Ultrastructural basis for alveolar-capillary permeability to protein. The intravenous injection into mice of small volumes (less than 0.1 ml) of peroxidatic enzymes of molecular weight of 40 000 daltons or greater results in little if any penetration of these probe molecules into endothelial junctions. The injection of cytochrome c (12 000 daltons), on the other hand, results in the localization of this tracer in some but not all endothelial junctions. When horseradish peroxidase (EC 1.11.1.7) is injected in a large volume of saline (0.5 ml), reaction product is present in endothelial junctions and basement membrane, but is prevented from entering the alveolar space by zonulae occludentes between epithelial cells. These experiments indicate that although endothelial junctions, under physiological conditions, are largely impermeable to molecules the size of horseradish peroxidase, and presumably most serum proteins, they are labile and susceptible to stretching if intravascular pressure is increased. Freeze-fracture studies show that pulmonary capillary endothelial junctions are composed of one or at the most two strands which show areas of discontinuity. Epithelial junctions, by contrast, are composed of a continuous, complex network of anastomosing fibres. These observations confirm physiological experiments which indicate that it is the pulmonary epithelium rather than the endothelium which determines the permeability properties of the alveolar-capillary membrane to lipid-insoluble molecules. Bidirectional pinocytic transport is an additional mechanism whereby lipid-insoluble molecules are transported across both endothelial and epithelial layers. The relative contribution of this transport mechanism to the total amount transported remains to be established.


The control of insulin release by sugars. The interaction of glucose, the major physiological regulator of insulin secretion, with the beta-cell involves the recognition of glucose as a signal, the transduction of this recognition into an intracellular event and the coupling of the event to the exocytotic discharge of insulin from secretory granules. The following aspects of this system are discussed: (1) the mechanism of insulin release; (2) the evidence implicating Ca2+ and cyclic AMP as coupling factors; (3) the main characteristics of glucose-stimulated insulin release; (4) gluco-receptor models and the evidence for them; (5) possible mechanisms for transduction of the response to glucose; (6) the extent to which the systems of the secretory response to sugars may also be involved in the control of proinsulin biosynthesis; (7) whether starvation induces specific changes in the glucoreceptor system.


The mode of action of adenosine 3':5'-cyclic phosphate in the regulation of insulin secretion. Changes in the pancreatic beta-cell concentrations of adenosine 3':5'-cyclic phosphate (cyclic AMP) may lead to changes in rates of insulin release, although little is known of the exact mechanism by which this nucleotide may influence the secretory process. Previous studies indicated that in the beta-cell, as in other mammalian cell types, the effects of cyclic AMP may be exerted by the activation of a cyclic AMP-dependent protein kinase, and we have attempted to identify possible substrates for this enzyme in beta-cells. Cyclic AMP stimulated the phosphorylation of specific non-nuclear protein substrates; this effect was observed both in intact cells preincubated with sodium [32P]phosphate to label intracellular ATP and in broken cell preparations incubated with [gamma-32P]ATP. The substrates for protein kinase in islets are unknown but as in other tissues might include microtubular protein and specific proteins of the granule and plasma membranes. In separate experiments cyclic AMP stimulated the efflux of calcium from an organelle-bound (probably mitochondrial) pool, and this may result in rapid changes of intracellular calcium distribution in the beta-cell; these might in turn play an important role in the regulation of secretion. These results suggest that cyclic AMP may directly affect cytosolic calcium concentrations in the beta-cell, as well as promoting the phosphorylation and activity of other components which may be necessary for the maintenance of adequate secretory responses.


Multiple forms of corticotropin (adrenocorticotropic hormone, ACTH) and their significance. Big corticotropin (adrenocroticotropic hormone, ACTH), an immunoreactive form of ACTH with low biological activity and which elutes in the void volume on Sephadex G-50 gel filtration, is found in plasma and extracts of human pituitary and tumour. Controlled tryptic digestion of big ACTH releases a product with full corticotropic activity which is indistinguishable from the (1-39) ACTH with respect to size, charge and susceptibility to tryptic digestion. Immunoreactive ACTH, predominantly in the big form, is found in virtually all tissue extracts of carcinoma primary to or metastatic from the lung, but not of carcinoma metastatic to the lung, and even in precancerous lung lesions. The absence of clinical Cushing syndrome in patients with carcinoma of the lung and moderate elevation of plasma concentrations of ACTH is due to the low biological activity of big ACTH. Prolonged survival (for more than two years) of patients with lung carcinoma has been observed only in those whose plasma ACTH is low before therapy or after resection of the lung tumour. Rabbit, rat and mouse pituitaries contain an intermediate sized ACTH but the usual 1-39 peptide predominates in the pituitaries of monkey, sheep, dog, cat and guinea pig, as well as man. The hormonal form of ACTH appears to be an important factor regulating the cortisol/corticosterone ratio in mammalian adrenal corticoid secretion because administration of porcine ACTH to rabbits alters the adrenal secretory pattern so as to decrease corticosterone production and increase cortisol production.


Membrane topology as revealed by the binding of macromolecules. Binding sites for specific molecules at the cell surface can be localized with the electron microscope in thin-section, freeze-etch and shadow-casting preparations. The receptors tested were binding sites to concanavalin A (Con A) labelled with haemocyanin and binding sites to insulin labelled with ferritin. Con A-binding sites were localized in endocrine cells of the pancreas and insulin-binding sites in isolated liver plasma membranes. The relevance of the topographical distribution of the binding sites to cell membrane organization is discussed.


Protein phosphorylation and hormone action. Although the scheme hormone leads to raised cyclic AMP levels leads to activated protein kinase leads to phosphorylated protein leads to physiological response may represent an outline for the action of several hormones, in the best understood example, namely regulation of glucogen metabolism in mammalian muscle, the picture is more complex. Modification of phosphorylase kinase by cyclic AMP-dependent protein kinase, after stimulation by adrenaline, leads to phosphorylation of the enzyme at two sites. Activation is associated exclusively with the phosphorylation of the primary site, but the secondary phosphorylation indirectly antagonizes the primary phosphorylation in that it is necessary to render the primary site susceptible to dephosphorylation. The recent separation of two distinct phosphorylase kinase phosphatases specific for the two sites shows that reversal of the hormonal stimulation is controlled by the relative activities of two enzymes with opposing functions. Glycogen synthetase, which is phosphorylated and inactivated by cyclic AMP-dependent protein kinase, is also under the control of insulin. Although insulin appears to stimulate glycogen synthetase by reversal of the inactivation catalysed by the cyclic AMP-dependent protein kinase, tissue cyclic AMP concentrations do not alter. The recent identification of a second glycogen synthetase kinase, unaffected by cyclic AMP, therefore raises the possibility that insulin action may also be mediated through phosphorylation-dephosphorylation mechanisms, which antagonize those mediated through cyclic AMP-dependent protein kinase.


Growth-promoting actions of peptide hormones. In common with other growth-promoting hormones, peptide hormones evoke multiple biochemical responses in their target tissues. These can be divided into two groups: (a) rapid effects involving permeability properties of the target cell to amino acids, sugars and ions or changes in key intracellular metabolites like cyclic nucleotides; (b) slow responses based on the stimulation of RNA and protein synthesis. The impossibility of explaining all the late events as the results of early changes raises the possibility that more than one species of hormone receptor exists. It is proposed that the final expression of growth and maturation results from the cooperative interaction of rapid and slow responses of the target cell to the hormone.


Prohormones of beta-melanotropin (beta-melanocyte-stimulating hormone, beta-MSH) and corticotropin (adrenocorticotropic hormone, ACTH): structure and activation. It is proposed that all peptide hormones and releasing factors are biosynthesized in the form of precursor molecules which are biologically inactive. Enzymic activation may take place by hydrolytic cleavage to release a terminal COOH group or by transmidation to form a COOH-terminal amide. Studies with pituitary prohormones and hormones are providing data that support this hypothesis. Evidence has been obtained that the 91 residue beta-lipotropin (beta-LPH) is the prohormone of beta-melanotropin (beta-MSH). The specificity of the pituitary enzymes involved in release of the hormone was demonstrated by the isolation of five constituent fragments of LPH, which were obtained in homogeneous form from the pituitary gland of the pig. The enzymes have specificities similar to trypsin and carboxypeptidase B; carboxypeptidase A and aminopeptidase activities do not appear to be involved. Mild digestion of beta-LPH by trypsin in vitro has confirmed the susceptibility of the peptide bond on the carboxy side of the paired basic residues at positions 59 and 60, adjacent to the COOH-terminus of beta-MSH, and tryptic digestion of a model peptide demonstrated the same specificity. The paired basic residues at positions 39 and 40 adjacent to the NH2-terminus of beta-MSH were more resistant to tryptic attack, both in LPH and in a model peptide. In the gland it is apparent that LPH is cleaved on the carboxy side of the paired lysyl residues at positions 39 and 40, whereas in the synthetic peptide cleavage takes place in between these residues. The activating enzyme may differ from trypsin; alternatively, explanation may be found in the conformation of the prohormone. Prediction of secondary indicates that both pairs of basic residues lie adjacent to beta-bends on the surface of the molecule and occupy sites accessible to enzymic attack. It seems likely that alpha-MSH and corticotropin (ACTH) share a common pro hormone. The release of ACTH could involve cleavage of a -Gly-Ser- bond in the prohormone to expose the NH2-terminus of the hormone. With alpha-MSH, a concerted acetylation and cleavage may take place to form the N-acetylserine residue; the COOH-terminus may be released as an amide by direct transamidation of a -Val-Gly- bond in the prohormone. Release of either hormone would be accompanied by the release of contiguous fragments of the prohormone. We have isolated two novel polypeptides from pig pituitary in substantial quantity and have determined the primary structures. They may represent fragments of a prohormone to alpha-MSH or ACTH.


