rather than extracellular origin. It may be that, via the action of cyclic AMP, such calcium can be mobilized from intracellular stores and shifted to a more tightly bound compartment where it can participate in the intracellular processes associated with secretion.


Innocuity and toxicity testing of non-viral components of foot-and-mouth disease vaccines. Some biological properties of 3 different adjuvants: saponin, DEAE-dextran and sodium azide applied in foot-and-mouth vaccines have been studied. As tested in guinea pigs, by dialysis of various batches of saponin, the author succeeded in reducing their toxicity by 80 to 90%, their hemolytic activity by 60 to 75% and their inflammatory capacity by 40 to 50%, whereas in cattle the inflammatory capacity of dialyzed saponin batches decreased by 80 to 90%. The inflammatory capacity of DEAE-dextran is about 50 to 60% less than that of the saponin. The effect of saponin and sodium azide has directly been studied in virus suspension also. Neither the saponin nor the sodium azide affected adversely the infectivity and immunogenicity of the antigen. The loss of potency due to the storage of the vaccine is not in relationship with the presence of the two above-named components.


Study on toxicity of Bordetella pertussis cultures and antigens. The toxicity of 201 Bordetella pertussis strains isolated in Belgrade has been studied. The strains were grown on a modification of Cohen and Wheeler's medium. LD50 of examined strains ranging between 1.06 and 1.95 billion bacteria in 35 (17.41%), between 2.04 and 4.83 billion bacteria in 92 (45.77%) and between 5.31 and 7.79 billion bacteria in 46 (22.88%) were found. Low toxic cultures of B. pertussis strains with LD50 ranging between 9.0 and 14.1 billion bacteria in 28 (13.43%) were obtained. The culture of the strains with low toxicity was 7 to 14 times more toxic than those prepared from the strains with low toxicity. In the cultures of 51 B. pertussis strains, toxicity for mice was not correlated with the number of viable cells. The toxic substances which influenced the loss of mouse weight were found in the culture filtrate of B. pertussis. A considerable detoxifying effect of heating at 56 degrees C for 30 min on liquid cultures prepared from highly toxic strains of B. pertussis was observed. The susceptibility of two different strains of mice for pertussis vaccine has been examined. The Albany strain of mice was less susceptible to toxicity control of the vaccine by mouse-weight-gain test than the Torlak strain.


An islet cell carcinoma containing glucagon and insulin. Chronic glucagon excess and glucose homeostasis. Described here is a patient who had an islet cell carcinoma containing both glucagon (glucagonoma) and insulin (insulinoma). Complete removal of the tumor was possible. Immunoreactive glucagon (IRG) could be extracted from all parts of the tumor (approximately 50 mug./gm.) and was shown to be fully bioactive. Immunoreactive insulin (IRI) could be extracted only from one section of the tumor (approximately 30 mug./gm.). The clinical and biochemical manifestations of the disease were dermatitis, diabetes, weight loss, anemia, hypoaminoacidemia, and hyperketonemia. The diabetes was characterized by low or normal fasting blood glucose concentrations and by impaired glucose tolerance (Kg = 0.4). After complete removal of the tumor, the dermatitis cleared, the catabolic state changed into an anabolic state, blood amino acid concentrations increased, and blood ketone-body concentrations decreased. Fasting blood glucose concentrations, however, rose above 200 mg./dl., and glucose tolerance declined further (Kg = 0.15). Hourly blood sampling for 24 hours, intravenous and oral glucose tolerance tests, intravenous arginine and tolbutamide tolerance tests with serial determinations of IRG, IRI, and blood glucose were performed preoperatively and again two weeks and two months postoperatively. The results of these studies demonstrated marked abnormalities in the stimulation and suppression of glucagon and insulin release. In addition, they failed to demonstrate a glycemic effect on the chronically elevated glucagon concentrations in this patient, while identifying insulin as the dominant factor determining blood glucose homeostasis.


