In vitro studies on the nature of vinblastine inhibition of thyroid secretion. The effects of vinblastine on thyroid secretion have been studied in mouse thyroid glands in vitro. Inhibition of the secretory response to TSH was observed within the first 30 min of exposure to vinblastine and was largely complete after 1 h. Inhibition of colloid droplet formation and hormonal radioiodine release was comparable for both TSH and cyclic AMP stimulation. No reversal of inhibition was seen when thyroids exposed to vinblastine were subsequently washed 2 or 4 h in vinblastine free mediu, nor could previous vinblastine inhibition be overcome by high levels of TSH or dibutyryl cyclic AMP. Ultrastructural effects of vinblastine (1 X 10(-5)M) were studied under conditions similar to those used to demonstrate inhibition of secretion. Microtubules present in thyroids incubated in control medium were reduced after 30 min and almost completely abolished after 1 or 2 h exposure to vinblastine. No other reproducible structural changes were seen. A higher concentration of vinblastine (5 X 10(-5)M), however, produced lysosomal clumping and the formation of numerous vinblastine crystalloids. Uptake and washout of [3H]-vinblastine were studied with both phases showing fast and slow components. The initial uptake phase (T1/2 approximately 12 min) correlates well with the disappearance of microtubules and the inhibition of thyroid secretion. A prolonged washout component (T1/2 = 10-18 h) may account for the irreversibility of vinblastine inhibition of in vitro thyroid secretion. It is concluded that inhibition of thyroid secretion produced by vinblastine is consistent with its actions on thyroidal microtubules.


Luteotropic regulation of dispersed rat luteal cells in early pregnancy. Viable and functional luteal cells were prepared, using a combination of hyaluronidase, collagenase, and a low concentration of trypsin in a Dulbecco's modified Eagle medium containing 0.5% bovine serum albumin and 3.3 mM Ca++, from corpora lutea taken from 2-day pregnant rats. The viability and functional capacity of the dispersed cells were evaluated by electronmicroscopy and by measuring steroidogenic capicity during perifusion. Dispersed luteal cells previously exposed in vivo to biphasic prolactin (PRL) surges were found to respond during perifusion to as little as 0.5 ng/ml LH by increased steroid secretion. The net progesterone synthesis and secretion remained elevated over a time course of 2 1/2 hours perifusion, and the magnitude of the luteotropic stimulation was dose dependent on LH. However, luteotropic stimulation of LH could not be maintained beyond 2 1/2 h without renewed (in vitro) PRL exposure. PRL by itself maintained the low initial secretion rate of progesterone but demonstrated no stimulatory effect. Different steroidogenic responses were noted during the in vitro administration of LH alone and the administration of LH plus PRL. In the former case, the decreasing rate of progesterone secretion was accompanied by an increasing 20 alpha-dihydroprogesterone secretion, suggesting that luteal 20 alpha-hydroxysteroid dehydrogenase activity was not suppressed. In the latter case, progesterone secretion was maintained and 20 alpha-dihydroprogesterone secretion fell suggesting an inhibitory action by PRL against 20 alpha-hydroxysteroid dehydrogenase activity. Dispersed luteal cells, preincubated at 36 C in medium containing only PRL, retained viability and functional capacity in response to LH-PRL stimulation for periods of time up to 48 h. Preincubation with LH alone did not prolong cell viability.


Water-soluble specific growth hormone binding sites from cultured human lymphocytes: preparation and partial characterization. Cultured human lymphocytes (IM-9 cell line) have specific binding sites or receptors for human growth hormone (hGH). Under appropriate conditions, this specific binding material is spontaneously released into the incubation medium and is solubilized without the use of detergents. This water-soluble preparation binds [125I]iodo-hGH with the same specifictiy as receptors on the intact cell. Unlabeled hGH, but not porcine growth hormone or insulin, competes with labeled hGH for binding to the soluble preparation. Growth hormone preparations of varying purity compete for binding to the soluble binding preparation in the same rank order as they compete for binding to the intact IM-9 lymphocyte. [125I]iodo-hGH incubated with, but not bound to, the soluble preparation is partially degraded, while the [125I]iodo-hGH that is bound to the soluble preparation is protected from degradation, and its ability to rebind to fresh cells is enhanced. The [125I]iodo-hGH-soluble binding preparation complex can be dissociated by the addition of large quantities of unlabeled hGH or by lowering the pH, and [125I]iodo-hGH in both instances remains intact and undegraded. The soluble binding preparation did not sediment when centrifuged at 200,000 X g for 4 hours and was not retained on 0.20 micron Millipore filters. The soluble binding preparation was not retarded on Sephadex G-200. Binding activity was abolished by tryptic digestion. These studies demonstrate that hGH-binding sites, like previously reported insulin binding sites, can be spontaneously solubilized from cultured human lymphocytes, without the use of detergents; these soluble binding preparations are of high molecular weight and are, at least in part, protein in nature.


Effects of parathyroid hormone in vivo on the protein kinase activity in rat kidney. Parathyroid hormone (PTH) was infused into thyroparathyroidectomized rats, and the protein kinase activity of the kidney was studied. When the tissue was homogenized in a buffer containing 5 mM potassium phosphate (pH 7.0), 2 mM EDTA, 1 mM mercaptoethanol, and 5 mM theophylline, the total protein kinase activity (measured in the presence of 5 muM cAMP) in the cytosol was decreased by the infusion of PTH, exhibiting an inverse relationship to cAMP level in the renal tissue. The decrease of total activity was accounted for by a decrease of cAMP-dependent kinase activity, and such a change was induced also by the infusion of calcitonin or dibutyryl cAMP. A substantial enzyme activity was solubilized from the particulate fraction with a buffer containing KC1. The infusion of PTH increased the kinase activity (activities measured in both the presence and absence of cAMP) solubilized from the particulate fraction, suggesting the translocation of activated enzyme from cytosol to some particulate cell component(s). However, when KC1 was added to the homogenization buffer in concentrations up to 150 mM or even higher, the total protein kianse activities in the cytosols of control and PTH rats were similar and there was simply an increase in the fraction of cAMP -independent activity. These observations indicate that the hormonally-induced increase of cAMP in vivo activates protein kinase of the kidney, and the activation of kinase results in apparent translocation of the enzyme from the soluble to the particulate fraction when the tissue is homogenized in buffers of low ionic strength. The physiological significance of this phenomenon, however, cannot be evaluated, due to the fact that the increased association of activated kinase with particulate component(s) is reversed by employing a homogenization buffer containing what is probably a physiological concentration of salt.


Daily variation in concentration of plasma corticosteroid in hypophysectomized rats. A daily variation in the concentration of plasma corticosteroid hormone is present in hypophysectomized rats implanted with pellets of adrenocorticotropic hormone (ACTH) and thyroxine. Although ACTH is necessary to maintain a functional adrenal, and thyroxine is required to permit the expression of the plasma corticosteroid rhythm, a normal daily variation in plasma corticosteroid concentration does not appear to depend on the periodic release of pituitary hormones.


Possible sequential transformation of a series of acidophils in the pituitary autografts in the renal capsule of male rats in association with ACTH cell. In our electron microscopy, acidiphils in the pituitary autografts placed in the renal capsules of immature male rats underwent a sequential transformation with the lapse of time: Within 3 and 6 days, all the somaotrophs packed with the large granules of about 350 mmu diameter dispersed. The size and number of the granules in somatotrophs were quickly and markedly reduced with severe modification of cell shape. There was evidence during this time course that Siperstein's or Moriarty's corticotrophs might be synonymous with the stellate shape of acidophils with the arrangement of small granules 150-200 mmu in diameter along the cell acidophils. The "acidophils of the small granule type" possibly related to ACTH production according to Yoshimura et al. (1974) were frequently detected in the grafts as elongated or irregularly shaped cells. Their minute granules 100-150 mmu in diameter were also distributed in row in the cytoplasmic peripheral area. Gradual loss of the minute  granules below 100 mmu in diameter eventually made the acidophils to transform into agranular cells. Our own idea that ACTH secretion might correlate with a series of cells transforming along the acidophil-axis was indirectly supported by the present observation on pituitary grafts. On the other hand, basophils rapidly degenerated and died away. Ten and 20 days after autografting, the graft cells which might be principally composed of the cells of acidophil origin enormously proliferated through mitotic division, showing the homologous fine structure, without the normal cell individuality. They always contained three different size and shape of granules simulataneously. Significance of such a rapid and strong response of acidophils to the ectopic replacement in the immature male rats was discussed from the view-point of hypothalamic regulation to simple protein hormones.


Reduction of 5'-nucleotidase activity in rat thyroid and adenohypophysis following methylthiouracil treatment. 5' -Nucleotidase activity was determined in rat thyroid and some other organs employing a specific assay method. During the course of methylthiouracil (MTU) treatment, thyroid 5'-nucleotidase activity decreased significantly. This decrease was specific for this enzyme since the activity of neutral phosphatase did not change and the activity of alkaline phosphatase and Mg2+-activated adenosine triphosphatase increased markedly. The 5'-nucleotidase activity of the adenohypophysis also decreased following MTU treatment. This enzyme activity of the liver, heart and whole brain remained unchanged after the treatment. The role of this enzyme was discussed in relation to tissue growth and increased contents of RNA and DNA in the thyroid and adenohypophysis.


Role of sodium and potassium in aldosterone binding of kidney receptors in the rat. An increase of the sodium concentration produced a diminished binding of labeled aldosterone to kidney receptors in both in vivo and in vitro conditions. The elevation of potassium level did not change the binding capacity of kidney nuclear receptors for aldosterone. Hypernatraemia-induced decrease of aldosterone uptake was significant in the nuclear fraction of both medullary and cortical region of the kidney but did not show remarkable changes in the uptake of cytosol fraction. The transfer of aldosterone from cytosol into the nuclear compartment seems to be changed by the alteration of extracellular sodium concentration.


Steroid production by quartered and capsular rat adrenal gland in response to haemorrhage. 60 min after rapid bleeding (1.5--2.0 per cent of b. w.) both aldosterone and corticosterone production rate by quartered rat adrenals were found to be elevated. However, no difference was observed in the rate of aldosterone and corticosterone production by capsular adrenals of sham operated and hypovolaemic rats. Corticosterone production rate by decapsulated adrenals was much more higher after haemorrhage than in the control group. The same alterations could be observed incubating adrenal tissue with ACTH (0.3 mug per ml). Steroid production rate by quartered adrenals of sodium deficient rats was not affected by high in vitro concentration of angiotensin II (2.5 mug per ml). It is concluded that the effect of acute blood loss on corticosteroid biosynthesis of the rat is mediated by ACTH alone.


