Interaction of papaverine and isoproterenol in the vascular system of the dog and intestinal smooth muscle of the rabbit. It was found that the effect of isoproterenol on the vasculature of the anesthetized dog can be potentiated by previous administration of papaverine. This effect is apparent in spite of concomitant potentiation of inotropic and/or chronotropic effects, which tend to obscure the vasomotor effect. Evidence is presented supporting the hypothesis that papaverine interacts with receptors of the vascular tree, which are usually classified as beta receptors and are responsive to isoproterenol. Evidence is presented showing almost complete inhibition of the chronotropic effects of papaverine by propranolol, and a synergism of papaverine with the chronotropic effects of isoproterenol. Evidence was obtained using the intestinal smooth muscle of the rabbit in support of the hypothesis linking mediation of the beta adrenergic system to papaverine effects.


Peyote, a potential ethnopharmacologic agent for alcoholism and other drug dependencies: possible biochemical rationale. The authors examine folk psychiatry among Native American Church members from an enthnopharmacologic viewpoint. Alcohol and opiate abuse among Indian and non-Indian are presented in case histories proving to be asymptomatic under Indian guidance and through participation in the peyote ritual. The biochemical alkaloids common in the peyote cactus, rather than just the psychoactive substances (mescaline), are purported to be pharmacologically similar to the neuroamine-derived alkaloids found in the brain during alcohol intoxification. Evidence is reviewed that points out possible common features of alcohol and opiate dependence leading to the speculation for a common mode of treatment may reside in plants rich in isoquinoline alkaloids.


Allergic contact dermatitis to a photopolymerizable resin used in printing. Organic photopolymerizable resins increasingly used in printing make it possible to produce flexible, lighter plates. A certain number of skin lesions and general symptoms appeared upon manipulation of plates made by the Letterflex process. In the workshop where this study was carried out, 12 out of 15 people suffered various degrees of skin lesions. The epicutaneous tests and biopsies performed indicate an allergic type reaction to one of the resin's constituents, polythiol. Experiments with animals confirm the strong allergenicity of this compound.


Adrenocortical function after stimulation with synthetic ACTH. In order to assess the effect of ACTH on the adrenal cortex when used as a therapeutic or diagnostic agent, adrenocortical function was tested before and 2 hours after 0.5 mg tetracosactrin given intramuscularly in 31 healthy children, divided into 4 groups according to age and sex. Mean plasma cortisol levels were shown to be 184% higher than baseline values after administration of the drug, and there were no significant differences related to age or sex. It is suggested that this simple test can be of practical value in the diagnosis of adrenocortical function in young people.


Ultrastructural features of human galactosaemic fibroblasts: effects of different nutritional environments. The ultrastructural characteristics of galactosaemic human fibroblasts exposed to media with various sugars were compared. Considerations of known biochemical pathways suggested that cells without the galactokinase enzyme and exposed to galactose would be functionally equivalent to cells in a medium without any sugar source. We found the ultrastructural characteristics of such cells to be consistent with that hypothesis. In addition, we unexpectedly found that homozygous galactosaemic fibroblasts, exposed to medium with glucose as the energy source, showed evidence of degeneration, such as autophagic vacuoles. Degenerative changes were not found in fibroblasts heterozygous for the galactokinase enzyme exposed to glucose. The implications and uses of these findings are discussed.


Effect of vitamin D3 on duodenal absorptive cell plasma membranes: freeze fracture replication study. Vitamin D3 induced changes in the number of 5.8 nm sized randomly scattered particles on the EF faces of plasma membranes of chick duodenal absorptive cells. Their number increased as compared to those in rachitic animals. Also vitamin D3 seemed to alter the nature of the zonula occludens. These changes appear to be related to increased active absorption and passive diffusion of calcium respectively under vitamin D3 influence.


[Biological availability of fluoride after combined administration with vitamin D3 in man (author's transl)]. Six adults ingested 1 mg fluoride, first as NaF in a watery solution and, secondly, in tablet form (D-Fluoretten 1000). Fluoride concentration was almost identical in the two series. After initial values within the normal range concentrations rapidly rose to a maximum (six times normal) reached on average after 30 minutes, followed by an, at first steep then more gradual, fall in concentration over eight hours. The area under the curve was similar in the two series. These results indicate that the biological availability of fluoride is not influenced by vitamin D3 in the adult and, presumably, in the small child as well.


Metoprolol: a review of its pharmacological properties and therapeutic efficacy in hypertension and angina pectoris. Metoprolol is a beta1-selective adrenoceptor blocking drug. In hypertension, its duration of effect is longer than expected from its half-life and it is suitable for twice daily administration. There is some evidence that once daily administration may be possible in treating hypertension. It is similar in efficacy to other beta-adrenoceptor blocking drugs in angina pectoris and essential hypertension, when given in equiactive beta-blocking dosages. Metoprolol is well tolerated and side-effects have not proved a problem. It has some pharmacodynamic and pharmacokinetic differences from other beta-adrenoceptor blocking drugs and may prove useful in cases where these differences are shown to be clinically important.


[Participation of cyclic-3',5'-adenosine monophosphate in the pathogenesis of myocardial hypertrophy in experimental hypertension]. The authors examined the concentration of cAMP in the myocardium of rats with experimental hypertension during various periods after modeling of the process. The changes in the values of cyclic nucleotide corresponded to the dynamics of the development of the arterial hypertension. There was na increase of the level of cAMP in the myocardium on the third day after modeling of hypertension, accompanying the initial elevation of the arterial pressure. The maximal values of cAMP were established on the 30th day, which corresponded to the maximal development of hypertension. Cyclic 3,5-adenomonophosphate began to diminish parallel to the lowering of the arterial pressure during the chronic phase 90th day. The obtained results give a possibility to discuss the participation of cAMP in the pathogenesis of myocardial hypertension, occurring in experimental hypertension.


[Rest-activity-cycle and stereotyped hand waving of a mentally defective child (author's transl)]. Stereotyped acts of a mentally defective girl. 6;6 years old, were observed from 6 a.m. till 8 p.m. during 16 days. Alternating phases of more or less frequent stereotyped hand waving showed a prominent periodicity of about 90 minutes. Sleep stages were also determined polygraphically during 10 nights. As statistically proved the mean peak-to-peak interval of the stereotyped activity at days corresponded to the mean REM- to REM period of nights. This suggests, that the stereotyped behavior of this child was driven by the basic rest activity cycle already described by Kleitman. The mean periodicity of sleep stages was represented by modeled rhythms adapted to the scored sleep stages. If the model rhythms of all 10 nights were extended backward to the preceding days, they paralleled the stereotyped activity. Half periods found in phase with REM stages corresponded to the maxima of the stereotyped activity. It will be discussed that the basic rest activity cycle triggers sensory as well as motor mechanisms characterizing both the phases of enhanced stereotyped activity and REM stages.


Inhibition of cortiocosteroidogenesis by delta-9-tetrahydrocannabinol. ACTH, cholera toxin, cyclic AMP but not pregnenolone-induced steroidogenesis in Y-1 functional mouse adrenal tumor cells was significantly inhibited by delta-9-tetrahydrocannabinol, cannabidiol, and cannabinol. The inhibition of steroidogenesis could not be correlated with a general depression in cell function or viability. The data suggest that cannabinoids inhibit corticosteroidogenesis at a site between the synthesis of cAMP and of pregnenolone.


Identification of beta-adrenergic binding sites in rabbit myometrium. Rabbit uterine muscle may contract or relax with adrenergic stimulation depending on the hormonal milieu. This difference in contractile activity has been shown to be due to alteration of adrenergic response between alpha-adrenergic (contraction) and beta-adrenergic (relaxation). When rabbits are treated with estrogen followed by progesterone, norepinephrine produces myometrial relaxation. This effect is blocked by propranolol, indicating that it is mediated by beta receptor activation. A subcellular preparation of this myometrium has adenylate cyclase activity that can be stimulated by isoproterenol + guanyl-5'-yl-imidodi-phosphate (Gpp(NH)p). The radioligand [125I]iodohydroxybenzylpindolol binds to the same preparation. The binding is rapid, 80% maximal in 10 min, and readily reversible (t1/2 = 5 min). The binding is high affinity (Kd = 0.12 nM), low capacity (15 fmol/mg protein), and is to a single class of binding sites. Binding is competed for stereoselectively by beta adrenergic agonists and antagonists. The competition of beta adrenergic agonists for binding, isoproterenol = ritodrine greater than epinephrine greater than norepinephrine, is consistent with interactions at a beta2-adrenergic receptor.


Potent inhibitory activity of [D-Leu6, Des-Gly-NH2(10)]LHRH ethylamide on LH/hCG and PRL testicular receptor levels in the rat. Injection of male rats with 40-200 ng of [D-Leu6, des-Gly-NH2(10)]LHRH ethylamide for 7 days caused a maximal 80% reduction of testicular LH/hCG receptor level with one injection per day being as efficient as 3 daily injections. A similar inhibitory effect was observed on testicular PRL receptors. Testis and seminal vesicle weight as well as plasma testosterone levels were also significantly reduced by this treatment. These data indicate that a LHRH agonist, when given at a relatively low dose, is capable of reducing testicular LH/hCG and PRL receptor levels as well as testicular function, the effect being probably mediated by increased endogenous gonadotropin secretion.


Rat adrenal corticosteroidogenesis; effect of ACTH, cycloheximide and sterol carrier protein. Corticosterone formation was determined in the reconstructed rat adrenal system which consisted of the mitochondria and post-mitochondrial supernatant fraction (PM-fraction) supported by l-malate, and effect of ACTH and cycloheximide in vivo and cycloheximide, Ca++ and sterol carrier protein (SCP) in vitro were examined. Mitochondria isolated from adrenals of rats which received ACTH 15 min before sacrifice showed an elevated corticosterone formation. Cycloheximide administration 15 min prior to ACTH injection completely blocked the effect of ACTH but in vitro addition of this drug to the incubation mixture did not modify the rate of corticosterone production even at higher concentrations. Since the PM-fraction isolated from adrenals of rats received ACTH or cycloheximide or both did not change the mitochondrial capacity for corticosterone formation, factor(s) which influenced by ACTH administration seemed to be localized in mitochondria. The SCP-bound cholesterol was utilized for corticosterone formation more efficiently than the free cholesterol when added to the incubation mixture, and this might be due to, at least in part, higher rate of binding to the mitochondrial inner membrane of the SCP-bound cholesterol.