On the mechanism of inhibition by iodine of the thyroid adenylate cyclase response to thyrotropic hormone. Rats maintained on a low-iodine diet were hypophysectomized, and their diet was than enriched with iodide. Cyclic AMP (cAMP) concentrations achieved in their thyroids following in vitro TSH stimulation were significantly lower than those in the thyroids of control animals that did not receive dietary iodide enrichment. The addition of 0.1% methimazole  (MMI) or 1% KC1O4 to the diet abolished this inhibitory effect of iodide. The administration of triiodothyronine in the died did not reproduce the inhibitory effect of iodide. The effect of iodide in vitro on the thyroid cAMP response to TSH was then investigated using paired thyroid lobes obtained from intact rats fed a low-iodine diet. During a 15-min incubation period, concentrations of iodide up to 10(-3)M, together with TSH (125 mU/ml), did not affect the thyroid cAMP response to TSH. In contrast, the preincubation of the lobes in 5 X 10(-5)M Nal for 2 h preceding a final 15-min incubation in medium containing TSH alone resulted in final cAMP concentrations significantly lower than those in paired lobes not exposed to iodide. Basal cAMP concentrations in thyroids not subjected to TSH stimulation were unaffected by preincubation in iodide. The inclusion of TSH during the preincubation period augmented the inhibitory effect of iodide on the final thyroid cAMP concentration achieved. The inclusion of MMI together with iodide during the preincubation period abolished the inhibitory effect of iodide on the final cAMP concentration achieved by TSH stimulation. Direct measurement of newly formed organic iodine in vitro demonstrated it to be inversely proportional to the final cAMP concentration achieved by TSH stimulation. The preincubation of thyroid lobes in iodide was without effect on the subsequent stimulation of cAMP by PGE1, or on the stimulation by F- of adenylate cyclase activity in the thyroid homogenate. The data support the concept of an as yet unknown organic form of iodine that limits thyroid adenylate cyclase responsiveness to TSH stimulation. This may, in part, explain the diverse, and generally inhibitory, actions of iodide on thyroid function.


Effect of pharmacological blockade of ACTH and TSH secretion on the acute stimulation of prolactin release by exposure to cold and ether stress. Acute exposure of male rats to cold (5C)leads to a rapid increase of plasma levels of thyrotropin (TSH), prolactin (PRL), corticosterone, and L-thyroxine. Exposure to ether is similarly followed by a rapid increase of plasma levels of PRL and corticosterone, while TSH release is inhibited. Acute treatment with dexamethasone (500 mug) inhibits almost completely the PRL response to both exposure to cold and ether stress, while the plasma TSH response to cold is only delayed and the decrease of plasma TSH observed after ether stress is unchanged. Basal plasma levels of both TSH and PRL are lowered after treatment with the steroid. Thyroxine treatment lowers the plasma TSH concentration to undetectable levels without affecting the plasma PRL response to cold or ether exposure. The present data suggest that the rise of plasma PRL observed after cold exposure is not related to TRH and may suggest that common mechanisms control ACTH and PRL secretion during acute stress exposure.


Evidence that cyclic AMP is not involved in the chronotropic action of glucagon in the adult mouse heart. The effects of glucagon on the adult mouse heart have been studied. Glucagon (1 mg/kg) increased heart rate in the adult mouse. This effect was enhanced in animals which had pretreated with reserpine to deplete catecholamines. No change in the cardiac cyclic AMP concentration after the injection of glucagon was seen in the hearts of reserpinized animals. Moreover, adenylyl cyclase activity, measured in two different laboratories in whole homogenates, 600 X g pellets or washed particles from adult mouse hearts, was not activated by glucagon. Finally, the injection of epinephrine (1.6 mg/kg), but not of glucagon (1.0 mg/kg), increased the per cent of cardiac phosphorylase a activity and depleted cardiac glucogen. Since increased levels of cyclic AMP are associated with an increase in the per cent of the cardiac phosphorylase a activity, these experiments provide evidence for the hypothesis that cyclic AMP is not essential for the rate effects of glucagon in the adult mouse.


Glucocorticoid receptors: evidence for a second, non-glucocorticoid binding site. Previous studies on cytoplasmic glucocorticoid receptors and enzyme induction led to the classification of steroids as inducers (optimal or sub-optimal), antagonists, or inactive steroids, with respect to their activity as glucocorticoids. The receptor was postulated to exist in allosteric equilibrium between two conformational states, one "active" and the other "inactive". Steroids behaved as inducers (optimal or sub-optimal), antagonists, or inactive steroids depending on their relative affinity for the active and inactive conformational state of the receptor. Another possible model would invoke multiple binding sites on a single receptor with interactions between the binding sites depending upon the particular steroid bound. To test this latter possibility, an experimental technique was developed to measure the rate of dissociation of tritiated dexamethasone ([3H]DM) or tritiated aldosterone ([3H]A) from the glucocorticoid receptor of rat liver or kidney cytosol. The dissociation of the [3H]DM-receptor at 25 C was not due to irreversible denaturation, and minimal recombination of the receptor with [3H]DM occurred. Progesterone and a number of other steroids consistently and significantly increased the dissociation rate of [3H]DM-receptor complexes in both liver and kidney cytosol. An identical effect was seen with hepatic glucocorticoid receptors labelled with [3H]A, like dexamethasone an optimal inducer. All steroids which enhanced glucocorticoid-receptor dissociation were either antagonists or sub-optimal inducers. Thus, it is postulated that glucocorticoid receptors have at least two classes of binding sites, and that occupation of the second site increases the dissociation rate of agonists from glucocorticoid receptors.


The porcine ovarian follicle: III. Development of chorionic gonadotropin receptors associated with increase in adenyl cyclase activity during follicle maturation. Specific binding of human chorionic gonadotropin (hCG), and the hCG-sensitive adenyl cyclase of granulosa cells from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) porcine ovarian follicles have been studied. The number of hCG-binding sites per cell (n) increases during follicle maturation without a change in the binding affinity. The values for n were 300-400 for small, 1,000-1,600 for medium, and 8,200-10,000 for large-follicle cells. The dissociation constant is 2.4 X 10(-10)M for all cells. hCG-sensitive adenyl cyclase was demonstrated in porcine granulosa cells. The adenyl cyclase system of granulosa cells becomes increasingly responsive to hCG stimulation during follicle development. Maximal adenyl cyclase activation by hCG (1 mug/ml) was 240, 750, and 7,000 molecules of cyclic AMP formed/sec/cell, respectively, for small, medium, and large follicle cell. The concentration of hCG giving half-maximal stimulation (1.0 X 10(-9)M) was similar for both large and medium follicle cells. It is concluded that: 1) an increase in hCG receptor sites per cell occurs during maturation of the porcine ovarian follicle without change of binding affinity, and 2) the increase in the number of hCG receptors correlates well with hCG-sensitive adenyl cyclase activity during follicle development.


Renin activity in dog brain: enzymological similarity to cathepsin D. The distribution and biochemical properties of the renin activity present in the dog brain were compared with those of the lysosomal enzyme cathepsin D. Renin and cathepsin activity were present in all brain regions studied, in association with high angiotensinase activity. Brain renin activity was partially purified by ammonium sulfate fractionation and Sephadex gel filtration, resulting in the removal of angiotensinase activity. The specific brain renin activity increased approximately one hundred times during this procedure; cathepsin D activity accompanied the brain renin activity throughout the purification and showed a similar increase in specific activity. The renin and cathepsin activity in the partially purified preparation behaved identically during isoelectric focusing. The partially purified renin and cathepsin activity exhibited saturation kinetics with their respective substrates and were without activity above pH 6.0. Both enzyme activities were irreversibly inhibited by the pepsin inhibitor pepstatin, in nanomolar concentrations. These data, in conjunction with the literature concerning brain cathepsin, suggest that the renin activity in brain is due to cathepsin D, and that this renin activity exhibited by cathepsin D may be of limited significance under physiological conditions.


The effect of experimentally induced focal epilepsy on operant responding in the rat. Dose-response studies were performed on freely moving rats bearing chronically implanted electrodes. The effects of intracortical injections of penicillin, bicuculline, and conjugated estrogens (Premarin) were determined on EEG activity and motor performance. Bicuculline in doses of 0.025 and 0.05% regularly produced spike and afterdischarge (AD) patterns of rapid onset (less than 10 sec,) brief duration (less than 20 min), and marked intensity. Lower doses had little or no effect. Premarin 1% and penicillin 100 international units produced spike and AD patterns of slower onset (5 min), longer duration (up to 2 hr), and mild intensity. Smaller doses generally produced no AD activity, whereas larger doses produced epileptic patterns severe enough to preclude their use in operant studies. Penicillin and Premarin both produced a significant decrease in operant-response rate in animals trained to bar press on a FR 20 schedule. A marked variability was noted in the behavioral responses of individual rats to the disruptive effects of the epileptogens.


Biphasic effects of imipramine in experimental models of epilepsy. In a variety of laboratory models of experimental epilepsy, imipramine exerts a biphasic action on the CNS as manifested by antiepileptic properties at low doses and convulsant effects at higher doses. In mice, imipramine (17.5-25 mg/kg, i.p.) blocks maximal electroshock seizures while exerting little or no effect on pentylenetetrazol-induced seizures. In cats, imipramine (2.5-15 mg/kg, i.v.) reduces penicillin and estrogen-induced epileptiform discharge, shortens afterdischarge duration and elevates afterdischarge threshold. Higher doses in mice induce neurotoxicity, including clonic seizures. In cats, doses above 20 mg/kg intensify chemically and electrically induced seizures and induce spontaneous epileptiform episodes. Such a biphasic action of imipramine may limit the drug's clinical utility as an antiepileptic agent and may provide an interesting tool for studies of catecholamines and brain excitability.


Three major forms of adenylate kinase from adult and fetal rat tissues. The major enzymatic forms of adenylate kinase have been purified to homogeneity from fetal liver and adult brain of the rat. The two enzymes differ with respect to isoelectric points, Km (ATP), Km (AMP), and Ka (citrate). Antibody to adult liver adenylate kinase does not inhibit either enzyme, while entibody to adult skeletal muscle enzyme inhibits the brain enzyme but not the fetal liver enzyme. It is therefore probable that there are three major forms of adenylate kinases in fetal and adult rat tissues.