The cyclic AMP response to glucagon. Comparison of tissue and plasma cyclic AMP levels in the rabbit. The effects of glucagon on tissue and plasma cyclic AMP levels have been investigated in rabbits anesthetized with urethane. Glucagon (2 nmole/kg.) caused at least a twofold increase in hepatic cyclic AMP, which reached a peak within two minutes and declined to basal values after 40 minutes. Plasma cyclic AMP also increased at least twofold, reaching a peak at 10 minutes and declining to basal values after 60 minutes. Glucagon (20 nmole/kg.) stimulated hepatic and plasma cyclic AMP in a manner indistinguishable from that observed at the lower dose. Hepatectomy abolished the plasma cyclic AMP responses to glucagon, and no significant stimulation of cyclic AMP concentration was noted in the heart, adipose tissue, small bowel, or kidney. Cyclic AMP hydrolysis was estimated in blood taken before and after administration of glucagon. Glucagon (2 nmole/kg.) increased cyclic AMP hydrolysis slightly, but this was explained by the raised cyclic AMP levels. By contrast, cyclic AMP hydrolysis increased two-to-threefold in blood taken 20 and 40 minutes after glucagon (20 nmole/kg.). The higher dose of glucagon also stimulated cyclic AMP hydrolysis in crude liver homogenate, which could not be explained by increases in cyclic AMP concentration. The increase in cyclic AMP hydrolysis observed in blood and liver may partly explain the failure to show additional stimulation of hepatic and plasma cyclic AMP levels with the higher dose of glucagon. Despite the changes in cyclic AMP hydrolysis, a highly significant correlation was observed in individual rabbits between the hepatic and plasma cyclic AMP responses to glucagon (2 and 20 nmole/kg.), when these were calculated as incremental areas above mean basal levels. It is suggested that measurement of plasma cyclic AMP levels after stimulation by glucagon may be an accurate index of the hepatic cyclic AMP response to glucagon in vivo.


Hypoglycemia in man pathologic and physiologic variants. To determine if an effective method existed for distinguishing the physiologic hypoglycemia of fasting from pathologic hypoglycemia, 72-hour fasts were conducted in 60 women and 20 men of normal weight, in 16 obese subjects, and in six of 11 patient with insulinomas. Only the pattern of change of the immunoreactive-insulin-to-glucose ratio (the I/G ratio), calculated at major time intervals of the fast, provided a clear-cut distinction between these groups; plasma glucose values alone could not make this distinction. The mean fasting I/G ratio was calculated for each subject from that subject's I/G ratios at 12-hour intervals during the fasting period. In no single case did the mean I/G ratio during fasting for an individual of normal weight equal or exceed the control I/G. I/G ratios increased dramatically during fasting in each patient with an insulinomas. Normal obese patients (15% greater than ideal body weight) did not provide a diagnostic problem, since, regardless of sex, glucose values of less than 55 mg/dl. did not occur. Although the pattern of change of the I/G ratio was extremely useful, the basal I/G ratio alone was potentially misleading; this was due to overlap of basal I/G ratios between subjects with simple obesity and patients with insulinomas. In addition, absolute values for the I/G ratio varied with the technique employed for measuring glucose and insulin. Change of the I/G ratio, however, was independent of the techniques used for measuring glucose and insulin. DIABETES 26:161-65, March, 1977.


Transient stimulatory effect of sustained hyperglucagonemia on splanchnic glucose production in normal and diabetic man. Insulin can modulate glucagon-stimulated hepatic glucose production and is considered to be the major factor acting in vivo to exert a couterregulatory action to glucagon. The insulin-dependent diabetic, therefore, might be especially vulnerable to enhanced hepatic glucose production promoted by glucagon. To investigate this hypothesis, low-dose glucagon infusions were administered to normal and diabetic men to compare the effects of glucagon on net splanchnic glucose production (NSGP). Four normal and three insulin-dependent, ketosis-prone, hyperglycemic diabetic men (insulin withheld for 24 hours) underwent brachial-artery-hepatic-vein catheterization. Each received a 90-minute glucagon infusion at 5 ng/kg./min. Glucagon levels rose four-to-fivefold in both groups, plateauing at 300-600 pg./ml. In the normals, NSGP rose from 92+/-12 to 211+/-31 mg./min. at 15 minutes and returned to basal levels by 45 minutes. Insulin measured in the hepatic vein rose from 19+/-6 to 33+/-11 muU/.ml., while plasma glucose rose 17 mg./dl. In the insulin-dependent diabetics, NSGP rose from 78+/-24 to a peak of 221+/-33 mg./min. at 30 minutes and then fell sharply to 113+/-15 mg./min. at 60 minutes despite continuing hyperglucagonemia. Plasma glucose in the diabetics rose 21 mg./dl. These data suggest a mechanism that acts to rapidly diminish glucagon-induced hepatic glucose production in diabetic man but does not appear to be mediated by increased insulin secretion.


