Calcium ion uptake in isolated pancreas cells induced by secretagogues. 1. Secretagogues of pancreatic enzyme secretion: pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, caerulein and the Ca2+ ionophore A 23187 stimulate 45Ca uptake into isolated rat pancreatic cells, whereas adrenaline, isoproterenol, secretin, dibutyrylic cyclic adenosine 3',5'-monophosphate and dibutyrylic cyclic guanosine 3',5'-monophosphate have no effect on 45Ca uptake. 2. A graphical analysis of the Ca2+ uptake curves reveals at least two phases: a fast phase, probably due to binding of Ca2+ to the membrane and a slow phase representing Ca2+ transport into cells. Both phases are stimulated by pancreozymin and carbamylcholine. 3. The 45Ca-exchangeable pool size is increased by both carbamylcholine and pancreozymin, whereas a significant increase of total content of cell calcium was too small to be detected. 4. Atropine blocks the stimulatory effect of carbamylcholine completely but not that of pancreozymin. The Ca2+ antagonist D600 blocks the stimulatory effects of both carbamylcholine and pancreozymin only partially. 5. The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca2+ transfer into the cell most probably through an increase of the cell membrane permeability for Ca2+.


Involvement of sulfhydryl groups in the action of the insulin and radiation on thymocyte Na+-dependent amino acid transport. 1,p-Chloromercuribenzene sulfonate concentrations less than 10(-5) M stimulate the uptake by thymocytes of 2-aminoisobutyrate, a non-metabolized amino acid. At concentrations greater than 10(-5) M of this reagent, transport is impaired and cell viability is effected. In contrast, 5,5'-dithiobis-(2-nitrobenzoate) between 10(-4) and 10(-6) M produces only stimulation of 2-aminoisobutyrate uptake after treating for 10 min. 2. Treatment of thymocytes with 10(-4)M 5,5'-dithiobis-(2-nitrobenzoate) reveals at least three categories of reactive SH groups. Titration of the most rapidly reacting category, 4 - 10(7)-7 - 10(7)/cell, activates 2-aminoisobutyrate transport to the same extent as does p-chloromercuribenzene sulfonate. Cells treated with 10(-6) M insulin showed a 30-50% reduction in the number of sulfhydryl groups that could be titrated with 5,5'-dithiobis-(2-nitrobenzoate). In thymocytes treated with 10(-6) M p-chloro(203Hg)mercuribenzene sulfonate, addition of 10(-6) or 10(-9) M insulin before treatment with the sulhydryl reagent again reduces the number of titrable SH groups by 20%. 3. Insulin (10(-10)-10(-6) M) also stimulates 2-aminoisobutyrate uptake, but the effects of insulin and SH blocker are not additive. 4. Insulin, but not p-chloromercuribenzene sulfonate, prevents the impairment of 2-aminoisobutyrate transport caused by gamma-irradiation. Treatment of cells with p-chloromercuribenzene sulfonate prior to irradiation increases the radiation impairment of 2-aminoisobutyrate transport. 5. gamma-irradiation reduces the number of 5,5'-dithiobis-(2-nitrobenzoate) reactive sulfhydryl residues by 37%. 6. A model for the action of insulin and irradiation on 2-aminoisobutyrate transport is presented.


[Structural transitions in DNA, isolated from normal and neoplastic cells]. A decrease of temperature transition in spin-labeled DNA isolated from leucose cells compared with normal cells is found. It is shown by kinetic formaldehyde method that it may be the result of many defects in the secondary structure of macromolecule in DNA1. The spin label method is shown to be used for the analysis of DNA macromolecule damaged with small irradiation doses. Various radiosensitivity of DNA isolated from the blood of normal and leucose cells is observed.


[EPR- and IR-spectroscopic study of the processes of carbonization of amino acid crystals]. Initial stages of "low temperature" carbonization in crystal amino acids have been studied by ESR- and IR-spectroscopy. It was revealed that block polycondensation of amino acids with the formation of polypeptide chains took place. In the air oxygen also surface carbonization of crystals took place; it became stronger with the occurrence of radiation defects.


[Electron paramagnetic resonance study of photosensitized transport and localization of electrons in enzyme-substrate complexes. Lysozyme and its inhibitor]. Photosensitized electron transfer are studied in three systems: lysozyme, its inhibitors (oligosaccharides) and their enzyme-inhibitor complexes. Electron donors were either tryptophane amino acid residues of lysozyme or tryptophane. It is shown that N-acetyl group of inhibitor molecules is a single electron--acceptor group in the inhibitor molecule as well as in the lysozyme-inhibitor complex. It is stated that the localization of unpaired electrons in the mixtures of lysozyme modification products and substrate--inhibitors depends on the state of enzyme molecule.


[Mechanism of the formation of paramagnetic centers in proteins exposed to UV-radiation]. It is shown that the process of formation of paramagnetic centres in UV-irradiated at 77degreesK protein solutions in a biquantum one. The protein molecule in triplet state is an intermediate product.


[Accumulation of paramagnetic centers in protein solutions exposed to UV-radiation]. It is shown that concentration of paramagnetic centres (PC) in UV-irradiated protein solutions at 77degreesK approximates the limiting value. The limiting number of PC (n) per one molecule is in direct proportion to that of aromatic amino acid residues in it n(sigma)=2+0,1 sigma. The formation of PC slopps because all the energy absorbed by aromatic amino acid residues is transfered to the radicals formed.


[Spin label study of microviscosity and structural transitions in cell membrane lipids and proteins. I. Heat-induced structural changes in cell nuclei of mouse organs in normal conditions]. With the help of weakly bound spin probes the effect of temperature on structural transitions of protein and lipid components in the nuclei membranes of different organs of intact mice has been studied. Microviscosity of lipids in the membrane is found to differ sharply from proteins. Found by both probes breaks on Arrhenius curve of the correlation time dependence on reverse temperature within the temperature range of 25degreesC--27degreesC and 40degreesC-43degreesC seem to be connected with structural reconstructions of the whole membrane. The results obtained according to the behaviour of probes are identical for the nuclei of different organs.


[Properties of the thalamocortical nonspecific system]. A study has been carried out on how parameters of stimulation of thalamo-cortical non-specific system in the rabbit brain influence the amplitude and temporal characteristics of a recruiting response.


[Rate of interaction of ferricytochrome c with negatively charged liposomes from natural lecithin: effect of the physicochemical state of the membrane hydrophobic layer]. The absorption velocity of ferricytochrome c on the surface of liposomes from egg lecithin containing 10% of lauric acid was studied. Liposomes were prepared from lecithin of three fractions which differed by the composition of fatty acids, unsaturation and the lipid interaction decreased at the temperature below T phi pi for lecithin fractions containing larger quantity of saturated fatty acids. An opposite tendency was observed for the temperature above T phi pi. In the phase transition region  of lecithin of refractory fraction the local maximum of protein-lipid interaction was observed. Judging by the character of the changes of the values of energy activation, small additions of cholesterol in the membrane loosen the bilayer at the temperature below T phi pi and condense it at above T phi pi. The data obtained are discussed in terms of the effect of the state of molecule hydrophobic part on the velocity of protein-lipid interaction.


[EPR study of electron transport in higher plant photosynthetic systems. II. Effect of temperature on the kinetics of photoinduced oxidation-reduction transformations of P700]. A sharp fall of ESR signal I is observed at temperature decrease while switching on light of 600 nm on the background of light of 707 nm; kinetics of the initiation of the signal under the illumination of light of 707 nm gets more complicated; the induction phase induced by previous illumination with the light of 600 nm increases.


[EPR spectra of animal tissues in vitro]. The ESR spectra of animal tissues have been recorded at 13-43 degrees K. The reduction of the ESR signal intensities of the Fe-S proteins have been found in starving mice which is due to the substrate diminishing in these animal tissues.


[EPR study of electron transport in higher plant photosynthetic systems. I. Effect of illumination prehistory on the kinetics of photoinduced oxidation-reduction transformations of P700]. After the adaptation of leaves to darkness (2--4 min) the kinetics of the initiation of ESR I signal aquires a multiphase character: after an initial leap the stage of slower growth sets in. The "memory" of the system about the effect of light of 600 nm, which was realized as a delay of the growth of the ESR I signal under the effect of light of 707 nm, was preserved in the darkness for several minutes. If the light of 600 nm is switched on at the background of the light of 707 nm not earlier than 2-4 min after its switching off in the preceding cycle, unmonotonous transitional process with an overleap is observed.


[Incorporation of low molecular SH-containing compounds in nitrosyl complexes of nonheme iron in cell-free and cell preparations]. It has been shown that the endogenic low molecular SH-containing compounds in non-cellular liver and yeast preparates include in nitrosyl non-haem iron complexes at basic pH and in result of addition of iron salts into these preparates. The content of nitrosyl non-haem complexes with SH-containing low molecular compounds is not more than 0,5-1% of the quantity of identical non-haem iron complexes with RS-groups of proteins. The low content of these complexes appear to be due to as the low content of low molecular compounds with ionized RS-groups as the competitive action and RS-groups of proteins for iron and NO.


Purification and some properties of rabbit skeletal muscle fructose 6-phosphate kinase inhibitor. The loss of activity of rabbit skeletal muscle FPK on storage and its restoration by ATP, AMP and cyclic AMP has prompted us to look for an inhibitory unit of the enzyme. We have purified this inhibitory factor from the crude muscle extract and isolated from crystalline FPK; both proteins have the same Mw of about 68,000 (SDS). Carboxymethylation revealed species of lower molecular weight. It is suggested that two different kinds of FPK exist, one composed only of "active" subunits and another composed only of "inactive" (inhibitor) subunits. States of intermediate activity exist, created by dissociation, reassociation and exchange of subunits, because the inhibitor and FPK share several subunits. A model is proposed where one or several inhibitors of molecular mass 68,000 replace the corresponding number of active subunits of 93,000 daltons, the structure of the native molecule remaining tetrameric. It is shown that cyclic AMP exerts its activation function on FPK only in the presence of the inhibitory protein, probably by displacing the exchange of the subunit in favor of the active tetrameric species of 360,000. Ammonium chloride plays probably an opposite role in this exchange. The inhibitor coverts the Michaelian behavior with respect to F-6-P into a cooperative response (sigmoidal shape of the curve) characterized by a Hill coefficient of 2. The Michaelian response with respect to ATP is preserved, the corresponding constant being only slightly affected. In the presence of subsaturating concentration of inhibitor, mixed species are detected. As a first approximation one can propose that a reversible equilibrium exists between free and complex FPK subunits. The dissociation constant of this equilibrium being equal to 4 X 10(-8) M in moles of FPK protomers.