Poly(A) hydrolase of chick-embryo fibroblasts. 1. Homogenates of cultured chick embryo fibroblasts have been quantitatively fractionated by differential centrifugation. Using cytochrome c oxidase, succinate dehydrogenase, acid phosphatase and NADPH-cytochrome c reductase as marker enzymes, poly(A) hydrolase has been shown to be a mitochondrial enzyme. 2. To test the biosynthetic origin of mitochondrial poly(A) hydrolase and to demonstrate its cytoplasmic site of synthesis, we have treated the cells with ethidium bromide, inhibitor of mitochondrial transcription, and chloramphenicol and cycloheximide, inhibitors of mitochondrial and cytoplasmic translations respectively. The activity of poly(A) hydrolase has been compared to that of succinate dehydrogenase, an enzyme coded for by the nuclear genome and that of cytochrome c oxidase, an enzyme coded for partly by the nuclear genome and partly by the mitochondrial genome. The results obtained indicate that in chick embryo fibroblasts poly(A) hydrolase is an enzyme coded for by the nuclear genome. Further, the hydrolase is synthesized on cytoplasmic ribosomes and has a half-life much shorter than succinate dehydrogenase and cytochrome c oxidase.


Protein phosphorylation of the smooth and rough endoplasmic reticulum in normal and neoplastic liver of the rat. Smooth and rough endoplasmic reticulum from rat liver and hepatomas exhibited endogenous protein kinase activity independent of adenosine 3':5'-monophosphate. The phosphorylation of smooth membranes by this process was consistently higher than that of rough membranes. When histone was added along with the smooth endoplasmic reticulum, cyclic AMP stimulated protein phosphorylation. Analysis of membrane-phosphorylated proteins by gel electrophoresis showed 5 major phosphorylated bands with estimated molecular weights of 155 000, 62 000, 50 000, 46 000 and 43 000, whereas major bands having estimated molecular weights of 62 000, 50 000 and 43 000 were found in membranes of the smooth endoplasmic reticulum of the Morris hepatoma 5123 C. Since previous studies in this and other laboratories have demonstrated the similarity of the protein components of membranes of the endoplasmic reticulum of normal liver and hepatoma, our findings indicate an inability of the protein kinase of hepatoma intracellular membranes to phosphorylate protein species that are found in membranes of both liver and the neoplasm.


Nitrate reductase from Chlorella vulgaris. Reaction with manganese (III) pyrophosphate and with ferric o-phenanthroline. Millimolar concentrations of tervalent manganese pyrophosphate can partially activate nitrate reductase which has been inactivated with NADH and HCN. The tervalent manganese complex is nevertheless not reduced by NADH in the presence of the enzyme, that is, it is not a substrate for the diaphorase moiety of the nitrate reductase. Ferric o-phenanthroline, on the other hand, is a good diaphorase substrate, but fails to activate the inactive enzyme.


Glycopeptides from bovine liver basement membrane and plasma membrane. Proteolytic digests of liver plasma-cell membranes from the cow were fractionated to yield two homogeneous glycopeptides and a third preparation about 92% pure. The composition of the two homogeneous glycopeptides made it clear that they were derived from basement membrane material rather than the plasma membrane. Ruminants are unusual in having large amounts of basement membrane in the liver while other animals generally have little or none. Both basement-membrane-derived glycopeptides contained a glucosyl galactosyl disaccharide linked to hydroxylysine, the smaller one contained no other sugar structure but the larger one contained in addition an acidic heterosaccharide, the two chains probably being linked separately to the same molecule. Smith degradation and beta-elimination operations show that this heterosaccharide has an inner structure containing mannose and hexosamine, with the sugars galactose, N-glycollyl-neuraminic acid and fucose situated more peripherally. The amino-acid-heterosaccharide linkage is alkali stable. The third glycopeptide, which may be plasma-membrane-derived, differs from the heterosaccharide described above in that it contains no glucose and contains some O-seryl and O-threonyl amino-acid--sugar linkage. It, too, has a periodate-resistant structure of hexosamine and mannose.


Spectroscopic investigation of binary and ternary coenzyme complexes of yeast alcohol dehydrogenase. Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.


NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane. NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.


Limited proteolysis of cytoplasmic and nuclear uterine estradiol receptors yields identical estradiol-binding fragments. Limited tryptic hydrolysis of the estradiol cytoplasmic receptor from calf uterus has been demonstrated to yield in a high-salt buffer a stable estradiol-binding molecule with the following characteristics: sedimentation coefficient 4.0 +/- 0.1 S; Stokes radius 3.5 +/- 0.05 nm; molecular weight 60000 (for an assumed v value of 0.73 ml g-1) and frictional ratio 1.36. Nuclear KCl extracts, prepared from uteri preincubated at 37 degrees C with labeled estradiol, were analysed by Sephadex G-200 chromatography and sucrose density gradient centrifugation. The following molecular parameters were found for the estradiol-receptor complex: sedimentation coefficient 4.4 +/- 0.1 S; Stokes radius 4.12 +/- 0.02 nm; molecular weight 77000 and frictional ratio 1.47 (v = 0.73 ml g-1). Limited tryptic proteolysis of this extract gave an estradiol-binding fragment with molecular characteristics identical to the trypsin-modified cytoplasmic receptor. In addition, mild tryptic digestion of whole labeled nuclei allowed us to solubilize almost quantitatively the nuclear [3H]estradiol in a macromolecular bound form. The molecule thus obtained showed molecular parameters very similar to the 60000-dalton trypsin fragments obtained from high-salt cytoplasmic and nuclear extracts. These molecules were undistinguishable by gel electrophoresis analysis at six different acrylamide concentrations. These results in conjunction with those derived from dissociation kinetics experiments and ligand specificity studies indicate the cytosolic protein is a functional part of the nuclear receptor. Based upon these and other studies we suggest that proteolytic cleavage of the estradiol-receptor complex, which results in the removal of the estradiol-binding sites from the nuclear recognition sites of the molecule, could play a role in the inactivation of the estradiol receptor in vivo.


Carboxymethylated liver alcohol dehydrogenase. Transient kinetic studies and effect of substrate structure on alcohol oxidation. 1. The rate constants for NADH binding and dissociation for carboxymethylated alcohol dehydrogenase have been determined and compared to those for the native enzyme. 2. Steady-state and transient kinetic experiments have shown that the hydrogen transfer step is rate-determining for oxidation of ethanol by carboxymethylated alcohol dehydrogenase. The rate constant of 0.19 s-1 is considerably slower than that for the native enzyme. 3. The steady-state parameter, V/[E], was obtained for each of a series of alcohols and correlated with the Taft sigma parameter. The linear relationship obtained indicates that the same step, hydrogen transfer, is rate-determining for all the alcohols. The sigma value obtained is the same as for the native enzyme; the implications of this for the mechanism of hydrogen transfer are discussed.


Cardiac slowing and respiratory arrest in preterm infants. Changes in respiration and heart rate during sleep states have been recorded by a polygraphic device in healthy preterm infants. Cardiac slowing/bradycardia often coincide with respiratory arrest/apnea. Bradycardia starts early during apneic spells. The incidence of respiratory arrest and cardiac slowing and their simultaneous occurrence is significantly increased by the active or REM sleep state. The physiologic, inhibitory mechanisms of active sleep suggest a neurogenic etiology of episodes of cardiac slowing/bradycardia and/or respiratory arrest/apnea in prematures.


The influence of phenobarbital on carbohydrate metabolism in developing rat liver. The influences of phenobarbital (PB) on enzymes of the carbohydrate metabolism in rat liver are examined during pre- and postnatal development. The following results are obtained: (1) PB generally increases G-6-Pase before and after birth. (2) Both the active and the inactive forms of phosphorylase are increased significantly during the prenatal periods. During the postnatal periods, mainly the active form of phosphorylase is influenced by PB. (3) Total glycogen synthetase is increased by PB during prenatal development but decreased during postnatal development. (4) The activity of alpha-glucosidase is increased during the prenatal period. (5) The activity of F-6-PK and 6-PGDH are decreased during the prenatal periods and G-6-PDH activity is decreased during both pre- and postnatal periods.


Chromosome abnormalities in neurological diseases. The current status of research into chromosomal abnormalities in neurological diseases is reviewed. The only possible association between chromosome aberration and neurological disorder is found in ataxia telangiectasia and in tumours of the nervous system. In the remaining diseases reviewed, no specific association was confirmed. This was expected to some extent, since the majority of these diseases (spinal muscular atrophies, muscular dystrophies, etc.) are due to single gene defects.


Hyperlipidemic neuropathy and dementia.

Hyperlipidemic neuropathy and dementia. Mixed types of sensory and motor peripheral neuropathy and dementia occurred as neurological complications in patients with established type IV and type V hyperlipoproteinemia. These complications were remedial by control of the hyperlipoproteinemia with diet and/or clofibrate resulting in symptomatic improvement as well as restitution of nerve conduction velocities toward normal. Diabetes mellitus as well as systemic metabolic and toxic disorders which commonly produce neurological complications were excluded. Segmental demyelination with disorganization of myelin lamellae were striking morphological features found on sural nerve biopsy. Fluctuations in memory performance correlated inversely with plasma lipid levels and appear to be a characteristic feature of hyperlipidemic dementia. Hyperlipidemic neuropathy and dementia, although rare, are remediable neurological disorders which should be considered in patients with neuropathy and/or dementia of unknown origin.


Effects of intravenous phospholipid on low density lipoprotein turnover in man. The mechanism of the rise in plasma low density lipoprotein (LDL) levels following intravenous administration of a triglyceride-phospholipid emulsion (Intralipid) has been investigated by measuring LDL turnover in eight healthy subjects. The plasma half-life, and the absolute and fractional catabolic rates of LDL protein (apo-LDL) were unaffected by intragastric Intralipid, whereas apo-LDL half-life was prolonged and its fractional catabolic rate was decreased by intravenous Intralipid. Similar changes were observed after intravenous administration of the egg phospholipid constituent of Intralipid. Accompanying increases in the oleate: linoleate ratio of both high and low density lipoprotein cholesterol esters were secondary to phospholipid exchange between infused and endogenous lecithin. These results suggest that the increased concentration of LDL in plasma following intravenous administration of egg phospholipid-containing emulsions was due, at least in part, to a decrease in the fractional catabolic rate of apo-LDL. The data further suggest a possible relationship between apo-LDL catabolism and the fatty acid composition of LDL.


Hydrosoluble immunostimulants of bacterial and synthetic origins. The role of whole Mycobacteria, mycobacterial cell walls and waxes D as immunostimulants was well established many years ago. More recently three different research groups have shown that hydrosoluble components from mycobacterial and other bacterial origins were as active as waxes D or cell walls and were free of many side-effects. Studies concerning the relationship between structure and activity were achieved which led to the description of a small biologically active fragment and to a first series of synthetic compounds.


