Rat gastric mucosal cAMP and cGMP after adrenergic stimulation and blockade. The effect of adrenergic stimulation and blockade on the concentration of cAMP and cGMP was studied in the rat gastric mucosa. Adrenaline (0.5-2.0 mg/kg) elevated the gastric mucosal cAMP and cGMP levels up to 100% in a dose-dependent manner. Blockade of the beta-adrenoceptors with 4 mg/kg propranolol suppressed the adrenaline effect on the cAMP level and increased the effect on the cGMP concentration up to 500%. The opposite effect was found under alpha-adrenoceptor blockade. Phenoxybenzamine, 4 mg/kg, prevented the adrenaline effect on the cGMP level and increased the effect on the cAMP concentration up to 300%. Predominant stimulation of the beta-adrenoceptors by isoproterenol or of the alpha-adrenoceptors by phenylephrine caused smaller changes in the cyclic nucleotide concentration than did adrenaline. Their effect was more pronounced when the non-stimulated receptor was blocked. The data indicate that the rat gastric mucosa contains alpha- and beta-adrenoceptors. The effect of adrenaline on the gastric mucosal cAMP level is mediated by beta-adrenoceptors and that on the cGMP level by alpha-adrenoceptors.


The effects of brainstem priming stimulation on interhemispheric hippocampal responses in the awake rat. Evoked hippocampal responses to stimulation of the contralateral hippocampus were recorded in the awake rat. The effects of priming stimulation applied to the nucleus locus coeruleus and the raphe nuclei on averaged evoked hippocampal responses to the interhemispheric stimulation were measured. It was found that priming stimulation of these monoamine-containing nuclei caused the formation of a late component in the interhemispheric potential without affecting the magnitude of the initial response component. Drugs which selectively interfere with noradrenergic or serotonergic transmission, antagonized the locus coeruleus or raphe primed late response component, respectively. Parenteral administration of d-amphetamine or l-amphetamine caused the appearance of a late response component which was similar to that seen after brainstem priming stimulation. It is suggested that the monoamines modify responses to afferent stimulation of particular pathways in the brain.


The mode of synaptic linkage in the cerebro-ponto-cerebellar pathway investigated with intracellular recording from pontine nuclei cells of the cat. 1. Intracellular potentials of pontine nuclei (PN) cells were recorded in cats anesthetized with pentobarbitone sodium. 2. Stimulation of the cerebellar nuclear regions or the brachium pontis induced an antidromic action potential composed of IS-SD spikes, after-depolarization and after-hyperpolarization. 3. Cerebellar stimulation produced EPSPs only in a few PN cells. 4. Activation of the corticopontine or pyramidal tract produced in all PN cells an EPSP built up from the unitary components with variable amplitudes and time courses. Paired or repetitive activation revealed a property of the frequency potentiation of the EPSP. 5. Unitary EPSPs also occurred spontaneously. A great majority of these spontaneous EPSPs were cerebral in origin, and had amplitudes and time courses comparable with those evoked by stimulation of the corticopontine or pyramidal tract. 6. The half-width versus time of peak relationship of these unitary EPSPs suggested a dendritic location of the synapses with variable distances from the soma. It is assumed that large, proximal synapses serve for efficient relay of signals while small, distal synapses for their integration.


Information processing along the course of a visual interneuron. Locust ocellar retinal cells are innervated by giant second order cells, 2 mm long, which show discrete zones of integration along their course, including a major zone in the axonal length of the neuron. The complex synaptic arrangements which exist between higher-order afferent and efferent cells and these second order cells along their course suggests that transmission takes place by the electrotonic spread of slow potentials. The size and accessibility of these visual interneurons offers a unique preparation for examining mechanisms of graded synaptic transmission.


Some properties of the goat placental lactogen. Goat placental lactogen was partially purified from a medium collected after placental tissue incubation. The data obtained by disc electrophoresis and isoelectric focusing experiments, as well as by means of radioreceptor assay methods, provide evidence of the similarity between the goat and ovine placental lactogen.


Isethionate may not be an inert substitute for extracellular chloride in the central nervous system. Replacement of extracellular chloride with isethionate or methylsulphate causes an increased efflux of 1-[14C]-GABA from the in vivo superfused rat cuneate nucleus. This raises the question of the suitability of these anions as inert substitutes for chloride in studies on the ionic dependency of membrane phenomena in the central nervous system.


Binding of ferritin-lectin conjugates to C-type virus in intact cells. Ferritin-Ricin II and Ferritin-Concanavalin A bound to budding as well as mature C-type viral particles. No differencies in binding between the viral coat and adjacent plasma membrane were detected with either lectin conjugate. Aggregation of viral particles by lectin conjugates was observed, and linking of virus to the plasma membrane resulted in phagocytosis of viral particles.


Effects of ACTH and diabetes on phospholipid metabolism in adrenal mitochondria. Incorporation of 32P into adrenal mitochondrial phospholipids (PL) incrased in ACTH-treated rats, but it decreased in diabetics, inspite of the fact that these animals showed adrenal overacity. Since diabetics did not show increased 11 beta-hydroxylation. as opposed to ACTH-treated rats, it is suggested that the stimulation of this enzyme activity by exogenous ACTH is related to an increased turnover of PL at the mitochondrial membrane. The process is impaired in diabetics and prevents the stimulation of 11 beta-hydroxylation.


Immunological studies on the key enzyme of arginine biosynthesis in Pseudomonas aeruginosa. A method to manufacture specific antisera with a minute amount of prue enzyme is presented. The influence of antibodies on activity and inhibition of an allosterically regulated enzyme was studied.


The effect of pyrophosphate and diphosphonates on calcium transport in red cells. The effect of 0.5 mM pyrophosphate (PPi), disodium ethane-4-hydroxy-1,1-diphosphonate (EHDP) and disodium dichloromethane diphosphonate (Cl2MDP) on the ATP-dependent Ca2+ extrusion from the human red cell ghosts was studied. PPi and Cl2MDP had no effect, when introduced into the cells or added outside to the medium. EHDP slightly increased the calcium concentration in the released cells and slightly decreased the rate constant of the calcium transport, having opposite effects when it was inside or outside the cells. PPi and the 2 diphosphonates were not found to move easily across the red cell membrane.


Spin label studies of ATP phosphoribosyltransferase of E. coli. Covalently bound bromoacetamide nitroxides have been used to detect the conformational changes and enzyme association induced by its feedback inhibitor, histidine.


Effects of tricaine methane sulphonate (M.S. 222) on the blood glucose levels in adult salamanders (Diemictylus viridescens). Exposure to M. S. 222 results in hyperglycemia in adult newts (Diemictylus viridescens). A combination of higher anesthetic concentrations with reduced exposure periods reduces the magnitude of this form of stress response.


Enhancement of virus growth produced by thiols and disulphides. Several thiols and disulphides have been found able both to shorten the latency phase and to increase the growth of several virus strains in cell cultures.


High density lipoproteins in ischaemic heart disease. High density lipoprotein cholesterol concentrations were significantly lower in ischaemic heart disease patients than in healthy subjects when age and sex-matched. This difference was, however, not observed in the older age group (greater than 60 years).


Effect of vinblastine on pancreatic enzymes secretion induced by cyclic nucleotide derivatives. Vinblastine did not affect the basal secretion of enzymes from the rat pancreas, but it potentiates the secretory response to dibutyryl cyclic AMP. This potentiation is confirmed by the observation of numerous pictures of exocytosis at the apical part of the acinar cell. Dibutyryl cyclic GMP by itself, or associated with vinblastine, failed to modify the spontaneous release of enzymes or the secretion induced by dibutyryl cyclic AMP.


Adrenergic supersensitivity of the pupil in idiopathic headache. In idiopathic headache (IH) sufferers, phenylephrine and fenfluramine induce a pupillary dilatation respectively greater and lesser than in controls. The difference may be due to a supersensitivity of the iris alpha adrenoceptors caused by a deficiency of noradrenaline in the iris adrenergic nerve terminal of the IH sufferer. These findings seem to support the hypothesis of a brain receptorial, monoamine supersensitivity in IH.


Opposite effect of PGE2 on cAMP levels in human adrenal medulla and pheochromocytoma. PGE2 (10(-7) M) caused increased cAMP accumlation in 5 pheochromocytomas, while in 3 human adrenal medullae PGE2 caused a significant decrease of cAMP level on incubating slices in vitro. This finding is discussed in relation to the opposite effect of PGE2 on catecholamine release from human medulla and pheochromocytoma slices in vitro.


Effects of N2, O2'-dibutyril cyclic GMP on the nucleoside phosphotransferase activity of the retina of the chick embryos. In the retina of the chick embryo, 2 different forms of nucleoside phosphotransferase take part in the phosphorylation of thymidine. One is an unstable form with higher molecular weight. The other with lower m. wt is a stable form. This paper shows that N2,O2'-dibutyril cyclic GMP causes a marked decrement of the activity of the unstable nucleoside phosphotransferase.


Alterations in the levels of cAMP and cGMP after decentralization of the rat superior cervical ganglion. After cutting the preganglionic nerve trunk of the rat's superior cervical ganglion, the levels of cAMP and cGMP were measured in a postoperative period of between 3 and 21 dad by a partial recovery after 7 to 21 days.


Lysosomal enzyme release associated with the invasion of rat liver by Novikoff hepatoma. The lysosomes of both Novikoff heptoma and liver from Novikoff heptoma-bearing rats were found to be relatively intact structurally, lower in acid phosphatase activity, greatly depleted in number but with nearly normal membrane integrity when compared with normal liver.


Studies on the effects of hormones on cholesterol synthesis in mammalian cells in culture. The studies described here suggest the potential physiological role of polypeptide and corticosteroid hormones in the regulation of cholesterol synthesis. Evidence was shown for substantial differences between various cell types in their responses to these agents and for certain degree of independence of the effects on biosynthesis of cholesterol from those on protein and DNA synthesis. Cholesterol synthesis and HMGCoA reductase are stimulated in a number of diploid cell lines following an incubation with insulin or with glucocorticoids for 4 hr or longer. Stimulation of sterol synthesis by insulin and by dexamethasone requires protein synthesis, but the two hormones do not compete for the same site. Addition of glucagon or of dibutyryl cyclic AMP, or elevation of intracellular cyclic AMP by PGE1 does not inhibit cholesterol synthesis in skin fibroblasts. A possibility of a relationship between the mechanisms of the hormonal effects and of feedback control of cholesterol synthesis is suggested.


The activation of lipoprotein lipase by lipase co-protein (apo C-2). Kinetic analysis of activation of lipoprotein lipase by apo C-2 indicates that the lipase co-protein increase the rate of lipolysis by adsorption to enzyme at the lipid interface, with formation of a 1:1 molar complex, whose dissociation constant in the presence of triglyceride substrate is about 3 X 10(-13) moles cm-3. Activation by apo C-2, like that by the entire lipoprotein apoprotein moiety (Fielding and Fielding, 1976) is reversible by inorganic salts and the dependence of activation on medium ion-pair activity supports the concept that such inhibition is mediated through a single specific anion-binding site of the lipase co-protein.