The metabolism of 17beta-estradiol-3-glucosiduronate and estrone-3-glucosiduronate in the rabbit. [6, 7-3H]-17beta-Estradiol-3-glucosiduronate, [6, 7-3H]-estrone-3-glucosiduronate or [6, 7-3H]-estrone was administered intravenously into the rabbit, and analysis and identification of the urinary metabolites were carried out. In either case, the major urinary metabolite was found to be a diconjugate. The sequential enzymic hydrolysis indicated that this diconjugate was glucosiduronate-N-acetyglucosaminide of 17alpha-estradiol. From these results, the conversion of the estrogen glucosiduronate into a diconjugate was thought a rather universal phenomenon in the rabbit.


Theophylline stimulation of binding of radioiodinated rat prolactin to murine mammary gland. Characteristics of prolactin receptors of murine mammary glands were studied. Mammary gland exhibited low but specific binding to 125I-rPRL (rat prolactin). Receptors were heat susceptible and partly protein in nature. Theophylline--both in vitro and in vivo treatment--enhanced the binding of 125I-rPRL to receptors. It remains to be established whether this action of theophylline is mediated through cyclic AMP "Sparing" effect or through its direct effect on cell membrane stabilization.


Binding of thyroxine and triiodothyronine in solubilized nuclear extract of human leukocytes. An in vitro study was carried out of the interaction of thyroidal hormones with leukocytes and with a solubilized extract of the nuclear fraction of human leukocytes. The respective association constants characterising the binding of triiodothyronine (T3) and thyroxine (T4) to whole leukocytes are roughly equal (for T3, KA = 3.1 X 10(11) 1/mol and for T4, KA = 4 X 10(11) 1/mol) but a 0.4 KCl extract of the nuclear fraction exhibits a different affinity to T3 (KA = 2.16 X 10(11) 1/mol) in comparison with T4 (KA = 1.3 X 10(10) 1/mol). In the nuclear extract, both hormones are bound with the affinity higher for T3 than for T4. The soluble nuclear binding protein in human leukocytes had a molecular weight 46 000, was chromatographically homogenous in chromatography on Sepharose 2B and Sephadex G200, and exhibited a longlasting stability at -25 degrees C, without any marked change in the binding affinity to thyroidal hormones.


Effect of combined dopaminergic and GABA-ergic stimulation on ouabain-induced epileptiform activity. In this study we tested a hypothesis that dopamine and GABA receptor-stimulating agents given together might mutually potentiate their anticonvulsant activities. To test this hypothesis, experiments were performed on free-moving rabbits with epileptiform activity evoked by cortical application of ouabain. After combined treatment with apomorphine and GHBA 3 out of 6 animals displayed no EEG epileptiform activity, and in the remaining 3 rabbits this activity was greatly reduced. A combined pretreatment with apomorphine and GHBA abolished epileptiform EEG activity during the first hour and shortened it during the second hour, but not significantly. Apomorphine given together with amino-oxyacetic acid before ouabain completely abolished the epileptiform activity in 4 out of 6 animals. In the remaining 2 rabbits the latency of ouabain affect was prolonged, the epileptiform activity during the first hour was abolished, and it was markedly decreased during the next 2 hr (significantly from controls or groups receiving apomorphine or amino-oxyacetic acid). Phenytoin was less potent than apomorphine with GHBA or apomorphine given together with AOAA. The animals' behavior was observed.


Enzymes of ammonia detoxication after portacaval shunt in the rat. I. Carbamylphosphate synthetase and aspartate transcarbamylase. At high systemic blood concentrations ammonia may be partially deviated into the pathway of pyrimidine synthesis, as has been observed in different genetic defects of the urea cycle. The portacaval shunt (PCS) rat presents an animal model to study ammonia detoxication without an underlying enzyme defect in the urea cycle. Since ammonia may induce a deviation into the pyrimidine pathway by influencing enzymatic reactions involved in this pathway, the activity of carbamylphosphate synthetase and aspartate transcarbamylase in liver as well as the excretion of orotic acid in the urine were measured in rats 10, 20 and 30 days after PCS. The results suggest that in this experimental model ammonia may be channeled into the pyrimidine pathway leading to a stimulation of the first enzymatic step and to an increased  excretion of orotic acid.


Studies on the mechanism of action of histone kinase dependent on adenosine 3':5'-monophosphate. Evidence for involvement of histidine and lysine residues in the phosphotransferase reaction. The reaction of the phosphate residue transfer catalysed by histone kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) was studied. The phosphotransferase reaction was shown to obey the mechanism of ping-pong bi-bi type. After incubation of the catalytic subunit of histone kinase with [gamma-32P]ATP the incorporation of one mole of [32P]phosphage per mole of protein was observed. The tryptic [32P]phosphohistidine-containing peptide was isolated and its N-terminus and amino acid composition were determined. The 2',3'-dialdehyde derivative of ATP (oATP) was used as the affinity label for the catalytic subunit of cyclic-AMP-dependent histone kinase. The inhibitor formed an alidmine bond with epsilon-amino group of the lysine residue of the active site and was irreversibly bound to the enzyme after reduction by sodium borohydride with concurrent irreversible inactivation of the enzyme. After inactivation, about one mole of 14C-labelled inhibitor was incorporated per mole of the enzyme. ATP effectively protected the catalytic subunit of histone kinase against inactivation by oATP. Tryptic digestion of the enzyme-inhibitor complex led to the isolation of the 14C-labelled peptide of the active site of histone kinase. Basing on these results, the role of histidine and lysine residues in the active site of the catalytic subunit of histone kinase was suggested.


The Greig polysyndactyly-craniofacial dysmorphism syndrome. A 15-month-old male child with the Greig polysyndactyly-craniofacial dysmorphism syndrome is presented and the relevant literature is reviewed.


Congenital scalp defect with distal limb reduction anomalies. Congenital scalp defects and distal limb reduction anomalies can occur as separate entities or in combination with other anomalies. They also occur together in an apparently autosomal dominant syndrome, an example of which is described in the present paper. The literature on the subject is reviewed.


Increased growth of Rous sarcomas in chickens pretreated with formalinized syngeneic tumor cells. The effect of inoculating formalinized syngeneic or allogeneic Rous sarcoma cells on the growth of Rous sarcoma virus (RSV)-induced tumors in two related inbred strains of chickens was studied. Chickens from both strains that received three weakly inoculations of syngeneic tumor cells had a significant increase in tumor growth and mortality after subsequent challenge with RSV. Development of RSV-induced tumors in chickens pretreated with formalinized allogeneic tumor cells (i.e. incompatible for major histocompatibility complex (MHC) antigens) was similar to what we observed in nonpretreated control chickens. The finding that the tumor-host relationship is altered only in chickens pretreated with formalinized syngeneic RSV-transformed cells, suggests that tumor-associated antigens of Rous sarcomas are modified MHC antigens analogous to "altered self" antigens thought to be present on certain virus-infected cells. If this hypothesis is correct, the results we obtained with formalin-killed syngeneic tumor cells can be explained on the basis of three possible mechanisms: immunological enhancement, immune tolerance or induction of antibody to anti-tumor idiotype. The merits of each mechanism in accounting for the results are discussed.


Effects of pimozide on noradrenergic transmission in rabbit isolated ear arteries. In the rabbit ear artery both dopamine and noradrenaline inhibit stimulation-induced (S-I) transmitter noradrenaline efflux. Pimozide, which is reported to be a specific dopamine receptor antagonist, was used to further study the effects of dopamine on transmitter efflux. In a concentration of 0.2 micrometer pimozide blocked the inhibition of S-I efflux produced by 0.5 micrometer dopamine but not that produced by 0.5 micrometer noradrenaline. In a concentration of 10 nM, pimozide enhances transmitter release and vasoconstrictor responses to sympathetic nerve stimulation; this may be due to blockade of feedback inhibition of transmitter release by endogenous dopamine. In a concentration of 1 micrometer, pimozide reduced transmitter release and vasoconstrictor responses to sympathetic nerve stimulation. Vasoconstrictor responses to noradrenaline and histamine are antagonized by pimozide in a noncompetitive manner.


Effect of chlorpropamide and phenformin on rat liver: the effect on plasma membrane-bound enzymes and cyclic AMP content of hepatocytes in vitro. Chlorpropamide and phenformin inhibited (Na+ - K+)-ATPase and stimulated a high affinity cyclic AMP-phosphodiesterase of isolated liver plasma membrane when tested in vitro. In addition, the two drugs decreased the intracellular cyclic AMP content of isolated hepatocytes without being effective on plasma membrane-bound adenylate cyclase. The results suggest that the plasma membrane plays an important role in the mechanism of action of the two hypoglycemic drugs, but do not exclude the presence of intracellular targets.


Inhibition of angiotensin converting enzyme by SQ 14,225 in conscious rabbits. The cardiovascular pharmacology of SQ 14,225 (D-3-mercapto-2-methylpropanoyl-L-proline), a new orally effective inhibitor of angiotensin-coverting enzyme (ACE) was investigated in conscious normotensive rabbits. Intravenous administration of SQ 14,225 (3.1-310.0 microgram/kg) resulted in a dose related inhibition of the pressor responses to 310 ng/kg, i.v. of angiotensin I (AI) without diminishing the pressor responses to 100 ng/kg, i.v. of angiotensin II (AII). In fact, the responsiveness of the rabbits to AII was significantly enhanced by higher doses of SQ 14,225. This enhancement of the pressor effects of AII was found to be related to the inhibition of ACE and the resulting decrease in the levels of endogenous AII. In addition, SQ 14,225 (1.0 mg/kg, i.v.) markedly potentiated the magnitude and duration of the vasodepressor responses elicited by bradykinin (1.0 microgram/kg. i.v.). At a dose of 1.0 mg/kg. i.v., SQ 14,225 had no effect on the vasodepressor effects of intravenously administered isoproterenol (0.4 microgram/kg), acetylcholine (1.0 microgram/kg) or prostaglandin E2 (3.0 microgram/kg, i.v.). The pressor responses to norepinephrine (3.0 microgram/kg, i.v.) were similarly unaffected by SQ 14,225 (1.0 mg/kg, i.v.). In normal rabbits SW 14,225 (1.0 mg/kg, i.v.) caused a small but significant decrease in arterial pressure; it had no such effect in anephric rabbits. The observation of this study indicate that SQ, 14,225 is a specific inhibitor of ACE in conscious rabbits.


The effects of dibutyryl cyclic AMP, theophylline and papaverine on the release of 3H-catecholamines from rat brain striatal and cortical synaptosomes. Papaverine enhances the spontaneous release of 3H-dopamine from rat brain striatal synaptosomes and 3H-noradrenaline from cortical synaptosomes in a dose-dependent way. Neither dibutyryl cyclic AMP nor theophylline had any significant effect on either spontaneous or on potassium-evoked 3H-catecholamine release. It is suggested that the effect of papaverine on catecholamine release is not due to its phosphodiesterase inhibitory potency.