Effects of catecholamines on rat myocardial metabolism. I. Influence of catecholamines on energy-rich nucleotides and phosphorylated fraction contents. 1. The influence of catecholamines (adrenaline and noradrenaline) on energy metabolism of the rat myocardium has been studied by incubating slices of this tissue with these hormones and by following the levels of the different phosphorylated fractions and adenylic nucleotides. 2. Similar effects are obtained with both hormones, adrenaline being more effective. 3. Catecholamines decrease significantly the total amount of phosphate while Pi content increases during the first 10 minutes of incubation; labile and residual phosphate contents increase at the beginning of incubation and decrease to the initial values afterwards. 4. ATP and ADP levels decrease significantly with both hormones; however, the effect of noradrenalin on the ATP level needs a longer time of incubation. The ATP/ADP ratios decrease after 5 minutes incubation and the total adenylic nucleotide content is severely decreased (35 per cent with adrenalin, after 20 minutes incubation). 5. Similar results have been obtained with other tissues; these results can explain the decrease of aerobic metabolism we observed under the same conditions.


[Spin-labeled nucleosides, nucleoside-5'-mono-, di-, and triphosphates]. A general method of synthesis of spin-labeled nucleosides, nucleoside-5'-mono-, di- and triphosphates is developed. It is based on the acyllation of ribose OH-group with N-(2,2,5,5-tetramethyl-3-carbonylpyrollin-1-oxyl)-imidasole. Their ESR and UV-spectra are studied. While studying the products of interaction between nucleoside mono-, di- and triphosphates and N-(2,2,5,5-tetramethyl-3-carbonylpyrrolin-1-oxyl)-imidasole along with the formation of monoradicals due to the acyllation of 2'(3')-OH-groups of ribose in the case of AMP, GMP and GDP acyllation by the phosphate group with the formation of biradicals has been also observed.


Effects of catecholamines on rat myocardial metabolism. II. Influence of catecholamines on 32p-incorporation into rat myocardial adenylic nucleotides and their turn-over. 1. The influence of catecholamines (adrenaline and noradrenaline) on 32Pi incorporation into intracellular phosphate and adenylic nucleotides has been studied on rat myocardium slices; consequently, the turn-over of nucleotides could be determined and compared under the influence of these two hormones. 2. In order to specify the site of action of these catecholamines, several inhibitors and activators of energetic metabolism were included in the incubation medium: 3'5'-AMP, caffein, ouabain, oligomycin, rotenone + antimycin. 3. Both catecholamines favour Pi exchanges between intra and extracellular spaces; ATP turn-over is greatly increased, while ADP turn-over is slightly decreased, and 32P-incorporation into ADP is increased. 4. 3'5'-AMP and caffein are without effect on Pi penetration; however, caffein increases catecholamine effects on this penetration. ATP turn-over is slightly increased by 3'5'-AMP or caffein. 5. Ouabain decreases ATP turn-over but does not prevent the adrenaline induced acceleration. Inhibitors of oxidative phosphorylation and electron transport decrease ATP-turn-over severely; this inhibition is not released by catecholamines. 6. It is concluded that the catecholamine effects observed are dependent on the oxidative phosphorylations process. The increase of Pi exchange by catecholamines may be related to the increase of extracellular space and cation translocations we observed with the hormones.


[Need to measure the viscosity of solutions of globular proteins when studying their rotational mobility by the spin probe method]. It is shown that for correct determination of correlation time of protein rotation, at least for high concentration, it is necessary to measure the viscosities of investigated solvents, whereas for definition by method of extrapolation values Azz the viscosity of water solution of sucrose may be taken as the viscosity of investigated solutions.


[Effect of endogenous lipid peroxides on respiration in rat liver mitochondria]. Effect of Fe2+- and ascorbate-induced lipid peroxidation on mitochondrial respiration in 3 and 4 states (according to Chance) is studied. Peroxidation was shown to result in an increase of the oxygen uptake rate in 4th state, and in the KCl-containing medium (but not in sucrose medium) it caused a decrease of the oxygen uptake rate in the phosphorylating state, which partially reversed when cytochrom c was added. These effects were observed only after the development of peroxidation (after "slow" flash of chemiluminescence), and the value of and effect correlated with the content of peroxidation products unsaturated fatty acids. No considerable differences in the damaging effect of peroxidation were observed under incubation of mitochondria in KCl, NaCl and choline containing media.


[Kinetic studies of the formation of abortive ternary complex lactate dehydrogenase (isoenzyme h4)-NAD-pyruvate]. The rate constants and equilibrium constants for four stages of process of the abortive ternary complex lactate dehydrogenase (porcine isoenzyme H4)-NAD-pyruvate formation are determined. These stages are 1) the enolization of pyruvate, 2) the formation of binary complex enzyme-NAD, 3) the formation of "intermediate" ternary complex enzyme-NAD-enol and 4) the transformation of "intermediate" complex into "final" ternary one, accumulation of the latter being followed by spectrophotometric and fluorometric methods. The constants obtained were compared with the corresponding ones for porcine isoenzyme M4 determined in earlier work. It was shown that the greater stability of ternary complex and the greater initial rate of ternary complex formation in the case of H4 is due to greater affinity of isoenzyme H4 with respect to NAD, greater magnitude of reaction rate constant for transformation of "intermediate" ternary complex into "final" one and lesser magnitude of reaction rate constant for the reverse transition.


[Protamine as a specific inhibitor of electron transport between cytochromes c and a on the outer surface of the mitochondrial membrane]. The basic protein protamine is found to be a potent inhibitor of mitochondrial cytochrome c oxidase, while the oxidase activity of the "inside out" submitochondrial particles is only slightly affected by this polycation. The site of inhibition of mitochondrial respiration by protamine is localized between cytochromes c and a, protamine combining with cytochrome oxidase competitively to cytochrome, c, Ki=2,5 x 10(-6) M. The data obtained suggest that it is the outer side of the mitochondrial membrane where oxidation of cytochrome c by cytochrome a occurs.


[On different forms of glyceraldehyde-3-phosphate dehydrogenase]. Evidence is presented for the structural differences between rat and rabbit skeletal muscle glyceraldehyde-3-phosphate dehydrogenases. Glyceral-dehyde-3-phosphate dehydrogenases isolated from frozen and fresh rat skeletal muscle were found to have different temperature stability and chromatographic behavior on CM-cellulose. Different nucleotide content of the preparations (NAD on one hand, and products of its degradation - ADP-ribose, AMP and adenosine, on the other) is shown to account for the observed effects.


[Formation of polysomes in virus-specific RNA in the transcriptive complex of Sendai virus infected cells]. Transcriptive complex in the cytoplasm of Sendai virus infected cells included parental RNA in the form of ribonucleoprotein, nascent RNA and polysomes. Its buoyant density in CsC1 was 1.45 g/ml. The complex dissociated three hours after the infection, and nascent RNA was fully separated from the parental RNP. The template for polysomes within the complex was identified under conditions when the complex was dissociated in cycloheximide-treated cells. Polysomes were revealed in the association with nascent RNA. They sedimented in preribosmal region of sucrose gradient, and had a buoyant density in CsC1 of 1.49 g/ml. Mild treatment with ribonuclease split the polysomes into monoribosomes.


[Biosynthesis of collagen and non-collagen proteins on free and bound polysomes from chick embryo]. Synthesys of collagen and non-collagen proteins was investigated in a cell-free system in the presence of free and bound polysomes isolated from chick embryos. Of total radioactive proteins synthesized on bound and free polysomes the amount of peptides digested by bacterial collagenase comprised 25-40% and 5-7% respectively. These data showed that collagen was predominantly synthesized by bound polysomes. Free polysomes were found to be much more active than bound ones in non-collagen protein synthesis. When bound polysomes detached from membranes by detergent treatment were incubated in a cell-free system, a release of non-collagen proteins into the incubation medium increased sharply, but the release of collagen peptides was as negligible as in the case of untreated polysomes. Kinetic studies of collagen synthetizing activity of polysomes bound to or detached from membranes suggested the role of endoplasmic membranes in stabilizing collagenous polysomes.


[Isolation of highly purified ribonuclease from cobra (Naja oxiana) venom]. Dialysis, gel-chromatography on Sephadex G-75 (superfine) and chromatography on sulphoethylcellulose give high yield (68 per cent) of 162-fold purified ribonuclease from cobra venom. In ion-exchange chromatography, ribonuclease is eluted in two fractions. The fraction with the highest specific activity has a molecular weight of 15900 and is homogeneous in 15 per cent polyacrilamide gel electrophoresis at pH 8.9. Electrophoresis at pH 4.3 reveals a minor fast component of this fraction which also exhibits a ribonuclease activity. Sulphoethylcellulose chromatography fairly separates cobra venom phosphodiesterase and 5'-nucleotidase eluted as a single fraction in gel chromatography.


[Activation of carbonic anhydrase from rat gastric tissue as a result of phosphorylation by 3',5'-AMP-dependent protein kinase]. Two isoenzymes of carbonic anhydrase, one with high activity and the other with low activity, were isolated from rat gastric tissue. It was found that both isoenzymes were phosphorylated in vitro by protein kinase isolated from gastric mucosa and that this process was stimulated by 3',5'-AMP. The phosphorylation of highly active carbonic anhydrase isoenzyme is shown to result in the increase of its activity. The phosphorylation of low active carbonic anhydrase isoenzyme did not affect its activity or decreased it slightly. The results obtained suggest that the activation of carbonic anhydrase in vivo by gastrin (pentagastrin), histamine and 3',5'-AMP is due to the phosphorylation of highly active isoenzyme by 3',5-AMP-dependent protein kinase. It seems possible that in this process histamine and 3',5'-AMP act as sequential mediators of pentagastrin effect.


[Effect of bradkinin-potentiating snake venom peptides and C-terminal pentapeptide fragment of bradkinin on carboxypeptidase N and kiniase activities of human blood serum]. Effects of bradikinin-potentiating peptides (BPP5a and BPPB), which are identical to peptides from Bothrops jararaca and Agkistrodon halys blomhoffii snake venom, and pentapeptide C-terminal bradikinin fragment on carboxypeptidase N and kinase activities in human blood serum are studied. It is found that BPP5a and BPPB at concentrations of 5-10(-4) M and 2.5-10(-4) M respectively do not inhibit the hydrolysis of hippuryl-L-lysine and hippuryl-L-argininic acid by carboxypeptidase N and at a concentration of 2-10(-3) M they do not affect the rate of the release of C-terminal arginine residue from bradikinin. BPP5a and BPPB do not inhibit the kininase activity of diluted human blood serum in vitro. Unlike BPP5a and BPPB, C-terminal pentapeptide bradikinin fragment is a competitive inhibitor of carboxypeptidase N, and it inhibits (at a concentration of 5-10(-4) M) the hydrolysis of hippuryl-L-lysine and hippuryl-L-argininic acid by 50%. It also inhibits the hydrolysis of bradikinin with diluted human blood serum. BPP5a and BPPB have an inconstant constriction effect on isolated rat uterus at concentration exceeding 4-6 orders the concentration of bradikinin. BPP5a at concentrations from 4-10(-9) to 4-10(-8) g/ml increases the sensitivity  of rat uterus in 1.6--2.0 times.