[Studies of substances with antiviral activity. VI. Activity on Herpes simplex virus of bis-homoanalogs of nucleosides and of hydrazone derivatives of various aromatic substrates]. Derivatives of different chemical structures, namely hydrazones of aromatic compounds and bis-homoanalogues of pyrimidine and purine nucleosides, have been investigated for inhibiting activity on Herpes simplex virus infection of human HEp-2 cell cultures. Some thiosemicarbazones, nucleoside analogues and guanylhydrazones proved active [compounds (I), (II b), (II c), (III), (IV a), (IV b), (V a), (V b); Fig. 1]. An increase in the number of guanylhydrazone chains linked to a fluorene structure [from 1 to 3: compounds (II a), (II b) and (II c)] increased the "activity index" (i.e., toxic versus active concentrations: Table II). Further in vitro investigations have been carried out on bis-guanylhydrazone of 1-phenyl-2-chloro-3,7-diformylindole (I) and it was found that its activity is of the order of 1 microgram per ml (Fig. 3). The compound completely blocks low multiplicity infections while it only blocks cytopathogenicity, not viral replication, in high multiplicity infections.


[Effect of low molecular weight basic proteins on mannose transport in Aspergillus niger microsomes]. Basic proteins of low molecular weight activate the transfer of mannose to endogenous glycoprotein acceptors in microsomal membranes of Aspergillus niger. The enhancement of mannosyltransferase activity is linked to the activation of the transport of mannose across the membrane. The role of these polycationic proteins on the membrane permeability is discussed.


Cholinergic, adrenergic, and purinergic neuromuscular transmission. A general model of the autonomic neuromuscular junction is proposed which emphasizes muscle effector bundles with gap junctions (or 'nexuses') forming the low resistance pathways allowing electrotonic coupling between neighboring cells, and extensive terminal varicose nerve fibers with 'en passage' release of transmitter. Some variations in autonomic neuromuscular geometry are discussed. Junctional clefts vary from 15nm in densely-innervated tissues such as vas deferens and iris to 2,000 nm in some large elastic arteries. Postjunctional specializations take the form of subsynaptic cysternae (in vas deferens and iris) and aggregations of plasmalemmal vesicles (in circular intestinal muscle). Current views of the synthesis, storage, release, and inactivation of transmitter during cholinergic, adrenergic, and purinergic transmission are summarized.


Modulation of smooth muscle activity by catecholamines. The inhibitory and excitatory actions of catecholamines are compared in various types of smooth muscle. Inhibition is usually but not invariably associated with membrane hyperpolarization and a decrease in membrane resistance. It also has a metabolic component frequently involving an increase in tissue cAMP. In some cases, the metabolic component is related to a cation pump, but the nature of this pump is unclear. With the exception of intestinal muscle where inhibition results from the synergistic action of alpha and beta receptors, inhibition is caused by activation of beta receptors. Excitation is mediated by alpha activation and is usually accompanied by a decrease in membrane potential and membrane resistance. Only the uterus has a metabolic component. The specific ionic permeability changes accompanying excitation are different in various smooth muscles. Clarification of the mechanisms responsible for these differential actions in various types of smooth muscle is a challenge for future work.


[Effect of several catecholamines on pulmonary circulation]. In anesthetized dogs, i. v. injections of 0.1-1.0 mcg/kg adrenaline, noradrenaline, and isadrine evoked a raise of blood pressure in the right ventricle of the hear by 13-200%. The greatest pressor effects were obtained with isadrine, the smallest those--with noradrenaline. In experiments with closed chest animals with perfusion of lungs lobe by venous blood under constant pressure and recording of blood flow with photoelectric drop recorder, injections into perfusate of 0.02-32.0 mcg adrenaline and noradrenaline diminished blood flow and increased vascular resistance. In 1/3 of cases isadrine evoked the same reaction, in other cases it increased blood flow in perfused lobe and decreased resistance. The isadrine effect was blocked by beta--blocking agent inderal, whereas those of adrenaline and noradrenaline--by alpha-blocking drug phentolamine. Adrenaline and noradrenaline to constrict the pulmonary vessels by activating the vascular alpha-adrenoreceptors. Isadrine can dilate and constrict the lung vessels. In intact circulation isadrine seems to cause vasoconstriction and elevate blood pressure in lesser circulation.


[Role of ACTH and glucocorticoids in providing for non-enzymatic fibrinolysis in animals under immobilization stress]. The 30-min immobilization stress was shown to raise significantly the non-enzymatic fibrinolytic activity of blood in rats. Combined with ACTH the effect is still greater. I. v. administration of 0.2 ml 0.01% solution of protaiminsulphate prevented the non-enzymatic fibrinolysis induced by the stress. Administration of ACTH after proteminsulphate again raised the fibrinolysis. This suggests that ACTH stimulates release of heparin. As the hydrocortisone administration proved ineffective, the stimulating effect of ACTH seems to have no relation to activation of glycocorticoids secretion. Glycocorticoids in this case appear to have a permissive role. This is corroborated by the data obtained with administration of protaminsulphate in 48 hrs after adrenalectomy. Neither ACTH nor hydrocortisone separately stimulated the non-enzymatic fibrinolysis in these animals. This process can be normalized in adrenalectomized animals with combined ACTH and hydrocortisone.


[Effect of beta-adrenoreceptor blockade on cerebral circulation and extracranial vascular tone]. The influence of anapriline (propranolol) on the cerebral circulation rate, tone of the cerebral and femural vessels, pO2 in the cerebral tissue, was studied. I. v. administration of anapriline decreased the arterial blood pressure. The changes of tone of the vessels are often of the two-phase character: a decrease followed by an increase. The lowering of the arterial pressure is the most probable cause of the lowering cerebral circulation rate. pO2 decreases accordingly to the blood flow. The effect of anapriline was greater in anesthetized animals.


The acro-renal syndrome of Curran.

The acro-renal syndrome of Curran. A nine-year-old girl with symmetrical acral deformities and anomalies of the renal tract is described. There have been four previous reports of this association, all in phenotypic males. In each case there was moderate intellectual retardation and in two cases dermatoglyphic analysis revealed abnormalities. It is suggested that there is now sufficient evidence to give the acro-renal syndrome independent status.


Search for infectious Epstein-Barr virus-releasing cell lines, with particular reference to a new producer line, NHAd-60. In a search for infectious Epstein-Barr virus (EBV)-producer cell sources, 36 EBV genome-carrying lymphoblastoid cell lines originating from various sources in Japan were screened. Three cell lines derived from different sources were found to release infectious EBV into the culture fluid. EBV from these 3 lines possessed leucocyte-transformation activity but did not induce early antigen formation. One of the three lines, NHAd-60, originated from an adenoid tissue; it released a relatively large amount of infectious virus (10(2) to 10(3) of 50% of transforming dose of cord leucocytes). The NHAd-60 virus strain was further characterized. Cellular, virological, and immunological properties were described and discussed, and compared with EBV from known producer cell lines.


Suggestive evidence for relationship between sensitivity and repair capability in rat ascites hepatoma cells treated with the antitumor agent, 1-(gamma-chloropropyl)-2-chloromethylpiperidine hydrobromide. Antitumor activity of 1-(gamma-chloropropyl)-2-chloromethylpiperidine hydrobromide (CAP-2) was studied in vivo and in vitro, using various rat ascites hepatoma cell lines. Among eight ascites hepatoma cell lines, AH-13 was extremely sensitive both to in vivo antitumor and to in vitro lethal action of the agent, whereas AH-44 was resistant in both cases. The sensitivity of ascites hepatoma cell lines to CAP-2, nitrogen mustard N-oxide, 4-nitroquinoline 1-oxide, and ultraviolet ray in vitro was widely different but their relative sensitivities were very similar against these agents. For all the agents, AH-13 was inactivated very rapidly and AH-109A moderately, whereas AH-44 was relatively resistant. These results indicate that the sensitivity of the cells to CAP-2 may be closely related to their repair-capability of damaged DNA. Similar experiments using various DNA repair-deficient mutants of Escherichia coli B strain demonstrated that the repair-deficient mutants were several times more sensitive to CAP-2 than the wild type strain. From these results, it may be concluded that CAP-2 induces DNA lesions repairable by the same repair mechanisms that work on pyrimidine dimers.


Geographical distribution of the subtype of hepatitis B surface antigen in Chinese. Using the much more sensitive hemagglutination inhibition assay for subtyping of hepatitis B surface antigen (HBsAg), we examined the determinants a, d, w and r in 192 from 228 HBsAg positive adults who had been found after screening with reversed passive hemagglutination method. Sixty-four subtypable cases were asymptomatic carriers and the remaining 128 were liver disease patients. Among them there was no significant difference of the subtypes, invariably with adw as the main subtype. Geographical difference was evident: adr was the main subtype (78 per cent) among the northern Chinese; while adw was dominant (76 per cent) among the southern Chinese with the Yangtze River as a boundary. Eight of the 18 adr-subtyped northern Chinese were born and live in Taiwan where 91 per cent of HBsAg positive Taiwanese were adw-subtyped. This was an indirect evidence that intra-familial spreading from parents played an important role in hepatitis B virus infection.


Acrodermatitis enteropathica, zinc, and the Paneth cell. A case report with family studies. An infant with acrodermatitis enteropathica was studied before and after starting zinc therapy. Clinical recovery was rapid, and the plasma zinc, serum and mucosal alkaline phosphatase activities returned to normal. Light microscopy of small intestinal biopsies showed normal mucosa. Electron microscopy of the Paneth cells initially revealed abnormal inclusion bodies which disappeared during therapy, suggesting that the abnormality is secondary to zinc deficiency, and not a primary defect. These abnormal inclusions may represent altered secretory granules and a proliferation of lysosomes. We were unable to define the heterozygous state biochemically or histologically.


Effect of adrenocorticotropic hormone on pure exocrine pancreatic secretion in man. The effect of adrenocorticotropic hormone (ACTH) on pure exocrine pancreatic secretion was studied in 7 subjects with external transduodenal drainage of the main pancreatic duct performed after biliary tract surgery. Intravenous injection of 0.25 mg of ACTH during a prolonged intravenous infusion of secretin (0.5 clinical units per kg-hr) plus cholecystokinin (0.5 Ivy dog units per kg-hr) significantly reduced protein and lipase (both concentration and output) without affecting volume and bicarbonate secretion. The reduction appeared soon after ACTH injection (peak inhibition in the first 15-min period) and lasted about 75 min. The adrenocortical response to ACTH reached its peak at the 60th min. The mechanism by which the pituitary hormone selectively inhibits pancreatic enzyme secretion without affecting volume and bicarbonate remains to be clarified.