Hormone receptor studies on frog macrophage cells by means of histamine, serotonin and indoleacetic acid. The phagocytotic activity of frog macrophage cells could be increased by 50% with histamine and by 24% with serotonin. These hormones have a similar effect at the various stages of phylogenetic development, to judge from the respective responses of the unicellular Tetrahymena which showed roughly the same percentual increases of phagocytosis as frog macrophages at roughly the same hormone concentrations. It is concluded that cytoplasmic membrane receptor patterns for a given function do not change in the course of phylogenetic development and the receptors have a capacity for selection, preferring histamine to serotonin, and the latter to the chemically closely related plant hormone indoleacetic acid.


The role of the adrenal gland in the lipid accumulation process in the liver of rats bearing an acth and prolactin producing tumor. The role of the adrenal glands in the development of fatty liver was investigated in rats bearing a transplantable pituitary mammotropic tumor which produces large quantities of ACTH and prolactin. The biochemical and histochemical and histochemical evidence obtained has demonstrated that the adrenal glands, particularly glucocorticoids, are essential for lipid accumulation in the liver of rats with tumor.


Protective value of dietary copper and iron against some toxic effects of lead in rats. Both dietary iron and copper were inversely related to lead absorption as indicated by erythrocyte and kidney lead levels, dietary iron having the greatest effect. Kidney copper values were depressed when dietary iron was low, a condition which was worsened by lead. Lead tended to lower heart cytochrome c oxidase especially when dietary copper was low, but also when dietary copper and zinc were high. Lead interfered with hematopoiesis when dietary copper and/or iron were low, the effect being expecially severe when both essential nutrients were low. These results show the importance of copper and iron nutriture and metabolism as factors which reduce lead toxicity, and emphasize the necessity of considering nutritional status in evaluating lead toxicity.


Undifferentiated patterns of key carbohydrate-metabolizing enzymes in injured livers. II. Human viral hepatitis and cirrhosis of the liver. Activities of key carbohydrate-metabolizing enzymes were determined on biopsied liver tissues obtained from patients with acute and chronic viral hepatitis and postnecrotic cirrhosis of the liver. The results indicated that the activities of fetal or prototype enzymes, low-Km hexokinases, glucose-6-phosphate dehydrogenase and pyruvate kinase type M2 increased, while those of adult type liver enzymes, glucokinase, glucose-6-phosphatase, fructose-1, 6-diphosphatase and pyruvate kinase type L decreased in livers of these cases. Phosphofructokinase activity tended to increase only acute hepatitis. Principal component analysis revealed that the enzyme patterns of acute hepatitis and liver cirrhosis were most deviated from the control and closely resembled those of hepatocellular carcinomas.


Factors influencing the establishment of the normal values of the respiratory activities of human liver mitochondria. Some factors that influence the values of respiratory activities of liver mitochondria isolated from surgical biopsy specimens have been studied. By sedimentating of mitochondria at a lower centrifugal force (5,500 g) than usually used for rat liver mitochondria, and washing the mitochondrial pellet twice, the contamination with lysosomes and microsomes was lowered. At 37 degrees C, and in the presence of hexokinase and glucose, the oxygen uptake was greater than at 25 degrees C and in their absence. The respiratory control was good and the respiratory activities were rather stable during the first 3-4 h after isolation. The respiratory activities of mitochondria isolated from patients with duodenal or gastric ulcers, biliary diseases, and subjects with no digestive diseases (all having normal liver) were compared. Differences in oxygen uptake and acceptor control index values with some substrates were noted. The conditions for selection of controls in studies on subcellular fractions of human liver include: absence of any hepatic antecedents; no clinical evidence of liver involvement; no abnormality in routine liver function tests; a histologic aspect free of pathological conditions, and a normal aspect of the tissue during the homogenization and the fractionation procedure (absence of steatosis or fibrosis). These data provide a basis for the standardization of methods in establishing the reference values of mitochondrial activities for the modifications in a variety of diseases.


Plastocyanin: possible significance of quaternary structure. Some properties of the blue copper protein plastocyanin from the green alga Scenedesmus have been investigated and compared with that from spinach, including amino acid composition, isoelectric point and copper content. The protein from Scenedesmus contains two, that from spinach four copper atoms per molecular weight of 40000. A combination of sodium dodecylsulfate/polyacrylamide gel electrophoresis and quantification of sulfhydryl groups indicates a strong preference for a species composed of 4 polypeptide chains of identical amino acid composition representing the enzymically active entity. Due to various treatments the subunits of both plastocyanins are detected as either monomer species alone or as monomer and dimer in a molar ratio of 2 : 1 on sodium dodecylsulfate gels. The four -SH groups per molecule are of different reactivity: two -SH groups can be detected after destruction of the chromophore; two more (forming an S-S bridge in the dimer) become evident after appropriate reduction. A KCN treatment for production of apoprotein is reported and the use of electrodialysis to improve incomplete apoprotein formation. These studies lend support to the proposal of a quaternary structure. Apoproteins were subjected to dodecylsulfate gel analysis, which proved to be an effective means of estimating both the extent of apoprotein formation and its reconstitution to the haloprotein.


The influence of insulin on various enzyme activities in human and rat hepatoma cells. 1. Incubation of human and rat hepatoma cells with insulin (1 mU/10(6) cells) decreases their content of adenosine 3':5'-monophosphate by more than half after 1 h and by about a quarter after 4 h. 2. The activities of the ATP-metabolising enzymes, adenylate kinase and Mg2+-adenosine triphosphatase are significantly increased by insulin within 1 h and after 4 h. Activity of succinate dehydrogenase and lactic dehydrogenase showed no change at either time interval. 3. Insulin markedly stimulated glucose 6-phosphate dehydrogenase activity within 1 h but by 4 h the increase was less apparent. Glutamate dehydrogenase activity by contrast was not increased by 1 h but was elevated at 4 h.


The prosthetic group of citrate-lyase acyl-carrier protein. The acyl carrier protein of citrate lyase contains adenine, phosphate, sugar, cysteamine, beta-alanine and pantoic acid in a molar ratio of 1:2:2:1:1:1. Peptides containing these components in the same stoichiometric relationship were isolated after proteolytic digestion of acyl carrier protein. All components were linked together in a single prosthetic group. This was released from the peptide by mild alkaline hydrolysis. Under these conditions a phosphodiester bond is cleaved which links the prosthetic group to a serine residue of the peptide. Incubation of the prosthetic-group-containing peptide with phosphodiesterase I yielded 4'-phosphopantetheine and adenylic acid. The 5'-AMP was not free but was substituted by presumably an acidic sugar residue, which was released by mild acid hydrolysis yielding free 5'-AMP. It was concluded from these results that the prosthetic group of citrate lyase acyl carrier protein consists of a substituted isomeric dephospho-CoA. This is bound to the protein by the 5'-phosphate group of adenylic acid. The 4'-phosphopantetheine residue is bound by a phosphodiester linkage to the 2' or 3' position of ribose and the remaining hydroxyl group of ribose is substituted with presumably an acidic sugar residue. The structural similarities of this prothetic group and coenzyme A are discussed and related to the catalytic properties of citrate lyase.


Phosphatidic acid metabolism in rat liver microsomes. Rat liver microsomes contain phosphatidate phosphatases which split phosphatidic acid into inorganic phosphate and diacylglycerol and a system of phospholipases and lipases, which split phosphatidic acid into free fatty acids, glycerol and inorganic phosphate. In the presence of ATP,CoA and [1-14C]palmitate, part of the monoacyl-sn-glycerol 3-phosphate formed by phospholipase action is reesterified, yielding radioactive phosphatidic acid. The sum of di- and triacylglycerols formed from phosphatidic acid in the presence of ATP and CoA exceeded the amount of diacylglycerol formed in their absence. The yield of neutral lipids from sn-glycerol 3-phosphate and monoacyl-sn-glycerol 3-phosphate markedly exceeded that from phosphatidic acid. Comparison of the yields of di- and triacylglcerols from glycerol-labelled and fatty-acid-labelled phosphatidic acid was used to establish the extent of deacylation and reacylation. About 60% of the diacylglycerol was formed by direct dephosphorylation. The triacylglycerols, on the other hand, were formed almost exclusively from recycled phosphatidic acid.


Studies on the mechanism of site I energy conservation. 1. Of the several iron-sulfur centers detected in the site I segment of the respiratory chain, centers N-2 and N-1a alone exhibit apparent phosphate-potential dependent half-reduction potentials, indicating their possible involvement in energy conservation. 2. At high phosphate potential, the apparent half-reduction potential of center N-2 shifts positively by 125 +/- 20 mV, while center N-1a shifts negatively by approximately 60 mV. 3. The redox state of individual iron-sulfur centers in various metabolic states was analyzed. Center N-2 is highly reduced (greater than 90%) in "state 4" mitochondria, while center N-1a stays mostly oxidized. 4. In a submitochondrial system, ATP addition induced reduction of center N-2 if the Eh of the suspension was poised from the high potential side of site I using the succinate/fumarate couple. In contrast, center N-2 was oxidized upon energization, if the Eh of the system was poised from the low potential side using the NADH/NAD couple. 5. Based on these redox behaviors of center N-2, a tentative hypothesis of site I energy transduction was proposed.


Preparation and properties of 3-halopyridine--adenine dinucleotides, NAD+ analogues and model compounds. The preparation of model compounds 1-(2',6'-dichlorobenzyl)-3-halogenopyridinium and the study of their properties were achieved. Their chemical reduction to the corresponding 1,4-dihydropyridines is proved by spectroscopic analysis. 3-Iodopyridine--adenine dinucleotide was prepared by enzymic transglycosidation while the 3-chloro, 3-bromo and 3-iodo pyridine--adenine dinucleotides were synthesized from 3-amino-pyridine--adenine dinucleotide. The 3-halogenopyridine--adenine dinucleotides were proved to be active as hydrogen acceptors with alcohol as a substrate. The absorption band at 290 nm of cinnamaldehyde appeared to be a very sensitive tool for studying the enzymic reaction. With the alcohol dehydrogenase from yeast, only slight activity was detected. 3-Halogenopyridine--adenine dinucleotides are competitive inhibitors with respect to nicotinamide--adenine dinucleotide with alcohol dehydrogenase from yeast, lactate dehydrogenase and malate dehydrogenase. The use of 3-iodopyridine--adenine dinucleotide as a heavy-atom derivative for X-ray structure determination is proposed.