Effects of 4-aminopyridine on mechanical activity and noradrenaline release in the rat portal vein in vitro. 4-Aminopyridine (4-AP) increased the spontaneous mechanical activity of the isolated rat portal vein. Since denervation and adrenergic receptor blockade failed to prevent this effect of 4-AP it is suggested that the drug enhances the electrical excitability of the muscle membrane. 4-AP significantly increased the response of the muscle to electrical nerve stimulation in most experiments but had little effect on the response to applied noradrenaline (NA). Both spontaneous and evoked release of 3H-activity, following preincubation in 3H-noradrenaline, were increased in the presence of 4-AP (10(-3) M). The present results with 4-AP can be explained by its known ability to block the transient potassium conductance which accompanies the action potential in excitable tissues.


Potentials in mammalian skeletal muscle from collagenase-treated tissue. Transmembrane potentials were recorded from skeletal muscle fibres dissected with the aid of collagenase perfusion. Collagenase treatment had little or no effect on the action or resting potentials.


Proposed effects of brain noradrenaline on neuronal activity and cerebral blood flow during REM sleep. We propose that the observed increases of both neuronal activity and cerebral blood flow seen throughout the brain during REM sleep may be effects of decreased central noradrenaline release.


Multiple oncogenesis of neural crest cells by steroids in suckling mice. In suckling mice injected with steroids, multiple tumors occurred in the sites where neural crest cells normally are present. It is speculated that the phase of the cell cycle of the neural crest cells may have something to do with its cell surface and cellular phenotypic expression in the system mediated by cyclic AMP.


Effect of dibutyryl cAMP and theophylline on cultured rat embryonic shields. Effects of N6, O2 dibutyryl adenosine 3',5'-cyclic monophosphate (db-cAMP) and theophylline (Th) on cultured rat embryonic shields were studied. After addition of db-cAMP to the culture medium, an increase of the weight of explants and of the incidence of the skeletal muscle was observed. Theophylline seems to be ineffective.


From deoxynucleotides to DNA synthesis.

From deoxynucleotides to DNA synthesis. The deoxyribonucleotides required for DNA synthesis are provided by reduction of ribonucleotides. Our studies aim at an understanding of the interplay between the processes involved in the production of deoxyribonucleotides and in their consumption. The enzyme system reducing ribonucleotides has been characterized in some detail, and its regulatory aspects were investigated with pure enzymes. Knowledge gained in this way made it possible to interfere specifically with the activity of ribonucleotide reductase in intact cells and to study effects on DNA synthesis. In this connection, the replication of polyoma DNA was used as a model that allowed dissection of DNA synthesis into different steps involved in the initiation and elongation processes.


Macrophage-induced reversal of immunosuppression by leukemia viruses. Suppression of the humoral immune response by several murine leukemia viruses has been well documented. However, the mechanism of suppressed immunoresponsiveness is not well characterized. Macrophages have been reported to be intimately involved in host-tumor relationships, and were therefore examined for their role in reversing suppression in two leukemia virus-induced tumor models. In vivo studies with the Friend leukemia virus (FL virus) were unsuccessful in demonstrating any role for stimulated peritoneal exudate (PE) cells, rich in macrophages, in restoration of antibody function in leukemic mice. However, in vitro studies with FL virus demonstrated that proteose-peptone stimulated PE cells from normal syngeneic mice in restricted numbers (1-3 x 10(5)), when added to 5 x 10(6) FL virus infected spleen cells, could partially restore immunity. Furthermore, using the Rowson-Parr virus (RP virus) model system it was shown that PE cells from RP virus-infected mice, as well as normal PE cells, were capable of restoring immunocompetence. Neither splenic adherent cells nor lymphoid cells from other tissues, when added to RP virus-infected spleen cells, were able to induce recovery of the immune response. In addition, treatment with antitheta serum plus complement had no effect on the ability of PE cells to restore immunity, implying that macrophages were solely responsible for reversal of immunosuppression. An alteration of antigen "processing" or "focusing" may be an important mechanism by which recovery of immune competence is achieved.


Effects of aging on the hypothalamic-hypophyseal-gonadal axis in female rats. The failure of reproductive function in aged rats could be due to deficiencies at the level of the ovary, pituitary, hypothalamus, or higher brain centers. The classic explanation that the ovary is depleted of follicles does not receive adequate support on the basis of histologic studies of aged ovaries. Basal serum gonadotropin levels change with increasing age in female rats. Serum follicle-stimulating hormone (FSH) levels rise while serum luteinizing hormone (LH) levels fall. Likewise, the characteristic response to castration is markedly altered in aged female rats with a reduced secretion of FSH and a minimal elevation of LH. However, the pituitaires of these animals are still caapable of responding to exogenous LH-releasing hormone with a delayed LH response whose magnitude simulates that seen in younger female animals. With increasing age there is decreased pituitary and/or hypothalamic sensitivity to the feedback action of estradiol. These data are consistent with the postulation that there is an altered hypothalamic-hypophyseal function in aged rats.


[Enzymes of the pentose phosphate pathway in the brains of rats during long-term deprivation of the paradoxical stage of sleep]. The selective paradoxical sleep (PS) deprivation in rats entailed during first 2 days a drop in activity of glucose-6-phosphatehydrogenase by 80 and 60%, and of 6-phosphogluconatdehydrogenase -- by 20--35 and 40--50% in the brain-stem and the cortex, respectively. Activity of the transketolase decreased by 27--29% on the 2nd day of PS deprivation only. On the 4th day, normalizing of activity of all the enzymes under study except transketolase, was observed in both regions of the brain. The consumption of riboso-5-phosphate by the homogenate and level of phosphoketopentoses formation in it reflected, mainly, the dynamics of transketolase activity changes.


[Effect of corticotropin and hydrocortisone on the contractility and heat production of skeletal muscles]. The ACTH was shown to increase whereas hydrocortisone entailed no changes of the muscle contraction amplitude, the duration of the muscle work to fatigue in the regimen of solitary rhythmic contractions being, however, shortened in both bases. The heat production for a unit of the muscle work remained the same after ACTH and decreased after hydrocortisone. The corticotropin increases whereas hydrocortisone decreases the efficiency of the energy substances usage by the working muscles.


[Studies on the biosynthesis and secretion of parathyroid hormone in monolayer cultures of bovine parathyroid cells (I) (author's transl)]. We have developed a preparation of monolayer cultures of bovine parathyroid cells in order to elucidate the control mechanism of the biosynthesis and secretion of parathyroid hormone (PTH) at cellular level. Dispersion of parathyroid cells was performed by stirring minced bovine parathyroid tissues in Hanks' BSS containing 0.3 yields to 0.5 percent collagenase at 37 degrees C for 60 min. Dispersed cells were cultured at 37 degrees C in MEM-Hanks' BSS containing 10 percent fetal calf serum and 15 mM HEPES. On the 5th day of the culture, the medium was replaced with 1 percent BSA-MEM-Hanks-HEPES buffer, and the cells were incubated with 3H-leucine or in the media containing various concentrations of calcium, magnesium, PGE1, PGE2 or DBcAMP. At the end of incubation, the cells were detouched and homogenized in 8M urea, 0.2 N HCL and 0.01 M cysteine solution. The isolation of proparathyroid hormone (ProPTH) and PTH was performed through the preparation of TCA-powder followed by CMC column chromatography. PTH in the incubation medium was determined by radioimmunoassay. It was demonstrated that the monolayer cultures of bovine parathyroid cells were synthesizing ProPTH and converting it to PTH. The cultures exhibited linear secretion rates of PTH into the medium. The secretion of PTH was markedly increased by PGE1, PGE2 or DBcAMP in the range of 10(-7) yields to 10(-5)M in the former and 10(-5) yields to 10(-3)M in the latter, while calcium or magnesium changed secretion rate in the range of 0.3 yields to 4.4 mM.


[The effect of calcium on steroidogenesis in isolated human adrenal cells (author's transl)]. The present experiment was planned to verify the effect of calcium on adenyl cyclase in isolated human adrenal cells. Normal adrenal glands were obtained surgically from patients with primary aldosteronism and advanced breast cancer. Isolated adrenal cells were prepared by the modified Haning's method. They were incubated at 37C under a gas mixture of 95 percent O2: 5 percent CO2 in calcium-free Krebs-Ringer bicarbonate buffer solution containing 0.2 percent glucose and 0.5 percent fatty acid-free bovine serum albumin, to which various doses of CaCl2 or ACTH were added. Thirty minutes later, cyclic-AMP was measured by cyclic-AMP assay kit (The Radio-chemical Center, Amersham). 11-OHCS was estimated fluorometrically by the modified Silber's method after incubation for 2 hours. In the calcium-free incubation medium, productions of 11-OHCS and cyclic-AMP were negligible. In the concentration of 2.54 mM/L of calcium, 11-OHCS production increased with significant difference statistically, while the increase of cyclic-AMP production was not significant. In the concentration of 12.70 mM/L of calcium, however, cyclic-AMP production increased remarkably. When ACTH was added to the incubation medium containing 2.54 mM/L of calcium, productions of 11-OHCS and cyclic-AMP also increased remarkably. These results indicate that adenyl cyclase of human adrenocortical cells is directly stimulated by calcium and suggest that calcium acts as the second messenger of ACTH.


[Secretory regulation and episodic secretion of aldosterone in patients with primary aldosteronism (author's transl)]. In nine patients with primary aldosteronism (PA), plasma aldosterone was measured in order to evaluate a role of the renin-angiotensin system, ACTH and potassium in the secretion of aldosterone. The circadian rhythm and the episodic secretion were also studied in three patients. The results were as follows: 1) In seven out of nine patients, plasma aldosterone levels (PAL) decreased after the two-hours standing, and it run parallel with plasma cortisol levels (PCL), but not with plasma renin activity (PRA). 2) An intravenous infusion of saline of 2000 ml during four hours failed to suppress PAL in all of five patients and the change of PAL was parallel with that of PCL. 3) PAL decreased markedly after the oral administration of 1 mg of dexamethasone (Dexa), and there was a significantly positive correlation (r = 0.898, p less than 0.01) between basal PAL and the its decrement. 4) Following an intravenous infusion of angiotensin-II at a rate of 0.015 microgram/Kg/min after the pretreatment with Dexa, no significant increase of PAL was observed in any of the five patients. 5) Following a single injection of 0.25 mg of ACTH after the pretreatment with Dexa, PAL rose rapidly, and its peak level was found 60 minutes after the injection. In four out of eight patients this rise was greater than that in the normal subjects. 6) An intravenous infusion of 20 mEq of KCl after the pretreatment with Dexa, produced normal response i.e., an increase of PAL in all seven of the patients. 7) The circadian rhythm and the episodic secretion of PAL in the patients were synchronous with those of PCL throughout twenty four hours.