Suppression of basal insulin secretion by adrenalin in normal man and in patients with insulinomas. Basal insulin secretion has been thought to be via a different mechanism from stimulated insulin secretion, partly because it is not similarly suppressed by adrenalin. However, adrenalin normally causes hyperglycaemia, but if it is infused while the plasma glucose is kept constant there is marked suppression of insulin secretion. Sympathetic stimulation modulates basal insulin secretion, and alpha adrenergic blockade impaired the suppression of insulin secretion in response to hypoglycaemia. Four of five benign insulinomas had marked suppression of insulin secretion by adrenalin, but one malignant and one benign insulionoma had little suppression. Both had a raised proportion of their basal plasma insulin as proinsulin, and the impaired suppression of secretion by adrenalin probably signified an undifferentiated tumour.


Effects of various modifiers of insulin release on the lanthanum-nondisplaceable 45Ca2+ uptake by isolated pancreatic islets. The uptake of 45Ca2+ by a lanthanum-non-displaceable pool in pancreatic islets was studied; Raising the extracellular D-glucose concentration from 3 to 20 mM stimulated the 45Ca2+ uptake in hand-dissected islets of ob/bo-mice as well as in collagenase-isolated islets of ob/ob or normal mice. The effect was dose-dependent in the range of 0-20 mM D-glucose and was seen throughout a wide range of extracellular calcium concentrations (16 mumol-2.56 mmol of Ca2+ added per litre of medium). The 45Ca2+ uptake was also enhanced by other known insulin secretagogues (D-mannose, L-leucine, tolbutamide) and was uninfluenced by compounds lacking insulin-releasing capacity (3-O-methyl-D-glucose, L-glucose, D-galactose, D-leucine). The stimulatory effect of D-glucose was blocked by inhibitors of glucose-induced insulin release (D-mannoheptulose, diazoxide, L-adrenaline). The results support the view that the lanthanum-nondisplaceable calcium pool is related to the insulin-releasing mechanism, although the exact nature of this relationship is still unclear.


Enhanced development of metastatic foci in thymectomized, irradiated, and bone marrow-reconstituted mice. The effect of depletion of immunocompetence of metastasis of a mouse hepatoma MH-134 was studied in syngeneic C3H/He mice. (2) Development of metastasis in the lung and lymph nodes was remarkably enhanced in the mice which were thymectomized, irradiated, bone marrow-reconstituted (Tx-X-BM), and grafted intramuscularly with tumor cells. (2) Number of tumor cells detected in the blood stream was within the same range in the control mice and the Tx-X-BM mice after intramuscular tumor-grafting. (3) Metastatic foci were not detected in the stomach and intestines, not only in control mice but also in the Tx-X-BM mice after intramuscular tumor-grafting. However, tumors grew progressively in the subserosal tissues of these organs of the Tx-X-BM mice, but not of the control mice, when a small number of tumor cells were directly inoculated into such tissues. (4) On 9th day after an intravenous injection of a large number of tumor cells, tumor growth was detected to almost the same extent in the lungs of the control mice and the Tx-X-BM mice. Tumors grew progressively in the Tx-X-BM mice thereafter, whereas the tumors regressed in the control mice. These results suggest that inhibition of metastasis by immune response depends primarily upon suppression of proliferation of the tumor cells lodged in individual organs.