[Isolation of exonuclease A5 preparation free of phosphomonoesterase contamination]. A method is described of the isolation of exonuclease from cultural medium of Actinomyces sp. of coelicolor type, strain 5. The enzyme preparation is practically free from phosphatase activity (the ratio between exonuclease and phosphatase activities is about 10000). Chromatography on DEAE-Sephadex after the heating of the enzyme preparation at 37 degrees C and pH 5.5 in the presence of 2-mercaptoethanol was used to elimate the phosphatase contamination. The preparation of exonuclease A5 is suitable for different structural studies of nucleic acids, it can be used (instead of snake venom PDE) in  a large scale preparation of 5'-mononucleotides from nucleic acids as well.


Granule enzymes of polymorphonuclear neutrophils: A phylogenetic comparison. The functional significance of granule enzymes in polymorphonuclear leukocytes (PMN) is not fully understood because of the multiplicity of the enzymes and the rare occurrence of deficiencies in man. In order to select appropriate laboratory animals for functional studies, a phylogenetic comparison of enzyme levels in animal and human PMN was undertaken. Neutrophils were obtained from a variety of laboratory animals and man; the activities of alkaline phosphatase, lysozyme, myeloperoxidase, and beta-glucuronidase were determined by histochemical and analytical techniques. Marked interspecies differences in enzyme activity were found; many species were deficient in alkaline phosphatase or lysozyme. Differences in pH optima and metal requirements of alkaline phosphatase were not of sufficient magnitude to explain the variations of this enzyme.


Defective polymorphonuclear leukocyte metabolism and function in canine cyclic neutropenia. Humans and grey collie dogs with cyclic neutropenia are known to suffer from an increased rate of bacterial infection. Because of the previously described microanatomic abnormalities of lysosome formation found in the polymorphonuclear leukocytes (PMNs) of dogs with canine cyclic neutropenia, studies of these cells were undertaken. PMNs from grey collie dogs were found to have significant metabolic and functional abnormalities when compared with normal collie PMNs. These included abnormally increased postphagocytic C1-glucose oxidation, decreased iodination of trichloroacetic acid-precipitable protein in the resting and phagocytizing state, decreased levels of intracellular myeloperoxidase,and a bactericidal defect against a variety of bacteria. Phagocytosis was normal. These abnormalities appear to differ from those previously described in the PMNs of patients with chronic granulomatous disease of childhood and the Chediak-Higashi syndrome and more closely resemble those seen in hereditary myeloperoxidase deficiency. Thus, the studies reported here demonstrate defective PMN function in a disease state previously believed to be a model only of periodic hematopoiesis.


Multiple myeloma with intramedullary masses of M-component. The bone marrow of a patient with light chain myeloma and amyloidosis was substantially infiltrated with basophilic globular particles. The globules, which were confined to the bone marrow, ranged in size from tiny intracytoplasmic inclusions to large (100 mu) extracellular particles. Both the intra- and extracellular globules stained with fluorescent antibody directed against the light chain (kappa) produced by the patient's abnormal clone, and not with other fluorescent antiserums. By electron microscopy, even the largest extracytophasmic globules were bounded at least in part by rough membranes, suggesting that their extreme size was the result of cellular synthesis rather than extracellular coalescence.


[Influence of the pituitary-adrenal axis on the brown fat tissue of the rat and golden hamster]. The suppression or reduction of the adrenal secretion in the Rat and in the golden Hamster results in a reduction of the weight of brown adipose tissue and of the volume of intra cytoplasmic vacuoles. When corticotropin is given to normal and hypophysectomized animals, the intra cytoplasmic vacuolar system and the corticosterone concentration of brown fat increase. After sham hypophysectomy and short ether intake, a considerable lipidic depletion and a high level of corticosterone are observed. The authors speculate that the by adrenaline. The morphologic responses are much more intense and rapid lipid content of brown adipose tissue is increased by corticotropin and reduced by adrenaline. The morphologic responses are much more intense and rapid in brown adipose tissue than in white adipose tissue.


The pharmacokinetics of pyridostigmine and 3-hydroxy-N-methylpyridinium in the rat: dose-dependent effects after portal vein administration. 1 The elimination kinectis of [14C]-pyridostigmine iodine and [14-C-methyl]-3-hydroxypyridinium bromide (3-OH NMP) have been studied in the rat. 2 For pyridostigmine, at a given dose level, the fraction of the dose eliminated unchanged was reduced and the metabolite fraction was increased after portal vein administration when compared to jugular vein administration. This indicates that pyridostigmine is subject to metabolism during the first passage through the liver. 3 When doses of pyridostigmine 1.25 mumol/kg and higher were injected via the portal vein, the proportion excreted in urine as unchanged drug remained constant; in contrast, the percentage of the dose eliminated as the metabolite was significantly reduced. This indicates that a dose-dependent process is involved in the urinary excretion of 3-OH NMP. 4 This conclusion was supported by studies involving the portal and systemic venous injection of 3-OH NMP at different dose levels. After 4 h, approximately85% of the lowest dose was eliminated unchanged in ug this period. The proportion of the dose eliminated in urine was not related to the route of administration. 5 After the injection of pyridostigmine into the jugular vein, the initial rate of drug excretion fell rapidly for approximately 10 min; in contrast, after injection into the portal vein, the rate of excretion of the drug rose to a maximum at 30 minutes. This suggests that the hepatoportal system behaves as a distinct region during the distribution of this drug.


In situ intraduct carcinoma of the breast: a long term follow-up study. Twenty-eight patients with in situ intraduct carcinoma of the breast are reviewed. Two patients had bilateral tumours. The numbers of patients treated by wide local excision, simple mastectomy and mastectomy with axillary dissection were approximately equal; none of these patients died of carcinoma. The conclusions regarding prognosis and treatment of in situ intraduct carcinoma of the breast are discussed.


Weight gain and the sleeping electroencephalogram: study of 10 patients with anorexia nervosa. The relation between reduced nutritional intake, with consequent weight loss, and sleep disturbance was studied by comparing certain sleep encephalogram patterns in a group of inpatients with anorexia nervosa before, during, and after a regimen of refeeding with a normal diet to a matched population mean weight. At low body weights patients had less sleep and more restlessness, especially in the last four hours of the night. During refeeding and weight gain slow-wave sleep initially increased and then tended to decrease during the final stage of restoration of weight back to matched population mean levels. With the overall weight gain, however, there was a significant increase in length of sleep and rapid eye movement sleep, the latter increasing especially during the later stages of weight gain. These results reaffirm that insomnia, and especially early morning waking, is associated with low body weight in anorexia nervosa, and their implications are discussed with particular reference to a hypothetical association between various anabolic profiles and the need for differing components of sleep.


Identification of radioactive 17beta-estradiol-17-beta-D-glucuronide and 17alpha-estradiol-17-beta-D-glucuronide in the urine of the domestic fowl after intramuscular injection of [4-14C]estrone. Two previously uncharacterized radioactive estrogen conjugates, 17beta-estradiol-17-beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17beta-D-glucopyranosiduronate) and 17alpha-estradiol-17beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17alpha-yl-beta-D-glucopyranosiduronate), have been identified in small but significant amounts in avian urine and in a ratio of approximately 2:1 after intramuscular injection of [4-14C]estrone.


Cysteine biosynthesis in Salmonella typhimurium: the presence of ATP-sulfurylase and APS-kinase in various cysteine-requiring mutants. Enzymatic tests were performed on a series of cysteine-requiring mutants for the presence of the sulfate activating enzymes. ATP-sulfurylase (sulfate adenylyltransferase EC 2.7.7.4) and APS-kinase (adenylylsulfate kinase EC 2.7.1.25). The enzymatic products adenosine 5'-[35S]sulfatophosphate and adenosine 3'-phosphate 5'-[35S]sulfatophosphate were identified by paper electrophoresis and measured quantitatively without elution from the paper. Cys mutants mapping in cistrons, A, H, I, J, G, and Ea contain both enzymes. Mutation in the D cistron leads to the loss of ATP-sulfurylase. Mutants mapping in the C cistron lack APS-kinase. Ba, Bb, and Bc mutants lack both enzymes. The control of the synthesis of these enzymes by cysteine was examined. Both enzymes are missing when cells are grown on cysteine.


Studies on adenylate cyclase-cyclic AMP system of the myopathic hamster (UM-X7.1) skeletal and cardiac muscles. Cyclic AMP content, adenylate cyclase (EC 4.6.1.1) activity and phosphodiesterase I (EC 3.1.4.1) activity of the hind leg skeletal muscle and cardiac muscle in 60- and 150-day-old normal and myopathic (UM-X7.1) hamsters were examined. In 60-day-old myopathic animals, cardiac cyclic AMP levels were higher and phosphodiesterase I activity was lower, without any changes in the basal adenylate cyclase activity, whereas in 150-day-old myopathic hamsters, cardiac cyclic AMP and basal adenylate cyclase activity were lower, without any changes in the homogenate phosphodiesterase I activity. On the other hand, basal adenylate cyclase and phosphodiesterase I activities in the skeletal muscle homogenate from 60- and 150-day-old myopathic animals were not different from the normal values but the skeletal muscle cyclic AMP levels were significantly less in 60-day-old myopathic hamsters only. The plasma cyclic AMP levels in 60-day-old myopathic hamsters, unlike 150-day-old myopathic animals, were higher than the normal. Although these results reveal differences in myopathic cardiac and skeletal muscles, it is concluded that changes in adenylate cyclase-cyclic AMP system in myopathy are dependent upon the degree of disease.


Evidence for 16alpha-hydroxylation of estrone 3-sulphate by guinea pig liver slices. [6,7-3H,35S]Estrone 3-sulfate (E13S) of 3H/35S = 3.57 was incubated with female guinea pig liver slices. Small amounts of free steroid and estrone-3-glucuronide, each containing 3H, were found. In addition, E13S, 17beta-estradiol 3-sulfate, and a 'disulfate' fraction, with 3H/35S = 4.4, 4.3, and 4.7, respectively, were also isolated from the incubated tissue. The latter fraction was a major metabolite and about 45% of it consisted of 'disulfates' of 16alpha-hydroxyestrone and estriol, thus providing strong evidence for 16alpha-hydroxylation in guinea pig liver slices.