[A case of medullary thyroid carcinoma with ectopic ACTH syndrome (author's transl)]. A 53-year-old male with Cushing's syndrome due to ectopic ACTH production from medullary carcinoma of the thyroid was reported. The clinical course and results of detailed endocrinological studies and immunohistochemical findings about the cancer tissue were described. An abnormally high concentration of calcitonin, ACTH and beta-MSH in both plasma and cancer tissue (thyroid, lymph nodes and liver) were documented by radioimmunoassay. Urinary 17-OHCS was as high as 38.4 mg/day and showed no supression following dexamethasone 8 mg/day administration. ORAL METYRAPONE (3 G/DAY) CAUSED NO RESPONSE IN URINARY 17-OHCS. Parallel increments in plasma calcitonin, ACTH and beta-MSH were observed following calcium and gastrin loading. Total thyroidectomy with modified radical neck dissection caused minimal changes of plasma levels of calcitonin, ACTH and beta-MSH and no improvement in the clinical manifestations of Cushing's syndrome. An aortogram revealed metastatic tumors in the liver. A second operation, total adrenalectomy, resulted in an improvement of the clinical and laboratory findings such as hypokalemia, high blood pressure, muscle atrophy and moon face. Immunofluorescent study showed different distribution patterns in calcitonin- and ACTH-positive cells in the primary focus but similar patterns in the liver metastasis.


Thyroxine secretion by isolated hog thyroid cells: a cyclic AMP independent pathway. The release of 131I-labeled thyroxine (T4) from isolated hog thyroid cells was increased 1.5--2-fold by thyrotropin (TSH). Dibutyryl cyclic AMP failed to reproduce this TSH action. In this in vitro system another cell activity, T4 synthesis, was stimulated in an essentially identical fashion by TSH and dibutyryl cyclic AMP (time course of action, dose-response relationship). 3-Isobutyl-1-methylxanthine (IBMX), 0.5 mM, did not alter the basal [131I]T4 release whereas it enhanced the [131I]T4 synthesis. TSH, 60 MU/ml, increased the intracellular cyclic AMP concentration 3-4-fold. Chlorpromazine (5 X 10(-4)M) abolished the TSH stimulation of cyclic AMP accumulation but did not alter the TSH-induced increase in [131I]T4 secretion. It is concluded that the TSH action on [131I]T4 secretion by isolated thyroid cells is not mediated by the adenylate cyclase-cylic AMP system.


Inhibitory effects of cyclic AMP on vasopressin release from the rat neural lobe in vitro. The basal release of vasopressin from the isolated neural lobe of the rat decreased in the presence of exogenous cAMP, 8Br-cAMP, diB-cAMP, theophylline, SQ 20,009 and RO20-1724. The concentration-related decrease in vasopressin release, in the presence of phosphodiesterase inhibitors, was accompanied by a progressive increase in cAMP concentration in the neural lobe. The findings suggest a local modulation of vasopressin release from the neural lobes.


Primary action of steroid hormone at the surface of amphibian oocyte in the induction of germinal vesicle breakdown. An insoluble steroid derivative was prepared by the coupling of desoxycorticosterone with aminoethylated agarose beads. When the naked full-grown Xenopus oocytes were incubated in contact with the steroid-bound agarose beads, the dissociation of oocyte nuclei or the initial step in the maturation of amphibian oocyte was induced in 30-100% of total oocytes. The induction did not occur in the oocytes covered with follicle cells and when the naked oocytes were placed apart from the steroid-bound agarose beads in the medium. The above findings confirmed that the oocyte surface was a primary site for this particular steroid action, eliminating the possibility of participation of free steroid artifactually released from the agarose. The 105,000g supernatant fraction of oocytes showed no sign of the presence of steroid receptor. This was not inconsistent with the assumption mentioned above.


10-years experience with a 28,800 roller bottle plant for FMD vaccine production. The roller bottle plant at the Istituto Zooprofilattico of Brescia is described and the following topics are discussed: (1) results of ten years cell and virus culture; (2) critical approach to advantages and disadvantages of applied technology, and (3) FMD vaccine requirements and cell substrate characterization.


Relative sensitivity of three cell substrates to the Sabin poliovirus strains. Poliovirus types 1, 2 and 3 (Sabin strains) were titrated using microtissue culture plates in three different cell substrates: Hep 2C, Vero and MRC-5 human diploid cells. Each type of poliovirus was titrated 60 times. The virus dilutions were inoculated in the three cell substrates and the titres (TCID50/ml) calculated by the Spearman-Kärber method. The results were analyzed by the U-test (Mann and Whitney procedure) in a Wang 600 computer. For poliovirus type 1 the differences in titres observed between Hep 2C and Vero cells were not statistically significant at p = 0.01, but the differences in titres between Hep 2C and MRC-5 human diploid cells and between Vero and MRC-5 cells were statistically highly significant (p = 0.001). For poliovirus types 2 and 3 the observed differences in titres were highly significant (p = 0.001) between Hep 2C and Vero cells, Hep 2C and MRC-5 and also between Vero and MRC-5 cells, with higher titres on Hep 2C cells. Accordingly with these results Hep 2C and Vero cells showed the same susceptibility to poliovirus type 1. In MCR-5 cells the average titre (G.M.T.) was 1 log10 below the average titres (G.M.T.) from Hep 2C or Vero cells. With poliovirus types 2 and 3 the higher titres were obtained on Hep 2C cells with significant differences to the titres observed in Vero and MRC-5 human diploid cells, which showed less susceptibility.


The contribution of cytochemistry and electron microscopy to the detection of contaminants in the cell material used for the production of virus vaccines. For the production of virus vaccine it is essential to use cellular material free of contaminants that could reach the final product. It is also important to check initial tissue for possible inherent infections. Studies on primary culture of chick embryo fibroblasts have shown in several cases that cultures which appeared normal by current cytological methods had a strongly positive reaction when investigated by the Feulgen reaction for nuclear DNA and acridine orange method proving intense RNA synthesis. Comparison of electron microscopic (EM) pictures of cell sections with results obtained from negatively stained preparations of identical cell material after pronase digestion have shown the presence of viruses, thus elucidating the nature of the inclusions. The combined approach of the above-mentioned problem by cytochemical and EM methods can usefully enlarge the rage of tests employed for the definition of cell populations acceptable for virus vaccine production.


Replication of poliovirus (LSc 2 ab) in perfused cultures of MRC-5 diploid cells. Application of a new perfusion culture system to the propagation of poliovirus type 1 (LSc 2 ab) in MRC-5 cells is described. Virus production was compared in perfused and stationary cultures of cells at the 26th to 30th population doubling. Yield of virus per cell and unit growth surface area were found to be consistently higher in the perfused system by factors of three to five fold respectively. The influence of experimental conditions on these results and the potential of perfusion for large scale vaccine manufacture are discussed.


Cellular changes in primary rabbit kidney cells resembling those caused by certain viruses. Changes suggestive of being caused by a virus have recently been observed in primary rabbit kidney cells. In their nature and development they resembled those of parvoviruses. The changes were found in cultures from rabbits from various sources within and outside The Netherlands. If the changes are caused by a viral agent, this agent is most probably of rabbit origin, as exogenous infection, for example through trypsin, could almost certainly be excluded. However, no virus has yet been isolated nor could particles be made visible by electron microscopy in affected cells. Hemagglutination could not be demonstrated nor could antibody in rabbits. The possible nature of these changes will be discussed.


Lipolysis and cyclic AMP levels in epididymal adipose tissue of obese-hyperglycaemic mice. Glycerol release from epididymal fat fragments of young adult (3-month old) ob/ob mice was three times lower than normal, on a tissue weight basis. Dose-response curves in response to isoproterenol and ACTH-(1--24) indicated that the capacity of the lipolytic process was reduced. However, the sensitivity to both hormones was normal, i.e. greater for ACTH than for isoproterenol. The burst of cyclic AMP observed at 7 minutes was affected even more than the lipolytic capacity in adipose tissue from obese mice. This was already observed in 1-month old animals, i.e. at a time when total body weight was still normal. It is concluded that the adenylate cyclase system is defective in adipose tissue of ob/ob mice. Besides, glucagon, vasoactive intestinal polypeptide, and secretin failed to stimulate glycerol release and cyclic AMP accumulation in both ob/ob, ob+/ob+, and HA-ICR mice, suggesting that mouse adipose tissue does not possess receptors for this group of hormones.


Cytomegalovirus vaccine prepared in WI-38. Human fibroblasts are the only cells that regularly produce cytomegalovirus of adapted laboratory strains and release fair amounts of cell free virus. One such strain, Towne, was adapted to WI-38 and could release 10(6) to 10(7) p.f.u. per ml tissue culture fluid. The maximum cell-free virus is released at 5-7 days after infection, when cytopathic effect has reached a peak. The virus harvest must be combined with stabilizer rapidly and rapidly lyophilized or frozen, for the virus is unstable even at 4 degrees C. With regard to control, cytomegalovirus is poorly neutralized by antibody-containing serum, and complement is required for maxium neutralization. Frequent addition of fresh antiserum may be necessary to keep cytomegalovirus from replicating in cells inoculated to detect other viruses. Another special problem is the putative association of oncogenicity with herpes group viruses. Tests must be done in newborn hamsters to show that live or inactivated virus does not cause tumors. Towne vaccine tested in man has produced asymptomatic infection with antibody response. No urine excretion has been observed, and antibodies persist for at least 2 years.


Use of polyethyleneglycol-treated serum for animal cell cultures. Although many proteins will be removed from sera by precipitation with 10% polyethyleneglycol (PEG) the growth-promoting properties of such PEG-treated sera for many cell lines and cell strains are hardly reduced. Among the precipitated proteins are the macroglobulins which are difficult to remove from cell cultures and which may cause allergic reactions if incorporated into vaccines. The gammaglobulins are removed in this way as well. If the sera are contaminated with viruses or phages the titres will be reduced by approximately 4 logs and thus the incidence of virus contamination will be reduced by the PEG-treatment. For foot-and-mouth Disease (FMD) vaccine production PEG-treated sera from vaccinated cattle were applied for cell and virus cultivation. The virus obtained was subsequently precipitated with PEG and collected by filter-aid filtration. A concentrated virus product was obtained, which was practically devoid of serum proteins.