[Resistance of a line of Djungarian hamster cells to 6-mercaptopurine]. A cell line resistant to 6-mercaptopurine (6-MP) is isolated from Djungarian hamster embryonic fibroblasts transformed with SV-40, 6-MP resistance is due to the absence or complete inhibition of GGPRT activity. Initial and resistant cell cultures are similar in the growth rate and in the inoculation efficiency. Caryological analysis (differential chromosome staining--S-bands) revealed considerable caryotype rearrangements in both resistant and sensitive lines as compared with Djungarian hamster normal chromosome set, and also the appearance of specific chromosome markers.


[Identification of the mink alpha2-lipoprotein Lpm-allotypes by the method of preparatory ultracentrifugation]. The density class accessory of 8 mink alpha2-lipoprotein allotypes are determined by means of preparative ultracentrifugation. It is found that Lpm1, Lpm2, Lpm2, Lpm3, Lpm4, Lpm5, Lpm7 and Lpm8 are determinants of lipoproteins with density exceeding 1.210, i.e. they are VHDL. The allotypic marker 6, which has been earlier assigned by other criteria to Lpm group, belongs to mink lipoprotein, which distributes during ultracentrifugation at the region of low and, partially, high density.


[Analysis of mutations induced by alkylating compounds in Drosophila sperm. I. Character of visible changes induced by N-nitrosoethylurea and ethylmethanesulfonate in a succession of Drosophila generations]. Newly appearing hereditary traits were observed in several generations of Drosophila melanogaster: changes in eye and body colour, wing shape, number and shape of chaetae. The results obtained may be interpreted as the evidence for the realization of potential changes induced by NEU and EMS into visible mutations during 1-4th generations. X-irradiation (at a dose of 1000 r) of males from the first generation did not affect the realization of long-living potential changes induced by NEU.


Syndactyly--a review and long term results. Syndactyly is classified and the principles of its surgical treatment discussed. The notes of eighty-five patients who had 222 webs between them were reviewed. A long term follow-up was carried out on thirty-two of these patients who had seventy-six clefts separated. The sexual and anatomical distribution of the syndactyly was investigated. The results of surgery were assessed including complications, and the relationship of complications to the type of graft used and the age at operation. It is suggested that complicated syndactyly is often separated at too early an age.


[Lymphography in malignant ovarian tumours (author's transl)]. 21% of 81 patients with malignant neoplasms of the ovaries had lymphographic evidence of metastases in regional iliac and aortic lymph nodes. The frequency of lymphatic metastases in various stages was approximately the same (FIGO stage I = 17%, II = 21%, III = 19%). About one fifth of patients therefore had to be restaged. Lymphography should be used for staging routinely, even if a laparotomy is planned.


Limitations of angiographic differential diagnosis in major hepatic processes. In 11 instances (6%) of 183 consecutive hepatic angiograms done for subsequently proven pathologic hepatic processes, either the diagnosis was incorrect (4 patients) or a satisfactory differential diagnosis was not established (7 patients), despite obvious angiographic abnormalities. Incorrect diagnoses were made in patients with suppurative hapatocarcinoma and liver cyst, macronodular regenerative cirrhosis, and multiple intrahepatic abscesses. Satisfactory differential diagnoses could not be established in patients with enlarged intrahepatic ducts, acute viral hepatitis, recurrent cirrhosis and acute liver necrosis. On analysis, means for minimizing diagnostic failure include the use of enhancement techniques such as infusion angiography and pharmacoangiography and an unbiased, detailed analysis of the angiographic findings. Changes secondary to the pathologic process and the coexistence of multiple processes, however, can occasionally prevent an accurate diagnosis.


[Angiographic findings in benign liver tumours (author's transl)]. Difficulties in the differential diagnosis of benign liver tumours and their differentiation from malignant liver tumours are discussed. Two patients with benign lesions were described, one with a liver cell adenoma, the other with a mixed cholangiohepato-adenoma (local nodular hyperplasia).


[Angiography and differential diagnosis of malignant liver cell tumours (author's transl)]. The angiographic appearances of malignant liver cell tumours are described. The differential diagnosis between hepatoblastomas and hepatocellular carcinoma is discussed with reference to ten patients seen by the authors, the difference depending on the maturity of the liver tissue undergoing malignant change.


[Limitations in the internistic treatment of ulcerative colitis and Crohn's disease]. Aetiology of ulcerative colitis and Crohn's disease is unknown. Therefore causal therapy is not possible. Conservative treatment for both diseases is of antiinflammatory, symptomatic and substituting nature. Surgical intervention is indicated if conservative treatment fails or if dangerous complications occur. The right moment for an operation can only be determined in close cooperation between internist and surgeon.


Effect of hyperthermia on epididymal cyclic AMP levels in diabetic non-diabetic and hypophysectomized rats. The effect of elevated body temperatures on the concentrations of epididymal cyclic AMP levels in non-diabetic, diabetic and hypophysectomized rats was studied. Cyclic AMP levels were increased during hyperthermia in all animals examined. This increase in epididymal cyclic AMP concentration was not seen in animals that had been supplemented with exogenous insulin prior to the experiment. The effect of pituitary lipolytic hormones on epididymal cyclic AMP levels was also investigated. Significant elevations of epididymal cyclic AMP levels were observed in hypophysectomized rats during hyperthermia indicating that pituitary hormones are not essential in causing these increases. Extrapituitary hormones, such as glucagon, might be responsible for epididymal cyclic AMP increases. Increases in epididymal cyclic AMP levels may therefore be the result of the reduction of blood insulin and concomitant increases of lipolytic hormones of both pituitary and extrapituitary origins.


Effects of clofibrate on the human fat cell adenylate cyclase system. The effects of chlofibrate on the adenylate cyclase system of human adipocytes were studied. Clofibrate reduced basal as well as hormone-NaF)stimulated adenylate cyclase activities to about the same extent (45% inhibition at 1 mg/ml clofibrate). The relative extent of hormonal stimulation was not altered by this compound. The inhibitory action of clofibrate was non-competitive with respect to the substrate ATP and cofactors (Mg2+-ions). Inhibition of enzyme activity was detectable after 2.5 min. Our results suggest that the antilipolytic activity of clofibrate is mediated via inhibition of the catalytic subunit of the fat cell adenylate cyclase.


Critical evaluation of the 5-day metyrapone test. Different parameters of the response to the 5-day metyrapone test were evaluated for their diagnostic accuracy in tests given to 58 children and adolescents as part of an investigation for short stature. Insulin test, vasopressin test and ACTH test were used for diagnostic reference. A log transformation of the data was clearly appropriate. The maximal daily excretion of 17-ketogenic steroids was distinctly positively correlated with the basal excretion. Consequently, the most correct parameter of the response is the SD score of the deviation of the maximal excretion from the regression of the maximal excretion on the basal excretion in the reference population. This parameter, 'relative maximal excretion' was shown to be the diagnostically most accurate index of the response of 11 parameters studied.


Iodinated bovine prolactin. Characterization and binding to mammary gland cells. Iodinated bovine prolactin (2.6 iodine atoms/molecule; labelled with a trace of 125I to give a specific activity of 0.041 muCi/mg) was prepared by the chloramine T method. It was active in two bioassays (pigeon crop sac and dispersed mouse mammary cell), though somewhat less active than the unmodified hormone. In an immunoassay, iodinated prolactin was more effective than the unmodified hormone at displacing 125I-prolactin from antibody. High specific activity 125I-prolactin (1 iodine atom/molecule; 70 muCi/microgram) was used for autoradiographic studies on the binding of prolactin to mouse mammary cells. In vivo the labelled hormone found in the mammary gland was associated with membranes of mammary epithelial cells and with alveolar lumen contents. In vitro 125I-prolactin was shown to bind to dispersed mouse mammary epithelial cells.


Immobilized Clostridium perfringens neuraminidase. Substrate cleavage and enzyme release during incubation. Pure Clostridium perfringens neuraminidase was immobilized on Sepharose 4 B, azido-Sepharose 4 B and controlled pore glass (CPG)- glycophase using different coupling procedures. The immobilized enzyme showed increased stability under various conditions relative to the soluble enzyme. The low release of active enzyme from the supports under incubation conditions was quantitated using a highly sensitive radioactive assay. The activity of the immobilized enzyme was dependent on the nature of the support and the substrate. Activity decreased with increasing substrate molecular weight, but the enzyme showed improved cleavage with GD1a micelles and human erythrocytes, substrates having ordered surface properties. Uses of immobilized neuraminidase in biochemistry and cell biology are considered and evaluated relative to the measured release of enzyme from the supports reported and to the molecular size and organization of possible substrates.


Infectious mononucleosis: recognition and management. Differentiation from other common viral infections depends on a triad of clinical, hematologic, and serologic determinations. A distinctive feature is that while a number of circulating immunoglobulins are often detected, only Paul-Bunnell antibodies, found in 75% of cases, are specific for IM. Symptomatic care only is indicated for most patients, though severe cases or complications may require antibiotics or corticosteroids.


Sclerosing hemangioma of the lung: an endothelial or epithelial neoplasm? The ultrastructural features of a sclerosing hemangioma of the lung in a 34 year old woman are described. The basic cellular response is thought to be endothelial and not epithelial. These conclusions are based on the finding of Weibel-Palade bodies within the tumor cells in addition to other characteristics generally associated with endothelial cells.


Factors affecting tracheobronchial mucociliary transport. In vivo mucus transport rates were studied in humans and donkeys by external measurement of the rate of clearance of insoluble monodisperse gamma-tagged aerosols. The influence of temperature, environmental toxicants and drugs which affect the autonomic nervous system were studied by determining the changes in clearance produced by them in individual subjects. In donkeys, increased pretest ambient temperature accelerated clearance by greater than 1.7%/ degrees C. Smoking 2-7 cigarettes reduced the duration of bronchial clearance by approximately 50% in both humans and donkeys. Donkeys were exposed to higher doses; with progressive slowing of tracheal and bronchial clearance for greater than 10 cigarettes. HCN, at concentrations up to 1230 ppm, produced only a mild transient slowing of clearance. In man, atropine slowed clearance, while the adrenergic stimulating drugs, isoproterenol and epinephrine, both accelerated it by greater than or equal to 4 times, as did isoproterenol when given subsequent to atropine. The cholinergic stimulating drug methacholine increased mucociliary transport in the the donkey. Administration of a tap water aerosol for 10-15 min in humans increased bronchial clearance rates by approximately 25%.


Investigations into the determination of the cytotoxicity of quartz dust by physical methods. The methods of thermally stimulated luminescence (t.s.l), thermally stimulated exoelectron emission (t.s.e.e.), electron spin resonance (e.s.r), and infrared spectroscopy (i.r.) have been used to investigate whether the specific cytotoxic activities of SiO2 dust of various origins can be attributed to physical parameters. After interaction of toxic dusts with water an increase in the intensity of the t.s.l. peaks below room temperature has been detected whereas no effect could be observed in the case of inert materials. Dusts of weak cytotoxicity have been characterized by infrared absorption bands at 3610 cm-1 and 3685 cm-1. The results of the present studies emphasize the significance of silanol groups and adsorption properties in relation to the fibrogenic activity of quartz dusts.