The pathology of invasive breast cancer. A syllabus derived from findings of the National Surgical Adjuvant Breast Project (protocol no. 4). The inter-relationships of 32 pathologic and 7 clinical parameters encountered in the study of 1000 examples of invasive breast carcinoma have been presented. In some instances the biological significance of these associations is at present unclear. In others it is to be noted that there is no information provided as to the rank of their significance. Nevertheless, the associations that were encountered not only help further characterize the various forms of breast cancer but also provide information regarding the possible biological significance of some of their features. Although it is not our intention to minimize the possible significance of the inter-relationships of pathologic parameters, most emphasis in the summarizing statements which follow has been placed upon those correlations which may relate to prognosis. In this regard reference has been made to  short-term treatment failure, vis a vis local recurrence and/or metastases, which may not necessarily accurately reflect patient survival, although generally such a relationship exists. Information in this regard as well as to the rank of the significance of these pathologic features shall be forthcoming when sufficient time has elapsed since the inception of this study to allow for such conclusions, i.e. survival or long-term treatment failure rates. Lastly, it becomes evident that the guidelines followed in the examination of these specimens appear to represent at least the minimum requirements necessary for a meaningful pathologic evaluation of breast carcinoma.


Primary oat cell carcinoma of the larynx: an ultrastructural study. An extrapulmonary oat cell carcinoma arising in the larynx is described. Ultrastructurally the tumor is identical to the oat cell carcinoma of pulmonary origin. The pressure of neurosecretory granules in our case strongly suggests that the tumor is derived from Kulchitsky-like cells, a histogenesis previously proposed for oat cell carcinoma of the lung.


Ultrastructural study of a postinflammatory "tumor" of the lung. A typical postinflammatory "tumor" of the lung was resected from an asymptomatic 22-year-old white woman. Light and electron microscopic studies showed the lesion to be composed of metaplastic pneumonocytes, proliferated stroma, and chronic inflammatory cells. These findings confirm the inflammatory nature of the lesion and serve to distinguish this lesion from the neoplastic pulmonary sclerosing hemangioma.


Cellular immunity in neoplasia. Antigen and mitogen responses in patients with bronchiogenic carcinoma. Cellular immune responses of patients with histologically confirmed lung carcinoma were assessed in vivo using cutaneous response and in vitro with a microlymphocyte blastogenic transformation (LBT) assay. In addition, correlation of the cutaneous response with the migration inhibitory factor (MIF) assay and LBT response was examined. The results indicated that cutaneous responses seen in patients with cancer of the lung were consistently lower than similar responses in normal controls (p less than 0.001). Similarily, the percentage of positive cutaneous responses seen with patients included in this study was lower than the frequencies reported by others. Stimulation of cells from lung cancer patients by PHA-M was also depressed when compared to similar lymphocytic responses in normal volunteers (p less than 0.001). The correlation between cutaneous response to tuberculin and the in vitro assays was high. The few instances of disparity demonstrate the need to utilize more than one assay in evaluating cellular immune functions. These data would support the work of others that indicate a depression of cellular immunity in advanced malignancy.


The urinary excretion of corticosteroid sulfates by cancer patients. Urinary excretions of free cortisol and corticosteroid sulfates were determined in 31 female controls, 77 breast cancer patients, 14 cases of colonic cancer, and 7 patients with bronchial carcinoma. Elevated corticosteroid sulfate excretion was present in 38% of patients with locally recurrent breast cancer and 30% of those with distant metastases, but in only 13% of the "early" breast cancer cases. A similar abnormality was seen in colonic cancer. Urinary free cortisol was usually normal. ACTH stimulation in a normal subject produced marked increases of both urinary free cortisol and corticosteroid sulfates. It is concluded that elevated corticosteroid sulfate excretion in cancer patients arises from an increased cortisol production rate combined with increased sulfurylation of the steroid. In bronchial carcinoma patients, changes similar to those occurring in the ACTH-treated normal subject may have resulted from ectopic ACTH production in the tumor.


Primary malignant fibrous histiocytoma of bone. A review of over 400 primary bone tumors yielded 11 lesions with features allowing classification as malignant fibrous histiocytoma on a purely histological basis. Correlation with roentgenograms and clinical data indicated that these tumors occurred as osteolytic lesions associated with a high incidence of pathologic fractures in the metaphyses of skeletally mature long bones. Nine patients developed pulmonary metastases and 3 developed lymph node metastases within 21 months following diagnosis. The average survival was 12 months in 6 of the 9 patients who had no secondary treatment in their metastases. Treatment of 3 patients with metastatic lesions with chemotherapy or radiotherapy has been clinically encouraging. Some evidence was obtained that either or both the histiocytic and fibroblastic component may metastasize. There is a need for biopsy of metastatic lesions and autopsy followup to more accurately assess the mode of spread and histologic type of the metastases.


Childhood Hodgkin's disease in Israel. A study of 17 cases. A study of 17 children suffering from Hodgkin's disease (H.D.) is presented. The special ethnic distribution with predominance in Arab and Oriental Jewish children is noted. A high incidence of mixed cellularity histology in Arab children is emphasized. A conservative approach to therapy, i.e. extended field regional radiotherapy only, resulted in a long first complete remission period in lymphocyte predominance and nodular sclerosis H.D. Stages IA and IIA. All children in Stage IIIB and IV were treated by six courses of combined chemotherapy, and were maintained either on chlorambucil and vinblastine or on MOPP. Patients with mixed cellularity H.D. Stage IIA and all patients in Stage IIIA were treated by four courses of MOPP after the completion of extended regional radiotherapy. The 5 children who did not receive the schedules treatment died. The remaining 12 are well, with a survival of 8 to 104 months.


Investigations on the turnover of adrenocortical mitochondria. III. Stereologic and autoradiographic observations on the effect of chronic treatment with dexamethasone on the rat Zona fasciculata mitochondria. The effects of chronic administration of dexamethasone (for up to 15 consecutive days) on both the morphology and DNA-synthesis of the mitochondria of the rat adrenal zona fasciculata were investigated by stereologic and autoradiographic techniques. Up to the 3rd day of continuous dexamethasone treatment, the average volume of mitochondria did not change, whereas the number of mitochondria per cell was significantly decreased. From the 3rd to the 15th day of hormonal administration both the volume and number of mitochondria were found to decrease in proportion to the duration of treatment. Autoradiography showed that after the 3rd day of dexamethasone administration there is virtually no incorporation of 3H-thymidine into the mitochondrial compartment. These findings are discussed in the light of evidence indicating that dexamethasone blocks ACTH-release by inhibiting the hypothalamo-hypophyseal axis. The results confirm the view that ACTH controls the maintenance of growth and proliferation of rat adrenocortical mitochondria.


The pituitary gland of the coelacanth fish Latimeria chalumnae Smith: general structure and adenohypophysial cell types. The pituitary gland of Latimeria chalumnae is situated rostroventral to the telencephalon. The hollow pituitary stalk is bent forward and is ventrally connected to a saccus-vasculosus-like organ, rostrally to a neurointermediate lobe. The infundibular lumen protrudes far into the neurohypophysial lobules. The elongated principal part (pars cerebralis) of the pars distalis is partly embedded in a dorsal depression of the pars intermedia and caudally invaded by the neurohypophysis. It may be divided into rostral and proximal pars distalis and includes a ramified hypophysial cleft, which continues rostrally as a duct with adjacent islets of pars distalis tissue (parts of a pars buccalis). The adenohypophysis consists of cell cords and follicles. Eight tinctorial cell types can be distinguished: in the rostral islets: large basophils with acidophil globules, in the rostral pars distalis: small basophils, large basophils with amphiphil characters and erythrosin-, orange G-positive acidophils; in the proximal pars distalis: orange G-positive acidophils and small and large basophils, having similar staining properties; in the pars intermedia: one amphiphil cell type.


Variability of the effects of serum-free medium, dibutyryl-cyclic AMP or theophylline on the morphology of cultured new-born rat astroblasts. The effects of serum deprivation, of dibutyryl-cyclic AMP (dBcAMP) and of theophylline on the morphology of cultured new-born rat astroblasts have been studied using Eagle's basal medium (BME) or Eagle's minimum essential medium (MEM) as culture media. Serum deprivation had no effect on cells cultured in BME, while in MEM, deprivation induced a rapid morphological transformation involving the appearance of multiple processes. This phenomenon was rapidly reversible when serum was again added. In serum-supplemented BME, dB-cAMP (1 mM) and theophylline (1 mM) had no effect. In serum-supplement MEM, theophylline (1 mM) had no effect while dB-cAMP (1 mM) induced a slower and poorly reversible morphological alteration. On the other hand cells in serum-free BME showed multiple processes after addition of dB-cAMP (1 mM) or theophylline (1 mM). This rapid alteration was completely reversed either by removal of dB-cAMP and theophylline or by addition of serum.


Monoamine fluorescence in the median eminence of the Japanese quail, Coturnix coturnix japonica, following medial basal hypothalamic deafferentation. Monoamine fluorescence was examined in the ventral hypothalamus of the Japanese quail, Coturnix coturnix japonica after medial basal hypothalamic deafferentation. In sham-operated control birds, numerous yellow-green fluorescent fibers were observed in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, a number of fluorescent fibers and cell bodies were observed. In birds with deafferented hypothalami, fluorescence disappeared both in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, which was within the area of deafferentation, fluorescence of neuronal perikarya did not change, but fluorescent fibers decreased markedly in number. Disappearance of monoamine fluorescence in the median eminence and the nucleus tuberis is discussed in relation to the tanycyte absorptive function and gonadaly development.


Subcellular compartmentation of free and esterified cholesterol in the interstitial cells of the mouse testis. The subcellular distribution of free and esterified cholesterol in mouse testis and the changes occurring in cholesterol content of whole testes and cell fractions after inhibition of gonadotropins with methallibure (ICI 33, 828) and restimulation with human chorionic gonadotropin (HCG) are reported in the present paper. In subcellular fractions, the bulk of free cholesterol is associated with organelles sedimented in the microsomal fraction while esterified cholesterol is mainly stored in isolated lipid droplets. The latter compartment increases in methallibure-treated mice 2.3 fold and is remarkably depleted after administration of HCG. There is a close parallelism between the changes in esterified cholesterol content and the variation in the numbers of lipid droplets found in electron micrographs of interstitial cells of mice receiving similar treatments. By contrast no significant changes were noticed in either free cholesterol concentrations of the microsomal fractions or in the fine structure of organelles associated with this fraction. The dynamic nature of steroidogenesis in the microsomal fraction requires the existence of a free cholesterol pool with a high turnover rate for use as an intermediate in androgen synthesis. On the other hand, the large content of free cholesterol in microsomes and its stability under different conditions suggest the presence of a cholesterol compartment with a slow turnover, as a constituent of the membranes.


Isolation of the plasma membrane from sea urchin embryos. A method for isolation of sea urchin embryos plasma membranes is described. Purification of the obtained fraction was assayed by several enzymatic markers and electron microscopy. The isolated plasma membranes appear to be pure from contamination of other cell membranes (endoplasmic reticulum and mitochondria), and they can therefore be used for analytical studies on the composition and structure of plasma membrane.