Hoplonemertine worms -- a new source of pyridine neurotoxins. Two pyridine bases were isolated from the marine hoplonemertine Amphiporus angulatus (Fabricius) and identified by mass and PMR-spectroscopy as 2, 3'-bipyridyl and 3, 2'; 3', 2"; 4", 3"'-tetrapyridyl (Nemertelline). Nemertelline, the first tetrapyridyl natural product to be reported, shows a structural resemblance to the tobacco constituent nicotelline. The crustacean toxicity of 2, 3' -bipyridyl is very similar to that of nicotine, but its mammalian toxicity is negligible.


The orthodox-paradoxical sleep cycle in the rat. Under the postulated existence of a mechanism regulating the NREM sleep- REM sleep sequence and a reset of this mechanism by long awakenings, the variability of sleep cycle in the rat was studied. Awakenings of various durations were included in the definition of sleep cycle boundaries. Results show that an intervening awakening of 1 min is close to the limit under which the same cycle seems to be resumed after the awakening and above which the previous cycle is abortive and a new cycle will start after the next sleep onset.


Altered brain cyclic AMP-responses in rats reared in enriched or impoverished environments. The accumulation of radioactive cyclic AMP elicited by various neurohormones has been examined in adenine-labeled telencephalon slices from rats raised in enriched or impoverished environments. Basal levels of cyclic AMP and responses of the brain slice cyclic AMP-generating systems to norepinephrine, isoproterenol and adenosine did not differ between the two group of rats, while responses to prostaglandin E1 were significantly greater with the impoverished group and responses to histamine appeared to be greater with the enriched group.


Effects of taurine on the transmembrane ionic currents of rat and guinea pig ventricular fibres. The effects of taurine on electrical properties of isolated myocardial ventricular fibres were studied in rat and guinea pig. The action potential amplitude was decreased in these species while the plateau and the repolarization phase were not altered. This suggests that simple inspection of the plateau phase is not sensitive enough to pick up changes in outward current since voltage clamp experiments showed that taurine decreased both potassium current and fast inward current. However, the slow inward calcium current remained unchanged with taurine in each species. These results suggest that the inotropic effects of taurine on rat and guinea pig heart contraction already described, were not due to modifications of the cell membrane permeability for calcium. It seems more reasonable to assume that the cardiac actions of this compound result from intracellular changes in calcium availability.


The effect of egg phospholipid administration upon liver enzymic activities during ethionine treatment. Female rats were injected subcutaneously with ethionine, and enzymic activities of liver membranes (Na+-k+-stimulated ATPase, Mg2+-stimulated ATPase, glucose-6-phosphatase, NADPH: cytochrome c oxido-reductase and NAD-nucleosidase) examined at proper intervals, during the intraperitoneal treatment of an egg phospholipid preparation (EPL). It is shown that EPL is unable to overcome the enzymic changes due to severe ethionine treatment, but is able to facilitate the recovery times after drug withdrawal for all the enzymic activities, except for NAD-nucleosidase. At lower dosage of the drug, the ethionine treatment is able to prevent the observed change of the glucose-6-phosphatase activity but not that of the Mg2+-ATPase. It is suggested that the EPL treatment may modify the chemical composition ahd/or architecture of liver membranes, altered by the ethionine injection, thus acting, at least partially, on the enzymic changes.


Comparison of the evolution of heme binding and NAD binding proteins. Of the 85 three-dimensionally characterized residues of cytochrome b5, 51 are structurally and topologically equivalent to the globin fold. When these proteins have been superimposed, the heme irons are found to be less than 1.4 A separated and the heme normals are inclined by less than 9.5 degrees. Comparison of minimum base changes per codon between heme binding and NAD binding proteins are of the same order.


Do evolutionary changes in cytochrome c structure reflect functional adaptations? Following the demonstration that the rate of evolutionary change in the amino acid sequences of cytochromes c of eukaryotic species was not constant either for a single line of phylogenetic descent during different evolutionary intervals or for separate lines of descent, the concept that neutral mutations account for the vast majority of the evolutionary variations could no longer be accepted. Previous studies had shown that all eukaryotic cytochromes c tested appeared to be functionally indistinguishable in their reaction with mitochondrial respiratory chain components. However, an examination of the kinetics at low ionic strength led to the discovery of a high affinity reaction of cytochrome c with cytochrome c oxidase that revealed large differences in activity between the cytochromes of the horse, baker's yeast and the protist Euglena. Observed Km values for this reaction of 10(-7) to 10(-8) M appear to represent actual dissociation constants, as demonstrated by direct binding studies of cytochrome c with purified cytochrome c oxidase. The high affinity reaction is sensitive to ionic strength and inhibited by ADP and ATP in the range of physiological concentrations, ATP being three times as effective as ADP. The possibility is discussed that this effect of ATP on cytochrome c binding to its oxidase could provide the basis of a mechanism for mitochondrial respiratory control. The demonstration of differences between cytochrome c of various species in this kinetic system opens the way to a systematic study of the possible evolutionary adaptations of cytochromes c to their oxidases.


The origin and evolution of protein superfamilies. The organization of proteins into superfamilies based primarily on their sequences is introduced: examples are given of the methods used to cluster the related sequences and to elucidate the evolutionary history of the corresponding genes within each superfamily. Within the framework of this organization, the amount of sequence information currently and potentially available in all living forms can be discussed. The 116 superfamilies already sampled reflect possibly 10% of the total number. There are related proteins from many species in all of these superfamilies, suggesting that the origin of a new superfamily is rare indeed. The proteins so far sequenced are so rigorously conserved by the evolutionary process that we would expect to recognize as related descendants of any protein found in the ancestral vertebrate. The evolutionary history of the thyrotropin-gonadotropin beta chain superfamily is discussed in detail as an example. Some proteins are so constrained in structure that related forms can be recognized in prokaryotes and eukaryotes. Evolution in these superfamilies can be traced back close to the origin of life itself. From the evolutionary tree of the c-type cytochromes the identity of the prokaryote types involved in the symbiotic origin of mitochondria and chloroplasts begins to emerge.


The mechanism of action of the copper intrauterine device. The effects of copper ions on the binding of steroids to receptors revealed that the inhibitory effect of Cu++ was apparent at 10(-6)M, ANd the binding capacities decreased to 10% at 10(-2)M Cu++. The kinetic study demonstrated that Cu++ was a competitive inhibitor of steroid hormone-receptor binding (Ki divided by 2.7 X 10(-5)M to estrogen receptor; Ki divided by 5.1 X 10(-6)M to progesterone receptor). These results indicate that copper ions interfere at the steroid-binding site of receptor and that progesterone receptor is more affected by copper ions than is estrogen receptor. The sedimentation pattern showed the dissociation and aggregation of receptor macromolecules by copper. These phenomena may indicate the biologic inactivation of receptor. In fact, morphologically, progestational proliferation was severely inhibited and estrogenic action seemed to be inhibited. The Timm stain showed copper uptake by endometrial epithelium and superficial stromata. The copper content apparently increased in the cytoplasm of uteri bearing a copper intrauterine device, compared with controls. In vivo, the concentration of cytoplasmic copper was approximately 1.4 X 10(-6)M, which was obviously inhibitory to steroid hormone-receptor interaction. However, complete morphologic suppression of the progestational effect by copper cannot exclude the coexistence of some other mechanism in these phenomena.


Gonadotropin binding sites in human postmenopausal ovaries. Glucose-6-phosphate dehydrogenase (G6PD) activity and gonadotropin binding sites were localized within seven postmenopausal ovaries. G6PD was localized in the cells of cortical stroma and hilus using a histochemical technique for the reduction of the tetrazolium salt, Nitro-Blue tetrazolium. Gonadotropin binding sites were localized by autoradiography following incubation of ovarian sections with (125I-hLH) and 125I-labeled follicle-stimulating hormone (125I-hFSH) were identified in the cortical stroma and hilus cells. Since these cells contain G6PD and other enzymes necessary for steroidogenesis and also have the capacity to bind both hLH and hFSH, steroidogenesis in postmenopausal ovaries appears to be controlled by circulating gonadotropins. Blood vessels within postmenopausal ovaries also bound both gonadotropins, but 125I-hFSH binding was often more intense than 125I-hLH binding.


[Investigations of the morphology and the function of human Sertoli cells (author's transl)]. Ultrastructurally and enzyme histochemically two types of activities of Sertoli cells are to be proved. By means of closest membrane contact and intensive energetic changes of substances, the active cells participate largely in the ripening of spermatozoa. Steroid biosynthesis in Sertoli cells is possible according to their pattern of organelles and enzymes. The ultrastructure of Charcot-Böttcher's crystalloids and the other characteristic inclusions in the cytoplasma of the Sertoli cells as well as implications to their function are described.


Morphological and biochemical changes in the hepatic endoplasmic reticulum and golgi apparatus of male Xenopus laevis after induction of egg-yolk protein synthesis by oestradiol-17 beta. This report describes morphological and biochemical changes accompanying oestrogen induced synthesis of the egg-yolk protein precursor, vitellogenin, in male Xenopus liver. Extensive proliferation of the rough and smooth endoplasmic reticulum and the Golgi apparatus occurs between 3 and 9 days after administration of oestradiol-17 beta. Subcellular fractionation showed that microsomal fractions have an increased number of ribosomes available for protein synthesis, hormone treatment enhances the in vitro protein synthetic capacity per unit of RNA; both in microsome and ribosome preparations. Polypeptides synthesized in vitro by ribosome preparations show an enrichment in serine content after hormone treatment and an increased proportion of ribosomes can be immunoprecipitated by antibodies directed against vitellogenin. Our data are consistent with the proposal that vitellogenin is synthesized on the ribosomes of the rough endoplasmic reticulum and processed and packaged for secretion in the smooth endoplasmic reticulum and Golgi apparatus. Response to hormonal induction of vitellogenin involves an early phase in which membrane proliferation occurs in order to increase the cellular capacity to synthesize, process and secrete large quantities of egg-yolk protein precursor.