Metallocytochromes c: characterization of electronic absorption and emission spectra of Sn4+ and Zn2+ cytochromes c. Tin (Sn4+) and zinc (Zn2+) derivatives of horse heart cytochrome c have been prepared and their optical spectra have been characterized. Zinc cytochrome c has visible absorption maxima at 549 and 585 nm and Soret absorption at 423 nm. Tin cytochrome c shows visible absorption maxima at 536 and 574 nm and Soret absorption at 410 nm. Unlike iron cytochrome c in which the emission spectrum of the porphyrin is almost completely quenched by the central metal, the zinc and tin derivatives of cytochrome c are both fluorescent and phosphorescent. The fluorescence maxima of zinc cytochrome c are at 590 and 640 nm and the fluorescence lifetime is 3.2 ns. The fluorescence maxima of Sn cytochrome are at 580 and 636 nm and the fluorescence lifetime is under 1 ns. The quantum yield of fluorescence is Zn greater than Sn while the quantum yield of phosphorescence is Sn greater than Zn. at 77 K the fluorescence and phosphorescence emission spectra of Sn and Zn cytochrome c show evidence of resolution into vibrational bands. The best resolved bands occur at frequency differences 750 cm-1 and 1540--1550 cm-1 from the O-O transition. These frequencies correspond with those obtained by resonance Raman spectroscopy for in-plane deformations of the porphyrin macrocycle.


Specific interactions of 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase with coenzymes. The binding of oxidized and reduced coenzyme (NAD+ and NADH) to 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase has been studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated sturgeon enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25%) and the induction of a difference spectrum in the ultraviolet absorbance region of the enzyme. Both of these spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme-NAD+ complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to four NAD+ molecules are bound to the enzyme tetramer. No anticooperativity can be detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of four binding sites with a dissociation constant of 25 muM at 10 degrees C, pH 7.0. The binding of NADH to the acylenzyme has been characterized spectrophotometrically. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. In addition, a large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme-DADH complex, since it disappears upon arsenolysis of the acylenzyme. The binding affinity of NADH to the acylated enzyme has been estimated by performing simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD+ to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD+, the reduced coenzyme NADH appears to be relatively strongly bound to the acylated enzyme, the dissociation constant of the acylenzyme-NADH complex being estimated as 2.0 muM at 25 degrees C. In addition a large quenching of the NADH fluorescence (about 83%) is observed. The comparison of the dissociation constants of the coenzyme-acylenzyme complexes and the corresponding Michaelis constants suggests a reaction mechanism of the enzyme in which significant formation and dissociation of NAD+-acylenzyme and NADH-acylenzyme complexes occur. Under physiological conditions the activity of the enzyme can be regulated by the ratio of oxidized and reduced coenzymes. Possible reasons for the lack of anticooperativity in coenzyme binding to the acylated form of the enzyme are discussed.


Interaction of apoprotein from porcine high-density lipoprotein with dimyristoyl lecithin. 1. The structure of the complexes. The morphology and structural organisation of the complexes formed from the apoprotein of porcine high-density lipoprotein and dimyristoyl phosphatidylcholine (lecithin) have been studied using the technique of small-angle X-ray scattering. Scattering measurements made in solvents of varying electron density were interpreted in terms of a scattering-equivalent model for the structure of the complex. This model is described by an oblate ellipsoidal morphology with dimensions at 20 degrees C: major axis 11.0 nm, minor axis 5.5 nm. Within this overall shape the lipid hydrocarbon chains are organised in an apolar core whilst the lipid polar head groups and protein are located in a outer shell 0.85 nm in thickness. The oblate morphology demonstrates that the structure of the complex is directed by the fundamental bilayer organisation of the lecithin. The dimension of the minor axis (5.5 nm) indicates that phospholipid hydrocarbon chains are orientated perpendicular to the interface.


Interaction of apoprotein from porcine high-density lipoprotein with dimyristoly lecithin. 2. Nature of lipid-protein interaction. The detailed molecular structure of the complex formed by the apoprotein from porcine high density lipoprotein and dimyristoly phosphatidylcholine (lecithin) has been investigated by a range of physical techniques. The complex, an oblate ellipsoid with major axis 11.0 nm and minor axis 5.5 nm (see the accompanying paper), is comprised of a section of lecithin bilayer with apoprotein at the surface. The main site of interaction between protein and lipid is in the lipid glycerophosphorylcholine group region; as with native high density lipoprotein the surface of the particle consists of a mosaic of lecithin polar groups and protein. The formation of this mosaic reduces the cooperativity of the lecithin chain motions and changes the curvature of the lipid-water interface, as compared to a bilayer. Otherwise, there are no major changes in lecithin motions indicating that no strong binding of lipid to protein occurs. The interaction involves the intercalation of amphipathic, 60% alpha-helical, apoprotein molecules among the lecithin molecules so that the protein residues at the lipid-water interface. The apoprotein has a high affinity for the lipid-water interface but specific lipid-protein interactions are not involved.


Properties of enzyme fraction A from Chlorella and copurification of 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase, adenosine 5'phosphosulfate sulfohydrolase and adenosine-5'-phosphosulfate cyclase activities. Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.


Arginyl-tRNA synthetase from baker's yeast. Purification and some properties. Arginyl-tRNA synthetase from baker's yeast (Saccharomyces cerevisiae, strain 836) was obtained pure by a large-scale preparative method, which involves four chromatographic columns and one preparative polyacrylamide gel electrophoretic step. The enzyme has a high specific activity (9000 U/mg) and consists of a single polypeptide chain of molecular weight approximately 73000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Amino acid analysis of the enzyme permitted calculation of the absorption coefficient of arginyl-tRNA synthetase (A(1 mg/ml 280 nm)=1.26). Concerning kinetic parameters of the enzyme we found the following Km values: 0.28 muM, 300 muM, 1.5 muM for tRNA(Arg III), ATP and arginine in the aminoacylation reaction, and 1400 muM, 2.5 muM, and 50 muM for ATP, arginine and PP(i) in the ATP-PP(i) exchange reaction. Arginyl-tRNA synthetase required tRNA(Arg III) to catalyse the ATP-PP(i) exchange reaction.


Electron-spin-resonance studies of a chlorpromazine derivative bound to DNA fibres. Solutions of DNA, spin-labelled with the radical cation of chlorpromazine, were used to produce oriented species of fibres pulled from a gel obtained by ultracentrifugation. The electron spin resonance spectra, recorded at X and Q band frequencies, are given for both gel and fibres; 14N hyperfine coupling parameters were obtained by computer fitting. The spectra are explained in terms of a strongly immobilized label having one principal hyperfine tensor axis parallel to the axis of the DNA helix, and the preferential orientation of the chlorpromazine ions with their planes perpendicular to the DNA helical axis.


Partial purification and some properties of oxalacetase from Aspergillus niger. 1. Oxalacetase from Asperigillus niger was found to be an inducible enzyme, the induction being dependent not only on neutralisation of the acidic growth medium but also on the presence of carbonate. An explanation is proposed. 2. Three methods were established for the quantitative determination of oxalacetase activity. These are based on the determination of the product acetate, on the absorbance of oxaloacetate and on coupling the hydrolysis of oxaloacetate to the oxidation of malate by NAD in the presence of malate dehydrogenase. 3. Oxalacetase was purified about 50-fold from cell-free extracts of A. niger and used to determine some of its properties such as kinetic constants. 4. 2S-[U-14C, 3-2H2] Malate in the presence of oxalacetase, NAD and malate dehydrogenase was partially converted to acetate and oxalate. The 3H/14C ratio of the isolated acetate was nearly twice as high as that of the malate used initially. The result demonstrates that the keto form of oxaloacetate, not the enol, is the substrate of the enzyme. 5. Equimolecular mixtures of 2S, 3S-[3-2H1] malate + 2S-[2-2H1] malate (mixture 1) and 2S, 3R-[3-2H1, 3H1] malate + 2S, 3R-[2-2H1, 3-3H1] malate (mixture 2) were prepared from 2S-[3-3H2] malate by incubation with fumarase in normal and tritiated water, respectively. The isolated mixture 1, in the presence of oxalacetase, NAD and malate dehydrogenase was incubated in tritiated water for formation of acetate and oxalate; the isolated mixture 2 was treated likewise in normal water. 6. The mixtures of symmetrically labelled [3H1] acetate and chiral acetates thus produced were isolated and the configuration of the [3H1, 3H1] acetate specimens was determined in the sequence acetate leads to malate leads to fumarate, as usual. The [2H1, 3H1] acetate derived from 2S, 3S-[3-2H1] malate (present in mixture 1( yielded a malate which on incubation with fumarase retained 65.0% of its total tritium content. This chiral acetate, therefore, had the R configuration. The [2H1, 3H1] acetate derived from 2S, 3R-[2-2H1, 3-3H1] malate produced a malate which retained 35% of its total tritium content, and therefore had the S configuration. 7. It was concluded that the detachment of the oxaloyl residue from oxaloacetate and its replacement by a proton proceed with inversion of configuration at the methylene group which becomes methyl during the hydrolysis.


The cytochrome bc1 complex of yeast mitochondria. Isolation and partial characterization of the cytochrome bc1 complex and cytochrome b. We have isolated the cytochrome bc1 complex and some of its constituent polypeptides from bakers yeast and have studied its spectroscopy, electrophoresis and amino acid analysis. The isolated complex contained 6 mumol of b heme and approximately 3 mumol of c1 heme per g of protein. The electron paramagnetic resonance spectrum was similar to that of the beef-heart preparation. The complex consisted of 7 polypeptides with mobilities on sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to Mr 44,000, 40,000, 32,000, 32,000, 17,000, 14,000 and 11,000. One of the polypeptides with Mr 32,000 was identified on sodium dodecylsulphate gels as cytochrome c1 by porphyrin fluorescence. Cytochrome b was isolated from the complex by treating it with guanidine hydrochloride; it had a purity of 20 mumol per g of protein and consisted of a polypeptide with Mr 32,000 plus two minor bands with Mr 14,000 and 11,000. We have isolated the polypeptide of Mr 32,000 from cytochrome b and the polypeptides of Mr 44,000 and 40,000 ("core proteins") from the complex, both by preparative sodium dodecylsulphate gel electrophoresis and determined their amino acid composition. Only the b polypeptide of Mr 32,000 shows the low proportion of polar amino acid residues that is considered typical of membrane proteins.