Characterization of monolayer cultures of type II alveolar pneumonocytes that produce pulmonary surfactant in vitro. In lung, type II cells are the site of synthesis of phosphatidylcholine (PC), a major component of pulmonary surfactant. Clonal culture methods permit isolation of an epithelial cell strain (L-2) derived from type II cells of intact rat lung capable of active PC production in vitro. Isotopic labeling of these monolayer cultures show that the choline incorporation pathway is the predominant biosynthetic route for PC production. This same pathway is utilized for PC production in whole lung. Three enzymes of this pathway are readily detected in L-2 cells. Determination of specific enzyme activity in monolayers and whole lung indicate that clonally derived L-2 cells are enriched in choline kinase (10-fold), cholinephosphate cytidyl transferase (10-fold) and cholinephosphotransferase activity (5-fold). Our second approach was to develop an in vitro system in which cellular inter-relationships and cell to cell contacts present in whole lung are maintained. This organotypic system is formed by reaggregation of monodispersed fetal rat lung cells into alveolar-like structures (ALS). The ALS are comprised primarily of type II cells as evidenced by the presence of osmiophilic lamellar bodies in the cells. Biosynthesis of these lamellar bodies occurs de novo and type II cells in the ALS continue to synthesize lamellar bodies. Both systems permit study of a variety of agents of pulmonary surfactant production in vitro.


Analyses of stage-specific multiple forms of lactate dehydrogenase and of cytochrome c during spermatogenesis in the mouse. Spermatogenesis is a programmed developmental process characterized by the inactivation of certain genes and the activation of other, testis-specific genes. Synthesis of unique gene products such as LDH-C4 and cytochrome ct occurs only at precise stage of germ cell formation. The developmental sequences of gene activation for these proteins was observed by immunohistochemical procedures. LDH-C4 is first detectable during mid-pachytene of the primary spermatocyte. The C subunits appear to be uniformly distributed throughout the cytoplasm of the spermatocyte. The mid-pachytene stage also marks the first appearance of cytochrome ct. The association of this electron transport protein with spermatocyte mitochondria is reflected in a granular fluorescence of specific antibody-treated sections of testis. Neither the C subunit of LDH, nor cytochrome ct appear in leptotene or early pachytene primary spermatocytes. These analyses indicate that there is stage-specific protein synthesis in the primary spermatocyte which is characterized by differential activation of the LDH-C locus and of the gene coding for cytochrome ct.


Combination chemotherapy of 6-thioguanine with various antitumor agents against murine leukemia L-1210. Antitumor activity of 6-thioguanine against L-1210 leukemia was studied in combination with various antitumor agents in clinical stages, such as mitomycin-C, carbazilquinone, cyclophosphamide, 3,3'-dimesyloxydipropylamine tosylate (864T), 4-hydroperoxyisophosphamide, and 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chlorethyl)-1-nitrosourea hydrochloride (ACNU). The combination treatments with 6-thioguanine and each of six agents, especially with ACNU, showed a distinct therapeutic effect against the early L-1210 leukemia at dosage levels not producing any significant antitumor activity with each agent alone (ip-ip). The excellent antitumor activity of the combination of 6-thioguanine with ACNU was also proved in various sites of tumor inoculation and routes of drug administration systems, i.e., ip-iv, iv-ip, and iv-iv systems. Moreover, the effectiveness of the combination of these two drugs was clearly shown against advanced L-1210 leukemia which was refractory to each agent alone.


Antitumor effect of 1,1'-polymethylene-bis (1-nitrosourea) and related compounds. Polymethylene-bis(1-nitrosourea), polymethylene-bis(1-nitroso-3-nitroguanidine), and polymethylene-bis(1-nitroso-p-toluenesulfonamide) derivatives were tested for antitumor effect against rat ascites hepatoma AH-13 and mouse leukemia L-1210. Bisnitrosoureas were effective against AH-13 and L-1210, bisnitrosoguanidines were effective against AH-13 alone, and bisnitrosotoluene-sulfonamides were ineffective against both tumor lines. Of all these compounds, 1,1'-ethylene-bis(1-nitrosourea) (EBNU) was the most effective. The antitumor effect of EBNU was compared with that of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Intraperitoneal administration of EBNU according to the schedule, day 1, days 1 and 5, and days 1, 5, and 9 after intraperitoneal inoculation of L-1210 showed marked prolongation of host survival, although the effective doses used were a few times higher than those used in BCNU to obtain a similar effect. The minimum effective dose (MED) of EBNU on AH-13 cells was estimated as 1 mg/kg, which was 10 times less than that of BCNU, suggesting that EBNU was more effective than BCNU against AH-13.


Role of leucocytes in ascites in the production of factor(s) stimulating differentiation of mouse myeloid leukemia cells. Although the ascitic fluid of animals bearing various tumors and that of mice induced by complete Freund's adjuvant had high activity for inducing differentiation of myeloid leukemic cell line (M1) from an SL mouse to macrophages and granulocyte-like cells, the activity in the ascitic fluid of syngeneic mice bearing the M1 cells was markedly reduced. Macrophages and granulocytes were abundant in the active ascites of animals bearing tumors (8 to 12% of the total ascites cells) while in the ascites of syngeneic mice bearing the M1 cells they were not (0.1 to 0.7% of the total ascites cells). Appearance of lymphocytes in the ascites of both types was not significantly different. Although the conditioned media of the Ehrlich tumor cells, M1 cells, and whole ascites cells with the M1 cells were not active in inducing differentiation of the M1 cells, the conditioned media of all the ascites cells with Ehrlich tumor cells and those of peritoneal macrophages and granulocytes in mice did show a high activity. These results indicate that the peritoneal macrophages and granulocytes in the ascites are responsible for the production of factors stimulating differentiation of the M1 cells.


Studies on tumors produced by cells transformed with herpes simplex virus type 2. Biological, histological, and ultrastructural studies were made on tumors produced in weanling hamsters inoculated with hamster embryo fibroblasts (HEF) transformed by herpes simplex virus type 2 or with cultured cells (tumor cell lines T1 and T2) derived from the tumor tissues. The malignancy of transformants increased through passages in vitro and in vivo. Histologically, two tumor cells lines (155-4T1 and U-15T1) produced fibrosarcomas and one (U-26T1) produced lesions resembling "malignant fibrous histiocytomas." Ultrastructurally, fibrosarcomas produced by 155-4T1 and U-15T1 consisted mainly of fibroblast-like cells with extracellular collagen fibers, whereas malignant fibrous histiocytoma-like lesions produced by U-26T1 consisted of undifferentiated cells, multinuclear giant cells, histiocyte-like cells, and fibroblast-like cells. Frequently, various kinds of "nuclear bodies" were found in the nuclei of tumor cells. Several herpes virus-like particles (120 approximately 140 nm in diameter) were detected in some nuclei of undifferentiated tumor cells produced by U-26T1.


Isozyme patterns of branched-chain amino acid transaminase in human tissues and tumors. The isozymes (enzymes I and III) of branched-chain amino acid transaminase (EC 2.6.1.42) from various human tissues were separated by DEAE-cellulose column chromatography. Their distributions were found to be considerably different from those in rat tissues reported before. In rats, all tissues examined contained enzyme I and in addition enzyme II was found in the liver and enzyme III in the brain, ovary, and placenta. In humans, however, enzyme II was not found in any tissues examined including the liver, and enzyme III was found in many other tissues besides brain, ovary, and placenta. All normal human tissues except the lung, ovary, and brain contain less enzyme III than enzyme I. Various human cancers of the liver, kidney, stomach, pancreas, and uterus showed significantly higher ratios of enzyme III to enzyme I than those of the corresponding normal tissues. Fetal liver and kidney also contained much higher concentrations of enzyme III than adult liver and kidney. These findings suggest that change of the isozyme pattern of this enzyme in cancer is similar in humans and rats, and that cancer tissues tend to express more immature phenotypes than normal tissues. Enzymes I and III of human tissues showed the same substrate specificities for valine, leucine, and isoleucine, and these amino acids competed for the active site of the enzyme. Thus the hereditary diseases, hypervalinemia and hyperisoleucine-leucinemia, may be due to genetic alteration of the enzyme protein, resulting in change of its substrate specificity.


Properties of a transposon conferring resistance to penicillins and streptomycin. R938 carries a transposon (TAbeta) of approximate molecular weight 9.5 Megadaltons (Mdal, 10(6) daltons). This contains genes for a beta lactamase of type TEM-1 and for streptomycin phosphatransferase (SPT). There is a ten-fold difference in the efficiency of transposition in different strains of E. coli K12.


Chromatin transcription. I. Quantitative determination of gene-specific RNA by use of bacterial plasmids containing the eukaryotic DNA sequence of viral DNA. Specific gene transcription from chromatin has been examined in two different systems by using direct measurement of specific sequences by hybridization with excess DNA probe. An improved procedure has been used for hybridization of DNA immobilized on filters. This procedure is sensitive to less than one part in 10(5). Polyoma RNA sequences were detected a level of 0.004% in the overall transcript from chromatin of ts-a-transformed 3T3 fibroblasts. By the use of constructed plasmid DNA carrying rabbit globin sequences, globin RNA was titrated in the RNA transcribed from chromatin of bone marrow cells. Its relative frequency was 0.01%. Results obtained with this new approach confirm that DNA in chromatin is not randomly transcribed. They further illustrate that a reproducible assay system is now available for studying control elements active on gene expression at the transcriptional level, taking advantage of the constructed plasmids containing eukaryotic gene DNA fragments.


The influence of dihydroergotoxine mesylate on the low-Km phosphodiesterase of cat and rat brain in vitro. Dihydroergotoxine mesylate (DHET; active substance of Hydergine) was shown to be a stronger inhibitor of the low-Km than of the high-Km phosphodiestrase (PE) in cat and rat brain homogenates. The inhibition due to DHET was greater when a purified low-Km PE preparation originating from a sonicated pellet (100,000 g) of rat brain tissue was used. The drug alters the kinetic properties of the enzyme, producing a lowering of the negative cooperativity effect. This kind of PE inhibition is of particular importance at normal cellular cAMP levels, but does not interfere with cAMP production by hormonal stimulation.


Dopamine-mimetic activity of ergot derivatives, as measured by the production of cyclic AMP in isolated retinae of the rabbit. The effects of several ergot alkaloid derivatives on the concentration of cyclic AMP in intact rabbit retinae were investigated in vitro. All compounds were found to be potent inducers of cyclic AMP production, half-maximal stimulation being obtained at 10(-6)M, and maximal stimulation at 10(-4)M concentrations. The range of effective concentrations was very well comparable with that of dopamine (or apomorphine) in the same experimental conditions. Furthermore, the accumulation of cyclic AMP induced by the ergot derivatives was blocked by fluphenazine, at 10(-4)-5 X 10(-4)M, or lithium, at 5 X 10(-2)M concentrations. Both antipsychotic drugs were found to be very potent inhibitors of dopamine-(or apomorphine-) induced production of cyclic AMP in the same preparation. A potential dopamine-mimetic activity of ergot derivatives (or of other dopamine-like drugs) can be specifically tested on isolated rabbit retinae in vitro, either before undertaking experiments in vivo or in correlation with behavioural studies.