Different effects of cycloheximide and chloramphenicol on corticosterone production by isolated rat adrenal cells. Cycloheximide and chloramphenicol both inhibit the stimulating effect of adenocorticotropic hormone (ACTH) on adrenal steroid production. To test whether these inhibitors had andy effect on adrenal steroid production, independent fromthe mechanism of action of ACTH we investigated their effect on the conversion of 25-hydroxycholesterol into corticosterone in isolated rat adrenal cells. Cycloheximide, both in the absence and in the presence of ACTH, had no effect on this conversion. Chloramphenicol inhibited the conversion of 25-hydroxycholesterol into corticosterone whether ACTH has no direct efeect on the cholesterol side-chain cleaving system. The inhibition by chloramphenicol of the ACTH-stimulated steroid production is at least partly due to inhibition of one or more of the processes involved in the conversion of 25-hydroxycholesterol into corticosterone.


Cryoprotein complexes and peripheral neuropathy in a patient with chronic active hepatitis. A cryoprotein complex was isolated and characterized from a patient with chronic active hepatitis and a severe peripheral neuropathy. This cryoprotein was composed of IgM, IgG, and hepatitis B surface antigen (HBsAg) and had a concentration of approximately 36 mg per 100 ml of serum. Electron microscopic examination of the cryoprotein demonstrated aggregates of Dane particles in close association with the antigenically related tubular and spherical forms of HBsAg. HBsAg, IgM, and IgG were detected by immunofluorescent staining in the intima of small arteries and veins. The association of a high serum level of cryoprotein and deposition of the cryoprotein components in small blood vessels suggests a role for the cryoprotein in the pathogenesis of peripheral neuropathy in this patient with chronic active hepatitis.


Hepatitis in an adult caused by Herpes simplex virus type I. Disseminated infection caused by Herpes simplex virus (HSV) type I is described in a previously healthy adult. The clinical course was characterized by progressive hepatic failure, fever, hypotension, atypical lymphocytosis, and disseminated intravascular coagulation. No skin lesions were present and the causal agent was not suspended premortem; however, HSV-I was isolated from autopsy liver, and characteristic histopathological lesions of herpes infection were observed in liver and esophagus.


Syndactyly and split hand.

Syndactyly and split hand. Careful analysis of clinical and radiological features of both webbing of normal fingers and typical split hand, suggests that these two types of malformations should not be classified separately. Study of human embryos and experimental malformations of the apical ectodermal ridge, supplies a resonable explanation for the close relation between webbing of normal fingers and typical split hand.


Corticosteroid effect on eosinophils in vitro: ultrastructural studies. The effect of corticosteroids, ultracortene and ACTH, on the eosinophilic cells of a patient with hypereosinophilic syndrome has been studied in vitro. Both drugs caused a marked decrease in the number of specific granules, a disappearance of their surrounding membrane and an almost complete destruction of their crystals. In addition, vacuolization of the cytoplasm, swelling of the mitochondria and distortion of their cristae were found. Incubation of the eosinophils with the antihistaminic drug mepyramine, did not produce ultrastructural alterations.


Cytostatic treatment of polycythaemia rubra vera. Comparison of the effects of some cytostatics in 100 patients in a period of five years. Experience with cytostatic treatment performed in patients with polycythaemia rubra vera is reviewed. The effectivity and side effects of the drugs applied are evaluated. Mannosulfan and mitobromitol were the drugs most sutiable for treatment. In certain special cases, 5-hydroxyurea was also satisfactory, while mitolactol was the least suitable.


Biochemical characterization and visualization of plasma membrane-DNA-protein complexes from Bacillus subtilis. Subcellular fractions containing plasma membranes with bound DNA and associated proteins were isolated from two different cultures of Bacillus subtilis 168 growing exponentially at different rates. Differences in the contents of individual between the dissociated complexes, established electrophoretically, can be explained by dynamic binding of the proteins to DNA, resulting in a control of DNA, resulting in a control of DNA replication. Electron microphotographs of isolated complexes display, in addition to unit membranes, associated filamentous structures in different arragement. Patterns obtained after treating the complexes with nucleases suggest a polydeoxyribonucleotide character of the filamentous structures.


Studies on the pituitary "fettstoffwechselhormon". VI. Preparation of two highly purified lipolytic active peptides from hog pituitary glands. The preparation of two highly purified lipolytically active hog pituitary peptides, called P-LF II C and P-LF II D is described. The two peptides are free of other pituitary hormone activities. In isolated rat and porcine adipose tissue, both fractions are lipolytically much more active than every other lipolytic active pituitary peptide described to date. By fraction P-LF II D, the first pituitary peptide was isolated which has lipolytic activity in isolated rat adipose tissue than corticotropin, the lipolytically most active pituitary hormone known so far. On isolated porcine adipose tissue, fraction P-LF II D as well as P-LF II C showed without doubt higher activity than corticotropin.


The effects of estradiol administration of the hepatic activities of some enzymes of carbohydrate, amino acid and lipid metabolism in the immature pullet. Two experiments were performed to examine the effects of intramuscular estradiol administration on the hepatic specific activities of some enzymes of lipid, carbohydrate and amino acid metabolism in the immature fowl. Estradiol increased the specific activities of the hepatic lipogenic enzymes, ATP citrate lyase and malate dehydrogenase (decarboxylating) (NADP), but had no effects on the activities of the glycolytic, gluconeogenic and amino acid metabolising enzymes except for pyruvate kinase and glutamate dehydrogenase which were reduced in activity in both experiments. The results indicate that the estrogen-induced increase in hepatic lipid biosynthesis is due to a specific effect on lipid metabolism and not to a general increase in liver metabolism.


Demonstration that cyclic adenosine 3',5'-monophosphate mediates the lipolytic action of parathyroid hormone. Studies were carried out with rat epididymal fat pads first to compare the effects of the synthetic N-terminal 1-34 peptide of bovine parathyroid hormone and of the native hormone to determine whether this portion of the molecule is responsible for the lipolytic action of the hormone and second to determine whether this biologic action of parathyroid hormone is mediated by cyclic adenosine 3',5'-monophosphate. The N-terminal polypeptide was as effective as the native hormone in stimulating lipolysis in the concentration range between 10(-8) M and 10(-6) M. Parathyroid hormone stimulated lipolysis by isolated fat cells. The concentration of cyclic adenosine 3',5'-monophosphate in the fat pads was significantly increased by the hormone (10(-6)M). Lipolytic stimulation by parathyroid hormone (10(-6)M) was diminished by insulin (100 muU/ml) and prostaglandin E1 (1 mug/ml), both of which are known inhibitors of lipolysis. The findings indicate that the amino-terminal 1-34 peptide portion of parathyroid hormone is responsible for the lipolytic action and that this effect is mediated through cyclic adenosine 3',5'-monophosphate.


[Virus infections of the respiratory tract of dogs]. Canine respiratory infections are reviewed with special reference to those caused by agents, other than distemper and canine infectious hepatitis viruses, but producing, both naturally and experimentally, the so-called distemper-like syndromes. The existence of canine infections caused by Adeno-Herpes-,Reo-, and Paramyxoviruses is reported, and for each of these agents the physicochemical characteristics, pathogenicity for dogs and other animal species, incidence and methods of dissemination are described. As regrads the prophylaxis of the individual infections that appear particularly widespread in kennels, the possible availability in the short or long term of immunizing preparations is indicated, along with the basic measures of hygiene for their control. The availability of immunizing preparations depends, firstly, on a more exact knowledge of the aetiological agents involved in canine respiratory conditions, and secondly, on a precise determination of the mechanisms by which the immune reactions are elicited. It is, however, obvious that the incidence of respiratory infections could be substantially reduced by the application of all the hygienic measures that can ensure a high environmental standard for the animals.


The role of calcium ion in epinephrine activation of lipolysis. Adipocytes were prepared by collagenase digestion of rat epididymal adipose tissue and incubated for 5, 15 or 30 minutes in Krebs-Ringer bicarbonate buffer containing albumin (40 mg/ml), glucose (1 mg/ml) and epinephrine. Calcium ion was present in some incubations at concentration of 2.5 mM and omitted from others; media with no added calcium contained 1.0 mM EGTA thereby producing a final calcium concentration of less than 10(-7) M. Glycerol release and accumulation of cyclic AMP were measured. Basal lipolysis and cell cyclic AMP levels were increased slightly but not significantly when adipocytes were incubated in calcium free media. Lipolysis could be activated with epinephrine in the absence of calcium but the sensitivity of the lipolytic response was greatly reduced; however, the maximum lipolytic response to epinephrine was not decreased in calcium free media. Similarly, incubation of adipocytes in calcium free media resulted in decreased accumulation of cyclic AMP in response to epinephrine but only when sub-maximum concentrations of the catecholamine were present. Varying the extracellular calcium concentration showed that a concentration of at least 10(-5) M was optimal for epinephrine activation of lipolysis. These observations are considered in accord with the view that activation of adenylate cyclase is facilitated by calcium ion.


Cyclic AMP level of human thyroid cells in monolayer culture. TSH induced refractoriness to TSH action. Thyroid cells from euthyroid patients with Graves' disease were cultured in a chemically defined medium. The cells preserved the ability to respond to TSH with 8-fold increase in cyclic AMP concentration. This cyclic AMP response to TSH was diminished by prior exposure of cells to TSH. The decrease in cyclic AMP response to TSH induced to TSH was reversible, was not associated with a similar decrease to cyclic AMP response to PGE1, and could not be attributed to increased phosphodiesterase activity or to decreased adenyl cyclase activity. The partial resistence to TSH stimulation of thyroid cells previously exposed to TSH may be due to changes in the TSH receptor, possibly caused by TSH itself.


The different modes of action of thyrotropin and prostaglandin E1 on cyclic adenosine 3',5'-monophosphate synthesis in human thyroid, as studied by sequential stimulations. PGE1 was equally effective in increasing 3H-cyclic AMP in normal and in toxic thyroids, whereas TSH was less effective but over a longer time in the toxic thyroids. Stimulation by a large second dose of TSH could not be elicited after prior stimulation by large doses of TSH. Similar results were obtained with regard to the effect of PGE1. However, stimulation by a large dose of PGE1 was still effective after the slices became refractory to TSH. Similarly, stimulation by a large dose of TSH was still effective after the slices became refractory to PGE1. It is suggested that the site and/or mode of action of TSH is quite different from that of PGE1.