Cushing's syndrome or obesity. Bilateral adrenal hyperplasia in a boy 10 years of age. A rare case of the Cushing syndrome, due to bilateral adrenal hyperplasia is described. Because of generalized obesity, normal height, normal bone age and family history of obesity, a boy 10 years of age had at first been misdiagnosed as simple obesity. A reduction in height velocity, advancement of bone age and development of precocious pseudopuberty led to a more detailed biochemical investigation and to the correct diagnosis. The clinical picture can be misleading in the differential diagnosis of Cushing's disease versus simple obesity. From a comparison of reports on pediatric Cushing's syndrome, it is obvious that in most pediatric cases fat distribution differs from that of adult Cushing's syndromes. Stunted growth is reported to be more frequent, but normal and excessive height in Cushing's syndrome is not uncommon. In adrenal hyperplasia a retardation of bone age is usually expected. Interpretation of endocrine functions must take into account that simple obesity leads to secondary endocrinopathies, which are similar to the findings in Cushing's syndromes. Plasma cortisol at midnight and 12 hourly excretion of free cortisol in urine as well as overnight Dexamethasone suppression of morning plasma cortisol are judged to be good screening parameters. Plasma ACTH assays can help in the initial diagnosis and are mandatory during the follow up for an early detection of Nelson's syndrome.


Intrathecal cytostatic chemotherapy of meningitis carcinomatosa. Clinical manifestation and cerebrospinal fluid cytology in a case of metastatic carcinoma of the breast. Effects of intrathecal cytostatic chemotherapy on the clinical symptoms and cerebrospinal fluid (CSF) findings are being demonstrated in a case of metastatic carcinoma of the breast with sighns of meningeal irritation and cranial nerve involvement. Clinical symptoms disappeared after repeated intrathecal applications of methotrexate. CSF cell counts were reverted to normal but tumor cells were found to be persistant in CSF over a period of 19 months. Risks of combined intrathecal and systemic cytostatic therapy and possible effects of parenteral citrovorum factor applications are being discussed. The importance of continued intrathecal chemotherapy and of regular and thorough examination of CSF cells in meningitis carcinomatosa is emphasized. Cytological methods included investigations on viable CSF cells.


Positive chronotropic effect of dialysable peptides derived from plasmin digestion of bovine fibrinogen preparations. Low-molecular weight dialysable peptides, obtained by plasmin degradation of purified bovine fibrinogen preparations, have been shown to increase the chronotropic activity of isolated rat atria. This effect was dose dependent and was inhibited by inhibitors of glycolysis (NaF and 2-deoxy-D-glucose), but not by an inhibitor of oxidative phosphorylation (2, 4-dinitrophenol). Propranolol, a beta-blocking agent, was also ineffective. Fibrinogen-derived peptides increased both cAMP levels and phosphorylase alpha activity in stimulated atria. The increase of these parameters was transitory and appeared to precede the occurrence of the positive chronotropic effect. In the test situation used, the biochemical and functional modifications induced by fibrinogen-derived peptides were similar to those induced by glucagon.


Biochemical and mechanical effects of phenylephrine on the heart. Injections of phenylephrine into isolated perfused guinea pig hearts increased cyclic AMP and phosphorylase a. Reserpine pretreatment or propranolol decreased or abolished the phenylephrine-induced biochemical changes without affecting the contractile response. Phentolamine, on the other hand, shifted the phenylephrine dose-response curve for contractility to the right without affecting the other parameters. The biochemical effects of phenylephrine are apparently due to the release of noradrenaline while the contractile effect is mediated directly through stimulation of alpha-adrenoceptors.


A noradren aline sensitive adenylate cyclase in the rat limbic forebrain: preparation, properties and the effects of agonists, adrenolytics and neuroleptic drugs. A method for the preparation of a noradrenaline sensitive adenylate cyclase from homogenates of the rat 'limbic' forebrain is described using Krebs--Ringer as the homogenising medium. Some of its properties resemble those reported previously by other workers, using slices. Its response to agonists show that it has the characteristics of a beta1-receptor i.e. the potency of 1-isoprenaline exceeds that of 1-noradrenaline which exceeds that of 1-adrenaline. Structure--activity analysis of the response of the adenylate cyclase to a range of adrenergic agonists shows a strict requirement for a catechol moiety and a beta-hydroxyl group. The activation of the enzyme by 1-noradrenaline is sensitive to stereoselective inhibition by 1-propranolol. The effect of a number of neuroleptic drugs was examined. Promazine was the most effective agent tested in antagonising the stimulation produced by 50 muM 1-noradrenaline, whilst the potent dopamine receptor antagonist, alpha-flupenthixol was only weakly active. Furthermore, there was no stereoselectivity in the antagonism produced by alpha- and beta-siomers of flupenthixol. Pimozide was not found to be a potent antagonist. Thus the spectrum of antagonism produced by neuroleptic drugs was quite different from that seen in the dopamine sensitive adenylate cyclase of the rat corpus striatum.


Alterations in splanchnic cyclic nucleotide levels in splanchnic artery occlusion shock and their modification by dexamethasone. The effects of dexamethasone sodium phosphate (DSP), 5 mg/kg, administration on the biochemical alterations in hepatic tissue subsequent to the production os splanchnic artery occlusion (SAO) shock was investigated. Following the induction of SAO shock, DSP-treated dogs exhibited a significantly improved cardiovascular status compared to placebo-treated shocked dogs, 2 hr after release of the occlusion, biopsies of the liver were taken and analyzed for beta-glucuronidase (BG), adenosine-3',5'-cyclic monophosphate (cAMP) and guanosine-3',5'-cyclic monophosphate (cGMP) content. SAO shock produced a significant increase in hepatic free BG activity which was reversed by DSP pretreatment. Additionally, SAO shock decreased hepatic cAMP levels, increased cGMP levels and significantly lowered the hepatic ratio of cAMP/cGMP. These changes in cyclic nucleotide levels were reversed by DSP administration and were found to be inversely related to changes in hepatic free BG activity. Thus, the ratio of cellular cAMP/cGMP may function as a regulatory mechanism for lysosomal enzyme release secondary to ischemia and hypoxia. Further, DSP may act to maintain lysosomal integrity in ischemic tissues by preservation of cAMP/cGMP ratios.


Stimulation of ribonuclease activity and its isoenzymes in germinating seeds of cowpea (Vigna sinensis) by gibberellic acid and adenosine-3',5-cyclic monophosphate. Application of GA3 and cyclic AMP to cowpea seedings caused a 2-3 fold stimulation of RNAase activity, together with the augmentation of RNAase isoenzymes. Inhibitor studies indicated the requirement of fresh RNA and protein synthesis for enzyme stimulation.


Cytokinin ocntents and cAMP metabolism during growth of Escherchia coli. During Escherichia coli growth, we found an inverse correlation between free cytokinin content and cAMP level. The rates of synthesis of adenylate-cyclase and cAMP-phosphodiesterase were practically constant.


Influence of cyclic nucleotides on protein synthesis in vascular smooth muscle. The incorporation of leucine-14C into protein in bovine mesenteric arteries was augmented by cyclic GMP (10-3 M) and decreased by cyclic AMP (10-3 M). There was no effect of 5'AMP (10-3 M). The phosphodiesterase inhibiting drugs theophylline (10-3 M) and papaverine (5 x 10-5 g/ml) both decreased the leucine-14C incorporation.


Myopathic changes at the end-plate region induced by neostigmine methylsulfate. Administration of large dose of neostigmine caused very quickly marked myopathic changes at the motor end-plate region. With continued injections, however, some recovery of the structural features did occur suggesting the reconstructive changes in the affected regions.


Plasticity of the hormone receptors and possibility of their deformation in neonatal age. Gonadotropin or TSH treated newborn animals reacted to TSH treatment in their adult age in a lesser degree than control ones. This suggests the plasticity of hormone receptors and the possibility of their deformation in neonatal age.


Hormonal induction of glutamate dehydrogenase in rat liver. Glutamate dehydrogenase rapidly increases in microsomes and appears in the cytoplasm after administration of cortisone, cAMP, hydrocortisone-acetate. Prolonged administration of ACTH maintains high level of enzyme in the mitochondria and microsomes. Hydrocortisone-acetate, insulin and cortisone decrease drastically enzyme in mitochondria.


Immunohistochemical study of the pars intermediate of the mouse pituitary in different experimental conditions. Alpha-MSH, beta-MSH and ACTH have been localized in the cells of hypophyseal intermediate lobe by fluorescence histoimmunological technics. Elaboration and excretion of these polypeptides are enhanced after dehydration or adrenalectomy. The most evident variations are seen with alpha-MSH and ACTH after dehydration, with beta-MSH after adrenalectomy.


[Effect of autonomotropic agents and their combinations with amidopyrine on aesptic inflammation and vascular permeability]. Experiments staged on rats demonstrated the display of antiexudative activity by phepracet, deseryl, chlorpromazine, phentolamine and sanotension in an inflammation produced through subplantar introduction of the Difco agar. Some of the above agents suppressed the reaction in response to dimethyl sulfoxide injection. The antiedematous and stabilizing the cellular membranes effects of amidopyrine were potentiated by using the latter in combination with sympatholytics and also with benactyzine. No synergism with respect to changes of the vascular permeability and kinnase activity of the blood was revealed.


[Effect of catecholamines on the activity of rabbit kidney microsomal (na+--k+)-activated atpase]. The significance of alpha- and beta-adrenoreceptors and the role of cyclic 3', 5' -AMP in the mechanism of the activation by catecholamines of the microsoma (Na+ - K+)-activated ATP-ase of the rabbits kidneys were investigated. Phentolamine did not obstruct the activating effect of epinephrine in the cortical and medullar layer. Inderal inhibited the reaction of the enzyme in response to epinephrine in the renal cortex and fully prevented any rise of the enzymatic activity in the medullar substances. Isopropylnorpinephrine and cyclic 3',5' -AMP stimulated the (Na+ - K+)-activated ATP-ase in the cortical and medullar layer. An inference is drawn to the effect that the activation of the enzyme occurring under the effect of catecholamines comes as a consequence of a beta-adrenoreceptors stimulation. It is suggested that the cyclic 3',5'-AMP may be directly involved in the mechanism accounting for stimulation by catecholamines of the activity displayed by the microsomal (Na+ - K+)-activated ATP-ase of the rabbits kidneys.