Contrasting effects of hypoglycemia on plasma renin activity and cyclic adenosine 3',5'-monophosphate (cyclic AMP) in low renin and normal renin essential hypertension. Insulin-induced hypoglycemia previously has been shown to provoke a beta-adrenergic response that normally results in an increase in plasma renin activity (PRA). In our study, hypoglycemia induced definite increases in PRA in a group of five patients with normal renin essential hypertension but failed to do so in a group of six patients with low renin essential hypertension. In both groups, plasma cyclic adenosine 3',5'-monophosphate (cyclic AMP; cAMP) increased more than 2-fold during hypoglycemia, but the response in the low renin group was significantly less than that previously observed in normal subjects under the same conditions. Plasma cortisol increased to an equal extent in both groups of hypertensive patients during hypoglycemia. Infusion of the phosphodiesterase inhibitor, theophylline, resulted in definite increases of PRA in patients with normal renin hypertension but not in patients with low renin hypertension. Because changes in the level of plasma cAMP during hypoglycemia have been thought to reflect adrenal catecholamine release, our finding of a blunted increase in plasma cAMP during hypoglycemia in patients with low renin hypertension may suggest that there is a generalized alteration in adrenergic responsiveness in this condition.


Combined para-aminosalicylic acid and dietary therapy in long-term control of hypercholesterolemia and hypertriglyceridemia (Types IIa and IIb hyperlipoproteinemia). The hypolipidemic effect of PAS-C-diet treatment was studied in 63 patients with Types IIa and IIb hyperlipoproteinemia for 6-36 months. Serum lipids and body weights of all patients were stabilized by a low cholesterol-saturated fat-refined carbohydrate diet before the initiation of an eight-week placebo-drug single-blind crossover study. During the placebo period the plasma lipids levels, mean +/- SD: cholesterol 355 +/- 63.5 mg%, triglyceride 141 +/- 68.7 mg%, and LDL-cholesterol 279 +/- 56.8 mg% were lowered to 274 +/- 53.1 mg+, 98 +/- 40.6 mg%, and 209 +/- 52.9 mg%, respectively (P less than 0.001 in each instance), with 7.5-11.0 grams of PAS-C/day given in one to three divided doses. In ten patients who have completed three years of treatment similar results were obtained. They showed no tendency to develop drug tolerance. Eight had watery diarrhea during the initial period which promptly subsided with interruption of drug therapy. Reintroduction of PAS-C in smaller dose (4.5 g/day) with gradual increment to effective dosage level was tolerated by all. No hematologic, hepatic, and ophthalmologic abnormalities were demonstrated by periodic monitoring. The hypoplipidemic effect of the drug was found to be diminished by alcohol and caloric excess.


Serum beta -- lipoproteins: studies in a patient and in guinea pigs after the ingestion of organophosphorus compounds. A patient admitted to this hospital with Parathion poisoning had a marked reduction in serum cholinesterase activity and concentration of low density lipoprotein. The enzyme and the lipoprotein levels returned to normal with clinical recovery. Guinea pigs treated with phospholine iodide showed depressed levels of serum cholinesterase and beta-lipoprotein. It is suggested that the decrease in beta-lipoproteins is due to the decrease in cholinesterase activity.


Enzymatic determinations of cholesterol in high-density-lipoprotein fractions prepared by a precipitation technique. An enzymatic method for cholesterol in serum [Clin. Chem. 20, 470 (1974)] was initially found to be unsatisfactory for measuring cholesterol in high-density-lipoprotein fractions prepared by precipitation with Mn2+. A fine precipitate formed in the cuvette and cholesterol values were falsely increased. We describe a simple, convenient method for circumventing these problems. An ethylenediaminetetraacetate solution is used to reconstitute the enzymatic reagent. Cholesterol values by this procedure correlated with those obtained by the Lipid Research Clinic's procedure for the same lipoprotein fraction preparations (regression slope, .998; Y-intercept, 8.9 mg/liter; correlation coefficient, .984; standard error of the estimate, 16.8 mg/liter). Precision of the assay, including the precipitation step, was calculated. The SDwithin day was 9.7 mg/liter and SDoverall was 23.7 mg/liter. Results for total cholesterol with the modified reagent were linearly related to concentrations exceeding 4 g/liter, thereby permitting determination of high-density-lipoproteins and total cholesterol in a single run.


Inhibitory effect of ascorbic acid (vitamin C) on cortisol secretion following adrenal stimulation in children. Measurements of plasma cortisol values before and at 4 and 6 hours after intramuscular administration of a depot preparation of synthetic b1-24 corticotrophin were carried out in 12 healthy children (group B) before, as well as on the 5th day of continuous ascorbic acid (AA) administration (1 g t.i.d. orally). Comparison of the results in group B with those of 8 healthy children similarly treated with corticotrophin but not given AA (group A) showed that, on the 5th day of AA administration the mean cortisol values after ACTH were significantly lower than the corresponding values in group A (p less than 0.02), or the post-ACTH values in group B observed on the 1st experimental day, i.e., before AA administration (p less than 0.001). On the other hand, AA administration had no significant effect on the fasting plasma cortisol values. These data suggest that AA excess following adrenal stimulation with ACTH exerts an inhibitory effect on cortisol secretion and consequently it may be of no benefit in conditions of stress.


On the metabolism of lipoprotein-X (LP-X). The characteristic low-density lipoprotein of cholestasis (LP-X) earlier described for humans is found with identical properties in dogs and rats after experimental cholestasis. After ligation of the common bile duct, LP-X may be detected in the plasma within the first 20 hours. A period of marked increase in concentration is followed by decreasing plasma concentrations and LP-X becomes undetectable 7-10 days after ligation of the bile duct in rats. High plasma bile salt concentration may alter the structural integrity of LP-X and may in part be responsible for its disappearance after long-lasting and severe biliary obstruction. Plasma decay curves for isolated LP-X injected intravenously into healthy animals revealed a rapid early fall in concentration followed by a gradual decline. The calculated fractional catabolic rate of LP-X was found to be 0.450 +/- 0.069 for dogs and 1.553 +/- 0.096 for rats corresponding to a mean biological half life of 37.7 +/- 6.4 h or 10.7 +/- 0.6 h, respectively. In vitro LP-X degradation occurs in post-heparin plasma, however, it seems to be too early to speculate on the enzyme activity and on the mode of action responsible for this disappearance.


A rapid, enzymatic assay for measurement of inorganic pyrophosphate in biological samples. An enzymatic assay for measurement of inorganic pyrophosphate (PPi) in biological samples is described using the UDPG pyrophosphorylase reaction and coupling it with three other enzymes in a system of phosphorylation and double reduction to form NADPH, which is measured fluorometrically. The method is specific for PPi in the presence of inorganic orthophosphate and can be used on small volumes of deproteinized ultrafiltrates of fluids or cell extracts. The assay was used here to determine the concentration of PPi in human plasma and serum, and the intracellular content of PPi in human skin fibroblasts, chondrocytes, and red blood cells. The mean PPi concentration of plasma was found to be 2.72 +/- 0.14 muM (mumo/l +/- S.E.M.); for serum it was 6.09 +/- 0.36 muM. The range (95% confidence limits) was 2.14-3.30 muM for plasma and 4.62-7.51 muM for serum. The average intracellular PPi content (pmoles PPi per 10(6) cells +/- S.E.M.) was as follows: (a) skin fibroblasts, 332 +/- 66; (b) articular chondrocytes, 655 +/- 46; red blood cells, 1.74 +/- 0.28.


Action of metabolic inhibitors on the release of intracellular enzymes from human and rat lymphocytes and human erythrocytes. A number of inhibitors of glycolysis and uncouplers of oxidative phosphorylation have been shown to increase the leakage of intracellular enzymes from preparations of rat lymphocytes and human lymphocytes and erythrocytes. The effect of each reagent on all three cell preparations is reversed in the presence of ATP in the medium. ADP is somewhat less effective. AMP exerts a slight protective effect on the human cells, but causes an increase in enzyme efflux from the rat cells. This species difference appears to be related to the concentration of adenylate kinase activity in the cells. The results are interpreted as supporting the theory that membrane permeability to enzymes and other intracellular proteins is dependent upon the energy content of the cell.


Two cases of the chromatin positive variety of ovarian dysgenesis (XO/XX mosaicism) associated with hGH deficiency and marginal impairment of other hypothalamic-pituitary functions. Plasma hGH response to provocative tests (insulin-induced hypoglycaemia and arginine infusion) appears normal in Turner's syndrome. Two cases are reported of the chromatin positive variety of ovarian dysgenesis (XO/XX mosaicism) with unusual absence of plasms hGH response to provocative tests (arginine infusion, insulin-induced hypoglycaemia). Study of other pituitary functions supports the view that a hypothalamic-pituitary impairment is present in these cases. In fact, in these cases we observed low values of gonadotrophin excretion and limited responses of plasma ACTH and plasma corticosteroids to provocative tests (insulin-induced hypoglycaemia, metopyrone). Moreover, markedly abnormal plasma TSH response to TRF was observed in Case 2. The results are discussed with reference to the significance of this rare association.


Induction and in Vitro demonstration of cellular immunity to DNA and RNA viruses in guinea-pigs. Guinea-pigs were immunized with different cells infected with vaccinia virus, herpes simplex virus type 1, herpesvirus saimiri, and the virus of vesicular stomatitis. Development of cellular immunity against these viruses was observed with transformation of blood and spleen lymphocytes and with the migration inhibition test using peritoneal exudate cells. Cellular immunity against vaccinia virus was first seen 6 days after the inoculation of cell-bound vaccinia virus by lymphocyte transformation. The avtivation of the vaccinia virus specific cellular immune response could be induced with tissue culturrus. Since infectious virus particles are not synthesized within this time period, it is likely that virus-induced antigens in the cell surface are active in production of cellular immunity. Vaccines from heterologous host cells were more effective inducers of an immune response than syngeneic cell cultures. For in vitro testing of cellular immunity to viruses, viral antigens could be used in both infective and inactivated form. Delayed hypersensitivity to viral antigens was always accompanied by immune reactions to the host cells used for virus propagation.


E and EAC rosetting lymphocytes in patients with carcinoma of bronchus I. Some parameters of the test and of its prognostic significance. Using peripheral blood lymphocytes separated by a Ficoll method and suspended in saline, means of 77-1% (s.d. 5-2) E rosettes (T lymphocytes) and 20-1% (s.d. 6-7) EAC rosettes (B lymphocytes) have been obtained with normal healthy donors. Poorer E-rosette formation resulted from higher centrifugation speeds during the washing of lymphocytes or erythrocytes, insufficient chilling, or rough handling. The presence of 5% albumin in the final mixture stabilized the rosettes and brought a constant subpopulation of B lymphocytes into rosetting. In patients with bronchial carcinoma who, at the time of diagnosis, had E-rosette percentages below 1 s.d. of the mean for normal donors, the length of survival was significantly shorter than in those with normal or high values. The same was true for those in whom null cells were detected. In each case the correlation effect was mainly found in the group of patients with squamous carcinoma.