Hepatic clearance of Salmonella typhimurium in silica-treated mice. Scanning electron microscopy demonstrates that crystalline silica destroys liver Kupffer cells but has no other obvious deleterious effects on the liver. Silica-treated livers still retain the ability to trap large numbers of bacteria perfused through the portal vein even though the rate of clearance is well below normal. In vivo, silica treatment decreases the rate of bacterial clearance from the blood, alters the in vivo organ distribution of cleared bacteria, and decreases the mean lethal dose of Salmonella typhimurium over 100-fold. Cumulatively, the data indicate that silica treatment enhances susceptibility to gram-negative infection, probably by destruction of macrophages.


Interaction of inflammatory cells and oral microorganisms. IV. In vitro release of lysosomal constituents from polymorphonuclear leukocytes exposed to supragingival and subgingival bacterial plaque. The deposition of bacterial plaques on tooth surfaces appears to be responsible for the initiation and progression of periodontal disease. In this study, human peripheral blood polymorphonuclear leukocytes (PMNs) actively released lysosomal constituents upon in vitro exposure to either viable or irradiated, supragingival or subgingival dental plaque. Plaques were obtained from the PMN donors (autologous plaque) or from pooled samples (homologous plaque) secured from patients with periodontal lesions. Fresh sera from PMN donors amplified the release reactions to supragingival and subgingival plaques. Heated (56 degrees C, 30 min) sera also enhanced release reactions, but not as consistently as fresh serum. It was postulated that modulation of PMN release by serum is mediated by complement components and/or antibodies to plaque bacteria. Electron microscopic observations indicated that degranulation and discharge of PMN lysosomal enzymes may be associated with phagocytosis of gram-positive and gram-negative plaque bacteria and with reverse endocytosis of lysosomes from cells contacting relatively large masses of aggregated plaque bacteria. These data suggest that PMN lysosome release in response to plaque may serve as a potential mechanism of tissue injury in the pathogenesis of gingival and periodontal inflammation.


Effect of Staphylococcus aureus delta toxin on Chinese hamster ovary cell morphology and Y-1 adrenal cell morphology and steroidogenesis. Since Staphylococcus aureus delta toxin previously had been shown to increase the cyclic adenosine 3',5'-monophosphate (cAMP) content of guinea pig ileum, the effect of delta toxin on such cAMP-mediated responses as morphogenesis and steroidogenesis in cultured tissue cells was examined. In contrast to cholera toxin, delta toxin did not cause spindling of Chinese hamster ovary cells. Unlike adrenocorticotropin or cholera toxin, delta toxin was unable to cause rounding of Y-1 adrenal cells or to promote steroid production by the cells. S. aureus alpha toxin and enterotoxin B were also unable to cause rounding of Y-1 adrenal cells. Omission of Ca2+ from the media still allowed for increased steroid production by adrenocorticotropin but not by delta toxin. Delta toxin at concentrations greater than 10 micrograms/ml did cause lysis of both Chinese hamster ovary and Y-1 adrenal cells. These findings suggest that the increase in intestinal cAMP levels caused by delta toxin is mediated through a mechanism different from that initiated by cholera toxin.


Evidence for a mouse pathogenicity locus in certain temperature-sensitive mutants of foot-and-mouth disease virus. Serial tissue culture passaging of three foot-and-mouth disease temperature-sensitive mutants demonstrated the stability of their temperature sensitivity and mouse avirulence characteristics. Recovery of mouse-virulent temperature-sensitive viruses after passage of the mutants in mice suggested that these were not covariant expressions of the same locus, but were under the control of different genes.


Immune response and latent infection after topical treatment of herpes simplex virus infection in hairless mice. Treatment of herpes simplex virus (HSV)-infected hairless mice with a 2% phosphonoacetic acid (PAA) ointment prevented the appearance of virus-induced skin lesions and subsequent central nervous system (CNS) involvement. Treatment started 24 h after infection significantly reduced the intensity of the skin lesions and also prevented CNS involvement. After four to six applications of PAA ointment, a moderate skin erythemia developed, followed by scaling and complete healing 7 days after cessation of the treatment. Mice treated early after HSV infection had low or undetectable levels of virus-specific antibodies but were completely resistant to reinfection. Early treatment prevented the development of a latent ganglionic infection, but treatment initiated 24 h after infection could not prevent the establishment of the latent infection. PAA-treated and HSV-infected mice with nondetectable levels of antibodies did not develop, with a single exception, a latent ganglionic infection unpon reinfection. The cell-mediated immune response determined by levels of [14C]thymidine incorporation in Ficoll-Hypaque-purified spleen lymphocytes cultures was low in PAA-treated mice; it increased slightly after challenge infection but was strong in mice that proved to harbor a latent HSV infection in the ganglia.


Biological characterization of avian osteopetrosis. Chicks infected as 12-day-old embryos with an end-point purified derivative of avian myeloblastosis virus developed a rapidly progressive osteopetrosis that manifested within 1 week of hatching. A detailed comparison of osteopetrotic chicks and normal hatchmates revealed the following. (i) Osteopetrotic chicks exhibited a stunting syndrome, growing at a mean rate that was 26% of the control rats. (ii) At autopsy, the mass of the lymphoid organs was reduced, whereas the mass of the heart, pancreas, kidneys, lungs, brain, liver, and bones of osteopetrotic chicks was increased. Edema was likely responsible for most of the increase in organ weight. (iii) Infected chicks exhibited a normochromic, normocytic anemia that was virus dose dependent and was not required for the development of osteopetrosis. (iv) Bone collagen content was normal. (v) Osteopetrotic bone was initially hypomineralized, but later became more fully mineralized. (vi) The concentrations of alpha, beta, and gamma globulins in the plasma were elevated in osteopetrotic chicks, whereas albumin concentration was decreased. (vii) The level of plasma alkaline phosphatase was elevated in osteopetrotic chicks, yet the level of acid phosphatase was unchanged. (viii) Body and bone temperatures were unchanged.


Terminal electron transport in Treponema pallidum. Reduced-minus-oxidized difference spectra of sonically treated virulent Treponema pallidum disclosed cytochromes of the b anc c types as well as large amounts of flavoprotein. Difference spectra of the carbon monoxide-binding pigment identified cytochrome o as the terminal oxidase. Physiological reduction of the cytochromes indicated that the cytochrome system was functional and established the capability of T. pallidum for aerobic respiration. The potential significance of these findings is discussed.


Cellular basis of persistent lymphocytosis in cattle infected with bovine leukemia virus. Peripheral blood lymphocytes from 14 cattle infected with the bovine leukemia virus (BLV) and 14 BLV-free cattle were examined by the membrane immunofluorescent antibody technique to detect surface immunoglobulin (S-Ig) and by the erythrocyte-antibody-complement (EAC) rosette test for the detection of complement receptors. Direct comparisons of the percentages of S-Ig-bearing cells and EAC rosette-forming cells in both infected and BLV-free animals showed no evidence for the presence of a substantial population bearing one surface marker but not the other. The data showed that cells with surface markers characteristic of B lymphocytes are responsible for most of the increase in peripheral blood lymphocytes which may accompany BLV infection. The release of infectious BLV and the spontaneous uptake of thymidine by short-term cultured peripheral blood lymphocytes from BLV-infected cattle were also studied. The results indicate that both of these activities are function of B lymphocytes.


Properties of density gradient-fractionated peripheral blood leukocytes from cattle infected with bovine leukemia virus. Discontinuous bovine serum albumin gradients were used to fractionate peripheral blood leukocytes from bovine leukemia virus (BLV)-free and BLV-infected cows. The release of infectious BLV and spontaneous incorporation of [3H]thymidine were not properties of density gradient-fractionated leukocytes from a BLV-free cow. When leukocytes from BLV-infected cattle were fractionated, B lymphocytes which spontaneously incorporated [3H]thymidine could be separated as a distinct subpopulation from B lymphocytes which replicated infectious BLV. Density gradient fractionation of leukocytes from a cow with lymphosarcoma is also reported. A fall in lymphocyte count at the time of tumor development is attributed to the loss of B lymphocytes which spontaneously incorporate [3H]thymidine.


Protection from oral herpes simplex virus infection by a nucleic acid-free virus vaccine. The effect of immunization with inactivated herpes virus vaccines, including a vaccine free of all nucleic acid, was investigated in a mouse model system. Protection against oral lesions induced by herpes simplex virus type 1 was demonstrated by several criteria: (i) reduction in the incidence and severity of primary oral lesions; (ii) decrease in acute and latent infection of the regional sensory ganglia; and (iii) protection from viral encephalitis and death. The immune response of mice to the vaccine and to subsequent virus challenge was measured by following serum-neutralizing antibody titers.


Immunoglobulin classes of antibodies in milk of swine after intranasal exposure to pseudorabies virus or transmissible gastroenteritis virus. Experiments were conducted to evaluate whether infection of the respiratory tract of pregnant swine with pseudorabies (Pr) virus would induce the secretion of immunoglobulin A (IgA) antibodies in their milk as was observed after enteric infection with transmissible gastroenteritis (TGE) virus. The immune response of sows to Pr virus inoculated intranasally and to TGE virus inoculated orally/intranasally or via a natural infection was studied. Emphasis was placed upon titers and Ig classes of Pr and TGE virus-neutralizing antibodies in colostrum and milk. All animals exposed to Pr virus (alone or in combination with TGE virus) developed Pr-neutralizing antibody titers in both serum and milk. Pr antibody titers were generally higher in colostrum than in serum, but the opposite was true in milk compared with serum, with milk titers declining markedly during lactation. In contrast, TGE antibody titers in milk from experimentally or naturally infected sows usually remained higher than the corresponding serum titers and persisted at relatively constant levels throughout lactation. Gel filtration studies of milk indicated that the antibody activity against Pr virus was associated almost entirely with IgG fractions, with small amounts of antibody detectable in IgM fractions in colostrum from two of nine sows. By comparison, TGE antibodies were primarily of the IgA class, with varying but lesser amounts of antibody associated with the IgG class. Such results suggest that viral infection of the intestinal tract of the sow, but not the upper respiratory tract, stimulates the secretion of IgA antibodies in the milk.


Genetics of macrophage-controlled resistance to hepatitis induced by herpes simplex virus type 2 in mice. The genetics of innate resistance of mice to hepatitis induced by herpes simplex virus type 2 (HSV-2) was analyzed by crossing resistant male GR to susceptible female BALB/c mice and backcrossing females of this F1 generation to susceptible male BALB/c mice. By scoring of macroscopic liver lesions and virus isolation studies from the liver 4 days after intraperitoneal inoculation of HSV-2, it appeared that the resistance was governed by one X-linked dominant gene or closely linked gene complex, as F1 female mice were resistant and F1 male mice were susceptible and the trait segregated in a ratio close to 1:1 in the backcross mating. A cellular expression in vitro of virus resistance was found in the macrophage population of the mice as measured by differences in the restriction of HSV-2 replication in macrophage cultures prepared from individual mice. In contrast to what was seen in macrophage cultures, virus replicated equally well in embryonic fibroblast cultures from susceptible and resistant strains of mice.