Effect of dihydroergotoxine on cyclic-AMP-generating systems in rat cerebral cortex slices. In slices of rat cerebral cortex, cyclic-AMP formation is stimulated in response to added noradrenaline (NA), isoproterenol (ISP) or adenosine. The effect of ISP could be antagonized only by the selective beta-adrenoceptor-blocking agent pindolol, whereas the stimulating effect of NA could be antagonized by both alpha- and beta-adrenoceptor-blocking agents. Dihydroergotoxine (DHET) at low concentrations (10(-8)M) antagonized the stimulating effect of NA but not of ISP or adenosine, suggesting that this substance has a high affinity for central alpha-adrenoceptors. We could also show that DHET is accumulated in rat cerebral cortex after repeated oral application. In slices of cortex obtained from rats after treatment with DHET for 3 and 6 weeks, but not after 1 week, NA had a significant lower effect on cyclic-AMP formation than in slices from corresponding controls.


Abnormal regulation of adrenal function in rats with streptozotocin diabetes. The factors that control adrenal steroid secretion and metabolism were investigated in rats made diabetic with Streptozotocin (65 mg/kg) and used one month after treatment. Diabetic animals possessed high resting levels of plasma corticosterone accompanied by adrenal hypertrophy; the showed an increased response to the stress of i.p. cold water injection. Moreover, the pituitaries of diabetic rats seemed to be releasing ACTH continuously and not storing it. Upon adrenal inhibition with Aminoglutethimide the expected increase in adrenal cholesterol and weight was of a smaller magnitude than in controls. The activity of liver enzymes that reduce ring A of corticosterone showed decreased activity in diabetics, which suggests that more corticosterone rather than its inactive metabolites were available to--but not able to suppress--the steroid feedback sites. The half-life of corticosterone in blood was similar in diabetes and controls. These results suggest that (a) diabetic animals were in a chronic stress condition; (b) the threshold for steroid feedback was less sensitive to variations in plasma corticosterone; (c) there is an abnormal peripheral disposal of corticosterone, but that other factors, besides the liver, regulate the clearance of the hormone from the circulation in the diabetic animals.


Studies on the occurrence and distribution of HI antibodies against some arboviruses in the serum of domestic mammals in Puglia. The virological and serological studies previously carried out on arboviruses in Italy are reviewed. The presence of antibodies to 11 arboviruses was investigated in the serum of various domestic animals (100 horses, 107 pigs, 102 sheep, 205 goats, 100 cattle and 200 dogs) from some areas of Puglia. The techniques are described. The results, given in tables and discussed in detail, support the hypothesis that in this region also there are arboviruses circulating, particularly those of group B.


Triiodothyronine receptors in lymphocytes of newborn and adult rats. Cytoplasmic and nuclear incorporation of 125I triiodothyronine by thymic lymphocytes has been demonstrated by electron microscopic autoradiography. Incorporation was significantly more pronounced in the newborn than in the adult age. The information emerging from the experime,tal observations contributes further evidence to the more precise interpretation of receptor maturation.


Presence of Aujeszky's disease virus in organs and materials from experimentally infected dogs. The presence of Aujeszky's disease virus was investigated in various organs and materials from experimentally infected dogs, employing the fluorescent antibody test (FAT), tissue culture and biological tests in rabbits. The FAT appeared less sensitive for virus detection than virus isolation in cell culture and rabbit tests. The virus distribution in the central nervous system was related to the site of inoculation.


Hyperinsulinemic hypoglycemia: effect of epinephrine suppression. The inhibitory effect of epinephrine on basal and tolbutamide-stimulated insulin release was studied in 5 patients with hyperinsulinemic hypoglycemia. Epinephrine inhibited both basal and tolbutamide-induced insulin release in patients with beta-cell adenoma and hyperplasia, but failed to inhibit insulin release in a patient with beta-cell carcinoma. The inhibition of basal insulin with epinephrine was maximum in patients with beta-cell hyperplasia. This differential inhibitory effect of epinephrine on insulin release may prove to be a useful screening test in the preoperative diagnosis of the nature of the lesion producing hyperinsulinemia.


Effect of hyperprolactinemia on pituitary-adrenal function. Three patients with hyperprolactinemia (2 with pituitary tumors and 1 with a hypothalamic disturbance) underwent evaluation of their hypothalamic-pituitary-adrenal axis using ACTH, metyrapone and in one case repetitive insulin tolerance tests. With the exception of a suggestion of a slight increase in adrenal responsiveness to pharmacological doses of ACTH in one of the patients, there was no alteration in the adrenal responsiveness of the 3 patients evaluated. We conclude that hyperprolactinemia probably does not systematically modify the responsiveness of the hypothalamic-pituitary-adrenal axis.


Fetal mouse lung in circumfusion system cultures. Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderatley dense secretory granule in the center of the whorl of the endoplasmic reticulum.


Enhancement of antibody-dependent cell-mediated cytotoxicity of herpesvirus-infected cells by complement. An investigation was made of the effects of complement on the levels of antibody-dependence cytotoxicity (ADCC) mediated by bovine leukocytes against herpesvirus-infected target cells. Neutrophil-mediated ADCC was considerably enhanced upon the addition of low levels of complement that alone failed to induce lysis of antibody-sensitized target cells. This enhancement was most apparent under suboptimum conditions such as at low effector-to-target cell ratios, low levels of sensitizing antiserum, and short-duration assays. Furthermore, cells and classes of immunoglobulin unable to induce ADCC could do so in the presence of complement. The action of complement is considered in terms of a more tenacious bond formed between effector and target cells. The implications of the results are discussed in terms of the part that complement might play in enhancing antiviral recovery processes.


Purification of beta-toxin from Clostridium perfringens type C. Beta-toxin was purified about 340-fold from culture supernatant fluid of Clostridium perfringens type C with a yield of about 24% in terms of biologically active beta-toxin. The purification involved ammonium sulfate fractionation, gel filtration through Sephadex G-100, isoelectrofocusing in a pH 3 to 6 gradient, and immunoaffinity chromatography. The purified beta-toxin gave a single band on polyacrylamide gel electrophoresis.


Alteration of host defense mechanisms by murine cytomegalovirus infection. An animal model of a sublethal infection, utilizing murine cytomegalovirus (MCMV), was developed to determine whether immunological factors could contribute to the establishment of a persistent viral infection. Adult female C3H mice inoculated intraperitoneally with 10(5) plaque-forming units of MCMV developed splenomegaly 5 to 12 days after infection. Virus replicated to peak titers (10(3) to 10(6) plaque-forming units per g of tissue) in liver, spleen, lung, kidney, and salivary gland tissue during the acute phase of the infection (3 to 12 days); it then decreased to undetectable levels in all tissues except salivary gland. Serum interferon was detected as early as 12 h after infection, peaked at 36 h (1,093 U/ml), and was undetectable by 4 days after infection. MCMV-infected animals were hyporeactive to interferon induction with New castle disease virus on days 5 to 9 of the infection. Splenic lymphocyte reactivity to phytohemagglutinin and lipopolysaccharide was normal early during the course of the infection, was suppressed during the acute phase of the infection, and had returned to normal by day 18. These data indicate that several parameters of host defense are transiently suppressed during the course of a MCMV infection. The capacity of cytomegaloviruses to alter host resistance may be one factor that contributes to the establishment of a persistent infection.


Replication of parainfluenza type 3 virus in alveolar macrophages: evidence of in vivo infection and of in vitro temperature sensitivity in virus maturation. Approximately 2% of cultured alveolar macrophages (AM), originally lavaged from the lungs of parainfluenza type 3 virus (PI-3V)-infected calves, were observed to contain viral antigen (by fluorescent antibody method) or viral nucleocapsids (by electron microscopy). Plaque assays, however, indicated that virus titers were generally low when cultures were incubated at 37 degrees C for 10 days. AM, obtained from "in vivo infected" and "noninfected" calves, were found to be equally susceptible to further in vitro PI-3V infection when cultures were incubated at 37 degrees C. AM that were obtained from the lungs of normal calves, cultured at 37 degrees C, and inoculated with PI-3V were observed to produce relatively high virus titers when the incubation temperature was shifted down to 32 degrees C. Results from hemagglutinin assays showed that considerable amounts of hemagglutinin were detected when AM cultures were incubated at 32 degrees C, but only limited amounts were detected at 37 degrees C. Results from electron microscopic examinations at both temperatures substantiated the results of plaque and hemagglutinin assays. The PI-3V, isolated from AM cultures incubated at 32 degrees C, grew well in Madin-Darby bovine kidney cells at 32 degrees C, but little virus was produced at 37 degrees C. In contrast, parent PI-3V grew equally well at both temperatures. The results are discussed in terms of host susceptibility, temperature-sensitivity and virus maturation, and surface viral antigens and persistent viral infection.


Respiratory infection in mice with sialodacryoadenitis virus, a coronavirus of rats. Sialodacryoadenitis virus (SDAV), a coronavirus of rats, evoked both serum neutralization and complement fixation antibody responses when inoculated intranasally in mice. Weanling gnotobiotic CD-1 mice inoculated intranasally with 10(3.0) mean tissue culture infective doses of SDAV remained asymptomatic. Virus was recovered from the nasopharynx, trachea, and lung from day 2 to day 7. Viral antigen was readily detected by indirect immunofluorescence in the lung but rarely in the nasopharynx. Infected mice developed interstitial pneumonia. Susceptible mice contact exposed to experimentally infected mice developed antibody to SDAV. Epizootiological studies indicated that retired breeder mice can have complement-fixing antibody to SDAV and mouse hepatitis virus (MHV) in the absence of MHV infection. These studies show that SDAV is infectious for mice and can be a pathogen for the respiratory system. Thus, SDAV infection of mice may be responsible for spurious seroconversions to MHV.


Interferon induction by vesicular stomatitis virus and its role in virus replication. Antibody against human fibroblast interferon increased the yields of vesicular stomatitis virus in human cells. The results show that endogenous interferon produced in the course of multicycle vesicular stomatitis virus infection depresses virus yields.