Pituitary and extrapituitary effects of somatostatin in normal man. The effects of synthetic linear somatostatin on basal circulating levels on several pituitary and pancreatic hormones, and of glucose and free fatty acids (FFA) were studied in 6 normal men after an overnight fast. A priming intravenous infusion of 250 mug of somatostatin in 18 sec was followed by a constant infusion of 500 mug over a period of 60 min. A decrease in plasma values of GH, prolactin, TSH, insulin and glucagon and in blood glucose was observed during somatostatin infusion, while FFA levels increased progressively. Plasma IRI and blood glucose increased rapidly when the somatostatin infusion was stopped, while FFA decreased progressively; GH, prolactin, TSH and glucagon remained low as compared to basal levels for one hour after the end of the infusion, i.e. until the end of the experiment. A slight but significant increase of LH and ACTH was observed after the end of the infusion.


Variation of several erythrocyte enzymes in the Dayaks of Sarawak. The Land and Sea Dayaks of Sarawak were surveyed for several erythrocyte enzymes. The gene frequency of 6PGDC in 132 Land Dayaks and 127 Sea Dayaks were 0.045 and 0.047, respectively. The gene frequency of PGM1-1 IN 285 Land Dayks and 240 Sea Dayaks were 0.716 and 0.779, respectively. The ADA2 gene frequency in 283 Land Dayaks and 188 Sea Dayaks were 0.154 and 0.090. ADA 5-1 was found once in the Land Dayaks and once in the Sea Dayaks. AK 2-1 was found once in 221 Sea Dayaks but not in any of 270 Land Dayaks. No PHI, LDH or CA variants were found among the Land or Sea Dayaks.


The formation of histotypic structures from monodisperse fetal rat lung cells cultured on a three-dimensional substrate. Enzymatically dissociated lungs from rat fetuses at 19-days gestation yield single cells which reaggregate to form alveolar-like structures when cultured on gelatin sponge discs. These structures form within 2 days and have been maintained in vitro for as long as 6 weeks. They are composed primarily of type II pneumonocytes as characterized by large, lightly stained nuclei and cytoplasmic inclusion bodies. The lamellar structure of these inclusion bodies has been confirmed by electron microscopy. The dynamic formation of inclusion bodies is suggested by the presence of lamellar bodies in the extra-cellular space and the appearance of new inclusions in the cytoplasm of the type II pneumonocytes. The formation and long-term maintenance of histotypic lung structures in vitro provides a model system for the study of lung development and synthesis of surfactant by type II alveolar pneumonocytes.


Suggestive evidence for both stimulatory and inhibitory domains on human lymphocytes, as indicated by phospholipid turnover studies with wheat germ agglutinin and other lectins. Short term effects (1 hours or less) of various lectins on phospholipid turnover in human lymphocytes were studied. As expected, concanvalin A and phytohemagglutinin produced 1,5-4.0 fold increases in incorporation of 32PO4 radioactivity into phospholipids (primarily phosphatidylinositol). Wheat germ agglutinin, a nonmitogenic lectin, not only failed to produce a response but actually inhibited phospholipid turnover, both in the presence and absence of PHA or con A. Since wheat germ agglutinin did not appear to be cytotoxic, as defined by a failure to see changes in vital dye uptake, and other evidence from our laboratory indicates that this lectin also inhibits aminisobutyric acid transport and DNA synthesis in human lymphocytes we would tentatively interpret its negative action as indicating the existence of specific inhibitory domains on the cell surface.


Immunological and biochemical studies of meningococcal C polysaccharides isolated by diethylaminoethyl chromatography. Meningococcal C polysaccharides isolated from the broth supernatant of C1+ and C1- strains by a combination of Sepharose 4B and diethylaminoethyl-52 chromatography are antigenically identical to the group C antigen isolated by the Cetavlon procedure. Immunizations result in the induction of precipitating, hemagglutinating, and bactericidal antibodies in rabbits and sheep and hemagglutinating and bactericidal antibodies in rabbits and sheep and hemagglutinating and bactericidal antibodies in gerbils. Selective destruction of C1- polysaccharides with neuraminidase has demonstrated that these antibodies are directed against the C polysaccharide. Neuraminidase digestions of the C1- polysaccharides resulted in release of up to 84% of the antigen as N-acetylneuraminic acid. C1+ polysaccharides proved to be more resistant to neuraminidase, requiring greater concentrations of enzyme and longer intervals of exposure for digestion. Chemical analysis of the C1+ and C1- polysaccharides indicated that they contain less than 0.5% protein and less than 0.5% nucleic acid.


Thermal inactivation of rabies and other rhabdoviruses: stabilization by the chelating agent ethylenediaminetetraacetic acid at physiological temperatures. Thermal inactivation of rabies and several other rhabdoviruses was studied using virus suspended in several different diluents. Rabies serogroup viruses were more stable than Kern Canyon or vesicular stomatitis viruses. Limited studies of two fish rhabdoviruses requiring low temperatures (less than 33 C) for replication indicated that they were not markedly more thermolabile than rabies virus. Bovine serum protein components in complex cell culture media stabilized virus at 56 C, but at temperatures of less than or equal to 37 C, sodium tris (hydroxymethyl)-aminomethane (NT) buffer containing ethylenediaminetetraacetic acid (EDTA) (NTE) was a much more efficient stabilizer of virus infectivity. Chelating agents EDTA and ethyleneglycol-bis-(beta-aminoethyl ether)tetraacetic acid were equally efficient in protection of rabies virus infectivity; the effect of each was lost when excess Ca2+ was added. Bovine serum in NT or NTE buffers produced a thermostabilizing effect at 37 C not provided by the same serum concentration in complex cell culture media. Bovine serum was more efficient than EDTA in stabilizing virus infectivity during repeated cycles of freezing and thawing.


Effect of passive antibody on parainfluenza virus type 3 pneumonia in hamsters. Both parainfluenza virus type 3 and respiratory syncytial virus may produce life-threatening pneumonia or bronchiolitis in infants less than 6 months old. Almost all infants in this age group possess passively acquired maternal antibodies to both viruses. It has been suggested that maternal antibodies may actually participate in the pathogenesis of these diseases in early infancy. This investigation examined the effect of moderate levels of passive antibody on the development of pneumonia in hamsters infected intranasally with parainfluenza virus type 3. The pneumonitis produced in this model was not enhanced by the presence of moderate levels of serum antibody to this virus. Furthermore, reinfection after an initial "sensitizing" infection under the cover of passive antibody did not result in a more severe pneumonitis. These studies do not support either of the two hypotheses that have been advanced to explain the pathogenesis of infections with respiratory syncytial virus in early infancy.


Stimulation of lymphocyte proliferation by killed mycobacteria and other bacterial species. Killed mycobacteria and some mycobacterial fractions induced spleen cells from normal C3H mice to incorporate tritiated thymidine to a relatively high degree. Thymocytes under the same conditions were not activated. However, incorporation of thymidine was significantly increased when mixtures of thymus and spleen cells were cultured in the presence of the inducers. A similar activation of spleen cells was displayed in the presence of gram-positive and gram-negative non-mycobacterial species.


Influence of feed on onset of disease in subacute myelo-optico-neuropathy virus-infected mice. An enriched feed, specially prepared to affect reproduction of mice, significantly suppressed the onset of disease in subacute myelo-optico-neuropathy virus-infected mice, whereas low-calorie feed enhanced the incidence of disease.


Rescue of vesicular stomatitis virus from homologous and heterologous interferon-induced resistance in human cell cultures by poxviruses. In human cell cultures the ability of poxviruses to rescue vesicular stomatitis virus from human interferon-induced resistance was significantly more efficient than the ability to rescue it from simian interferon-induced resistance. The sensitivity of the poxvirus to interferon was not related to its ability to rescue vesicular stomatitis virus.


Lymphocyte transformation and interferon production in human mononuclear cell microcultures for assay of cellular immunity to herpes simplex virus. Interferon production and transformation in response to herpes simplex virus antigen were studied in microcultures of human mononuclear cells. Mononuclear cells consisting of monocytes and both T and B lymphocytes were purified by Ficoll-Hypaque gradients. Lymphocytes, predominantly T with 5% B, were obtained by passage of buffy-coat cells through nylon fiber columns. For some experiments, autochthonous macrophages and column-purified lymphocytes were stimulated with herpesvirus antigen. The effect of specific antibody and cell concentration on reactivity is described. Crude and purified antigens were compared as cell culture stimulants. Significant differences in transformation and interferon were observed between donors with a history of herpes labialis and donors with no detectable antibody, both in cultures prepared by Ficoll-Hypaque gradients and by column purification of lymphocytes. Cultures from seronegative donors prepared by Ficoll-Hypaque gradients produced interferon but did not transform when stimulated by herpes simplex antigen. "Immune" interferon production, that is, type II as opposed to type I, occurred only with autochthonous macrophage and column-purified lymphocyte cultures. Interferon produced by Ficoll-Hypaque-purified mononuclear cultures was type I, and its production was unrelated to immune status. Similarly, column-purified lymphocytes responded to herpes simplex virus antigen with type I interferon if obtained from a seropositive donor.


Severe hemorrhagic varicella with visceral involvement: virological and serological studies during treatment with cytosine arabinoside. A boy 13 year-old suffered an extremely severe and prolonged attack of hemorrhagic chickenpox with visceral involvement, the diagnosis being confirmed by isolation of varicella-zoster-virus (VZV). There was no other compromising disease. All preceding vaccinations including two against smallpox had been uneventful. The severity of the attack could not be ascribed to any persistent cellular or humoral immunodeficiency. The patient developed a good antibody response. The course of serological reactions to VZV infection was studied extensively using the different techniques of complement fixation and immunofluorescence for IgG, IgM, and IgA antibodies. Therapy was conducted cautiously using cytosine arabinoside (Ara-C) between the 10th and 17th day of disease; the temperature fell and VZV multiplication ceased, strongly suggesting a beneficial influence on the patient, who recovered completely.