Adverse reactions to Zn1-24 ACTH therapy associated with specific cellular immunity. Lymphocyte transformation to 1-24ACTH, as assessed by the incorporation of tritiated thymidine, has been demonstrated to be associated with severe adverse reactions occurring in patients receiving a Zn-linked 1-24ACTH preparation (Tetra cosactrin depot, 'Synacthen'). Antibodies measured with an isotope-binding assay occurred commonly in all patients receiving therapy and did not correlate with adverse reactions. Lymphocyte transformation with the 1-24ACTH polypeptide, a part of the naturally occurring ACTH molecule, has not been previously recorded. The significance of antibodies and cell-mediated immunity to this polypeptide hormone is discussed.


The complement system in cryoglobulinaemia. Interaction with immunoglobulins and lipoproteins. Serum from a patient with an IgM-lipoprotein cryoglobulin, both before and after removal of the cryoprecipitate at 0 degrees C, had extremely low levels of whole complement (C), C1, C4 and C2, while amounts of the remaining components were normal or only slightly reduced. The cryopredipitate, when added to fresh normal human serum, reproduced this pattern of C fixation. Separation of the patients's serum at 37 degrees C into its lipoprotein, IgG and IgM fractions revealed that the IgM alone would precipitate at 0 degrees C. This precipitation was unaffected by the patients's IgG, but was markedly enhanced by extremely small amounts of the patient's d less than 1-075 lipoprotein fraction or of homologous very low density lipoprotein (VLDL). Aggregation occurred even at 37 degrees C in the presence of VLDL. Fixation of semi-purified human C1 paralleled these results closely: it occurred with the patient's IgM alone at 0 degrees but not at 37 degrees C, while IgM in the presence of the patient's lipoprotein, or of VLDL from normal serum, fixed C1 strongly at 37 degrees as well as at 0 degrees C. Fab dimers and monomers prepared from the patient's IgM did not aggregate in the cold, even in the presence of lipoprotein, and did not inhibit the aggregation of intact IgM in the presence of VLDL, at any temperature. All three highly purified IgM cryoglobulins, and three of four IgG cryoglobulins, fixed C1 strongly. The IgG preparation which failed to fix C1 was the only one which had lost its cryoprecipitability during purification. Measurement of C3 or whole C levels may be an insensitive method for detecting C fixation in cryoglobulinaemia. It is suggested that analysis for C1, C4 or C2 should be employed instead.


Effect of oral contraceptives on plasma clearance. The effects of chronic steroid contraceptive therapy on drug clearance from plasma were studied by using plasma antipyrine, phenylbutazone, and cholecalciferol half-lives in women. After 3 mo of oral steroid therapy (Norinyl, 2 mg; norethindrone + mestranol), the antipyrine half-life was increased in 3 of 6 subjects, phenylbutazone half-life was not consistently altered, and vitamin D3 half-life was increased in 3 of 4 patients. After 1 to 7 yr of oral steroid theraphy, the antipyrine half-life was longer while taking the contraceptive than when the contraceptive treatment was discontinued in 4 of 6 subjects, whereas that of phenylbutazone was not consistently altered.


Effects of repeated doses of scopolamine on the electroencephalographic stages of sleep in normal volunteers. Single doses of scopolamine markedly alter sleep patterns in man. This study intended to evaluate the persistence of these changes during continued administration. The design consisted of a sequence of habituation, no-medication, saline (control), scopolamine (0.006 mg/kg intramuscularly 3 consecutive nights), and saline. The first dose of scopolamine markedly retarded the onset of stage rapid eye movement (REM) sleep (p less than 0.005) and diminished the total amount of REM sleep during the night (p less than 0.025). A decrease in total number of eye movements (p less than 0.05) and an increase in body movements (p less than 0.025) were also observed. Changes after the second dose of scopolamine were less marked but still significant. The third dose of scopolamine produced less marked changes than the preceding two. When compared with the first scopolamine night, the onset of stage REM was retarded to a lesser extent (p less than 0.05) and the total amount of REM sleep was increased (p less than 0.05). An increase in the duration of the first REM period was also observed. Rebound effects on the appearance of the first REM period (p less than 0.01), number of eye movements (p less than 0.001), total amount of REM sleep (p less than 0.01), and body movements were observed in the last saline night.


Calcium balance in drug-induced osteomalacia: response to vitamin D. The effect of oral vitamin D3 therapy on calcium balance was compared in 18 institutionalized subjects with drug-induced osteomalacia and in 18 similar subjects without osteomalacia. The subjects with osteomalacia were receiving standard doses of phenytoin and phenobarbital. Diagnosis of osteomalacia was based on low serum calcium and phosphorus, elevated alkaline phosphatase, and appropriate roentgenographic bone changes. The study group achieved positive calcium balance at approximately 975 IU of vitamin D3 per day, while the control group achieved positive calcium balance at approximately 380 IU of vitamin D3 per day. The difference is highly significant (p less than 0.001). These data support previous observations that the osteomalacia of patients receiving anticonvulsant drugs is related to the drugs and that these patients require supplemental doses of vitamin D.


Variations in thyroid function and sleep in healthy young men. 1. The total serum thyroxine, tri-iodothyronine resin uptake, total plasma protein concentration and the free thyroxine index (FTI) were determined repeatedly, at 07.15, 13.00 and 22.30 hours over 4 days, in six healthy young men. 2. There was a significant diurnal variation in the total serum thyroxine concentration but this reflected changes in the binding capacity of serum proteins and in the total plasma protein concentration which could be explained by changes of posture. The FTI, and presumably therefore the free thyroxine concentration, varied very little with the time of day. 3. The FTI varied significantly from day to day in three of the six subjects, presumably as a result of changes in thyroxine secretion because the serum binding capacity did not vary. 4. The subjects' sleep at night was assessed by electro-encephalogram. In days when the FTI was highest for a particular subject his sleep was more fragmented by spontaneous awakenings, the amount of rapid-eye-movement sleep was reduced and that of delta-wave sleep was increased, implying that variations in thyroid function over a period of a few days in healthy subjects can be of physiological significance. The cause of these variations is uncertain.


The diagnostic value of serial brain scanning. A survey of the literature pertaining to several serial brain scanning procedures has been presented. These procedures include rapid brain imaging, sequential brain imaging, delayed from imaging, and follow-up brain imaging. Applications of these techniques to specific clinical problems have been stressed and the reported results reviewed. Thus, it has been indicated that rapid brain imaging is most useful in detecting lesions secondary to cerebrovascular disease but may also provide some helpful information pertaining to the differential diagnosis of other C.N.S. lesions demonstrated on subsequent static brain scans. Sequential brain imaging is a time-consuming adjunctive procedure which, however, can be extraordinarily helpful in a highly selected group of problem cases which present with relatively small lesions adjacent to normal anatomic structures which themselves have considerable radioactivity. Delayed brain imaging has the distinction of detecting the greatest number of intracranial lesions but is attended by tactical problems in maintaining an optimal patient flow through the department and also has the undesirable consequence of reduced information density and diminished image quality, unless greater radiation doses are injected. Follow-up brain imaging is useful in the differential diagnosis of cerebrovascular and neoplastic disease and in the assessment of effectiveness of radiation therapy.


Radiation-enhanced murine sarcoma virus genome rescue activity. Although the doses of X-ray (312-2,500 R) used for irradiation of cells caused impairment of DNA synthesis and cell replication, co-cultivation of X-irradiated MuLV- carrier cells with un-irradiated nonproducer cells of MSV-induced tumour resulted in as much as 20-fold increase in MSV retrieval compared with the un-irradiated control. The enhancement was apparent also as a 3-fold increase in the number of cells producing MSV (infectious centers) in the co-cultivation plate. This suggested that the MSV genome rescue efficiency in terms of MSV per cell, as well as the number of cells producing MSV, increased markedly. By uridine-3H-labeling and focus assay experiments, evidence was presented which suggested that an increase in MSV/MuLV ratio in the culture fluid of co-cultivation plates was obtained when the MuLV-carrier cells were pre-irradiated. By contrast, X-irradiation of the nonproducer cells prior to co-cultivation caused only reductions of MSV genome rescue efficiency. However, use of X-irradiated MuLV-carrier cells for co-cultivation with X-irradiated nonproducer cells restored this efficiency to some extent. The dose-survival curve of the nonproducer cells was not much different from those of the MuLV-carrier cells after X-irradiation. It was suggested that the viability of nonproducer cells was required for replication of MuLV transferred from the carrier cells and for subsequent MSV genome rescue.


Anti-Epstein-Barr virus antibody in patients with cancer of various sites and control groups. Sera collected from patients with nasopharyngeal carcinoma (NPC, 321 cases), cancer of the other sites (297 cases), diseases of the ear, nose and throat (64 cases) and neighborhood controls matched for age and sex (817 cases) were titrated for antibodies to Epstein-Barr virus (EBV) by the indirect immunofluorescence antibody technique. High anti-EBV antibody titers (greater than or equal to 1:640) were noted in 55% in patients with NPC but only less than 31% were noted for the other 3 groups. The differences in the distributions of anti-EBV antibodies are statistically significant. The geometric mean levels of the antibody titers were 1:342 for the patients with NPC but less than 1:178 for the other 3 control groups. The relative risks shows that more than 40 times higher risk for those with equal to or higher than 1:640 antibody titers than those with lower than 1:40. The patients with NPC also show for higher ridit scales than the other 3 groups. The etiology of NPC was discussed with our findings.


[Peculiarities of primary hyperparathyroidism in childhood]. Following introductory remarks on the connected literature, a report is given on personal experience in the diagnosis and treatment of primary hyperparathyroidism (p.hpt) in a 14 years old girl. Clinical symptomatology and predominant form of organic manifestation as well as special biochemical diagnostic procedures of p.hpt in childhood are discussed. The necessity of longterm postoperative controls until complete regeneration of skeletal lesions and of adequate therapeutic Ca++-substitution is stressed.


[Selective ACTH deficiency in two siblings (author's transl)]. In a previous report (Dtsch. med. Wschr. 97 [1972], 1943) the case of a 5-year-old boy had been described who, at autopsy, was found to have severe adrenocortical hypoplasia and absence of ACTH-producing R-cells of the anterior pituitary. On examining other members of his family, his 3 1/2-year old brother was found to have selective ACTH deficiency with secondary adrenocortical insufficiency. This is the first published report of two siblings with selective ACTH deficiency, presumably on a hereditary basis.