Effect of input multiplicity on the establishment of simian virus 40 persistent infections in rhesus monkey kidney cells. Monolayer cultures of LLC-MK2 rhesus monkey kidney cells become persistently infected with simian virus 40 after infection at input multiplicities of 100, 10, or 1 plaque-forming unit per cell. After 3 weeks, all cells of the cultures infected at a multiplicity of 1 plaque-forming unit per cell produced the simian virus 40 T antigen. In contrast, 8 to 11 weeks elapsed before all the cells in the cultures infected at a multiplicity of 100 plaque-forming units per cell produced T antigen. Defective interfering particles and interferon production were not evident during this time.


Characterization of the allergenic components of the house dust mite, Dermatophagoides farinae. Characterization and purification of the allergenic components of the house dust mite, Dermatophagoides farinae, were investigated. It was found that the mite antigen was heat-stable, pronase-sensitive, and periodate-resistant, which suggested that the antigenic determinants of the mite antigen resided mainly in the protein component. Purification of the mite antigen was tried using Sephadex G-150, DEAE cellulose, and isoelectrofocusing columns. The most potent allergenic fraction was around 20,000-30,000 in molecular weight, although the allergenic activity spread in a wide molecular weight range. Prausnitz-Küstner type skin reactions were performed on rats using mouse homocytotropic antibodies for allergenic evaluation, and the clinical usefulness of this method is discussed.


Chromosome banding, isoenzyme studies and determination of Epstein-Barr virus DNA content on human Burkitt lymphoma/mouse hybrids. Four independently fused hybrid clones derived from a cross between the mouse mammary carcinoma TA3Ha and the human Burkitt lymphoma line Daudi were tested for the EBV-determined nuclear antigen (EBNA), EBV-DNA and the presence of human chromosomes, in the course of serial propagation in vitro. EBNA and EBV-DNA were lost in parallel with the loss of human chromosomes. It seems that the persistence of EB-viral genetic information does not require the presence of a specific human chromosome(s) in this particular hybrid combination.


Mechanism of the establishment of Epstein-Barr virus genome-containing lymphoid cell lines from infectious mononucleosis patients: studies with phosphonoacetate. A concentration of disodium phosphonoacetate (PA) has been defined which will reduce the synthesis of infectious EB virus in a producer cell line to 1% of control values but which will not affect the growth of EB virus-transformed cells in a 12-week colony-forming assay. When total mononuclear cells or T-lymphocyte-depleted mononuclear cells from the blood of acute IM patients were cultured in the presence of PA at the above concentration, the regular establishment of EB virus genome-containing cell lines seen in control cultures was almost totally abolished. In further experiments, when T-lymphocyte-depleted IM mononuclear cells were co-cultivated with foetal cells of the opposite sex in the presence and absence of PA, cell lines of mixed or of exclusively foetal origin were obtained not only from control co-cultures but also on those rare occasions when transformed foci developed in PA-treated co-cultures. The results suggest that all cell lines derived from the blood of IM patients are initiated in culture by a two-step process of virus release and secondary infection, and argue against the occurrence of any direct outgrowth of IM cells transformed by the virus in vivo.


EBV specific secretory IgA in saliva of NPC patients. Presence of secretory piece in epithelial malignant cells. Saliva samples from 59 patients with nasopharyngeal carcinoma (NPC) and from 20 normal individuals were studied to determine the nature and origin of the EBV-specific IgA in NPC saliva. About 50% of NPC saliva samples contained secretory IgA specific for EBV. The corresponding tumor IgA(alpha) was found in plasma cells surrounding the epithelial tumor cells and the secretory piece at the surface of epithelial cells. A slightly higher proportion of NPC saliva samples containing IgA was found in patients from Tunis than in samples from Hong Kong. Attention is drawn to the clinical value of the salivary IgA in diagnosis and monitoring of treatment of NPC.


An analysis of the accessibility and nonaccessibility of patients in a follow-up study. In a follow-up study of 184 emergency room patients experiencing acute drug reactions, several respondents were inaccessible. In this paper the 83 accessible and 101 nonaccessible patients are compared in order to determine why respondents are often difficult to locate in a follow-up study. Those patients who were the most accessible proved to be primarily young, White males who had been referred to a traditional drug treatment program from the hospital emergency room. Those patients who were the least accessible proved to be primarily White females between 24 and 29 years of age who had not been referred for further treatment.


Methadone maintenance and narcotic blocking drugs. Drugs which specifically antagonize certain of the actions of opioids are reviewed. These antagonists include nalorphine, levallorphan, naloxone, naltrexone, and cyclazocine. Programs involving antagonist treatment are discussed.


Plotting regression lines for a new beta-lactam antibiotic (mecillinam). Regression lines for mecillinam (FL 1060) were established according to the recommendations of th ICS. The investigation involved 477 Gram-negative and Gram-positive organisms with the widest possible spectrum of sensitivity which were isolated from fresh material for examination. Solid and liquid Müller-Hinton media (Merck and BBL) were used as nutrient media. The Difco Müller-Hinton medium is not suitable for this subtance because of the high ionic content. A general regression line for mecillinam is not admissible. The considerably restricted sphere of indication shown by our studies is presented with reference to the blood levels attainable in vivo. At the same time, the good sensivity of salmonella is pointed out.


Hyperdynamic beta-adrenergic circulatory state. A case report. A 22-year-old Japanese male with hyperdynamic beta-adrenergic circulatr without premedication. An abnormal electrocardiographic pattern was observed during the dental treatment, which was analyzed from the standpoint of beta-adrenergic receptors and drugs. During treatment, local anesthetics without adrenergic beta-stimulant would be desirable but premedication with beta blocker may be enough even when anesthetic agents with beta-stimulators are used. Furthermore, premedication with minor tranquilizers is also effective.


E.s.r. of spin-trapped radicals in aqueous solutions of amino acids. Reactions of the hydrated electron. The reactions of hydrated electrons (eaq-) with amino acids were investigated by the spin-trapping method and by electron spin resonance. Tertiary nitrosobutane was used as a spin-trap to stabilize the short-lived radicals. Hydrated electrons were produced by gamma-radiolysis of de-aerated aqueous solutions of amino acids in the presence of sodium formate or tertiary butanol to scavenge OH. Radicals produced by reductive deamination of 19 amino acids were identified. Radicals formed by scission of the CH3-S- and -S-CH2- bonds of methionine as well as by deamination were observed. In the case of phenylalanine the radical formed by electron addition followed by proton transfer was identified. The reaction of proline and of hydroxyproline with eaq- resulted in the opening of the cyclic structure.


Thiamine pyrophosphokinase activity in liver, heart and brain crude extracts of control and thiamine deficient rats. The activity of thiamine pyrophosphokinase has been measured in liver, heart muscle and brain of normal and thiamine deficient rats. The activity measurement has been performed by use of thiamine-35S as substrate and separation of the reaction products by high voltage electrophoresis. KM has been determined as 7.1.10(-6) mol/l. The activity of thiamine pyrophosphokinase is reduced in liver and heart muscle of thiamine deficient rats significantly, whereas no decrease of the enzyme activity has been found in the brain. The content of thiamine pyrophosphate has been measured in the liver of normal and thiamine deficient rats. Injection of thiamine to deficient rats normalized the content of thiamine pyrophosphate in the liver within 6 hours. Our data suggest that thiamine pyrophosphokinase is an adaptive enzyme, the activity of which depends on the thiamine content of the cells.


Photochemical inhibition of poliovirus replication by 4,5',8-trimethylpsoralen plus light. Poliovirus replication in HeLa cells is significantly inhibited by a 30-min pulse of infected cells with 4,5',8-trimethylpsoralen plus long wavelength ultraviolet light. When infected cells are exposed to psoralen plus light during peak viral RNA synthesis, formation of virus-specific RNAs is inhibited. Viral RNA species, formed in vivo in the presence of, or treated in vitro with, psoralen plus light, appear to have become degraded when analyzed by sedimentation in sucrose-dimethyl sulfoxide gradients. Treatment with psoralen plus light in vitro results in the loss of infectivity of single-stranded viral RNA.


Hemagglutination and hemagglutination-inhibition studies with a strain of Nebraska calf diarrhea virus (bovine rotavirus). A hemagglutinin has been prepared from Nebraska calf diarrhea virus (NCDV) propagated in BS-C-1 cell line. After cesium chloride centrifugation, the hemagglutinin of the bovine rotavirus was found to be associated with intact virions (density 1.355 g/ml) but not with virions lacking an outer capsid layer (density 1.375 g/ml). In hemagglutination-inhibition (HAI) tests, the hemagglutinin reacted specifically with NCDV serum, and HAI seroconversions were detected in some sera tested. Cross-reactions were observed in complement fixation tests between the human and bovine rotaviruses but were not demonstrated by HAI, suggesting that the hemagglutinin detects a specific rather than a group antibody response.


Detection of herpes simplex virus thymidine kinase polypeptides in cells labeled with 35S-methionine. To investigate the size of herpes simplex virus (HSV) thymidine kinase (TK) polypeptides, procedures have been devised to purify the enzyme from infected cells labeled with 35S-methionine by (i) affinity chromatography on Sepharose-5'-amino-5'-deoxythymidine; (ii) preparative isoelectric focusing or preparative PAGE; and (iii) glycerol gradient centrifugation. Portions of enzyme fractions, at each purification step, were also treated with an immunoadsorbent, Sepharose-anti-HSV-1 TK immunoglobulin (IgG). Labeled polypeptides eluted from the immunoadsorbent were analyzed by electrophoresis in SDS slab gels and autoradiography. The results demonstrate that the molecular weights of HSV TK polypeptides are about 40,000. TK-negative HSV-1 mutant B2006 failed to induce the 40 K dalton polypeptide.


Correlation between polyion effect on cell susceptibility to in vitro infection with murine C-type viruses and polyion effect on some membrane-related functions. Polyions were tested for effects on some membrane-related functions. Both polycations investigated reduced the negative surface charge of assay cells and enhanced in vitro infectivity of murine C-type viruses, but had no influence on leukemia-virus-induced XC cell syncytia formation. Three polyanions increased the net outer cell charge, while only one of four inhibited infectivity and two of three impeded syncytia formation. Polyions had a slight, probably toxic, effect on the transmembrane potential, independent of their charge. Cells treated with fluorescent DEAE-dextran showed diffuse staining, which 4 h later had been modified into a granular fluorescence with unstained areas now present. This change correlated with a loss of enhancement of viral infectivity. The only polyanion which inhibited viral infectivity had a strong antihyaluronidase activity, and hyaluronidase and Ca++ both increased viral infectivity. It is suggested, therefore, that polyions may in part work on virus-cell membrane interactions by influencing membrane enzymes and not necessarily by simply changing the net outer cell surface charge.