Surface markers on human B and T-lymphocytes. IX. Two-color immunofluorescence studies on the association between ebv receptors and complement receptors on the surface of lymphoid cell lines. Receptors for the third component of complement (C3) were demonstrated on the surface of established human lymphoid cell lines by a membrane fluorescence test with FITC- or TRITC-conjugated antibodies against human C3. Two-color fluorescence staining of EBV receptors and C3 receptors showed complete overlapping of green and red fluorescence. Capping of the EBV receptor induced co-capping of the C3 receptor and vice versa. There was neither overlapping nor co-capping when EBV or C3 receptors were examined in relation to Fc receptors, surface IgM or beta2 microglobulin. The kinetic pattern of EBV receptor capping was identical with the pattern of C3 receptor capping but differed from the pattern of IgM capping. These results suggest a close association between EBV and C3 receptors on the human B-lymphocyte.


Detection of Epstein-Barr virus DNA in American Burkitt's lymphoma. Twenty American patients with Burkitt's lymphoma (BL) were studied serologically and biochemically to detect an association with Epstein-Barr virus (EBV). A wide range of antibody reactivities was encountered. Five patients had serological profiles similar to those of African patients with BL: one and possibly two of these patients harbored EBV-DNA in their tumors. Fifteen patients had low or absent antibody to EBV: tumors from 10 of these patients lacked evidence of EBV-DNA. An unexplained association between elevated antibody (greater than or equal to 1:80) to EBV virus capsid antigen (VCA) and a favorable prognosis was noted in this series; this prognostic observation requires further substantiation. Thus, it appears that EBV-associated BL is inversely proportional to tumor endemicity. An etiological role for EBV in BL remains speculative.


Phenotypic mixing of vesicular stomatitis virus and D-type oncornavirus. After mixed infection of cells with vesicular stomatitis virus (VSV, thermolabile mutant tl 17) and oncornavirus type D, phenotypically mixed virions are formed containing the VSV genome and oncornavirus and VSV envelope. The virions are thermostable and have serologic characteristics of both viruses.


Epstein-Barr virus: experimental infection of Callithrix jacchus marmosets. Eight common marmoset monkeys (Callithrix jacchus) were inoculated with about 10(4) transforming units of B95-8 virus; seven of the marmosets died 50-111 days post inoculation and all seven showed microscopic and/or macroscopic lesions compatible with a diagnosis of lymphoproliferative disease. Low levels of anti-VCA antibodies were detected in plasma from six marmosets. Attempts failed to establish continuous EBV-carrying lymphoblastoid cell cultures by cultivation in vitro of circulating lymphocytes or minced lymphoid tissues obtained at necropsy.


A biochemical approach to the study of the transmission of mouse mammary tumor viruses in mouse strains RIII and C3H. Mouse mammary tumor virus (MMTV) proviral sequences were detected in the cellular DNA of mammary tumors and livers of RIII and C3H mice by molecular hybridization with radioactively labelled MMTV 60-70S RNA or tritiated MMTV complementary DNA (cDNA). By means of DNA:DNA reassociation kinetics, the DNA of the mammary tumor cells of these two mouse strains were found to contain more MMTV proviral sequences than the DNA of liver cells of these same tumor-bearing mice. Evidence is also presented that the DNA of the liver cells lacks a part (approximately 25%) of the MMTV proviral sequences found in the mammary tumor cells of these mouse strains. The relationship of the extra MMTV proviral sequences found in mammary tumor cells to the early mammary tumor-igenesis seen in these mouse strains is discussed.


Anti-EBV antibody titers in non-Hodgkin lymphomas. Antibody titers to Epstein-Barr virus (EBV)-related antigens, i.e. viral capsid antigen (VCA), the D and R components of the early antigen (EA) complex and the EBV-associated nuclear antigen (EBNA), were determined in a series of 86 patients with non-Hodgkin lymphomas and in 150 matched control subjects. The lymphoma patients belonged to four histological groups: diffuse, nodular, hyperbasophilic malignant lymphoma (HML) and unclassified. The EBV-related serological data were compared to the incidence of antibodies to other herpes viruses, i.e. cytomegalovirus (CMV), herpes simplex virus (HSV) and varicella zoster virus (VZV), and correlated with immune disorders, which are particularly frequent in the HML type of lymphoma. The results revealed a significantly higher incidence of anti-EA-D titers in lymphoma patients and slight but significant increases in the geometric mean anti-VCA titers in the HML and unclassified group of patients. These elevated anti-EBV titers in patients were not associated with an increase in titres of antibodies to other herpes viruses. They did not correlate with the signs of immune deficiency observed or with the incidence of auto-antibodies.


Production of lymphoid tumors in hamsters by direct implantation of normal human peripheral and umbilical cord leukocytes. Peripheral leukocytes from Epstein-Barr virus (EBV)-seropositive or seronegative normal adults, or human umbilical cord leukocytes infected or not infected with EBV, were directly transplanted into 50 newborn hamsters treated with anti-lymphocyte serum. Lymphoid tumors were produced after a latent period of 10-25 days in 39 animals: 8 of 14 recipients of seropositive donor leukocytes, 2 of 2 recipients of seronegative donor leukocytes, 13 of 15 recipients of EBV-infected cord leukocytes, and 16 of 19 recipients of non-infected cord leukocytes. EBV-determined nuclear antigen-positive human lymphoblastoid cell lines were established in vitro from tumors produced with seropositive donor leukocytes or EBV-infected cord leukocytes but not from tumors produced with seronegative donor leukocytes or non-infected cord leukocytes. The results indicate that EBV is not a prerequisite for in vivo production of tumors but necessary for in vitro establishment of lymphoblastoid cell lines from such tumors.


A quantitative analysis of the susceptibility of human leukocytes to transformation by Epstein-Barr virus. Susceptibility of lymphocyte-enriched cell fractions isolated from human umbilical cord blood and adult peripheral blood to transformation by the B95-8 strain of Epstein-Barr virus (EBV) was investigated quantitatively. Minimum multiplicity of input of virus (50% transforming dose) per cell (MOI) necessary to induce maximum level transformation of cord cells ranged from 0.02 to 0.2. The frequency of initially transformed cells (fraction of transformable cells) in the cord cell samples from two different individuals was estimated to be 2.6 to 6.2%. In this system, the appearance of cells positive for EBV-associated nuclear antigen (EBNA) paralleled the growth curve of transformed cells. About 70% of the latter were EBNA-positive. In adult cell preparations from two individuals, 1.8 and 0.03%, respectively, of the cells were transformable indicating larger individual variations in sensitivity to EBV than in cord cells. The EBV susceptibility was also determined by the transforming efficiency (TE) expressed as the negative log of the virus dilution which induces transformation in 50% of cell cultures infected at an MOI of 0.2. From the TE value, a minimum MOI which induces transfromation could be calculated. Also by this test it was shown that the EBV susceptibility of adult cells was not only lower but also much more variable between individuals than that of cord cells. There was no correlation between the susceptibility of cells and the titer of anti-EBV antibody in donors' sera. In cultures of mixed cord cells and adult cells known to have low EBV susceptibility, the minimum MOI increased in proportion to the amount of adult cells.


Independent regulation of two types of aryl hydrocarbon (benzo(a)pyrene) hydroxylase in mammalian cells. Aryl hydrocarbon (benzo(a)pyrene) hydroxylase induced by dibutyryl cyclic AMP (dcAMP), plus aminophylline (AHH I) can be ditsinguished from the hydroxylase induced by benz (a) anthracene (AHH II) by its lower Km for benzo (a) pyrene. Treatment with the combination of benzo (a) anthracene and dcAMP plus aminophylline induced both AHH I and AHH II activities. After optimal induction of AHH II activity by benz (a) anthracene, the addition of dcAMP plus aminophylline gave an induction of AHH I. Although AHH I activity declined to an almost basal level 24 h after treatment with dcAMP plus aminophylline, the addition of benz (a) anthracene prevented this decline. Inducibility by dcAMP plus aminophylline or by benz (a) anthracene varied in different cell lines. Some cell lines were induced by both substances, with a higher induction by benz (a) anthracene, while other lines were inducible only by benz (a) anthracene, and a third cell type was not inducible by either. Selection for resistance to benzo (a) pyrene of a cell line inducible by both compounds resulted in a fourth cell type which was more inducible by dcAMP plus aminophylline than by benz (a) anthracene. The results suggest that there is an independent regulation of hydroxylase AHH I and AHH II and that the induction of these two enzyme activities is determined by different genetic controls.


Meeting the psychosocial needs of pacemaker patients. A review of the literature concerning patients who have received permanently implanted cardiac pacemakers indicates that a substantial number of these patients experience difficulties in adjusting to their medical condition. Common feelings among these patients are anxiety and depression. It is suggested that difficulties often arise from the patient's misconceptions about the pacemaker and inadequate psychosocial support. To assist such patients in their adjustment, the Pacemaker Support Program was developed to provide psychosocial counseling and pacemaker education from the preoperative phase through to the outpatient pacemaker follow-up clinic phase. In the year of the program's operation a marked decline in adjustment problems has been observed, the program has been readily integrated into the hospital routine, and it has been enthusiastically accepted by both the hospital staff and the patients.


The role of the psychiatric consultant in the treatment of burned patients. Two cases of seriously burned patients are presented which differ in initial psychiatric presentation and subsequent course. In each case, the role of the psychiatric consultant is described. The role includes direct evaluative and psychotherapeutic contact with the patient and liaison with the surgical and nursing staff. This dual approach is necessary for a consistent rehabilitative effort, particularly in hospitals that do not have a psychiatric liaison team.


An EBV-genome-negative cell line established from an American Burkitt lymphoma; receptor characteristics. EBV infectibility and permanent conversion into EBV-positive sublines by in vitro infection. An in vitro line was derived from an American Burkitt lymphoma, designated Ra No. 1, which produced malignant tumors when inoculated into thymus-deficient nude mice. The cells have B-lymphocyte characteristics, with surface-associated mu and kappa chains and Epstein-Barr virus (EBV) receptors, and can be readily infected with EBV in vitro. B95-8 virus induced EBV-determined nuclear antigen (EBNA) but not early antigen (EA) in Ra No. 1 cells, whereas P3HR-1 virus induced both EBNA and EA, but the EA level was much lower than in the prototype Raji strain, EBNA and EA levels were comparable in Ra No. 1 and in the previously established, EBV-infection-sensitive BJA-B lymphoma. The two lines differed, however, because BJA-B could be converted into a permanent EBV-carrying line by the B95-8 but not by the P3HR-1 virus strain, whereas Ra No. 1 could be converted by the P3HR-1 virus. This demonstrates that different B-lymphocyte-derived lymphoma lines can vary with regard to the restrictive control they can exert on the same virus strain. Furthermore, virus strains vary in their sensitivity to the restrictions of the same host cell. Permanent EBV conversion of the Ra No. 1 cell did not appear to change the cellular controls, as judged by the unchanged sensitivity of the cell to viral antigen (EA, viral capsid antigen) induction by P3HR-1 virus superinfection.