Estrogen receptor in breast cancer of the Japanese. The estrogen receptor was assayed, using the 2,000g supernatant and dextran-coated charcoal method, in 243 tissue samples from human breast cancer, benign breast diseases, macroscopically normal breast tissues, normal uterine myometrium, and uterine myoma. The estrogen receptor was found to be positive in 52.1% of 98 primary breast cancer and in 54.1% of 24 metastatic tumors. The receptor in the breast cancer was found to be similar to that in normal uterine myometrium in the binding character; that is, the dissociation constant of 10(-9) approximately 10(-11) M and number of binding sites of 0 approximately 2,800 fmol/mg protein. There was no correlation between the presence of the receptor and some clinical factors such as menopausal status, age of the patient, urinary 17-ketosteroid excretion, clinical stage of cancer, tumor size, positive or negative axillary lymph node metastasis, histological type, metastatic site of the cancer, or disease-free interval. The estrogen receptor appeared to be retained by metastasis of cancer, and this may lead to the use of the receptor assay with mastectomy specimens for the prediction of response to hormonal therapy in future recurrence of malignancy. Furthermore, it may be possible by this assay to select patients suitable for adjuvant therapy with hormones at the time of mastectomy. A good correlation was found between the presence of the receptor and response to the major endocrine ablation therapy in patients with advanced or metastatic breast cancer. When the receptor was negative in the cancer tissue, the change of response to the endocrine therapy was minimum. On the other hand, if the cancer contained the receptor, approximately 60% of the patients with metastatic or advanced breast cancer responded well to the major endocrine ablation therapy. Thus, the estrogen receptor of breast cancer in Japanese patients appears to bear a close resemblance to that reported in Western patients in its incidence and the correlation to some biological characteristics of the cancer.


Depression of multinucleation by interferon in simian virus 40-transformed cells treated with cytochalasin-B. Multinucleation of simian virus 40-transformed mouse cells (mKS-A TU-7), which was induced by Cytochalasin-B, was depressed by the addition of crude interferon or by poly (riboinosinic)-poly(ribocytidylic) acid. In the absence of Cytochalasin-B, growth of the cells was not inhibited by interferon. Possible mechanism of this phenomenon is discussed.


Disseminated cytomegalic inclusion disease in an adult with cirrhosis of liver and review of literatures. We reported a 44 year old man with micronodular cirrhosis who eventually died from massive hematemesis and melena. At postmortem studies, there were disseminated cytomegalic inclusion bodies detected in various organs. In liver, intranuclear inclusion bodies were observed in cirrhotic liver. We discussed here possible pathogenesis of CMV (cytomegalovirus) infection in adults, in reviewing literatures.


Structure-activity relationships of gastrointestinal hormones: motilin, GIP, and [27-TYR]CCK-PZ. The docosapeptide and the tritetracontapeptide corresponding to the entire amino acid sequence of porcine motilin and gastric inhibitory peptide were synthesized, and in addition, an unsulfated form of cholecystokinin-pancreozymin (CCK-PZ) was prepared to cast some light on the structure-activity relationships of these gastrointestinal hormones. In a series of motilin peptides, elimination of the pentapeptide from the amino terminus decreased the activity (in vitro contraction of rabbit duodenal muscle) to 1/250 of the whole molecule, and subsequent removal of the tripeptide Thr-Tyr-Gly resulted in the complete loss of its effects, In a series of gastric inhibitory peptides, two fragments corresponding to positions 1 to 28 and 26 to 43 were both inactive. However, the nonacosapeptide (15 to 43) retained one-fourth of the activity of the tritetracontapeptide (suppression of the gastric acid secretion stimulated by tetragastrin in Heidenhain pouch dogs). Synthetic [27-Tyr]CCK-PZ exhibited 1/250 of the activity of natural CCK-PZ (amylase release from rat pancreas). This compound was smoothly and efficiently labeled with 125I (specific activity 200 to 250 muc per mug).


Role of DNA sequences in genetic recombination in the iso-1-cytochrome c gene of yeast. II. Comparison of mutants altered at the same and nearby base pairs. X-ray-induced mitotic recombination rates and spontaneous meiotic recombination rates have been determined in two-point crosses of various defined cyc1 mutants of the yeast Saccharomyces cerevisiae. All but one of the 17 cyc1 mutants chosen for this study contained either the addition, deletion or substitution of single base-pairs located within a defined segment of the gene that corresponds to the 11 amino acid residues at the amino terminus of iso-1-cytochrome c; approximately half of these mutants had alterations of the AUG initiation codon, some at the same base pair. Up to 66-fold differences in X-ray-induced recombination rates were observed when the same cyc1 mutant was crossed to cyc1 mutants having different alterations in the AUG initiation codon; over a ten-fold difference was observed in series of homologous crosses involving mutants with different changes at the same base-pair. Recombination rates that were associated with specific cyc1 mutants co-segregated with the particular alleles following meiosis, and comparable recombination patterns were also observed for independently isolated, identical mutations. With the mutants used in this study, the frequencies of meiotic recombination did not differ as markedly, suggesting a dissimilar dependence on specific DNA sequences for these two modes of recombination. These disproportionalities of recombination rates suggest that the nature of the mismatched bases influences the recombination process, but not in a way that can be simply interpreted.