[Etiology and prevention of recurring aphthous stomatitis]. New results concerning the etiology of recurrent aphthae are reported. According to own experiments of the authors, adenoviruses persisting in the epithelial cells of the oral mucosa may sensitize the aphthous patient. Adenovirus antigen was demonstrable in the oral mucosa cells in more than 50% of the patients and in the lymphocytes in approx. 1% of the patients. Lymphoblast transformation in the circulating lymphocytes could be demonstrated in vitro with heat-inactivated type 1 adenovirus, in 75% of the patients. This virus type has been isolated from 2 patients. Köbner's isomorphous phenomenon could be demonstrated in aphthous patients. The use of a disinfectant for mouth rinsing, the removal of foci as well as vitamin treatment are recommended for preventing recurrent aphthae.


[Extension implantations as an aid in defect-restoring prosthodontics]. Because of a lack of denture retention, great difficulties are often encountered in edentulous patients who require prosthetic treatment of defects in the maxillo-facial region. The use of enossal implants may enable temporary rehabilitation with regard to mastication and speech. The possible range of indication is considerably limited in cases of extensive defects of the alveolar process, advanced atrophy and by radiological therapy of malignant tumors. The duration of therapeutic success is contingent upon most accurate implantation techniques and prosthetic treatment as well as the patient's oral hygiene.


Characterization of the androgen receptor in the anterior pituitary of the rat. The specific androgen receptors for testosterone (T) and 5alpha-dihydrotestosterone (DHT) in the cytosol fraction of the anterior pituitary of rats have been further characterized using electrophoresis and isoelectric focusing in polyacrylamide gels. After labeling of the cytosol fraction in vivo and in vitro, we were able to demonstrate androgen-protein complexes moving with an electrophoretic mobility (Rf) of 0.5 in 3.25% acrylamide gels containing 0.5% agarose and 10% glycerol. This method was used for quantitative measurements of pituitary androgen receptors, allowing multiple samples to be run simultaneously with little or no non-specific binding. There was no measurable dissociation of the androgen-receptor complexes during electrophoresis. When radioactive testosterone (1 nM) was added to pituitary cytosol fractions in vitro, there was an increase in the binding up to 4 hours of incubation at 0 C and little or no increase between 4 and 24 hours. All the binding studies therefore were done by incubation overnight at 0 C. When cytosol fractions were incubated with increasing concentrations of radioactive testosterone, a typical saturation curve was found. Scatchard plot analysis showed a binding capacity of 12.0 femtomoles/mg protein and the equilibrium constant of dissociation was estimated to be 3.4 +/- 0.7 (SD) X 10(-10)M. Like other androgen-receptor complexes, the testosterone-receptor complex in the anterior pituitary gland had an extremely slow rate of dissociation at 0 C (t1/2 greater than 4 days). The steroid specificity of the cytoplasmic androgen receptors was tested in vitro by the competing efficiency of different unlabeled steroids for [3H] testosterone binding. T and DHT caused the same inhibition of [3H]T to the receptors. However, since metabolism, of DHT to 5alpha-androstane-3alpha,17beta-diol occurred even at 0 C, the affinity of DHT for the receptor is probably somewhat underestimated. Cyproterone acetate had approximately half the affinity for the receptor compared with T, whereas lower affinities were found for progesterone and 17beta-estradiol. Cortisol did not appear to have any affinity for the receptors. Isoelectric focusing in polyacrylamide gels showed a peak of bound radioactivity with an isoelectric point of 5.8. Thus, the characteristics of the cytoplasmic androgen receptors of the anterior pituitary gland are very similar to those of the androgen receptors described in the ventral prostate, epididymis, and testis.


Cytoplasmic and nuclear progesterone receptors in the guinea pig uterus. Although progesterone receptors have been studied in the uterine cytoplasm of many species, relatively little was known about the nuclear content of these binding proteins. In the present study, a nuclear progesterone receptor was deteced in the guinea pig uterus. The binding of progesterone to the nuclear receptor was hormone and tissue specific. Furthermore, the nuclear localization of the progesterone receptor complex in the uterus was both time and temperature dependent. Since the nuclear receptor was extracted in high salt buffer, the effects of KCl on several physical properties of the cytoplasmic and nuclear binders were studied. In the presence of high salt, cytosol and nuclear receptors were virtually indistinguishable. These proteins were clearly distinguished upon removal of the KCl by rapid dialysis: the nuclear receptor had a slower sedimentation rate, a faster rate of dissociation and a higher binding affinity than did the cytosol receptor for progesterone. We conclude that the cytosol and nuclear receptors for progesterone in the guinea pig uterus are distinct macromolecules. These observations are consistent with the postulate that the cytoplasmic receptor is a precursor of that in the nucleus.


Effects of hypophysectomy, ovariectomy, and cycloheximide on specific binding sites for lactogenic hormones in rat liver. We have previously shown that specific binding sites for lactogenic hormones are present at much greater levels in the liver membranes of female than of male rats. In the present studies [125I]iodo-hGH was used to study binding sites specific for lactogenic hormones in liver membranes. In male rats, a single injection of 2 mg estradiol valerate induced these binding sites. The induction was maximal by 9-12 days and was dose-dependent. Ovariectomy significantly reduced the specific binding of [125I]iodo-hGH from 9.7 +/- 0.7% in shamoperated to 6.9 +/- 0.3% in experimental rats (P less than 0.01) without a change in affinity. Fluctuations in specific binding of [125I]iodo-hGH were observed at different stages of the estrous cycle. Binding at estrus and diestrus I was significantly greater than at diestrus II and proestrus (P less than 0.05). The disappearance of binding sites following hypophysectomy was rapid, declining from 13.2 +/- 1.2% in intact rats to 6.0 +/- 0.8% and 2.2 +/- 0.4% 14 and 48 h, respectively, after surgery. In contrast, binding of insulin was slightly increased after hypophysectomy. Anti-estrogens (clomiphene, ICI 46,474, and nafoxidine) prevented the induction of binding sites in male rats given estradiol (E2). A single injection of 200 mug cycloheximide 11 days after an injection of 2 mg E2-valerate reduced binding by more than 90% in 3 h with a return to control levels by 48 h. The maximal decline in insulin binding was 54% during this entire period. These studies suggest that endogenous estrogen plays a role in regulating hepatic binding sites for lactogenic hormones. The level of these binding sites is critically dependent on the presence of an intact pituitary. The possible rapid turnover of these sites suggests that regulatory influences at the tissue level may have an important role in modulating hormone action.


Effect of intrathecal injection of melanotropic-lipolytic peptides on the concentration of 3',5' cyclic adenosine monophosphate in cerebrospinal fluid. Rabbits were anesthetized with urethane, and the concentration of 3',5' cyclic adenosine monophosphate (cAMP) in cerebrospinal fluid (CSF) was measured before and after injection into the cisterna magna of the following biologically active peptides and amines; adrenocorticotropin (ACTH), beta-melanocyte-stimulating hormone (beta-MSH), choroid plexus peptide IIF, arginine vasopressin, oxytocin, glucagon, epinephrine, serotonin, histamine, and acetylcholine. Only epinephrine and the lipolytic-melanotropic peptides ACTH, beta-MSH, and IIF influenced cAMP. Five to 500 mug ACTH caused a 3 to 10X increase in cAMP within 30 min; the concentration of nucleotide returned to baseline within 60-90 min after 5 or 50 mug, and remained elevated for at least 120 min after 500 mug. Effects of the same magnitude and tempo as those caused by 5 to 500 mug ACTH were produced by .1 to 10 mug beta-MSH and 5 to 500 mug IIF. Epinephrine at doses of 5 to 500 mug caused rises in cAMP of similar degree as the same dose of ACTH or peptide IIF, but the peak value was not reached until 60 to 90 min after injection.


The effect of elevated doses of thyrotropin on mouse thyroid. In the course of validating the McKenzie assay it was found that large doses of bovine or rat TSH have a retarding effect on hormonal secretion from the thyroid. TSH in doses less than 10 mU had a maximal effect on thyroidal secretion at about 2-3 h after administration. Doses of TSH in excess of 10 mU caused the peak of secretion to appear at about 9-10 h and caused, as well, a decreased thyroidal iodine release at earlier times. This effect was not due to changes in the pattern of iodocompounds secreted by the thyroid or to a change in the clearance of the blood [125I] thyroxine. An estimate of the integrated secretion with time was linear with log dose up to 100 mU TSH. This pattern of response to TSH is also seen when colloid droplet formation is the parameter studied. cAMP accumulation shows a different pattern of response to TSH. Control levels of cAMP were 1-2 pmoles/mg thyroid. Doses of TSH smaller than 5 mU caused no significant increase in cAMP. With a further increase in TSH up to 200 mU per mouse, cAMP levels increased linearly with the log of TSH and reached 400 pmoles/mg thyroid. Theophylline administered together with large doses of TSH increased the level of the thyroidal cAMP and further decreased the release of iodocompounds at 2 h. These results indicate that there may be an intrathyroidal mechanism which prevents a surge of secretion of thyroid hormones with acute changes in blood TSH levels. The mechanism sensitive to excess TSH is probably associated with steps in thyroidal activation between the accumulation of cAMP and colloid droplet formation.


Ovarian cyclic adenosine monophosphate-dependent protein kinase activity: ontogeny and effect of gonadotropins. The ontogeny of ovarian cyclic AMP-binding and protein kinase activities during the postnatal development of the rat, as well as the effect of LH and FSH administration on ovarian cyclic AMP-binding and protein kinase activities in 5-day-old and in hypophysectomized rats was examined. Ovaries of 4 to 8-day-old rats possessed little or no measureable cyclic AMP-binding and protein kinase activities. Subsequent postnatal development occurred in three distinct phases. During the first phase, ovarian cyclic  AMP-binding and protein kinase activities increased progressively from age 8 days to age 23 days, when adult levels were observed. Protein kinase activity declined markedly during the second postnatal developmental phase from days 24 to 26, lost its cyclic AMP-dependency, and became refractory to stimulation by cyclic AMP. Studies employing a heat-stable protein kinase inhibitor protein isolated from rabbit skeletal muscle suggest that ovarian protein kinase activity during the refractory period was largely of the cyclic AMP-independent variety. During the third postnatal phase, comprising days 30 to 40, ovarian cyclic AMP-binding and protein kinase activities increased to levels seen in sexually mature rats. Protein kinase cyclic AMP-dependency which was lost during the refractory second postnatal period was fully restored during the third phase. Administration of FSH or LH led to a marked increase of ovarian cyclic AMP-binding and protein kinase activities in 5-day-old rats. Hypophysectomy of 20-day-old rats caused a significant reduction of the cyclic AMP-binding and protein kinase activities in a 27,000 X g supernatant fraction, as well as in the mitochondrial, microsomal, and 105,000 X g supernatant fraction. The decreased cyclic AMP-binding and protein kinase activities of these fractions could be partially restored by FSH or LH treatment of the hypophysectomized rats. The results indicate that ovarian cyclic AMP-binding and protein kinase activities, as well as the ability of ovarian protein kinase to respond to cyclic AMP are gradually acquired after the first postnatal week. The postnatal development of ovarian protein kinase and cyclic AMP-binding activities presumably involves the participation of FSH and LH, although the precise mechanism of LH and FSH action remains to be established.