[Effect of polyvinylpyridine N-oxide on lipid metabolism indices in healthy and silicotic rats]. In the early period of investigations (in 1 month) polyvinylpyridine-N-oxide (PNO) causes a fall in the content of lipid compounds (total lipids, phospholipids, choesterol) in the liver and an inhibition of the lipolytic activity in the tissue of intratracheally quartzdust laden rats. Introduction of PNO into the lungs of dust-laden rats over a period of 4 months produces an appreciable diminution in the quantity of all the lipid components against the background of an increased lipolytic activity, recuced under the effect of quartz dust. In healthy rats PNO depresses the lipolytic activity of the pulmonary tissue and intermittently brings down the lipid compounds therein and in the spleen. In the blood of healthy and silicotic rats receiging PNO the level of the total and free cholesterol goes up. Early after administration of PNO the content of total lipids and beta-lipoproteids in the blood serum of dust-laden rats augments, while later on the concentration of total lipids goes down, while the level of summary ketone bodies rises.


[Changes in the functional state and structure of the adrenals in CC14 poisoning]. Introduction of CCI4 in doses of 0.1 and 0.2 ml/100 g to albino rats reduced the steroid production generated by adrenal sections. In 24 and 48 hours after poisoning with a small dose of CCI4 (0.1 ml/100 g) there could be seen an increased concentration of 11-oxycorticosteroids in the plasma. Morphological signs of a damage suffered by the gland were observed only in poisoning with a large dose of CCI4 (0.2 ml/100 g).


[Effect of psychotropic and anticonvulsant preparations on the conformational transitions in transport ATPase]. The effect of neuroleptics, antidepressants, somnifacients and antiepileptic drugs on Na+- and K+-dependent transitions in the preparation of the transport ATP-ase from renal tubules of the guinea pig were looked into by using the spinal probes procedures. Neuroleptics inhibit most strongly (by 100--67 per cent) the conformant transition in the Na+- K+-dependent ATP-ase, their action manifesting itself to a greater extent with respect to the K+-dependent conformant transitions. The influence of antidepressants is close to that of the neuroleptics, although it is noticeably less intensive (78--46 per cent). The action of hypnotics (barbituric acid derivatives) and of antiepileptic drugs of a different chemical structure closely approaches, but is different from the effect of neuroleptics and antidepressants, both as concerns its intensity and the lack of any essential differences in the action upon the Na+- and K+-dependant conformant transitions.


[Effect of aminazine and haloperidol on caudate inhibition of the conditioned reflex reaction of avoidance in cats]. The effect of neuroleptics on the avoidance behaviour under electric low-frequency stimulation of the caudate neucleus was studied in experiments with cats. Chlorpromazine (1 and 3 mg/kg) and haloperidol (0.5 mg/kg) were found to bring disorder in the behaviour of the animals and to intensify the inhibitory function of the caudate nucleus. Chlorpromazine proved in all respects to be a more active substance. The results obtained may be attributed to disruption of the central dopamine- and noradrenergic transmission.


[Excitability of the hypothalamus and limbic structures of the rabbit brain under the influence of glucocorticoids and ACTH]. The influence of glucocorticoids and ACTH on the excitability of the hypothalamus and limbic structures of the brain in rabbits was studied in chronic tests with electrochemotrodes preliminarily implanted into the brain. Results bearing proof to a dissimilar effect produced by ACTH and glucocorticoids on the excitability of the anterior and posterior hypothalamus, hippocampus and amygdala are presented. It is suggested that the mechanism of the retroinhibitory action of corticosteroids and ACTH provides for the participation of hypothalamic and limbic structures of the brain.


Malignant transformation-linked imbalance: decreased xanthine oxidase activity in hepatomas. Xanthine oxidase was decreased 2- to 10-fold in all examined rat hepatomas irrespective of the malignancy; growth rate and degrees of histological differentiation of the neoplasms. The affinity to substrate (KM=6-8 muM) and the pH optimum (8.0) of the liver and hepatoma enzymes were the same. The reprogramming of gene expression, as manifested in the decreased activity of this key purine metabolizing enzyme, appears to be specific to neoplastic transformation. Since glutamine PRPP amidotransferase activity was increased but the opposing enzyme, xanthine oxidase, was decreased in all the hepatomas, the reprogramming of gene expression results in an imbalance that favors synthesis against catabolism. This enzymatic imbalance should confer selective advantages to the cancer cells.


[Study of the process of ACTH and insulin interaction with the plasmatic membranes of rat liver cells in vitro using luminescence methods]. The process of interaction of ACTH and insulin with plasmatic cellular membranes of the rat's liver and artificial lipid membranes (liposomes) was studied by the method of proper proteinic fluorescence and by using hydrophobic fluorescent probes. This process is shown to be attended by conformative readjustments of the membranous proteins and also by definite structural changes in the lipid phase of the membranes. In its action ACTH involves the hydrophobic layer of the lipid phase to a greater extent than does insulin. A comparison of the data on the effect produced by ACTH and insulin on the plasmatic membranes as against the results obtained with liposomes suggests that for the hormones to display their activity these have first to combine with the specific membranous protein, while a change in the lipid phase comes only as a consequence of such an interaction.


Tubal plastic surgery at the Tel Hashomer Hospital: a critical study. The study presents 93 patients in whom tubal surgery was carried out for primary or secondary mechanical infertility. A classification of these patients into five groups is suggested. Twenty-four uterine pregnancies were recorded, eighteen of which reached term. A plea for enlarging the scope and number of interventions in more patients of the older group and of the more complex pathologic group is presented and discussed. This approach may lower the the over-all success rate but may benefit some of this group of patients. Last but not least, the follow-up of these patients, who constantly move from one physician to another, seeking more treatment, is difficult. Liaison with referring gynecologists and standardization of methods would be of great help in the follow-up.


[Active transport of fluorescein in the choroid plexus of the brain and regulation of its rate by cyclic 3',5'-AMP]. With the aid of luminescent microscopy and microfluorimetry in vitro, the transport of labelled fluorescine into the structures of the brain lateral ventricles' chorioid plexus was studied in rabbits. An against-gradient pumping of fluorescine into the cytoplasm of the neuroepithelium cells and into the lumen of adjucent capillaries was shown to occur in the chorioid plexus. Such accumulation of fluorescine proceeds in the active transport way with participation of a carrier. The active transport system is congenial to fluorescine - Km = 4-10(-5) M, which renders even small amounts of this substance to be quickly removed from the milieu. The fluorescine-transporting system fulfills transition of other organic acids as well. The system of organic acids active transport is, apparently, localized in the apical and basal membranes of the neuroepithelial cells surrounding the plexus' filia which makes the concentration of transported acid in the neuroepithelial cells higher than in milieu, and in the capillaries' lumen higher than in the cytoplasm. An enzyme system adenylcyclasa-phosphodiesterase was found in the chorioid plexus' neuroepithelium. The system participates in regulation of velocity of the organic acids transport by means of changing the intracellular concentration 3',5'-AMP.


[Analysis of an increase in the body's sensitivity to insulin]. In adult albino rats, increased sentivity of the organism to insulin action caused by systematic muscular exercises preserved in conditions of adrenergic blockade but was reduced by theophylline administration (inhibition of 3'-5'-AMP-phosphodiesterase activity). The sensitivity to insulin action 3'-5'-phosphodiesterase (in muscles, liver, and adipose tissue) increased during adaptation to muscular activity. Simultaneously the insulin inactivation in muscle and liver tissues also increased.


[The activity of 3',5'-AMP phosphodiesterase and inactivation of insulin during muscular activity]. In trained adult albino rats, more rapid mobilization of carbohydrates and FFA, more economical carbohydrates expenditure during muscular exercise, and more rapid resynthesis of glycogen during recovery period, were observed. This depends on both the changes of 3',5'-AMP-phosphodiesterase activity of muscles, liver, and adipose tissue, and the degree of insulin inactivation by muscle and liver tissues. Both these processes considerably differ in untrained and trained animals.


Detection of RNA-instructed DNA polymerase in the mitochondria of Rous sarcoma cells using sephadex G-150 thin-layer gel filtration. Analyses of the DNA polymerase present in the inner mitochondrial membrane and matrix fraction of the mitochondria isolated from Rous sarcoma cells and chick embryo cells, and in the lysate of Rous sarcoma virus were performed, using a modification of the thin-layer gel filtration on Sephadex G-150 superfine. The method allowed the simultaneous determination of the molecular weight of the isolated enzymes. In the mitochondria or Rous sarcoma cells an RNA-directed DNA polymerase, activated by poly(rA): oligo (dT) synthetic duplex, was detected, with the same molecular weight as the reverse transcriptase isolated from Rous sarcoma virus. Such enzyme was not found in the mitochondria of chick embryo cells.


Separation of time-defined avian myeloblastosis virus (AMV) using column cultivation of leukaemic myeblasts. A new technique is described of column cultivation of cells immobilized on Sial glass cullet. The technique ensures a controlled and continuous production of time-defined young virus. During the column cultivation the morphological characteristics of tho immobilized cells remain essentially unchanged.


Inhibition of lymphocyte blastogenesis by factor(s) in alloxan-diabetic rat plasma. Supplementation of rat lymphocyte cultures with plasma from alloxan-diabetic rats produced a dose-dependent suppression of mitogen-induced blastogenic responses. Viability measurements indicated that this inhibition was not due to a direct cytotoxic effect of alloxan-diabetic plasma on rat mononuclear leukocytes in vitro. This inhibition was not explained by hyperglycemia alone and was observed when blastogenesis was induced in vitro by phytohemagglutinin (PHA), concanavalin A (con A), or allogeneic cells. Peripheral blood lymphocytes from alloxan-diabetic rats appeared to be more sensitive than normal cells to the inhibitory effect of diabetic plasma. Heating at 56 degrees for 60 minutes produced only a partial loss of the inhibition by diabetic plasma. Alloxan-diabetic rat plasma promoted an increased accumulation of adenosine 3',5'-monophosphate (cyclic AMP) in mononuclear leukocytes. Insulin (1 and 100 muU./ml.) in vitro enhanced PHA-induced blastogenesis but failed to reverse the inhibition caused by diabetic plasma. Ultrafiltration through a cellulose dialysis membrane with an exclusion size of 12,000 molecular weight did not remove the inhibitory factor(s). These results indicate that a depressed cellular immune response is produced during an insulin-deficient diabetic state. The suppressed in-vitro blastogenic response of lymphocytes from alloxan-diabetic rats appears to involve some circulating inhibitory factor(s) in diabetic plasma. This inhibition may be explained, in part, by the ability of diabetic plasma to elevate cyclic AMP in mononuclear leukocytes.