Studies on viremia and antibody formation in ducklings and goslings after experimental infection with Lednice (Yaba 1) virus. A very low virus level was found for a short time in the blood of ducklings and goslings after experimental Lednice (Yaba 1) virus infection by subcutaneous (scut) and intranasal (inas) route and in blood of ducklings also after infection by intracerebral (icer) route. Subsequently virus neutralizing antibody formation was examined. Antibody formation was found in most of animals. In goslings onset of antibody formation seems to be slower than in ducklings after the same virus infection dose; vice versa, in ducklings antibody titer decreased after 6 weeks. Using low infection virus dose, neutralizing antibody level in ducklings was lower than in the former case. On examining the blood and the organs, the virus was detected in a very low amount and for a very short time in blood and spleen of goslings and only in blood of ducklings.


Activities of mitochondrial enzymes during aerobic synchronous growth of aerobically and anaerobically grown Saccharomyces cerevisiae. The mitochondrial enzymes cytochrome-c:O2-oxidoreductase (E.C.1.9.3.1), NADH:cytochrome-c-oxidoreductase (E.C.1.6.2.1), NADH: ferricyanide oxidoreductase (E.C.1.6.2.99), L-malate hydrolase (E.C.4.2.1.2) and L-malate:NADH-oxidoreductase (E.C.1.1.3.7) increase their activities during the aerobic synchronous growth of aerobically grown Saccharomyces cerevisiae in discrete steps and only once during the cell cycle. An identical phenomenon was observed during the aerobic synchronous growth of anaerobically grown yeast. The mechanism of completization of mitochondrial membranes is thus likely to be discontinuous and the same during both mitochondrial multiplication and the conversion of promitochondria to fully functioning mitochondria.


Cardiolipin synthesis during the cell cycle of the yeast Saccharomyces cervisiae. Cardiolipin synthesis was studied during the aerobic synchronous growth of aerobically grown yeast Saccharomyces cerevisiae. The time course of the synthesis was stepwise and the rise in cardiolipin level in cells coincided in time with the increase in cytochrome oxidase activity. This finding supports the notion of discontinuous completion of the inner mitochondrial membrane and hints at a close relation between cardiolipin and cytochrome oxidase activity.


Effect of iron-beryllium antagonism on the growth of Pseudomonas fluorescens type S. Studies of the growth of Pseudomonas fluorescens type S in acidified peptone nutrient broth supplemented with potassium dioxalatoberyllate show that the inhibitory action of beryllium on the lag phase can be strongly counteracted by an increase in the iron content of the medium. In terms of relative concentrations, Fe3+ is up to 250 times more effective than Mg2+ as an antagonist of beryllium under the conditions employed. Conversely, evidence of the effect of beryllium on iron-dependent constituents of the cell is provided by the fact that cultures of Pseudomonas fluorescens adapted to relatively high concentrations of beryllium show a marked decrease in cytochrome c content.


Dependence of yeast sporulation on mitochondrial protein synthesis. Sporulation in diploid Saccharomyces cerevisiae is not dependent on continued protein synthesis in the mitochondria. Using chloramphenicol, it is shown that proteins essential for respiration and sporulation are synthesized in mitochondria early during growth in a presporulation medium.


Eye movements during transcendental meditation. Characteristic changes in eye movements occurred during meditation with closed eyes in a proportion of subjects experienced in TM. The most common changes were an increase in slow, large-amplitude, 'rolling' eye movements and a concomitant decrease in rapid, low-amplitude, 'jerky' eye movements. Much variation occurred between individuals, however, some subjects showing no differences between TM and non-meditation. Any changes that occurred were not necessarily constant for the whole recording period nor consistent between sessions. The physiological effects of TM are far more variable than previously publicized. In general, the main changes in eye movements during TM are similar to those during passive hypnosis.