Characteristics of herpes simplex virus resistance to disodium phosphonoacetate. Herpes simplex virus (HSV), which was partially resistant to the inhibitory effect of disodium phosphonoacetate (PAA), could be recovered following four virus passages in the presence of 100 microgram/ml PAA. Resistant strains were isolated from both HSV type 1 and HSV type 2. Virus resistance to PAA was not complete, and in most isolations a significant proportion of the virus stock remained susceptible to the drug. Resistance was shown to be heritable and persisted through virus passage and cloning experiments. PAA inhibited the replication of virus-specific DNA in sensitive strains of HSV but not in resistant strains of HSV. In vitro experiments directly demonstrated that PAA inhibited the activity of the virus-specific DNA polymerase 10 times more effectively in PAA-susceptible HSV than in PAA-resistant HSV. The treatment of HSV-infected mice with high levels of PAA did not induce the formation of resistant virus strains.


Growth characteristics of acute hemorrhagic conjunctivitis virus in monkey kidney cells. III. Viral RNA synthesis at permissive and nonpermissive temperatures. Monkey kidney cells infected with the J 670/71 strain of acute hemorrhagic conjunctivitis (AHC) virus in the presence of actinomycin D synthesized two species of viral RNA. One species had properties similar to the virion RNA, sedimented in a sucrose gradient with a sedimentation coefficient of 34S, and was sensitive to RNase. The other species sedimented at 18S and was resistant to RNase. The inability of AHC virus to replicate at 39 degrees was attributed to a lack of virus-specific RNA synthesis in infected cells.


Application of the critical point dried whole cell technique to the study of animal rhabdoviruses. The critical point dried (CPD) whole cell technique was applied to the study of the morphogenesis and morphology of rabies virus and vesicular stomatitis virus (VSV) in mouse neuroblastoma and baby hamster kidney (BHK) cells. With the stereoscopic technique, progeny viruses at the cell surface and within the cytoplasm of the CPD whole cells were clearly visualized. The presence of many fine cellular processes and of virus budding from these processes were prominent features of the infected cells. Long strings of virus particles and virus apparently fused into different forms were often seen; a possible mechanism for the formation of these aberrant forms is discussed. Negative staining of the CPD whole cells clearly revealed the detailed structure of virus particles in the process of budding.


Reiteration frequency of feline type C viral genomes in homologous and heterologous host cell DNA. Differences in the multiplicity of proviral sequences of two feline endogenous viruses, RD-114 and FeLV, in homologous and heterologous host DNA were examined by viral cDNA hybridization using cellular DNA fractionated with respect to reiteration frequency. The endogenous proviral DNAs in cat cells were fractionated predominantly with intermediate-repeated sequences, while RD-114 proviral DNA in virus-infected human cells was fractionated together with unique-sequence DNA. These differences were consistent with the results of kinetic analysis using unfractionated DNA.


Serum angiotensin converting enzyme in sarcoidosis: clinical significance. The elevated activity of serum angiotensin converting enzyme (ACE) in sarcoidosis was usually reduced during steroid therapy but remained above the normal value (52 nmol-min-1-ml-1) in approximately a third of patients during the first year of therapy. ACE was elevated in 5 of 10 patients treated with steroids for at least four years. It was significantly higher in untreated patients (placebo group), who were subsequently placed on steroid therapy because of clinical deterioration, than in those remaining on placebo therapy. In general ACE tended to diminish with increasing duration of disease. The results suggest that serial study of ACE may be useful in the clinical management of sarcoidosis, including assessment of the degree of activity, the need for steroid therapy, therapeutic result and the prognosis.


Increased serum angiotensin converting enzyme activity in sarcoidosis. The activity of serum angiotensin converting enzyme (ACE), assayed fluorometrically with the substrate hippuryl-L-histidyl-L-leucine was significantly higher in 116 patients with sarcoidosis than in 415 patients with other diseases--pulmonary and nonpulmonary--and in 58 normal subjects. Serum ACE was elevated in 59% of 46 recently diagnosed, untreated patients with sarcoidosis and was significantly reduced following steroid therapy in three patients with sarcoidosis, but in only one of four patients treated with placebos. The results suggest that elevated serum ACE activity is a valuable, although not absolutely specific, diagnostic indicator of sarcoidosis. It should be stressed that a normal value does not rule out the disease, and that steroid therapy tends to reduce the serum ACE activity.


Infectious bovine rhinotracheitis: a review and update. Infectious bovine rhinotracheitis (IBR), a herpesvirus infection of cattle, has diverse clinical manifestations. Known mainly as a respiratory tract disease characterized by tracheitis, rhinitis, and fever, IBR plays a prominent role among causes of undifferentiated bovine respiratory disease and abortion. It also causes conjunctivitis, infectious pustular vulvovaginitis, balanoposthitis, and rarely, encephalitis. The virus is readily transmitted and has worldwide distribution. Some cattle develop a latent infection, which can be reactivated. The disease can be diagnosed by clinical signs and lesions and by a variety of virologic and immunologic techniques. Control of IBR is based largely on vaccination. A review is timely because efficient utilization of the wide assortment of available vaccines requires knowledge of recent developments in the diagnosis, immunology, and epidemiology of the disease.


beta-Lactamase and beta-lactam antibiotics resistance in acinetobacter anitratum (syn: A. calcoaceticus). The cephalosporin beta-lactamase (cephalosporinase) produced by Acinetobacter was studied. The enzyme was partially purified by means of column chromatography and its properties were investigated. The enzyme was induced by benzylpenicillin, 6-aminopenicillanic acid and cephaloridine. Its molecular weight is 30,000 its optimal temperature 40C, and its optimal pH 7.25 similar to 7.50. Substrate specificity studies using various cephalosporins and penicillins, showed that the enzyme functioned as a cephalosporinase rather than penicillinase.


Adrenocortical function in aging exercise-trained rats. Plasma corticosterone concentrations and in vitro adrenal secretion of corticosterone were determined in exercise-trained rats. Rats, 100, 200, and 300 days of age, were trained for a 10-wk period by treadmill running. Following the training program, rats were subjected to an acute bout of swimming. Acute swimming elevated plasma corticosterone concentrations in all age groups. At 170 days of age, the plasma corticosterone concentration following swimming was higher in exercise-trained rats than in controls. The opposite was true of acutely swum rats at 270 and 370 days of age. Acute swimming elevated the in vitro adrenal gland response to adrenocorticotropic hormone stimulation in control rats at all ages and in trained rats at 170 days of age. The in vivo relationship of epinephrine and the pituitary adrenal system is suggested as a mechanism which could have caused this response. The relationship of secretion rates to plasma corticosterone concentrations indicated that extra-adrenal mechanisms, such as decreased turnover, were also responsible for the elevated plasma corticosterone levels observed in response to acute swimming.


Oxidation of cerebral cytochrome aa3 by oxygen plus carbon dioxide at hyperbaric pressures. The reduction-oxidation level of cytochrome aa3 in the intact cerebral cortex of cerveau isolé or pentobarbital-anesthetized cats was monitored by means of dual-beam reflectance spectrophotometry. Respiratory gases containing varying fractions of carbon dioxide and oxygen were administered at increased pressures, allowing titration of the cytochrome redox state from maximum reduction during nitrogen ventilation to the completely oxidized state. We show that the fully oxidized state could be reached with about 5% CO2-95% O2 inspired at 4 ATA. Maximum oxidation was achieved only through the relaxation of oxygen-induced vasoconstriction of the cerebral vessels, as our data indicated that large blood volume responses accompanied the increases in inspired carbon dioxide. With out technique, we have established that under resting air-breathing conditions cytochrome aa3 is about 85% reduced in the cat cerebral cortex, and that the absorption peak for cytochrome aa3 in situ is located near 602 nm. A free energy change of an additional 8 kcal is calculated to occur with donation of an electron pair from 85% reduced cytochrome aa3 to oxygen as compared to a 1% reduction level.


Transport of antibiotics and metabolite analogs by systems under cyclic AMP control: positive selection of Salmonella typhimurium cya and crp mutants. Mutants in the cyclic AMP (cAMP) control system in Salmonella typhimurium (cya = adenyl cyclase, crp = cAMP receptor protein) were partially resistant to growth inhibition by 22 antibiotics (including fosfomycin, nalidixic acid, and streptomycin) and 29 inhibitory analogs of normal bacterial fuel/carbon sources. This resistance was used as the basis for an efficient positive selection of cya and crp mutants. We propose that these antibiotics and analogs enter the bacteria through transport systems normally used for transporting fuel/carbon sources and that this is accomplished because of a structural similarity between the antibiotic and the natural substrate of the particular transport system involved. We propose that these transport systems are all under positive control by cAMP and that cAMP acts as a signal molecule (alarmone) for fuel/carbon deprivation. Evidence is provided for a hierarchy within operons controlled by cAMP. The methodology is shown to be useful for analyzing both antibiotic transport systems and the cAMP super-control system.


Mapping of the mecillinam-resistant, round morphological mutants of Escherichia coli. Genes responsible for round morphology in mecillinam-resistant, round morphological mutants of Escherichia coli have been mapped. Three mutants, called rodX, mapped at around 14 min, and two, called rodY, mapped at around 70 min by P1 transduction. These are either the same or very close to the loci reported, respectively, for the rodA (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol. 115:436-442, 1973) and envB genes (B. Westling-Häggström and S. Normark, J. Bacteriol. 123:75-82, 1975). This suggests that mecillinam can be used very efficiently to select for found morphological mutants of rodA and envB after nitrosoguanidine treatment.


Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: glucose-negative mutant and regulation of intracellular cyclic AMP. Glucose-negative mutants of Mycoplasma capricolum were selected for growth on fructose in the presence of the toxic glucose analog alpha-methyl-D-glucopyranoside. The mutants are defective in the phosphoenolpyruvate:sugar phosphotransferase system for glucose. One mutant, pts-4, was studied in detail. It lacks the glucose-specific, membrane-bound enzyme II, IIGlc, as well as the general, low-molecular-weight, phosphocarrier protein, HPr. In place of the latter, however, it has a fructose-specific protein, HPrFru. Consistent with these changes, the mutant lost the ability to grow on glucosamine and maltose but retained its ability to grow on sucrose. In the glucose-negative mutant, glucose did not regulate the intracellular concentration of cyclic AMP. The intracellular concentration of cyclic AMP in M. capricolum is regulated by the presence of metabolizable sugars. In the wild-type, both glucose and fructose reduced the intracellular concentration of cyclic AMP; however, in the glucose-negative mutant, glucose no longer regulated the intracellular level of cyclic AMP.


Elevated cyclic AMP concentration in streptomycin-dependent Escherichia coli. Streptomycin-dependent Escherichic coli B and K-12 cultures, which have relaxed catabolite repression when grown to glucose-salts medium, have an elevated concentration of cyclic AMP.