Individual differences in infants' sleep. Individual differences in sleep patterns were repeatedly studied in 22 infants in a residential unit for mothers and babies at two, six, 12 and 20 weeks of age. Electroencephalogram, horizontal and vertical electro-oculogram, electromyogram of chin muscle, and respiration and body movements were recorded during the first two sleep cycles. The age factor was significant in all variables studied. The infants' individuality was significant in (1) the frequency of respiration during quiet sleep, (2) the frequency of rapid eye movement during paradoxical sleep, (3) the occurrence of regular EEG activity during paradoxical sleep, and (4) body movements during paradoxical sleep. Individual differences in these sleep variables persisted with considerable stability during the first five months of life.


Hepatoma occurring two decades after hepatic irradiation. Hepatoma has been related to chemical, dietary, metabolic, infectious, and genetic factors. therapeutic hepatic irradiation has never been associated with hepatoma. A case of hepatocellular carcinoma is presented that occurred two decades after radiotherapy for presumed hepatic hemangioma. Radiation-related liver disease is discussed in general, with emphasis placed on radiation hepatocarcinogenesis.


Inhibition by propranolol of bile acid stimulation of rabbit colonic adenylate cyclase in vitro. Bile acids, especially unconjugated deoxycholic acid, cause diarrhea by inducing colonic mucosal secretion of water and electrolytes. This effect has been shown to be mediated by adenylate cyclase (AC). Propranolol, a beta-adrenergic blocking agent which inhibits AC, may then prevent this action of bile acids on colonic mucosa. The aim of this study was to determine the effect of bile acids, catecholamines, and propranolol on AC activity in colonic mucosa. The in vitro effects of deoxycholic acid, taurocholic acid, NaF, epinephrine, norepinephrine, and propranolol on AC in rabbit colonic mucosa were determined. NaF, 10(-4) M, increased AC activity to 220% of control (P less than 0.01). Deoxycholic acid, 10(-4) M, increased AC activity to 178% of control (P less than 0.01). Lesser but significant (P less than 0.01) stimulation of AC occurred at both higher and lower concentrations of deoxycholic acid, with no effect at 10(-10) M. Taurocholic acid, 10(-4) M, and epinephrine and norepinephrine, 10(-2) M, 10(-4) M, 10(-6) M, and 10(-8) M, had no effect on AC. Propranolol, 10(-6) M, caused a 60% decrease (P less than 0.01) in the stimulated AC activity induced by 10(-4) M deoxycholic acid. Propranolol, 10(-4) M, decreased basal AC by 30% (P less than 0.01). (1) Deoxycholic acid, but not taurocholic acid, epinephrine, or norepinephrine, stimulates colonic AC activity. (2) Propranolol inhibited this deoxycholic acid stimulation of AC. (3) Catecholamines are not intermediaries in this action of propranolol on colonic mucosal AC activity.


Propranolol inhibits adenylate cyclase and secretion stimulated by deoxycholic acid in the rabbit colon. Bile acids cause diarrhea by inducing colonic secretion, probably mediated through the cyclic AMP system. The aim was to determine the effects of an adenylate cyclase inhibitor, propranolol, on deoxycholic acid (DCA) stimulation of net secretion and the cyclic AMP system in the colon. In each of 30 New Zealand white rabbits, 0.9% NaC1 as control and 6 mM and 8 mM DCA were injected in random sequence into three colonic loops in situ. Propranolol, 4 mg per kg was administered intravenously to 12 of the 30 rabbits 1/2 hr before preparation of the loops, i.e., 5 1/2 hr before the rabbits were killed. In the 18 untreated animals, 6 and 8 mM DCA significantly stimulated colonic net secretion and mucosal adenylate cyclase activity; 6 mM DCA caused no change in mucosal phosphodiesterase activity, whereas 8 mM DCA caused a 25% decrease (P less than 0.01). In propranolol-treated animals compared to untreated animals, the volume of luminal fluid in controls was not different, with 6 mM DCA it was 88% less (P less than 0.01), and with 8 mM DCA it was 45% less (P less than 0.01); adenylate cyclase activity in controls was 43% less (P less than 0.01), with 6 mM DCA it was 67% less (P less than 0.01), and with 8 mM DCA it was 65% less (P less than 0.01); phosphodiesterase activity in controls and with 6 mM DCA was not different and with 8 mM DCA it was 38% greater (P less than 0.02). In conclusion, propranolol prevented DCA stimulation of colonic net secretion and inhibited the cyclic AMP system. Propranolol, therefore, warrants investigation as therapy for diarrhea caused by bile acids in the colon.


Utilization of the quantitative assay of lipoprotein X in the differential diagnosis of extraphepatic obstructive jaundice and intrahepatic diseases. Quantitative determination of LP-X, abnormal serum low density lipoprotein, was performed on the sera of 620 patients with jaundice in two medical centers, one in Oklahoma City, Oklahoma, and the other in Birmingham, England. The results of serial assays over a period of 5 to 8 days after patient admission to hospital or after onset of jaundice, if this occurred in hospital, correlated best with the type and management of jaundice. In some cases of early cholestatic disease of extrahepatic origin LP-X may be absent, but after the observation period it was found that only 1 of 81 (98%) patients with obstruction of the extrahepatic bile duct system remained negative. Of the remainder, 74 (91%) had or developed levels of LP-X exceeding 300 mg per 100 ml. In addition, 43 (88%) of 49 subjects followed serially showed increases in LP-X concentration, with no change in 3 patients. Of 539 subjects with intrahepatic disease, 14 (26.5%) were LP-X positive and 27 (19.4%) of these had initial LP-X levels higher than 300 mg per 100 ml. During the follow-up period, 35 (74%) of 47 patients with intrahepatic disease showed a reduction of LP-X; of the remaining 12 patients 4 had mitochondrial antibody-positive primary biliary cirrhosis, and 6 had severe cholestasis associated with acute infectious hepatitis and high aspartate transaminase levels. Similar figures for alkaline phosphatase showed less consistent changes during the follow-up period. In this retrospective appraisal the trends and absolute levels of LP-X, in addition to the use of similarly followed levels of the routine liver function tests, allowed better differentiation of jaundice requiring surgical correction from that remediable by medical means exclusively than did the use of the routine liver function tests alone. In addition, LP-X is specific for liver dysfunction, whereas other routine liver function tests are not.


[Surgical treatment of ovarian tumors, especially ovarian carcinomas]. In every case of ovarian tumor or even if this possibility cannot be excluded by other means a diagnostic laparotomy is indicated. There are only a few exceptions to this rule. Before surgery every diagnostic step should be undertaken to determine the nature, extent and origin of the tumor. At laparotomy three situations may be found, 1. the tumor is obviously benign, 2. the nature of the tumor is doubtful and 3. the tumor is malignant. The diagnostic and therapeutic steps in all these situations and also in case of persistent or recurrent tumor are discussed. Treatment of ovarian cancer is radical surgery if possible and/or chemotherapy and radiation.


[Experimental study on aldolase isozyme during the development of hepatoma in the rat (author's transl)]. Aldolase (ALD) consists of three different types of isozyme, i.e. muscle or A type, liver or B type and brain or C type, their relative proportions being fixed for each tissue. In this study, isozyme profiles in the serum and DAB induced hepatoma as it grows were examined in comparison with those in the fetal liver, in the hope to obtain information for early detection of the malady. In the very early phase of hepatoma growth with no elevated enzyme activity, ALD-A fraction after electrophoresis was found to increase. In the course of hepatoma growth, ALD-B decreased while ALD-A increased with appearance of hybrid of ALD-A and ALD-C. In the normal adult liver, no hybrid of ALD-A and ALD-C was encountered. Serum profile of the rat with DAB induced hepatoma was similar to that of the cancer tissue. On the other hand, the fetal liver shows a profile with high ALD-A fraction and even with hybrid of ALD-A and -C. These features disappeared gradually as maturation proceeded and became close to the adult pattern. Thus it is clear that DAB induced hepatoma exhibits retrogressive change in hepatic differentiation in its isozyme profile. The results also indicate usefulness of isozyme profile for early detection of the hepatoma.


Hypoglycemia in diabetics.

Hypoglycemia in diabetics. Hypoglycemia in diabetes can be divided into 1) spontaneous hypoglycemic reactions due to absolute or relative overproduction of endogenous insulin or some other blood glucose-lowering substance, as in islet cell adenoma or carcinoma, latent or protodiabetic conditions, (extrapancreatic) tumors and pituitary and/or adrenal disorders; and 2) hypoglycemia caused by exogenous, i.e. therapeutic, measures. The problem of hypoglycemia in insulin-treated diabetics is far from being solved. As revealed by continuous blood glucose monitoring, nocturnal hypoglycemic attacks frequently escape attention especially in juvenile diabetics. Circadian variations in peripheral glucose utilization, rather than changes in plasma insulin activity, are likely to be involved in this mechanism. At least, this was the conclusion drawn from studies carried out by means of a glucose-controlled insulin and glucose infusion system (GCIGIS) -or artificial pancreas-which delivers short-acting insulin and glucose on demand intravenously. Hypoglycemic reactions in patients being treated with oral anti-diabetic agents, on the other hand, should be regarded primarily as one of the side reactions intrinsic to the mechanism of action of some of these drugs, e.g. sulfonylureas, which act mainly via stimulation of secretion of endogenous insulin reserves not responding properly to postprandial blood glucose increments. In the case of glibenclamide, at least partial resensitization of the defective glucose receptor of the beta-cell also becomes operative. A higher incidence of a characteristic type of hypoglycemic reaction was observed soon after glibenclamide therapy was introduced. Better understanding of the drug and dissemination of the information about it to doctors and patients has reduced the number of hypoglycemic reactions caused by glibenclamide to the same proportions as for other sulfonylureas. Hypoglyoemia following therapeutic hypophysectomy retains its position as one of the main hazards of this heroic therapy.