Effect of silica on the pathogenic distinction between herpes simplex virus type 1 and 2 hepatitis in mice. The role of macrophages in the difference in liver pathogenicity between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in mice was investigated by selectively blocking the macrophage function of the mice by silica. Intravenous administration of 3 mg of silica 2 h before virus inoculation partially abolished the difference between the two virus types, as judged by macroscopic and microscopic examination of the livers and by virus isolation studies. Intraperitoneal inoculation of 50 mg of silical before virus seemed more effective in suppressing the macrophage function, since this treatment almost completely eliminated the difference in hepatotropism between HSV-1 and HSV-2 as assessed by the number and size of the lesions appearing in the liver. The final outcome of the infection, death from encephalitis, was, however, not influenced by macrophage blockade.


Production by mixed lymphocyte cultures of a type II interferon able to protect macrophages against virus infection. In supernatants of mixed mouse spleen cell cultures established for 4 days, a species-specific inhibitor of virus replication with a broad antiviral spectrum was found. The inhibitor was destroyed by trypsin, was nondialyzable and acid labile, and was not neutralized by antibody to mouse L cell interferon. This indicates that in mixed lymphocyte cultures a type II interferon is made that has no immunological relationship with "fibroblast" interferon. This leukocyte product was shown to protect mouse hepatitis viruses. It is suggested that lymphocyte interferon may collaborate with macrophages in host defense against viruses, as a mediator of cellular immunity.


Commitment to deoxyribonulceic acid synthesis and the cell cycle in endotoxin-stimulated murine spleen cells. Events associated with endotoxin-induced mitogenesis in murine spleen cells were investigated. Commitment to deoxyribonucleic acid (DNA) synthesis, the onset of DNA synthesis, and phases of cell cycle were timed. Increased levels of DNA synthesis in murine spleen cells stimulated with endotoxin were observed 12 to 16 h after the addition of the mitogen. The total cell cycle time of stimulated B-cells was 11 to 14 h. The S-phase was 8 h. The G2-phase was 1 h, and the combined M-plus G1-phase of cycling cells was 2 to 5 h. A 1- to 4-h exposure to lipopolysaccharide elicited a significant increase in DNA synthesis. Progressively longer exposures to lipopolysaccharide, up to 24 h, produced further increases in first-cycle DNA synthesis. Polymyxin B, when added with endotoxin to cultures from the outset, inhibited first-cycle DNA synthesis. However, if addition of the antibiotic was delayed, progressive increases in first-cycle dna synthesis were observed. These data indicate a heterogeneity among B-cells in their responsiveness to endotoxin.


In vitro studies on the mechanism of herpesvirus plaque growth inhibition by sensitized lymphocytes. Inhibition of herpesvirus plaque growth was observed when herpes simplex virus (HSV)-sensitized rabbit lymphocytes were placed in contact with an HSV-infected human foreskin monolayer. This inhibition was obtained as early as 3 h when a ratio of 6 viable lymphocytes to target cells was used, and the supernatants of these cultures also demonstrated plaque size reduction when put onto newly infected cell monolayers. Interferon, which is present in this system, had no effect on HSV when tested on human foreskin monolayers, indicating that interferon was not the mechanism for plaque size reduction. Plaque growth inhibition was attributed to the T lymphocyte, because purified T cells reduced plaque growth and anti-rabbit thymocyte serum eliminated the effect of T cells. The specificity of this assay was determined by the facts that nonsensitized lymphocytes did not reduce the size of a plaque and the recognition of an infected cell by the sensitized lymphocyte was necessary for the release of a soluble mediator into the supernatant fluid. This cytotoxic lymphocyte was detected in the peripheral blood of rabbits as early as 4 days after initial corneal infection, with a maximum response at 7 to 10 days.


Role of viremia in the suppression of T-cell function during murine cytomegalovirus infection. The suppression of T-cell deoxyribonucleic acid (DNA) synthesis by serum from mice acutely with murine cytomegalovirus (MCMV) was investigated. Spleen cells from uninfected mice were exposed to concanavalin A in the presence of serum taken from mice at various times after infection with MCMV. The capacity of the serum to suppress DNA synthesis first appeared at day 3 postinfection and was associated with free infectious virus. Addition of MCMV to serum from uninfected mice also suppressed DNA synthesis. Ultracentrifugation of serum from mice acutely infected with MCMV removed most of the virus and aborgated the inhibition of DNA synthesis. However, in two of four experiments, serum from mice in weeks 4 and 5 postinfection did not contain infectious MCMB but did suppress. Therefore, it appears that MCMV itself can suppress DNA synthesis of T cells; however, this may not be the exclusive mechanism of suppression exerted by serum from MCMB-infected mice.


Preparative polyacrylamide gel electrophoresis purification of Clostridium perfringens enterotoxin. Preparative polyacrylamide gel electrophoresis has been used to purify the enterotoxin of Clostridium perfringens from Sephadex G-100 extracts. Purified toxin of high specific activity was eluted in 1 to 3 h, depending upon the length of the acrylamide gel used. Recovery of biological activity with this technique ranged from 80 to 90%. The purity and physical characteristics of the toxin were similar to those previously reported for the protein purified by other methods. Use of preparative electrophoresis will enable the production of larger amounts of high-specific-activity toxin in a shorter time than other currently available procedures. This method was also used to isolate a form of enterotoxin that has a mobility, relative to bromophenol blue tracking dye, of 0.87 to 0.90 in 7% acrylamide gels.


Role of T lymphocytes in recovery from murine cytomegalovirus infection. Congenitally athymic nude (Nu/Nu) mice inoculated intraperitoneally with murine cytomegalovirus (MCMV), in doses as low as 1.3 X 10(1) plaque-forming units succumbed to the infection. In contrast, the mean lethal dose for heteroxygous euthymic (Nu/+) littermates was 4 X 10(3) plaque-forming units. Though histopathological changes consistent with MCMV infection were found in the spleen, lungs, and adrenals of nude mice, there were only small focal areas of involvement in the liver. In contrast, Nu/+ mice dying from infection had pathological evidence of severe hepatitis. Spleen cells from immune and control BALB/c mice were injected intravenously into syngeneic mice that had been inoculated previously with lethal doses of MCMV intraperitoneally. Mice receiving 1 X 10(7) or more immune spleen cells were protected against the infection, whereas mice receiving 1 X 10(8) control spleen cells or immune serum were not. Treatment of immune spleen cells with anti-theta serum and complement significantly reduced their protective effect. Immune mechanisms associated with T lymphocytes appear to be critical for recovery from MCMV infection.


Human lymphoblastoid cell lines derived from individuals without lymphoproliferative disease contain the same latent forms of Epstein-Barr virus DNA as those found in tumor cells. The physical state of the intracellular Epstein-Barr virus DNA was characterized in four human lymphoblastoid cell lines, F-265, NC-37, U-303 L, and PG-1, derived from individuals without lymphoproliferative disease. For comparison, a previously investigated Burkitt lymphoma line, Raji, and a more recently established cell line of that origin, Rael, were also studied. The techniques employed were CsCl density gradient centrifugation, glycerol gradient centrifugation, and ethidium bromide-CsCl density gradient centrifugation in combination with nucleic acid hybridization, as well as electron microscopy contour length measurements of purified circular EBV DNA. All six cell lines contained multiple copies of covalently closed circular EBV DNA-molecules of the same size, as well as viral DNA with the properties of integrated DNA. No differences could be detected between the forms of EBV DNA present in cell lines derived from non-malignant sources and those present in lymphoma lines.


Transformation of lymphocytes by Herpesvirus papio. Cotton-topped (CT) or white-lipped (WL) marmoset lymphocytes were transformed in vitro with herpesvirus papio (HVP) into permanently growing lymphoblastoid cell lines (LCL). Five of 9 HVP-transformed CT cell lines contained cells with antigens reacting with antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and/or to EBV-induced early antigens (EA). None of 12 WL LCL revealed such antigen-producing cells. Cells from both groups of cultures failed to react with antibodies to the EBV-specified nuclear antigen (EBNA). Exposure of baboon circulating lymphocytes to X-irradiated HVP or EBV-carring cells, or to suspensions of EBV resulted in establishment of LCL which all contained VCA and/or EA-positive, but no EBNA-positive cells. Nuclear antigens were undetectable also with anti-VCA-positive sera from baboons, chimpanzees, or other non-human primates. DNA-complementary RNA (cRNA) filter hybridization with EBV cRNA showed that with one exception transformed CT or WL marmoset cells contained at least 1-2 virus genome equivalents per cell, while at least 12-25 virus genome equivalents per cell were detected in transformed baboon cells. These data need confirmation by DNA-DNA reassociation kinetics.


Productive Epstein-Barr viral infection of the human lymphoblastoid cell line 6410 with release of early antigen inducing and transforming virus. The human lymphoblastoid cell line 6410 was superinfected with P3HR-1 derived Epstein-Barr virus. Subsequent to the first cycle of infection, characterized by early (EA) and virus capsid (VCA) antigen synthesis, the culture, designated 6410-EBV, continued to synthesize VCA. Further immunofluorescence and electron microscopic examination over 18-24 months showed the 6410-EBV culture to be productively infected with EBV. Virus was recovered, in quantity, from the culture fluids and assayed for ability to induce EA synthesis in Raji cells and to transform human umbilical cord lymphocytes. The virus was found to possess both properties, in contrast to P3HR-1 virus which only induces EA. HLA and karyologic analyses of P3HR-1, 6410 and 6410-EBV cultures showed relatedness of 6410-EBV to 6410 cells and dissimilarity to P3HR-1. The biologic activity data coupled with the cytologic analyses confirm a productive infection of the target cells. The reason for differences in biologic activity between the infecting (P3HR-1) and progeny virus is unresolved. It may be speculated that the endogenous EBV genome of 6410 cells was activated and gave rise to a different strain of EBV or to a mixed progeny-parent population as a result of dual infection. Alternatively, the parent strain of virus may have been modfied as a result of interaction with the new host cell.