Interaction of spironolactone and digitalis with the 5 alpha-dihydrotestosterone (DHT) receptor of rat ventral prostate. The aldosterone antagonist, spironolactone, has been shown to block the effects of exogenously administered androgen in rat. This suggests that interaction of the drug with androgen at the target tissues may occur. In this paper we have studied the possible interaction of spironolactone with the 5alpha-dihydrotestosterone (DHT)1 receptor of rat ventral prostate. The competitive receptor assay used involves precipitation of the 105,000 X g supernatant of the homogenized tissue with protamine sulfate, removal of the unprecipitated cytosol, and incubation of the precipitate in the presence of the appropriate [3H]DHT steroid solution at 0 C for 18 hours. Using this method the Kd (dissociation constant) for DHT in the rat prostate was in the range of 1.9-4.0 X 10(-9)M and the binding capacity was 0.21 pmol/mg protein. Spironolactone was found to interfere with the binding of DHT to the precipitated cytosol and displayed an estimated Kd of 1.3-4.6 X 10(-8)M. Several digitalis preparations were similarly studied. Digitoxin and digitoxigenin also interfered with the binding of [3H]DHT and had an estimated Kd of 0.8-3.6 X 10(-8)M. Digoxin interacted less strongly and its estimated Kd was 10(-6)M. We believe these results suggest an interaction of spironolactone and digitalis with the DHT receptor and may help explain some of their antiandrogenic actions in the rat and in man.


Diethylstilbestrol-elicited accumulation of cyclic AMP in incubated rat hypothalamus. Diethylstilbestrol (DES) and 17beta-estradiol elicit significant increases in levels of endogenous and [14C]cyclic AMP in [14C]adeninelabeled hypothalami from immature female rats, but only after incubation of the hypothalami with these estrogenic agents for 40-50 min. Estriol has no effect. The 2-fold accumulation of cyclic AMP elicited by low concentrations (20 muM) of DES appears dependent on catecholamine-related mechanisms, since it is prevented by either alpha- or beta-adrenergic antagonists. Higher concentrations (100 muM) of DES elicit a 3-fold accumulation of [14C]cyclic AMP which is only partially blocked by adrenergic antagonists, haloperidol, or the adenosine antagonist, theophylline. These phenomena appear to be specific for the hypothalamus since incubation of cerebral cortical slabs with estrogens resulted in no significant increases in the levels of cyclic AMP. The slow time course in whole hypothalami and the complete lack of effects of low concentrations of estrogenic agents in chopped hypothalami suggest an indirect mechanism of action. Interaction of estrogens with receptors in cell bodies may result, after a latent period of 40-50 min, in enhanced release of catecholamines from distal synaptic terminals and in stimulation of catecholamine-sensitive adenylate cyclases at post-synaptic sites.


Aldosterone production by isolated adrenal glomerulosa cells: stimulation by physiological concentrations of angiotensin II. The production of aldosterone by isolated canine zona glomerulosa cells was measured after the incubation of cell suspensions with angiotensin II and ACTH, and during changes in extracellular potassium concentration. Adrenal cell suspensions were prepared by collagenase digestion and physical dispersion of the capsular layer of the dog adrenal cortex, and aldosterone production was determined by direct radioimmunoassay of cell incubation media. The isolated dog adrenal cells were highly responsive to angiotensin II, with aldosterone production significantly stimulated by concentrations of the octapeptide as low as 10(-11)M. Thus, the steroidogenic response of zona glomerulosa cells was consistently observed at peptide concentrations within the physiological range of angiotensin II in dog plasma, i.e., 2.0-5.0 X 5.0 X 10(-11)M. The maximum aldosterone response of 3-8 times the basal level of steroid production was induced by 3 X 10(-10)M angiotensin II, and a decrease in aldosterone production occurred at peptide concentrations above 10(-9)M. The aldosterone response of isolated adrenal cells to ACTH was consistently less sensitive than their response to angiotensin II, by a factor of 10-20 fold. Aldosterone production was significantly increased by 10(-10)M ACTH, and reached a maximum at 10(-8)M ACTH. By contrast with angiotensin II, ACTH usually evoked a higher maximal level of aldosterone production, and did not produce a decline in steroidogenesis at peptide concentrations above the level which caused maximum stimulation of aldosterone formation. Changes in the potassium concentration of cell incubation media were also accompanied by marked effects upon aldosterone synthesis which was abolished in the absence of potassium and became detectable in the presence of 0.5 mM K+. After remaining constant between 2.5 and 4.0 mM K+, aldosterone production rose sharply above 4.5 mM K+ and reached a maximum at 8 mM K+. These observations provide direct evidence that aldosterone production by zona glomerulosa cells is influenced by changes in angiotensin II levels within the normal plasma range.


Effect of ACTH, alpha-MSH, and beta-Lipotropin on calcium and phosphorus metabolism in the rabbit. Studies were undertaken to investigate the effects of synthetic 1-24 adrenocorticotropin (ACTH), bovine alpha melanocyte-stimulating hormone (alpha-MSH), and ovine beta lipotropin (beta-LPH) on plasma calcium and phosphate in rabbits. Equimolar concentrations of these hormones were infused intravenously in intact and thyroidectomized animals. In addition, ACTH was similarly administered to adrenalectomized rabbits. ACTH, alpha-MSH, and beta-LPH all lowered plasma calcium and raised plasma phosphate. These changes were not prevented by prior thyroidectomy. ACTH was equally effective in inducing hypocalcemia and hyperphosphatemia in the absence of the adrenal glands, while adrenalectomy alone raised plasma calcium. From these findings we have concluded that 1) ACTH, alpha-MSH, and betaLPH affect phosphate as well as calcium metabolism; 2) these hormones do not act by releasing calcitonin; and 3) ACTH exerts its hypocalcemic-hyperphosphatemic effect, at least in part, independently of its trophic action on the adrenal glands.


The covalent structure of collagen. The amino-acid sequence of alpha2-CB4 from calf-skin collagen. Sequencing of chymotrypsin, trypsin, collagenase- and hydroxylamine-derived peptides, using the automated Edman degradation procedure, yielded the complete amino acid sequence of alpha2-CB4 from calf skin collagen (321 residues). Together with the data from earlier work, an uninterrupted sequence in the helical region of the alpha2-chain from residues 1-393 is now known. Glycine is found in every third position of the peptide. Hydroxylation of proline and lysine occurs only in the Y-position of the triplet Gly-X-Y and is not complete in every position. Some residues, such as glutamic acid, leucine, phenylalanine and arginine, are distributed non-randomly between the X and Y-positions and this non-random distribution is different in the alpha1 and alpha2-chains. Comparison of the N-terminal 393 residues from the helical region of the alpha1 and alpha2-chains revealed a nearly identical distribution of charged polar residues arginine, lysine, glutamic and aspartic acids. The distribution of the triplet Gly-Pro-Hyp is simialr in both chains. The remaining residues in the alpha2-chain exhibit a high degree of substitutions when compared with those in the alpha1-chain. Approximately one in every two residues in both the X and Y-positions are substituted.


Investigations on the kinetic mechanism of octopine dehydrogenase. 2. Location of the rate-limiting step for enzyme turnover. The kinetic mechanism of octopine dehydrogenase has been investigated by stopped-flow and isotope replacement techniques. When the enzyme is saturated by substrate and coenzyme, both for NADH oxidation and NAD+ reduction, the stationary phase is preceded by a rapid burst. Under these saturation conditions, furthermore, the stationary phase shows a secondary isotope effect when 4S-[4(2)H]NADH is substituted for NADH and when (on the other reaction end) D-[2H] octopine is substituted for D-octopine. The data are taken to indicate that the rate-limiting step for enzyme turnover is a step following a very fast chemical transformation of the reagents. However, when the substrate concentration is lowered below the corresponding Km value keeping the coenzyme concentration at saturating levels, the time course of the reaction shows no burst and the stationary phase has a larger isotope effect. This indicated that under those non-saturating conditions, the enzyme turnover has a larger contribution than the hydrogen-transfer step. Changing the coenzyme concentration alone has very little or no effect on the amplitude of the burst or on the isotope effect. These features are discussed in terms of the other known kinetic properties of the enzyme, and in terms of analogous studies reported in the literature for other dehydrogenases.


3-Deoxy-2-octulosonic acid 5-phosphate: a component of the endotoxin of Bordetella pertussis. Upon hydrolysis with 2 N hydrochloric acid for 2 h, a 3-deoxy-octulosonic acid 5-phosphate was released from the endotoxin of Bordetella pertussis. The structure of the compound was established through chemical degradation. By periodate treatment of the intact endotoxin it was shown that positions 7 and 8 of the bound octulosonic acid phosphate were free, which, if present in a cyclic form, must be a pyranoside.


Electron paramagnetic resonance studies of cytochrome P-450 in plant microsomes. The technique of electron paramagnetic resonnance spectrometry has been applied to the study of plant microsomal electron-transport components. Only tulip-bulb microsomes were found to give strong enough signals to allow detailed study. At 77 K in the oxidised state, signals were observed at g values of 2.40, 2.25 and 1.93, characteristic of cytochrome P-450 in the low-spin state, and also at g = 4.27, attributable to ferric iron in a rhombic environment. The signals at g = 2.40, 2.25 and 1.93 disappeared upon reduction with sodium dithionite. At 10 K in the oxidised state, signals at g = 8.3 and 3.3 appeared, and these were attributed to high-spin cytochrome P-450. At this temperature a further signal at g = 6, due to cytochrome P-420, was seen in aged tulip-bulb microsomes. Redox titration of both high-spin and low-spin cytochrome P-450 gave the same apparent midpoint potential of -315 +/- mV at pH 6.8 and 25 degrees C. The significance of this value is discussed. Addition of "type I" or "type II" ligands to oxidized cytochrome P-450 caused an increase and a decrease, respectively, in the ratio of the high-spin to the low-spin form. A second effect of aniline, a type II ligand of cytochrome P-450, was to remove the g = 6 signal, suggesting that it also interacts with cytochrome P-420. No iron-sulphur proteins similar to those found in some other cytochrome P-450 electron-transport chains could be detected in any of the microsomes analysed.