[To the differential diagnosis of bronchiolo-alveolar-cell carcinoma in early state (author's transl)]. Prompt surgical treatment of the solitary form of bronchiolo-alveolar-cell carcinoma offers a good survival rate in comparison with bronchogenic carcinoma; the prognosis of the diffuse form is poor. Review of 28 cases of solitary BAC showed an airbronchogram in 36% and a tail sign in 85% of cases; both features were found in 32% of tumors. The air bronchogram seems to be specific for this type of tumours, the tail sign is found with granulomatous or inflammatory diseases as well. In the differential diagnosis of a peripheral coin lesion with air bronchogramm and tail sign, without calcifications and atelectasis, a BAC should be taken into serious consideration.


[Determination of autohemolysin formation using the plaque test. Demonstration of autoimmune phenomena following vaccinations and infections]. A simple and sensitive modification of the plaque assay for the evaluation of autohaemolysin-producing cells (APC) in the peripheral blood is described: Very small chambers, (volume less than or equal to 0.03 cc) made out of glass slides (75 X 25 mm), are filled with a diluted (1:7) blood cell suspension. After incubation for 26 hours the haemolytic plaques produced by APC in the thin monolayer of blood cells can be counted under the microcope at a magnification up to 320-fold. The number of APC in guinea pigs immunized with cholera vaccine and in monkeys infected with variola-virus was evaluated by means of this method. Cholera vaccination significantly raised the APC up to 7 days after immunization. In monkeys these cells increased markedly between 4 and 7 days after infection with variola-virus. The increase was less pronounced in animals partially protected by prior vaccination with attenuated vaccinia-virus (MVA-strain) and showing a mild course of variola. Autoimmune mechanisms may exist in apparently normal organisms. They may rise after tissue destruction or cell alteration by infectious or other exogenous agents. The method described might be useful to determine the degree of reactogenicity of bacterial and viral vaccines.


[Ovarian metastases in breast carcinoma]. The effect on prognosis of the presence of ovarian metastases was studied in patients oophorectomized in treatment of progressive breast cancer. At laparotomy, 28% (n = 42) were found to have ovarian metastases. This high percentage indicates that cases studied were in a relatively progressed state. In the cases with ovarian metastases, the clinical course of the disease was no different to those histologically free from metastases. It appears therefore that the presence of secondary ovarian metastases at oophorectomy is of no therapeutic or prognostic significance. The time interval between oophorectomy and mastectomy, however, apparently does have a bearing on the progress of the disease.


[Cyclic adenosine monophosphate, prostaglandin E and aggregation of blood platelets in man]. The relationship of cyclic AMP to platelet function has been examined directly by assay of the platelet content of the nucleotide and platelet aggregation. Platelet aggregation is favored by an decrease in platelet cyclic AMP and inhibited by an increase in cyclic AMP. The platelet aggregation is accompanied by reduction in cyclic AMP. beta adrenergic substances increase platelet cyclic AMP in vivo and simultaneously block platelet aggregation. Thrombin selectively causes human platelets to form and release PGE. The amount formed was proportional to the amount of thrombin added and high enough to influence platelet aggregation in vitro. Platelet prostaglandin formation by washed platelets and platelet-rich plasma was not proportional to the degree of platelet aggregation induced by stirring. We were unable to detect PGE-formation in response to ADP. Thrombin-induced synthesis of PGE is inhibited by ADP. Aspirin selectively inhibits PGE-production in human platelets. This reaction does not depend on the extend of platelet aggregation. These studies suggest that cyclic AMP is the key to regulation of platelet aggregation and that PGE, formed by platelets, through its effects on cyclic AMP may play a role in regulating platelet aggregation.