Tumour-specific complement-dependent serum cytotoxicity against a chemically induced rat hepatoma. A short-term 51Cr release test was used for the detection of complement-dependent cytolytic activity of syngeneic serum for transplanted aminoazo dye-induced rat hepatoma cells in suspension. Serum samples from rats bearing intraperitoneal implants of one hepatoma (hepatoma D23) were specifically cytotoxic for hepatoma D23 target cells, although this activity was not detected in sera from donors bearing subcutaneous tumour grafts. Other sera containing demonstrable IgG antibodies reactive in membrane immunofluorescence tests with individually distinct tumour-specific antigens or tumour-associated embryonic antigens were not cytotoxic for hepatoma D23 cells; and even though serum from donors carrying intraperitoneal tumour grafts contained tumour-specific IgG antibody, the complement-dependent reactivity was confined to the 19s region of fractionated tumour-bearer serum. These findings are discussed in relation to the development of humoral responses in the tumour-bearing host and with regard to the significance of the availability of an objective and reproducible assay for measuring humoral responses directed against the tumour-specific antigens associated with chemically induced rat tumours.


Possible two-stage transplacental liver carcinogenesis in C57BL/6 mice. A single SC injection of 2-acetylaminofluorene (AAF) was given to pregnant C57BL/6 mice on day 15 of gestation, and the offspring subsequently given twice-weekly injections of phorbol for 25 weeks. Control groups included: (1) untreated; (2) AAF-treated mothers (kept under observation for 18 months, as with all the other groups); (3) untreated offspring of untreated mothers; (4) untreated offspring of AAF-treated mothers, and (5) phorbol-treated offspring of untreated mothers. The incidence of hepatomas in the phorbol-treated offspring of AAF-injected mothers was 8/74 (11%), as compared with 2/80 (2.5%) in the untreated offspring of AAF-injected mothers. The AAF-injected mothers themselves developed 3/36 (8%) hepatomas; while all othe other control groups were free from liver tumours. The development of reticulum cell sarcomas, and of a few cases of lung adenomas, in the various groups, was presumably spontaneous. The results seem sufficiently encouraging, as a model for the study of systemic carcinogenesis, to warrant further attempts at two-stage transplacental carcinogenesis, using other potential initiators and promoters.


Strategy of goitre and cretinism control in Central Africa. Goitre prevalence in Ubangi Mongala (north-western Zaïre) ranges from 27 to 60 per cent in the male population and from 48 to 78 per cent in the female population; the prevalence of cretinism ranges between 0.7 and 7.6 per cent. This severe endemia affects 1.5 million inhabitants and constitutes a major public health problem. If administered to an entire population, single injections of slowly resorbable iodized oil can reduce goitre prevalence substantially, correct the iodine deficiency and restore normal thyroid function for a period of 3 to 7 years, depending on the parameter considered. The cost of programme of endemic goitre prevention based on this method can be estimated at 0.07 US dollars per person and per year of protection. A strategy of goitre and cretinism control in Central Africa, using iodized oil in an attack phase and iodized salt in a follow-up phase, is proposed. The organization of this programme will be dovetailed into other public health activities and will strengthen the basic health service framework.


Comparison of poliovirus detection in sewage and stool samples; a study in a crèche in the third week after vaccination. In a model experiment conducted in a Prague crèche, the detection rate for polioviruses in partially purified, non-concentrated sewage as sampled by means of gauze pads (Moore's method) was compared with that for individual stool samples. In the third week after the vaccination of children with live attenuated type 1 poliovirus (March 1975) and at an equal interval after their vaccination with a combination of types 2 and 3 (May 1975), corresponding poliovirus types were detected both in individual stool samples and sewage. The mean amount of sewage virus recovered during one week was directly proportional to the percentage of positive stool samples. After combined type 2 and 3 poliovirus administration, however, both types were only detected in one sewage sample, while in two samples type 3 only, and in one sample type 2 only, were identified. The results of the study indicate that poliovirus is detectable in sewage by the method employed if it is excreted by approximately one per cent of persons in the sewer catchment area of a small community. If a number of antigenic types are simultaneously present, their identification requires systematic examination of the sewage.


Studies on cytochrome C. XII. Synthesis of the protected undecapeptide (sequence 66-76) and pentadecapeptide (sequence 66-80) of horse heart cytochrome c. This paper is part of a series on synthesis of suitably protected peptides covering the 66-104 sequence of horse heart cytochrome c. It describes the preparation, by conventional procedures, of a partially protected N alpha-benzyloxycarbonyl-undecapeptide hydrazide corresponding to the sequence from 66 to 76 (Fragment F), which represents a building block for the synthesis of the entire 66-104 sequence. Moreover, the preparation is described of a partially protected pentadecapeptide corresponding to the sequence region 66 to 80, which represents the key peptide for the semisynthesis of the same COOH-terminal sequence utilizing the natural 81-104 N epsilon-trifluoroacetylated CNBr fragment.


Studies on cytochrome C. XIII. Synthesis of the protected undecapeptide (sequence 77-87) of horse heart cytochrome c. A solution synthesis of Z-Gly-Thr-Lys (Tfa)-Met-Ile-Phe-Ala-Gly-Ile-Lys (Tfa)-Lys (Tfa)-NHNH-Boc corresponding to the sequence 77-87 of horse heart cytochrome c is described. The protected undecapeptide was obtained from intermediate hepta- and tetrapeptide fragments by an azide coupling.


Studies on cytochrome c. XIV. Synthesis of the protected heptadecapeptide (sequence 88-104) of horse heart cytochrome c. A solution synthesis is described of the partially protected N alpha-benzyloxycarbonylheptadecapeptide Z-Lys (Tfa)-Thr-Glu-Arg-Glu-Asp-Leu-Ile-Ala-Tyr-Leu-Lys (Tfa)-Lys (Tfa)-Ala-Thr-Asn-Glu (OBu t)-OBu t corresponding to sequence 88-104 of horse heart cytochrome c. The synthesis is achieved through the preparation of two subunits H1 (sequence 88-96) and H2 (sequence 97-104) and their linkage by an azide coupling step.


Characteristics of dimer formation, excision and DNA strand growth in Yoshida sensitive and resistant cells after ultraviolet irradiation. The sedimentation properties of the nascent DNA of Yoshida sarcoma cells, sensitive and resistant to methylene dimethane sulphonate and cross-resistant to U.V. light, have been studied after irradiation with U.V. light at 11 and 22J/m2. It has been shown that the DNA formed immediately after irradiation with 11J/m2 is some eight to nine times longer than the calculated inter--dimer distance in both cell-lines. Differences were, however, observed between the two cell-lines, in that the absence of excision of dimers in the sensitive cells was accompanied by the formation of a DNA component of low molecular weight, whereas excision in the resistant line was not so accompanied. There are some similarities between the Yoshida tumour line sensitive to methylene dimethane sulphonate and the U.V.-sensitive line of Xeroderma pigmentosum.


Immunoelectron microscopic localization of herpes simplex virus antigens in rabbit cornea with antihuman IgG-antiferritin hybrid antibodies. Sheep antihuman IgG-antiferritin hybrid antibodies were used for the ultrastructural localization of herpes simplex virus (HSV) antigens in rabbit corneas from animals with herpetic keratitis. In animals with epithelial keratitis in which active viral replication is occurring (6 days after infection), viral antigen was found within nuclei, on nuclear membranes, and on cell surface membranes of epithelial cells. In animals with early necrotizing keratitis in which active viral replication cannot be demonstrated (14 to 21 days after infection), viral antigen was found in association with the cell surface of stromal keratocytes. Since lymphocytic cells in intimate contact with degenerating keratocytes have previously been identified in the cornea, these observations provide a basis for the view that cell-mediated immunopathogenesis is involved in the etiology of herpetic stromal keratitis.


Lysozyme tear level in patients with herpes simplex virus eye infection. The lysozyme level in tears of patients with HSV eye infection was examined and correlated with the clinical findings and presence of virus. The concentration of the enzyme in tears of patients during acute attack was 2.83 mg./ml. This value was significantly lower than that in tears from healthy controls (6.1 mg./ml.) and tears from the patient's healthy eye (4.46 mg./ml.). After termination of treatment with either IUDR or poly I:C, the lysozyme level rose to an average of 4.34 mg./ml. During the latent period of the disease the level increased (5.34 mg./ml.), but it remained lower than in healthy subjects who had never suffered from HSV eye infection. This may be an indicator of possible future recurrences.


Characterization of purely ecotropic and amphotropic naturally occurring wild mouse leukemia viruses. Two new strains of murine leukemia virus, one (strain 4996) purely ecotropic and the other (strain 1313) purely amphotropic, were isolated from spontaneous lymphomas in aged wild mice (Mus musculus). The 4996 virus is the first wild mouse field isolate which consists solely of ecotropic virus without the concomitant presence of amphotropic virus. The 1313 isolate is distinct in host range from seven other previously described wild mouse amphotropic isolates and is also the only murine leukemia virus shown to replicate in chicken cells.


Identification and characterization of a papillomavirus from birds (Fringillidae). From skin papillomas of the chaffinch (Fringilla coelebs), a virus has been purified and studied by physicochemical techniques and electron microscopy. The virions measure 52 nm in diameter and are composed of 72 morphological units arranged in a skew T = 7d surface lattice. A sedimentation coefficient of about 300S and a buoyant density of 1.34 g/ml in CsCl were determined for the particle. Its protein composition resembles that of human papillomavirus, and the circular double-stranded genome measures 2.6 micronm. This is the first demonstration of a member of the Papovaviridae family that affects a nonmammalian host.


A rapid method for detecting Junin virus viremia in the guinea pig. A method for detecting Junin virus viremia in guinea pigs is described. The method consists of infecting BHK-21 cells with blood samples from infected guinea pigs; 48 h later, Junin virus antigens are detected in the cells by indirect immunofluorescence. Application of this technique to patients with Argentine hemorrhagic fever may lead to the quickest method for the virologic diagnosis of this disease.


Application of enzymehistochemical methods to isolated subcellular fractions and to sucrose-ficoll density gradients. A contribution to the comparison of histochemical and biochemical data. To compare histochemical and biochemical determinations of enzyme activities, enzymehistochemical procedures are applied to sections of pellets of subcelluar fractions. These investigations are of value to determine the subcellular localization of histochemically demonstrable enzyme activities and to test the homogeneity of an isolated fraction. In homogenating duckling liver a great part of the endothelial cells is not destructed and consequently is found in the nuclear fraction. Kupffer cell lysosomes land in the heavy mitochondrial fraction, whereas hepatocyte lysosomes are chiefly found in the light mitochondrial fraction. beta-Glucuronidase activity shows a preferentially microsomal localization. Application of enzymehistochemical staining reactions to discontinuous gradients and comparison with biochemical data provides additional information about the validity of an enzymehistochemical reaction. In rat liver the tetrazolium reductases show a distinctly dual localization: activity in the mitochondrial band and in microsomal bands. As to their localization in different bands of the gradients non-specific esterases demonstrate a clear pH-dependency.


Gas-liquid chromatographic determination of residues of methanesulfonate of n-aminobenzoic acid ethyl ester in fish. In this gas-liquid chromatographic procedure for determining residues of methanesulfonate of m-aminobenzoic acid ethyl ester (MS-222) in fish muscle, homogenized tissue is extracted with distilled water, and proteins are removed by coagulation with trichloroacetic acid, centrifugation, and filtration. After careful pH adjustment of the filtrate, MS-222 is partitioned into benzene-ethyl ether and measured by alkali flame ionization gas chromatography. Tissues with known additions of 1-19 microgram MS-222/g were analyzed, with recoveries of 84-95%.