Human insulinoma tissue: in vitro studies of proinsulin/insulin biosynthesis and release. The rate of proinsulin/insulin turnover has been studied in human insulinoma tissue from ten patients. During the incubation of tumor tissue the ratio of immunoreactive insulin (IRI) release to content was significantly higher than that from isolated human pancreatic islets. The tumor cell cytoplasm (100,000 g supernatant S-100) contained approximately 45% of the IRI. Endogenous proinsulin and insulin were released throughout the incubation. Newly synthesized proinsulin was detected in the 15 min pulse microsomal, secretory granule, S-100 and media samples. These results suggest that defective hormonal storage and release mechanisms are operative in human insulinoma cells resulting in a higher turnover of proinsulin and insulin compared with pancreatic islet tissue.


Glucagon secretion by the transplantable islet-cell tumor of the Syrian hamster. The transplantable islet-cell tumor of the golden hamster has already been shown to produce hypoglycemia and hyperinsulinemia in the receptor animal. The present study demonstrates that the plasma pancreatic glucagon concentrations are significantly increased in the tumor-bearing animals but that this hyperglucagonemia is not abolished by administration of glucose or of diazoxide. It is also unresponsive to arginine administrations. In these animals, increased peripheral glucagon plasma concentrations are observed along with a reduced porto-aortic glucagon gradient. Moreover, plasma glucagon in the vena cava is usually higher than that in the aorta and a significant quantity of glucagon is found in the tumor. We conclude that glucagon release from the tumor is in fact responsible for the observed hyperglucagonemia.


Response of islet cell tumors to enterohormones. The insulin response to oral glucose, tolbutamide, arginine, pancreozymin and cerulein was studied in a group of subjects with insuloma and a group of normal control subjects. The same parameters were studied in a small number of cases after administration of secretin and gastrin. The glucagon response (IRG) to arginine, pancreozymin and cerulein was also studied. In subjects with insulomas plasma IRI values after oral glucose, tolbutamide and pancreozymin, starting from elevated basal levels, reached high absolute levels though the increase above basal levels did not differ significantly from normal. After cerulein plasma IRI values increased in some insuloma patients but not in normal subjects. After arginine the plasma IRI increase above basal levels was significantly lower than normal in patients with insulomas. The glucagon response to arginine was normal in the patients with insulomas; these patients showed a clearcut glucagon response to cerulein and a very irregular response after pancreozymin.


The diagnosis of insulinomas and other causes of fasting hypoglycemia. The decrease in insulin secretion in normal subjects in response to hypoglycemia provides a basis for suppression tests for diagnosing insulinomas. The majority of patients with insulinomas have hypoglycemia after an overnight fast, and inordinatley raised plasma insulin levels are diagnostic. A prolonged fast is a means of enhancing hypoglycemia. Fish insulin can be used to induce hypoglycemia over a short period, and provides a simple, dynamic suppression test which appears to give good diagnostic discrimination. A normal endogenous insulin response to fish-insulin-induced hypoglycemia, together with clinical assessment and a few simple investigations, virtually excludes the diseases which cause fasting hypoglycemia in adults.


Medical treatment of endogenous organic hyperinsulinism. There are several situations in which medical therapy of hyperinsulinism induced by islet cell tumors or hyperplasia is necessary and at present we have at our disposal several drugs which are capable of reducing endogenous hyperinsulinism. They are: -Streptozotocin, which represents today the most useful therapeutic agent for beta cell carcinoma therapy; -Diazoxide, which represents the drug of first choice for the treatment of most hypoglycemic syndromes caused by islet cell adenoma or hyperplasia; -Propranolol, Chlorpromazine, Diphenylhydantoin, which may be regarded as a useful alternative to diazoxide, although they are capable of giving rather inconstant results. These drugs may today effectively substitute for corticosteroids and glucagon in the medical treatment of almost every chronic hyperinsulinemic hypoglycemic syndrome, including malignant beta cell carcinoma.


Onto- and phylogenetical aspects on insulin-producing islet-cell tumors. When onto- and phylogenetical aspects are taken into account when studying insulin-producing islet-cell tumors, it becomes clear that the recently discovered "type IV" islet parenchymal cells (or "delta 1" -cells) may play an important role in various states of hypoglycemia, both of neoplastic and non-neoplastic origin. Moreover, it becomes obvious that increased attention to this kind of islet cell through intensified light-microscopical, histochemical, ultrastructural and experimental investigations covering a wide range of animals, may help to clarify several unsolved problems in the cytological composition of the islets of Langerhans. The occurrence of islet-cell tumors (or hamartomas) in the most primitive vertebrate islet parenchyma known, viz. that of the hagfish, Myxine glutinosa, is reported, as well as an additional case of mixed carcinoid-islet-cell tumor in a human pancreas.


High-molecular IRI (big, big insulin) in islet cell adenoma. In agreement with data previously reported (Yalow and Berson 1973), after gel filtration of acid-ethanol extracts of islet cell adenomas (performed in 3 M acetic acid) 1.4% to 1.8% of total immunoreactive insulin (IRI) eluted ahead of proinsulin. The high-molecular IRI (HM-IRI) was, however, found to be heterogenous in size and consisted of at least three components, the major one having an estimated molecular weight of about 50,000. Under certain conditions, HM-IRI was partially dissociated into proinsulin- and insulin-like components. We conclude that HM-IRI does not represent a precursor of proinsulin and insulin, but probably a self-association product of the peptides or an association of insulin and proinsulin to other proteins extracted from tumor tissue.


Oral glucose tolerance test (OGTT) during epinephrine infusion in three patients with insulomas. An epinephrine infusion (6 mug/min) in three patients with insulomas inhibited insulin secretion induced by OGTT and increased glucagon secretion in two patients, with an exceptional rise in a case of poly-adenomatosis.


Histochemistry, ultrastructure and hormone content of human insulinomas. Forty human insulin-producing tumors were investigated with histochemical, immunohistological and ultrastructural methods and extracted for insulin and proinsulin. These studies resulted in the following findings: 1) A variable number of tumor cells contained only a few or often no beta granules. 2) The insulin concentration was lower and the proinsulin percentage higher in insulinoma cells compared with normal beta-cells. 3) According to the ultrastructural appearance of the secretory granules four types of insulinoma were established. 4) Insulinomas frequently contained cells with atypical secretory granules which were Grimelius silver-positive. Their similarity to the Type IV islet cell and to cells regularly found in gastrinomas, Verner-Morrison tumors and glucagonomas suggests that all endocrine pancreatic tumors originate from a common precursor cell. 5) The morphological and biochemical findings support the theory that uncontrolled hormone release and/or decreased storage capacity are responsible for fasting hyperinsulinism in insulinoma patients. 6) The severity of hypoglycemia symptoms and the result of stimulatory tests were not related to the size, the insulin concentration and the total insulin content of the tumor.


The federal mental health program: past, present, and future. Dr. Brown traces the history of America's federal mental health program from its beginning in the early 1900s. NIMH, the institute he currently directs, was established in 1946 for the treatment and prevention of mental and emotional illnesses through research,training, and services. It is now one of the institutes of the Alcohol, Drug Abuse, and Mental Health Administration of the Department of Health, Education, and Welfare. Dr. Brown describes its continuous progress toward providing high-quality mental health care to the entire population, and discusses priorities for the future that include continuation of research on schizophrenia and depression and research that will benefit children and the elderly.


Homologous passive cutaneous anaphylaxis (PCA) in mice and heterologous PCA induced in rats with mouse IgE. A study was made of the effect of anit-histamine, antiserotonin and of different anti-anaphylactic drugs on PCA reactions induced in mice with IgG1 or IgE. Further, using the ability of mouse IgE to sensitize rat mast cells, a comparative study was also made of PCA reactions induced in mice and rats with mouse IgE. Antihistamines produced a partial inhibition of PCA reactions induced in mice with mouse IgG1 or IgE and in rats with mouse IgE whereas antiserotonin inhibited PCA reactions induced in rats with mouse IgE, but had no effect on PCA reactions induced in mice with mouse IgG1 or IgE. The simultaneous use of antihistamine and antiserotonin resulted in a total inhibition of PCA reactions induced in mice with IgG1 and in a marked but not total inhibition of PCA reaction due to IgE; PCA reactions induced in rats with mouse IgE were totally inhibited. Compounds known to change the intracellular level of cyclic AMP were found to have little or no effect on PCA reactions induced in mice with either IgG1 or IgE in spite of producing a complete marked inhibition of PCA reactions induced with mouse IgE in rats. Diethylcarbamazine or disodium cromoglycate were also very effective inhibitors of rat PCA reactions induced with mouse IgE although having no effect on PCA reaction induced in mice with this same antibody or with IgG1. Thus, in spite of sharing common mediators released from the same type of target cell sensitized with the same type of antibody, PCA reactions induced in mice and rats with mouse IgE reacted very differently to the pharmacological effect of most of the drugs tested. This fact seems to indicate that the physiological mechanism operating in mouse mast cells are different from those operating in rat mast cells.


Rat IgE production. II. Primary and booster reaginic antibody responses following intradermal or oral immunization. Primary and booster IgE antibody responses have been elicited in Hooded Lister rats by the intradermal injection or oral administration of very small quantities of egg albumin. Oral immunization was effected by giving antigen by stomach tube or in the drinking water. The minimum primary dose of antigen found to be effective was 1 mug intradermally and 10 mug orally, administered together with an intraperitoneal injection of B. pertussis adjuvant. In rats immunized with these doses secondary responses could be evoked by giving even smaller quantities of antigen, thus 1 ng intradermally or 1 mug orally without adjuvant. Smaller challenge doses were not tried. Large primary doses of antigen (greater than 100 mug) presented by these routes were, on the other hand, found to be inhibitory to the production of secondary IgE responses, this effect being similar to that observed in previously reported intraperitoneal immunization experiments. By contrast with previous experiments, however, tertiary responses could be obtained following immunization by these routes, and we believe this to be reflection of the absorption of smaller and therefore less inhibitory quantities of antigen. Our results are discussed in relation to the control of IgE antibody production, current concepts of the control of antigen absorption through mucosal barriers, and possible implications of the genesis of naturally occurring IgE responses in man.


Production of circulating reaginic (IgE) antibodies by oral administration of ovalbumin to rats. Reaginic antibody synthesis following parenteral and/or oral administration of ovalbumin and Bordetella pertussis organisms as adjuvant has been evaluated in LOU/M/Wsl inbred rats. These rats are able to produce high reaginic antibody serum levels after intraperitoneal injection of this antigen. Primary oral administration of ovalbumin doses between 10 and 100 mg with Bordetella pertussis organisms given as adjuvant by the intraperitoneal or the oral route led to characteristic reagnic responses. Secondary reaginic responses were obtained by oral administration of ovalbumin without any adjuvant in animals sensitized by the oral or the intraperitoneal route. A hundred micrograms of ovalbumin was enough to induce reaginic responses but more constant and higher reaginic levels were obtained with a 50 mg dose in the experimental model employed.