Use of highly specific antibodies against 17alpha-OH-progesterone in a simplified nonchromatographic RIA and in the simultaneous determination of four sex hormones in human plasma. A highly specific antiserum raised against the 3(O-carboxymethyl)oxime of 17alpha-hydroxyprogesterone (17-OHP) was produced in rabbit and used in the development of a specific radioimmunoassay (RIA) for 17-OHP which included a celite chromatography. Methods allowing the measurement of plasma 17-OHP levels either separately or in combination with that of several plasma androgens (testosterone, delta4-androstenedione and dehydroepiandrosterone) are described. Moreover, RIA meabsurement of plasma 17-OHP levels with or without chromatographic (celite column) purification gave comparable results (mean +/- SD) in 29 normal adult males (118 +/- 34 ng/100 ml) and 35 normal adult females (follicular phase: 46+/- 16 ng/100 ml); luteal phase: 241 +/- 71 ng/100 ml).


Protein synthesis in postnuclear supernatants from mengovirus-infected Ehrlich ascites tumor cells. The effect of mengovirus infection on the protein synthetic capacity of Ehrlich ascites tumor cells cultured in vitro was studied in vivo and in vitro employing postnuclear supernatants prepared at various times post-infection in the absence and in the presence of 1% Triton X-100. The amino acid incorporating activities of extracts obtained in the presence of the detergent were reduced by about 30% compared with the capacities of the corresponding postnuclear supernatants prepared in the absence of Triton X-100; but the course of the activity vs. time curve was not influenced by the detergent. Under the conditions employed, the postnuclear supernatants were unable to reinitiate protein synthesis once elongation of nascent polypeptide chains concomitant with ribosome runoff was completed. After mengovirus infection, a gradual disappearance of polysomes from postnuclear supernatants and a simultaneous accumulation of monosomes was observed. The protein-synthesizing activities of normal and infected cells were inversely proportional to the monosome concentrations of their corresponding extracts. Qualitatively, protein synthesis in intact cells and in postnuclear supernatants responded similarly to mengovirus infection. In both cases an initial reduction of host-specific amino acid incorporation was followed by a burst of viral protein synthesis. However, the two activity vs. time curves showed the following significant differences: 1) The activities of extracts from control cells and from mengovirus-infected cells nearly in the infectious cycle were low compared with the activities observed in vivo. 2) In the middle of the infectious cycle, the peak of viral protein synthesis occurred later and the activity was higher in vitro. 3) Finally, in the late period of the infectious cycle the postnuclear supernatants had considerable protein synthesizing activity, at a time when protein synthesis in vivo was nil.


Lipid-protein interactions between human apolipoprotein A-I and defined sphingomyelin species. A 13C-NMR spectroscopic study. Chromatographyically and immunologically homogeneous apolipoprotein A-I (apoLp A-I) from human serum has been recombined in separate experiments with three species of sphingomyelin. The differed in the degree of saturation of their fatty acyl residues, stearoyl (18:0), oleoyl (18:1) and linoleoyl (18:2). The lipoprotein complexes formed were purified by CsCl density gradient centrifugation between 1.07 - 1.09 g/cm3 and by gel filtration. Stearoylsphingomyelin does not recombine with the apoprotein A-I below its phase transition temperature (tc = 41.5 degrees C). The lipoproteins eluted with the following apparent molecular weights: 18:0-sphingomyelin apoLp A-I, 8.0 X 10(5); 18:1-sphingomyelin apoLp A-I, 4.0 X 10(5); and 18:2-sphingomyelin apoLp A-I, 4.0 X 10(5). In electron microscopy the particles appear as discs of 160 - 170 A diameter and 50 - 60 A thickness. Their tendency to form stacked aggregates of discs decreases with the degree of their unsaturation. CD measurements underline the considerable increase in alpha-helicity of the secondary structure of apo A-I after recombination with the phospholipids. This increase in order is equal for the three sphingomyelin species (alpha-helicity of apoLP A-I = 0.46, after recombination 0.89). If the three sphingomyelin species are used in equal molar amounts in the recombination experiment, no preference for any one sphingomyelin species is observed. Recombination of apoLp A-I with sphingomyelin, labelled with the isotope 13C in the choline group, C-14 of stearic or linoleic, or C-11 of oleic acid, were performed for spin lattice relaxation time (T1) experiments. Compared with sphingomyelin liposomes, the polar head groups of these lipids in the lipoprotein particles possess a considerably higher mobility, whereas the changes in T-1-times of the C-atoms in the centre of the fatty acid chains of the lipids refer to their interactions with the polypeptide side chains. A model of the lipoprotein complexes formed is proposed on the basis of the experimental data.