Morphological changes in chick embryo cell cultures induced by avian leukosis viruses. Morphological alterations were observed after 5-15 serial passages of cells infected with three different strains of avian leukosis virus: OK 10, an A subgroup virus isolated from a natural infection; RAV-1, an established laboratory strain of the A subgroup, and RAV-2, a laboratory strain of the B subgroup. The infected cells had a prolonged lifespan of approximately 28 passages, compared to 14 passages for control cells. However, the altered cells had none of the attributes of transformed cells, such as growth in soft agar, loss of contact inhibition, tumor formation in chickens, or loss of fibroblast surface antigen. Therefore, we refer to the changes as conversion rather than transformation. The morphological changes differed depending upon the subgroup of the inducing virus. Cells converted with the A subgroup viruses were uniformly epitheloid whereas cells converted with the B subgroup virus were less uniform in size and shape. We speculate that an event similar to conversion may take place in vivo and contribute to the oncogenicity of leukosis viruses.


Cytomegalovirus infection of human lung epithelial cells in vitro. Human lung epithelial cells were productively infected with human cytomegalovirus in vitro. Infectious virus was released up to 8 weeks postinfection. The cells retained their morphological characteristics throughout the period of observation, while simultaneously bearing all the features typical of cytomegalovirus infection.


Preliminary studies on an unusual poxvirus of the western grey squirrel (Sciurus griseus griseus) of North America. Particles of a poxvirus found in Californian western grey squirrels have overall dimensions indistinguishable from those of the poxviruses of the genera Orthopoxvirus and Leporipoxvirus, but have surface structures somewhat reminiscent of virions of members of the genus Parapoxvirus. Extracts of tissues infected with the squirrel poxvirus cross-react in gel-diffusion tests with extracts of tissues infected with Californian myxoma virus, but not with a similar preparation infected with a South American strain of myxoma virus.


Infectivity of Rous sarcoma cell DNA: comparison of two techniques of transfection assay. A DNA extracted from a clone of chicken cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D(SR-RSV-D), was assayed for infectivity by means of DEAE-dextran and calcium techniques. The calcium technique like the previously described DEAE-dextran procedure gave rise to viruses in transfection assays with both native and denatured (S1 nuclease susceptible) DNAs. The efficiency of these transfection techniques with native DNA was compared and found to be about the same provided that with the calcium technique carrier DNA was used to complement DNA concentrations lower than 2.5 mug/ml.


Effect of thyroidectomy and in vivo administration of triiodothyronine on DNA synthesis in isolated mitochondria. The experimental conditions were studied which allow hormonal levels to affect the incorporation of labelled deoxyribonucleosides triphosphates (dNTP's) into mitochondrial DNA by isolated liver mitochondria, obtained either from thyroidectomized young male rats (T) or from animals of the same age thyroidectomized and then treated with triiodothyronine (T + T3). It was demonstrated that: (a) extramitochondrial DNA, on which extramitochondrial DNA polymerase may act, was absent; (b) the permeability to dNTP's, the thymidine kinase activity, the energy supply, and the nuclease activities were unaffected by hormonal conditions; (c) the bacterial contaminations contribute for only 1% to incorporation. The characterization of incorporation product showed that: (a) such product was indeed DNA, as it was DNase-degradable for about 90%; (b) the labelled DNA was indeed mitochondrial DNA, as a 10 minutes preincubation with acriflavine or ethydium bromide (Eth. Br.) inhibited the synthesis by 90%.


Characteristics of immunity induced by neuraminidase-treated lymphosarcoma cells in C3H (MTV+) and C3H (MTV-) mice. The immunogenicity of 6C3HED lymphosarcoma ascites tumor cells was greatly enhanced upon treatment with Vibrio cholerae neuraminidase. As a result, mammary tumor virus (MTV)-free C3H mice which had been repeatedly immunized with the treated cells resisted a challenge of 10(6) untreated viable cells inoculated i.p., whereas all control animals died after receiving less than 10 lymphosarcoma tumor cells. In contrast, there was no increase in the refractoriness to challenge when MTV-infected C3H mice were immunized. Incubation of 6C3HED lymphosarcoma cells with serum of splenic lymphocytes from the immunized MTV-free C3H mice neutralized their tumorigenicity and protected recipient MTV-free and MTV-infected C3H mice against the disease. As shown by the 51Cr microcytotoxicity assay, the complement-dependent cytotoxic antibody titer reached 1,024; 29% of the target cells were lysed by lymphocytes obtained from immune MTV-free C3H mice. When the immune lymphocytes were pretreated with anti-theta serum they lost their ability to neutralize the tumorigenicity of the lymphosarcoma, failed to be stimulated by the T cell mitogens, phytohemagglutinin and concanavalin A, and failed to achieve cytolysis of the lymphosarcoma target cells in vitro. These observations establish the central role of the theta-antigen-bearing immune lymphocytes in cell-mediated immunity in this system. Failure of the immune system in the immunized MTV-infected C3H mice to cope with the challenging lymphosarcoma, even at low cell numbers, was reflected, not in the absence of humoral response, but in the relatively low level of cell-mediated immunity. This was manifested in both the neutralization and in the vitro cell-mediated immunity assays. This apparent immunological deficiency observed in the MTV-infected C3H mice could be countered by the transfer of lymphocytes from MTV-free C3H mice immunized with neuraminidase-treated 6C3HED lymphosarcoma cells. The recipient MTV-infected C3H mice then rejected a subsequent challenge with lymphosarcoma tumor grafts.


Structural investigation of the antibiotic ristomycin A. Synthesis of ristobiose and ristotriose. Proof is given by synthesis confirming the structure of ristobiose as 2-O-alpha-D-mannopyranosyl-D-glucose (IV) and ristotriose as O-alpha-L-rhamnopyranosyl (1 leads to 6)-O-[alpha-D-mannopyranosyl (1 leads to 2)]-D-glucose (X) which are obtained from ristomycin A upon mild acid hydrolysis. Both oligosaccharides, IV and X, have been detected for the first time as the components of an antibiotic.


Complete regression of a guinea pig hepatocarcinoma by immunotherapy with "tumor-immune" RNA or antibody to fibrin fragment E. Two novel immunotherapeutic regimens were developed for a uniformly lethal, intradermally growing transplantable ascites variant (line 10) of a diethylnitrosamine-induced hepatoma in strain 2 guinea pigs. In an apparently tumor-specific immunotherapy model, 32 guinea pigs were cured by the injection into the tumor area, five or seven days after tumor challenge, of syngeneic or xenogeneic RNA extracts obtained from lymphoid tissues of line 10-immune strain 2 guinea pigs or rhesus monkeys, as part of a total regimen which included syngeneic nonsensitive peritoneal exudate cells injected prior to, and tumor-specific antigen injected after, the RNA. In another immunotherapy model, not tumor-specific, 18 strain 2 guinea pigs were cured by the injection into the tumor area, 6 and 16 days after tumor challenge, of antibody specific for fibrin fragment E (FFE), an essential component in the formation of a fibrin matrix considered to be important in tumor development. When therapy was delayed to 12 days in the RNA test system, or to 16 days in the anti-FFE test system, complete abrogation of the tumors did not occur. The long-term survival of the 50 successfully treated animals and their immunity to further tumor challenge indicated that both immunotherapeutic procedures had systemic effects. To test this further, line 10 cells were injected intradermally simultaneously at two sites and only one site was treated. When the one tumor location was treated with anti-FFE, complete regression of the treated tumor and a 30% retardation in the development of the untreated tumor were observed. When this tumor location was treated with the RNA regimen, complete regression of the tumors occurred at both the treated and the untreated sites. Optimal conditions for both immunotherapeutic models and their combination have yet to be establshed. Nonetheless, both immunotherapeutic regimens were more effective than any other immunotherapy thus far reported for this tumor, including the use of BCG or its derivatives.


Interactions between lymphocytes and C-type oncornaviruses in mice. Several aspects of C-type oncornavirus-lymphocyte interactions in the mouse are reviewed: 1) immunological abnormalities induced by C-type viruses, particularly immunosuppression and autoimmunity; 2) activation or amplification of endogenous C-type viruses during immunological reactions; and 3) the immune responses to these endogenous oncornaviruses. A unifying hypothesis is proposed to relate various interactions between lymphocytes and C-type oncornaviruses.


Pathway of thiamine pyrophosphate synthesis in Micrococcus denitrificans. The pathway of thiamine pyrophosphate (TPP) biosynthesis, which is formed either from exogeneously added thiamine or from the pyrimidine and thiazole moieties of thiamine, in Micrococcus denitrificans was investigated. The following indirect evidence shows that thiamine pyrophosphokinase (EC 2.7.6.2) catalyzes the synthesis of TPP from thiamine: (i) [35S]thiamine incubated with cells of this microorganism was detected in the form of [35S]thiamine; (ii) thiamine gave a much faster rate of TPP synthesis than thiamine monophosphate (TMP) when determined with the extracts; and (iii) a partially purified preparation of the extracts can use thiamine, but not TMP, as the substrate. The activities of the four enzymes involved in TMP synthesis from pyrimidine and thiazole moieties of thiamine were detected in the extracts of M. denitrificans. The extracts contained a high activity of the phosphatase, probably specific for TMP. After M. denitrificans cells were grown on a minimal medium containing 3 mM adenosine, which causes derepression of de novo thiamine biosynthesis in Escherichia coli, the activities of the four enzymes involved with TMP synthesis, the TMP phosphatase, and the thiamine pyrophosphokinase were enhanced two- to threefold. These results indicate that TPP is synthesized directly from thiamine without forming TMP as an intermediate and that de novo synthesis of TPP from the pyrimidine and thiazole moieties involves the formation of TMP, followed by hydrolysis to thiamine, which is then converted to TPP directly. Thus, the pathway of TPP synthesis from TMP synthesized de novo in M. denitrificans is different from that found in E. coli, in which TMP synthesized de novo is converted directly to TPP without producing thiamine.