Analysis of fat-soluble vitamins. XVI. Antirachitic activity of 5,6-trans-vitamin D3 alone and in the presence of 5,6-cis-vitamin D3 resin, using chick bioassays. Two biological assays were conducted in which the antirachitic activity in chicks of 5,6-trans-vitamin D3 added to feed is compared with that of 5,6-cis-vitamin D3. On the basis of the results obtained it is concluded that the relative potency of the trans isomer is, at the most, 5% and that the antirachitic activity of the trans isomer is not markedly enhanced (an increase to a relative potency of 16%, at the most) if the cis isomer is also included in the diet. The results are not conclusive on the inhibition or lack of inhibition of the antirachitic activity of 5,6-cis-vitamin D3 by the presence of 5,6-trans-vitamin D3 in the feed.


Simple sensitive technique for detecting organochlorine pesticides on thin layer chromatograms. Chlorinated hydrocarbon pesticides can be quickly detected using commercially available thin layer chromatographic plates dipped in an acetone solution of silver nitrate. The limits of detection are functions of the pesticide, adsorbent, developing system, and concentration of the silver nitrate in acetone solution. On exposure to ultraviolet light, 0.002 microgram 2,4,5-T produced clear darkening within 30 min on precoated silica gel plates (polyvinyl alcohol binder) coated with a solution of 0.1% silver nitrate in acetone. For this system, a 60-min detection period was necessary for a 0.05% coating solution. On the silica gel plates (polyvinyl alcohol binder, 0.1% silver nitrate), 0.02 microgram lindane is detected within 75 min. For alumina plates (polyvinyl alcohol binder, 0.1% silver nitrate), 0.025 microgram aldrin is detected within 10 min. Darkening of this plate prohibits the detection of 0.012 microgram aldrin. On silica gel plates (polyvinyl alcohol binder, 0.1% silver nitrate), 0.015 microgram aldrin can be detected within 45 min. The method described provides sensitivities equal to or exceeding literature values.


High pressure liquid chromatographic separation and identification of vitamins D2 and D3 in the presence of fat-soluble vitamins in dosage forms. A simple and rapid qualitative method is described for determining the presence of vitamin D2 (ergocalciferol) and/or vitamin D3 (cholecalciferol) in various preparations by reverse phase high pressure liquid chromatography (HPLC). When both D2 and D3 are present, this method effectively separates and identifies each vitamin D form by its respective retention time. A significant difference between vitamins D2 and D3 exists in their antirachitic activity in poultry. Preparations can be tested rapidly by this method to ascertain that the correct D vitamin form has been added. Fat-soluble vitamins such as vitamins A, E, K1, and K3 do not interfere. Vitamins D2 and D3 were separated at the baseline in model preparations. As little as 2 ng each of vitamin D2 and vitamin D3 can be separated and identified.


Kinase replacement by a dehydrogenase for Escherichia coli glycerol utilization. A mutant of Escherichia coli that employs a glycerol:nicotinamide adenine dinucleotide 2-oxidoreductase (EC 1.1.1.6), instead of adenosine 5'-triphosphate:glycerol 3-phosphotransferase (EC 2.7.1.30), as the first enzyme for the dissimilation of glycerol was constructed. This mutant, like the wild-type strain, still cannot grow anaerobically on glycerol without an exogenous hydrogen acceptor.


Effect of carbon source and the role of cyclic adenosine 3',5'-monophosphate on the Caulobacter cell cycle. The expression of cell cycle events in Caulobacter crescentus CB13 has been shown to be associated with regulation of carbohydrate utilization. Growth on lactose and galactose depends on induction of specific enzymes. Prior growth on glucose results in a delay in enzyme expression and cell cycle arrest at the nonmotile, predivisional stage. Dibutyryl cyclic adenosine 3',5'-monophosphate (AMP) was shown to stimulate expression of the inducible enzymes and, thus, the initiation of the cell cycle. beta-Galactosidase-constitutive mutants did not exhibit a cell cycle arrest upon transfer of cultures from glucose to lactose. Furthermore, carbon source starvation results in accumulation of the cells at the predivisional stage. The cell cycle arrest therefore results from nutritional deprivation and is analogous to the general control system exhibited by yeast (Hartwell, Bacteriol. Rev. 38:164-198, 1974; Wolfner et al., J. Mol. Biol. 96:273-290, 1975), which coordinates cell cycle initiation with metabolic state. Transfer of C. crescentus CB13 from glucose to mannose did not result in a cell cycle arrest, and it was demonstrated that this carbon source is metabolized by constitutive enzymes. Growth on mannose, however, is stimulated by exogenous dibutyryl cyclic AMP without a concomitant increase in the specific activity of the mannose catabolic enzymes. The effect of cyclic AMP on growth on sugars metabolized by inducible enzymes, as well as on sugars metabolized by constitutive enzymes, may represent a regulatory system common to both types of sugar utilization, since they share features that differ from glucose utilization, namely, temperature-sensitive growth and low intracellular concentrations of cyclic guanosine 3',5'-monophosphate.


A method for incorporating labeled lacithin into serum low density lipoproteins in vitro. A sonicated dispersion of [14C]lecithin was incubated with high density lipoproteins (HDL) coupled to Sepharose. After washing the gels thoroughly with a buffer, the gels were incubated with low density lipoproteins (LDL); [14C]lecithin was transferred from the sonicated dispersion via HDL-Sepharose to the LDL. The LDL fraction thus prepared showed no contamination with lecithin dispersion or HDL. The lecithin:cholesterol  acyltransferase (LCAT) reaction could be completely inhibited during preparation, and the net recovery of radioactivity in LDL was 16% of that of the original lecithin dispersion. The [14C]lecithin in the washed HDL-Sepharose was shown to be a substrate of the LCAT reaction in vitro.


A latent collagenase from embryonic human skin explants. Collagenase released from embryonic and adult human skin explants has been studied with special reference to the latency of the enzyme. 1) Embryonic human skin explants showed a much higher capacity for collagenase production than did adult skin, on the basis of unit weight of tissue. 2) Culture medium from embryonic skin explants contained latent collagenase at almost twice the concentration of the active form. No appreciable amount of latent enzyme was observed in the adult skin system. 3) The molecular weights of active and latent collagenases were about 40,000 and 50,000, respectively. 4) The latent collagenase was found to be activated by simple passage through a Sephadex G-50 column after adding NaI to a final concentration of 3 M. The degree of activation produced by this treatment was as high as that by limited proteolysis with trypsin. It was concluded that no activating enzyme system was involved in the activation of latent collagenase during NaI treatment, and that the latent enzyme was composed of an enzyme-inhibitor complex. 5) The physiological significance of latent enzyme in the regulation of collagenase activity in vivo is discussed.


Studies on pig serum lipoproteins. IV. Isolation and characterization of glycopeptides from pig serum low density lipoprotein. Three glycopeptides were isolated from the pronase digest of the protein moiety of pig serum low density lipoprotein. The isolation procedure consisted of pronase digestion, gel filtration on Sephadex G-25 and G-50 columns, paper chromatography and DEAE-Sephadex A-50 column chromatography. Based on the carbohydrate analysis, the isolated glycopeptides were classified into two types. One type (GDI) consisted of mannose and N-acetylglucosamine residues in the molar ratio of 6:2 and had a molecular weight of about 2,300. The other type (GDII and GDIII) consisted of sialic acid, mannose, galactose, fucose, and N-acetylglucosamine residues in the molar ratio of 1:4:2:1:3 and 2:4:3:1:3, respectively. The molecular weights of GDII and GDIII were about 2,100 and 3,100, respectively. The results on the strong alkaline treatment of these glycopeptides suggested that all carbohydrate chains were linked to the peptide chains through N-acetylglucosaminyl-asparagine linkages. Of these glycopeptides and pig serum lipoproteins, only glycopeptide GDI and native LDL strongly interacted with concanavalin A.


Phosphodiesterase-phosphomonoesterases from Fusarium moniliforme. VI. A modified method of purification and identification of isozymes. Fusarium phosphodiesterase-phosphomonesterase was purified 1,630-fold with 19% yield from dried powder of the culture medium by a modified method consisting of seven steps. The purified preparation was shown to be devoid of inactive protein by disc electrophoresis. The preparation was homogeneous with respect to size as demonstrated by gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weights determined by gel filtration on Sephadex G-200 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 106,000 and 100,000, respectively. The sedimentation coefficient at infinite dilution was 5.71 S. Isoelectric focusing of the purified preparation showed the presence of at least four isozymes with isoelectric points of 6.6, 6.3, 6.2, and 5.9.


The mechanism of action of citrinin on rabbit kidney alkaline phosphatase activity in vivo. The effect of citrinin poisoning on rabbit kidney alkaline phosphatase was investigated. After seven days administration of citrinin (2 mg/kg body weight daily) the animals were sacrificed and the level of enzymes estimated in serum and kidney. Serum enzymes showed no variation in activity in the citrinin-treated animals, but in kidney, alkaline phosphatase activity decreased significantly. The decreased activity was mainly associated with the cytoplasmic fraction and in fractions Ib and II. The enzyme II obtained from citrinin-treated animal showed no kinetic difference in substrate specificity, inhibition by phenylalanine, phosphate, sodium-EDTA and Zn2+ ions, activation by Mg2+ ions, thermal inactivation and electrophoretic mobility to that of control Enzyme II. Immunological studies showed that the decrease in enzyme activity was due to existence of inactive enzyme protein. Hormones like cyclic AMP, prostaglandin E1 and parathyroid hormone reversed the decreased enzyme activity due to citrinin poisoning in mouse and rabbit. This study favours the possible existence of active and inactive forms of alkaline phosphatase in the system.


Intrinsic activity of guanosine 3',5'-monophosphate-dependent protein kinase similar to adenosine 3',5'-monophosphate-dependent protein kinase. I. Phosphorylation of histone fractions. Guanosine 3',5'-monophosphate (cyclic GMP)-dependent protein kinase partially purified from silkworm pupae reacts preferentially with H1, H2A, and H2B histones but not with H3 AND H4 histones. However, the latter can serve as substrates in the presence of a stimulatory modulator as described by Kuo and Kuo (J. Biol. Chem. 251, 4283-4286 (1976)). With H2B histone as substrate high Mg2+ concentrations (50-100 mM) are necessary for the maximum rate of reaction. Although effects of the modulator and Mg2+ vary significantly with the histone fractions employed, analysis on the phosphorylation of histone fractions provides evidence that cyclic GMP-dependent protein kinase possesses an intrinsic activity that is similar to that of adenosine 3',5'-monophosphate-dependent protein kinase.