The differential effect of cyclic AMP on lymphocyte stimulation by T- or B-cell mitogens. Dibutyryl-cyclic adenosine 3'5'-monophosphate (cAMP) added to mouse spleen cell cultures under serum free conditions inhibited the stimulation of [3H]thymidine incorporation induced by T-cell mitogens much more than the one induced by B-cell mitogens. The inhibition was most impressive with phytohaemagglutinin, followed by concanavalin A, pokeweed mitogen and the specific antigen Keyhole limpet haemocyanin (KLH). The response to bacterial lipopolysaccharide (LPS) was clearly less susceptible to suppression and the effect on the stimulation induced by trypsin and suramin, both B-cell mitogens, was marginal. Similar results were obtained by addition of isoproterenol or therophyllin to the cultures.


Two distinct lumphocyte-stimulating soluble factors (LAF) released from murine peritoneal cells. I. The cellular source and the effect of cGMP on their release. Culture fluids of murine peritoneal cells contain two distinct, non-dialysable priniciples (LAF) with thymocyte proliferation-stimulating properties. One of them is elaborated from phagocytic cells (presumably macrophages), the other is released by non-phagocytic cells (lymphocytes). cGMP added exogenously stimulates the production and/or release of LAF from phagocytic cells, but not from non-phagocytic cells. The phagocytic cells (but not the lymphocytes) require intact RNA and protein synthesis for LAF release. The action of LAF differs from those of cGMP itself by being non-dialysable, and unable to prevent the inhibitory action of cAMP on mitogen-stimulated lymphocyte proliferation.


Quatification of phagocytosis by human neutrophils. The use of radiolabelled staphylococcal protein A-IgG complexes. Human polymorphonuclear leucocytes suspended in a serum-free medium ingest radiolabelled insoluble staphylococcal protein A-IgG complexes, providing a quantitative measure of phagocytosis. The degree of uptake of protein A-IgG complexes is dependent on the cell concentration, the amount of immune precipitates and duration of incubation. The rate of phagocytosis is enhanced by the presence of serum. During the process of ingestion of immune precipitates, release of the lysosomal enzyme myeloperoxidase but not of the cytoplasmic enzyme lactic dehydrogenase occurs.


Marek's disease in chickens: development of viral antigen in feather follicles and of circulating antibodies. The infection of young chickens with Marek's disease herpesvirus (MDHV) leads to the early appearance of viral antigen in the bursa Fabricii, in the kidney tubular epithelium, and particularly in the epithelial cells of growing feather follicles (Calnek and Hitchner, 1969; Purchase, 1970). Viral antigen may persist in feather follicles over several months, whereas in neural lesions or in lymphoid tumors induced by MDHV viral antigen is either lacking or present only in a few cells (Calnek and Hitchner, 1969; Spencer and Calnek, 1970; Purchase, 1970). Haider et al. (1976) have pointed out the high diagnostic value of the double-diffusion agar-gel participitation test (Chubb and Churchill, 1968) using growing feathers of MDHV-infected chickens as antigen.


Some effects of silical treatment on Marek's disease. Treatment of newly hatched chicks with silica by the intraperitoneal route delayed the onset of mortalities due to the JM strain of Marek's disease (MD' virus inoculated at 6 days of age. During the 88-day observation period fewer silica-treated chicks died of MD, but this difference was not usually statistically significant. Silica treatment had no effect on the susceptibility of 4-week-old birds. Silica treatment reduced the antibody response to MD but, in general, not significantly. The antibody response to bovine serum albumin was significantly enhanced if measured by the indirect hemagglutination test but not if measured by the agar gel diffusion test, whereas the response to Brucella abortus was enhanced significantly in N-line (MD-resistant) chicks but not significantly in P-line (MD-susceptible) chicks. Five days after infection, silica-treated chicks had significantly less fluorescing antigen in thymus and bursa than did untreated chicks; no difference was observed in the spleen. After silica treatment the glass-adherent cell population in the buffy coat was increased by up to 10-fold compared with untreated chicks. It is suggested that silica treatment induced macrophage proliferation, with subsequent restriction of MD virus spread, yet allowed an adjuvant-type effect with other antigens.


Genetic control of natural immunity to ecotropic mouse leukemia viruses: production of endogenous immunogen. Mice of the AKR and C57L strains naturally produced low titers of antibody against ecotropic murine leukemia viruses (MuLV). The F1 hybrid of these strains produced anti-MuLV antibody in higher titer than mice of either of the parental strains. Progeny of the genetic backcross C57L X (AKR X C57L)F1 segregated for the production of infectious ecotropic MuLV (according to the Akv-1 and Akv-2 loci) and for the production of antibody against MuLV. All mice that contained infectious MuLV produced anti-MuLV antibodies. Thus, the persistent production of high-titered MuLV in these mice did not result in immunological tolerance towards viral antigens. In contrast, mice that did not contain infectious MuLV could be separated into antibody-producing and -nonproducing classes. The absence of detectable antibody to MuLV in an individual mouse was invariably associated with a virus-free phenotype. Antibody against MuLV reacted primarily with p15 and gp70 proteins of the viral envelope. It was concluded that overt production of endogenous ecotropic MuLV served as a major immunogenic stimulus for the production of anti-MuLV antibody in these mice.


Genetic control of natural immunity to ecotropic mouse leukemia viruses: immune response genes. Humoral immune response to ectropic leukemia viruses in AKR and C57BL/6 mice was controlled by a gene that mapped in linkage group IX. Mice of the AKR strain had an immune nonresponsive allele of this gene, whereas mice of the C57BL/6 strain had an immune responsive allele. Antibody against murine leukemia virus (MuLV) reacted primarily with p15 protein of the viral envelope. It was concluded that the failure to find antibody production in AKR mice was the result of a genetic immunological defect, rather than the result of immunological tolerance that was induced by the persistent viremia of endogenous MuLV.


Interaction of Chlamydia trachomatis organisms and HeLa 229 cells. The infection of HeLa 229 cells in monolayer culture with trachoma (B/TW-5/OT) and lymphogranuloma venereum (LGV) (L2/434/Bu) organism was studied in terms of two parameters: radioactivity counts of cell-associated tritium labeled organisms at the initial stage of inoculation for measurement of attachment, and inclusion counts of infection cells after incubation for measurement of growth. Factors affecting attachment and inclusion formation and correlation of the two are presented. It was shown that attachment is an important initial step in infection by Chlamydia trachomatis. The rate of attachment was temperature dependent. The attachment of LGV organisms was affected more profoundly by temperature than was that of trachoma organisms. Attachment and inclusion formation of trachoma and LGV organisms were inhibited by heparin. Diethylaminoethyl-dextran was again shown to enhance attachment and inclusion formation of trachoma but not LGV organisms. NaF had no effect on attachment, but inhibited inclusion formation of both trachoma and LGV organisms. Both attachment and inclusion formation of trachoma organisms were strongly enhanced by centrifugation of the inoculum onto the cell monolayer. Although inclusion formation of trachoma organism was much greater in susceptible cells (HeLa 229) than relatively insusceptible cells (fetal tonsil), attachment was only slightly greater. The results based on the test of two cell lines suggested that attachment prpbably is not a critical factor in determing a cell line's susceptibility to infection with trachoma organisms.


Quantitation of antibody to hepatitis A antigen by immune electron microscopy. A set of precipitin reactions was performed by immune electron microscopy (IEM) with hepatitis A antigen (HA Ag) and varying quantities of antibody to HA A q (anti-HA). Serial dilution of anti-HA resulted in progressive diminution in IEM antibody rating. These data, together with a highly significant correlation between IEM ratings and immune adherence hemagglutination (IAHA) titers on 92 coded serum samples, confirm that quantitative serology development can be performed by IEM. To demonstrate the different kinetics of antibody development by IEM and IAHA, we used both techniques to test for anti-HA in longitudinally collected sera from  a chimpanzee experimentally infected with hepatitis A virus. Detection of anti-HA was possible by IEM during acute hepatitis, but IAHA anti-HA was not observed until approximately 4 weeks later. Six weeks after acute illness, IEM ratings reached a plateau beyond which IAHA titers continued to rise gradually. Peak titers were achieved 11 months after inoculation.


Vaccinia virus meningitis in mice after intracerebral inoculation. The pathogenesis of experimental vaccinia virus infection in weanling mice after intracerebral inoculation was followed with virological, histological, and immunohistological methods. High-dose inoculation, virus spread from brain to thoracic and abdominal viscera probably by an undetected early viremia. Virus did rise to detectable levels in blood by day 5 and was found to be associated with the mononuclear cell fraction. By day 12, 30% of the animals had died and no further deaths occurred. Rise of neutralizing antibody correlated with disappearance of cell-free virus in blood, brain, and viscera. Virus was present in the brains of animals for 20 days after inoculation. This animal model may be useful to study mechanisms of persistent central nervous system virus disease relevant to man.


Comparison of central nervous system disease produced by wild-type and temperature-sensitive mutants of vesicular stomatitis virus. The pathogenicity of infection produced following intracerebral (i.c.) inoculation of wild-type vesicular stomatitis virus (VSV) or temperature-sensitive (ts) mutants of VSV was compared. ts mutants used were ts 31 (VSV complementation group II) and ts 41 (VSV complementation group IV). The i.c. injection of wild-type VSV in weanling Swiss mice produced a rapidly fatal encephalitis with death of mice in 2 to 3 days. Histopathologically, such mice exhibited minimal changes of encephalitis on light microscopy. In contrast to the highly virulent, rapidly fatal central nervous system (CNS) infection seen after i.c. inoculation of wild-type VSV, infection with ts 31 VSV produced a more slowly progressive CNS infection characterized by hind limb paralysis and death 6 to 9 days after infection. Histopathologically, CNS infection with ts 31 is associated with previously unreported extensive spongiform changes in the gray matter of the spinal cord. The inoculation of ts 41 i.c., on the other hand, did not result in either clinical illness or histopathological changes in the spinal cords or brains of infected mice. The absence of clinical and histopathological lesions following i.c. infection of ts 41 VSV suggests that the capacity to alter the pathogenesis of VSV CNS infection may be a function of only certain ts mutants of VSV.