Intrinsic activity of guanosine 3',5'-monophosphate-dependent protein kinase similar to adenosine 3',5'-monophosphate-dependent protein kinase. II. Phosphorylation of ribosomal proteins. Guanosine 3',5'-monophosphate (cyclic GMP)-dependent protein kinase purified from silkworm pupae reacts with rat liver ribosomal proteins when a stimulatory modulator (Kuo, W.N. & Kuo, J.F. 1976) J. Biol. Chem. 251, 4283-4286) is added to the reaction mixture. Judging from autoradiogram of the radioactive proteins separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide slab gel, the protein kinase utilizes the same proteins as those phosphorylated by adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase. Fingerprint maps of the tryptic phosphopeptides of radioactive ribosomal proteins, which are phosphorylated by these two classes of protein kinases, are very similar. These results suggest that cyclic GMP-dependent protein kinase possesses an intrinsic activity that is similar to that of cyclic AMP-dependent protein kinase.


A carbon monoxide-binding hemoprotein formed by heme accumulation in Escherichia coli. A CO-binding hemochrome was accumulated in Escherichia coli cells, when intracellular heme concentration was increased by aerobic incubation of resting cell suspensions with ALA. Reduced minus oxidized difference spectrum of the hemochrome showed peaks at 560, 530, and 430 nm and a shoulder at 575 nm. The peaks of CO reduced minus reduced difference spectrum were located at 572, 540, and 422 nm. The CO spectrum was similar to but not identical with the spectrum of cytochrome o, a known terminal oxidase in E. coli. SDS-polyacrylamide gel electrophoresis of the CO-binding hemochrome showed its molecular weight to be about 33,000. The hemochrome in crude cell-free extracts was oxidized by aeration and reduced by the addition of succinate or NADH. The reduction by succinate was inhibited by inhibitors of succinate dehydrogenase [EC 1.3.99.1], and the reduction by NADH was inhibited by 2-heptyl-4-hydroxy-quinolin-N-oxide, which is an inhibitor of electron transport in E. coli cells.


Enzymatic oxidation of isethionate to sulfoacetaldehyde in bacterial extract. Isethionate degradation in a bacterial extract was shown by the isolation of enzymes and by identification of an intermediate to take place in two steps; dehydrogenation to sulfoacetaldehyde and desulfonation leading to the formation of sulfite and acetate. The enzyme responsible for isethionate oxidation in the presence of FAD was particulate in nature and a solubilized preparation obtained by extraction with buffer of low ionic strength had oxidizing activities against only isethionate and n-butanol among compounds tested. The enzyme was inhibited by thiol and carbonyl reagents.


Oxidative pathway of choline to betaine in the soluble fraction prepared from Arthrobacter globiformis. One strain of bacteria which showed high H2O2-generating activity was isolated from soil and characterized as Arthrobacter globiformis based on its morphological, nutritional, and physiological characteristics. The activities of H2O2 generation, NAD reduction and oxygen consumption in the bacterial cells were examined using choline, betaine aldehyde or betaine as substrate. Choline was oxidized to betaine aldehyde under aerobic conditions in a reaction coupled with H2O2 generation and oxygen consumption. On the other hand, betaine aldehyde seemed to be oxidized to betaine through two distinct oxidative reactions, H2O2 generation (oxygen consumption) under aerobic conditions and NAD reduction under either aerobic or anaerobic conditions. These enzyme activities were found in the supernatant fraction of the sonicated cell preparation.


Purification and properties of an acid phosphatase of Micrococcus denitrificans distinct from thiamine phosphate phosphatase. To determine whether the acid phosphatase in Micrococcus denitrificans participates in hydrolysis of thiamine phosphate in the synthesis of thiamine pyrophosphate, acid phosphatase was purified 280-fold by conventional procedures, which removed thiamine phosphate phosphatase completely. Studies showed that this acid phosphatase is a different protein from thiamine phosphate phosphatase and that it has no binding site for thiamine phosphate on its active site.


An ESR study of the Mn(II)-heavy meromyosin system. The Mn(II)-heavy meromyosin system was studied by measuring the ESR spectrum of Mn(II). The temperature dependence of the line width parameter W(1, t) of a freshly prepared sample changes at around 7-10 degrees C, where W(1, t) is the reciprocal of the peak-to-peak height of the lowest magnetic field component of the hyperfine structure. It is shown that the change in the slope of W(1, t) at 7-10 degrees C is due to a change in the structure of Mn(II)-heavy meromyosin or a change in the interaction between Mn(II) and heavy meromyosin without ATP. This result is in accord with the recently reported observations that heavy meromysin ATPase activity showed different temperature dependence above and below 10 degrees C in the presence of Mn(II). The characteristics of the spectrum of the Mn(II)-heavy meromyosin system in the liquid state between 2 degrees C and 20 degrees C are compared with those of a frozen sample of Mn(II)-heavy meromyosin in a low temperature region (-50-0 degrees C) and with those of the lyophilized material. The forbidden transitions are observed, and hence the zero field splitting parameter can be obtained. It is 115 +/- 15 gauss at -50 degrees C, and decreases with increase of the temperature to 70 +/- 15 gauss at 20 degrees C.


Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states. Adenylate cyclase in liver membranes was solubilized with Lubrol PX and partially purified by gel filtration. The partially purified enzyme was susceptible to activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Studies on the binding of [3H]Gpp(NH)p to various fractions eluted from the gels revealed that an upper limit of 1% of the Gpp(NH)p binding sites is associated with adenylate cyclase activity stimulated by the nucleotide. The glucagon receptor, pretagged with 125I-glucagon in the membranes, solubilized with Lubrol PX, and fractionated on the same gel columns, eluted in a peak fraction that overlaps with, but is separate from, adenylate cyclase in its Gpp(NH)p-stimulated form. Addition of GTP to the solubilized glucagon-receptor complex caused complete dissociation of the complex, as has been shown with the membrane-bound form of the complex. Since the GTP-sensitive form of the glucagon receptor complex separates from the Gpp(NH)p-sensitive form of adenylate cyclase, it is concluded that the receptor and the enzyme are separate molecules, each associated with a distinct nucleotide regulatory site or component. These findings are discussed in terms of the possible structure of the hormone-sensitive state of adenylate cyclase.


Stimulation of adenosine 3':5'-monophosphate formation by prostaglandins in human astrocytoma cells. Inhibition by nonsteroidal anti-inflammatory agents. Prostaglandins (PG) of the E series and catecholamines stimulate adenosine 3':5'-monophosphate (cAMP) formation in human astrocytoma cells (1321N1). These two classes of effectors activated adenylate cyclase upon interaction with different receptor systems. No evidence for a mediatory role for PG in the action of catecholamines was found. PG interacted with 1321N1 cells with an order of potency of PGE1 = PGE2 greater than PGA1 greater than PGF2 alpha. The effect of combinations of the various PG indicated that all efficacious PG interacted with a common receptor. 7-Oxa-13-prostynoic acid and indomethacin were shown to be competitive inhibitors of the effect of PGE1 with Ki values of 4 and 150 micron, respectively. These two compounds did not inhibit the effect of isoproterenol. Polyphloretin phosphate caused a complex pattern of inhibition of the effects of PGE1 and at higher concentrations also inhibited the effects of isoproterenol. The mefenamate class of nonsteroidal anti-inflammatory agents was found to inhibit the effects of PGE1 with a potency order of meclofenamic acid greater than flufenamic acid = mefenamic acid. The inhibitory action of meclofenamic acid was complex involving specific, but partial, insurmountable antagonism of PGE1 as well as competitive inhibition of PGE1 effects. At higher concentrations of meclofenamic acid a nonspecific inhibition of the effects of both PGE1 and isoproterenol was observed. These studies suggest that the inhibition by nonsteroidal anti-inflammatory agents of the physiological effects of PGE1 in animals may occur, at least in part, at the level of adenylate cyclase. The possibility that multiple classes of adenylate cyclase-linked PGE receptors might exist in nature is discussed.


On the structure of the prosthetic group of citrate (pro-3S)-lyase. The prosthetic group of citrate (pro-3S)-lyase from Klebsiella aerogenes as well as Streptococcus diacetilactis was obtained eigher by beta elimination or pronase digestion of the enzyme and purified by DEAE-cellulose chromatography. The compound was shown to contain 3 mol of PO4, 2 mol of ribose, and 1 mol of sulfhydryl/mol of adenine. 5'-AMP and dephospho-CoA are components of the prosthetic group. The evidence obtained so far support our proposed structure of 3' (or 2') leads to 1''-(5''-phosphoribosyl)dephospho-CoA for the prosthetic group of citrate lyase. The presence of one phosphomonoester group in the compound isolated after beta elimination and the absence of the same in the compound isolated after pronase digestion indicated that the prosthetic group is attached to the enzyme through a phosphodiester bond. Analyses of the pyruvate released by beta elimination and subsequent acid hydrolysis of the peptide-bound prosthetic group and its degradation products showed that the phosphodiester linkage is between the hydroxyl group of a serine residue of the protein and the 5''-PO4 group of the second ribose.


Ethionine and the phosphorylation of ribosomal protein S6. Ribosome phosphorylation was studied by monitoring the phosphorylation state of small subunit protein S6 as visualized on two-dimensional electrophoretograms of ribosomal proteins isolated from rat liver. No phosphorylation of S6 was observed under conditions of ethionine-induced inhibition of protein synthesis. Moderate phosphorylation, detected as the appearance of S6 and four or five phosphorylated derivatives, was observed in saline-treated animals. Reversal of ethionine-induced inhibition of protein synthesis by treatment with adenine led to extensive phosphorylation of S6. A model for protein synthesis which includes requisite phosphorylation of ribosomes during initiation is proposed. Cyclic adenosine 3':5'-monophosphate concentration was significantly elevated in liver of both ethionine- and ethionine plus adenine-treated rats, relative to that of saline-treated animals.


Calcium stimulation of plasminogen activator secretion/production by swiss 3T3 cells. Actively growing Swiss 3T3 cells secret high levels of plasminogen activator which decreases after the cells become confluent. In contrast, simian virus 40-transformed 3T3 cells secrete large amounts of plasminogen activator independent of cell density (Chou, I.-N., O'Donnel, S.P., Black, P.H., and Roblin, R.O. (1977) J. Cell. Physiol. 91, 31-38). These results suggest a correlation between active cell multiplication and plasminogen activator secretion in both 3T3 and simian virus-transformed 3T3 cells. The data reported herein indicate that treatment of both subconfluent and confluent Swiss 3T3 cells with high concentrations of Ca2+ (final 3.0 to 4.9 mM) increases the amounts of both secreted and cell-associated plasminogen activator in a dose-dependent manner. In addition, the ionophore A23187 (19 to 95 nM) in the presence of a normal level of Ca2+ (1.8 mM) stimulates both production and secretion of plasminogen activator from growing 3T3 cells. The Ca2+ stimulation of plasminogen activator production/secretion may be related to the mitogenic effect of Ca2+.