Mechanistic studies of polyene enhancement of interferon production by polyriboinosinic-polyribocytidylic acid. The production of interferon by polyriboinosinic-polyribocytidylic acid [poly(I) . poly(C)] and poly(I) . poly(C)-diethylaminoethyl-dextran in L929 cells was enhanced from 10 to 100 times by polyene macrolides, including amphotericin B (AmB), AmB methyl ester, nystatin, and filipin. AmB and its water-soluble methyl ester were the most effective; retinol, a nonmacrolide polyene, was ineffective. Interferon induction by Newcastle disease virus was not enhanced by AmB. The kinetics of interferon production were not markedly altered by AmB. Polyenes and poly(I) . poly(C)-diethylaminoethyl-dextran did not need to be present on cells simultaneously to enhance interferon production. Pretreatment with polyenes was as effective as simultaneous addition. Even treatment of washed cells, several hours after removal of poly(I) . poly(C)-diethylaminoethyl-dextran, resulted in enhancement of interferon production. AmB did not appear to form a macromolecular complex with poly(I) . poly(C) in that neither the ultraviolet absorption spectrum nor the melting point of poly(I) . poly(C) was altered by mixing with AmB. Isotopic studies indicated that AmB did not enhance binding of poly(I) . poly(C) to cells. Since the macrolide polyenes have been demonstrated to bind to cell membrane sterols with subsequent alterations in membrane permeability barriers, they may enhance interferon production by increasing cell penetration of poly(I) . poly(C).


Inhibition of herpesvirus replication and herpesvirus-induced deoxyribonucleic acid polymerase by phosphonoformate. Phosphonoformate was found to be an inhibitor of the deoxyribonucleic acid polymerase induced by the herpesvirus of turkeys. The apparent inhibition constants were 1 to 3 muM. Phosphonoformate was also able to block the replication in cell culture of Marek's disease herpesvirus, the herpesvirus of turkeys, and herpes simplex virus. It was as effective as phosphonoacetate. Phosphonoformate was not an effective inhibitor of a phosphonoacetate-resistant mutant of the herpesvirus of turkeys nor of its induced deoxyribonucleic acid polymerase.


Antiviral effects of amphotericin B methyl ester. The methyl ester of amphotericin B (AME) is water soluble, retains antifungal activity, and is significantly less toxic in mammals than amphotericin B. In contrast to amphotericin B, which is not water soluble, AME exhibits antiviral effects against vesicular stomatitis virus, herpes simplex virus types 1 and 2, Sindbis virus, and vaccinia virus in a plaque reduction assay. No antiviral effects could be demonstrated against the unenveloped adenovirus type 4 or echovirus type 11. The extent of virus inactivation was found to be dependent upon the AME concentration, contact time, and temperature. No consistent effect of the virus concentration on the probability of plaque-forming unit inactivation could be determined. The antiviral effects of AME were partially antagonized by the presence of serum. Binding of AME to vesicular stomatitis virus was demonstrated by the comigration of drug and virus in linear sucrose gradients. AME represents a new class of antiviral agents with activity at concentrations relevant to therapeutics. Sterol components of the host cell membrane that become incorporated into the viral envelope are postulated as the site of reaction with AME.


Effect of amphotericin B methyl ester on vesicular stomatitis virus morphology. The water-soluble methyl ester of amphotericin B inactivates vesicular stomatitis virus in association with morphological alterations of the envelope.


Treatment of a murine cytomegalovirus infection with exogenous interferon, polyinosinic-polycytidylic acid, and polyinosinic-polycytidylic acid-poly-L-lysine complex. Treatment of a cytomegalovirus infection of mice with exogenous murine interferon did not alter final mortality or mean day of death. Pretreatment with two interferon inducers significantly reduced mortality, but treatment initiated after infection was not effective.


Role of sediment in the persistence of enteroviruses in the estuarine environment. The survival of four enteroviruses commonly found in sewage effluents was examined when the viruses were adsorped to marine sediments in estuarine water and compared with virus survival in estuarine water alone. Echovirus 1, coxsackieviruses B3 and A9, and poliovirus 1 survived longer when associated with marine sediment. When the estuarine water was polluted with secondarily treated sewage effluent, virus survived for prolonged periods in sediments, but not in the overlaying estuarine water.


New modification of Willis and Hobbs' method for identification of Clostridium perfringens. A modification of the medium of Willis and Hobbs is described. All strains of Clostridium perfringens grown on it gave a positive lecithinase reaction. Some gave a negative reaction in the origin medium.


Viral infection as a precipitant of wheeze in children. Combined home and hospital study. Sixteen children with asthma were studied for one year and viral isolation attempted during all episodes of wheezing. In 91 episodes investigated, 13 viruses were isolated (isolation rate 14%); whereas only one virus was isolated from 120 specimens taken when the children were symptom free. Rhinovirus was the commonest isolate and most were obtained during August, September, October. Episodes of wheezing associated with virus infection were not clinically different not more severe than those due to other precipitants.


Neonatal hyperammonaemia with complete absence of liver carbamyl phosphate synthetase activity. Two newborn infants, male (A) and female (B), with lethal hyperammonaemia are described in the same family. In both, symptoms started on the second day of life. Lethargy and hypotonia were the most prominent initial findings and were followed by convulsions and coma. In both, blood ammonia levels rose to 570 mumol/u (795 microgram/100 ml) a few hours before death, which occurred on the third and fourth day of life respectively. Assay of liver urea cycle enzymes in baby B showed a complete absence of mitochondrial carbamyl phosphate synthetase activity.


The surgical treatment of Wilms' tumor: results of the National Wilms' Tumor Study. Surgical data derived from the 606 patients in the National Wilms' Tumor Study have been analyzed to determine the effect of surgical technique on results of treatment. In addition to surgical excision of the tumor, patients were treated with chemotherapy and radiation therapy according to the study protocol. Under these controlled conditions, certain aspects of surgical technique which have traditionally been thought to be important for success appear to be irrelevant. Physical characteristics of the tumor, preoperative rupture and vascular invasion by tumor were not associated with higher relapse rates. Large tumors, those with capsular infiltrations, and tumors with spread to lymph nodes higher recurrence rate. Operative spill increased the chance of abdominal recurrence. There was no evidence that early ligation of the renal vein was of value in prevention of recurrence, nor was incomplete removal of tumor associated with an increase in relapse rate. Although several critical factors of surgical technique were not studied, it is clear that others are not significant and need not be continued.


Cholecystosonography for the diagnosis of cholecystolithiasis. The gallbladder can be visualized by ultrasound (cholecystosonography) and gallstones seen as echo producing densities. Under cholecystosonographic observation the gallbladder can be demonstrated to contract following stimulation by cholecystokinin. This establishes patency of the cystic duct and excludes a diagnosis of acute obstructive cholecystopathy. The gallbladder has been identified in 84 of 86 patients. Stones have been identified sonographically in 64% of 42 patients with proven gallstones (75% of the last 20 cases). The gallbladder contracted following stimulation in 18 of 20 cases with a patent cystic duct. Cholecystosonography is simple, safe and economical. Cholecystonography with cholecystokinin stimulation is the first diagnostic study to be performed when cholecystolithiasis is suspected and the following circumstances exists: a) an acute right upper quadrant (RUQ) syndrome consistant with acute obstructive cholecystopathy. b) cholestasis or hepatic dysfunction. c) a history of allergy to contrast media. Cholecystosonography may detect gallstones in a gallbladder visualized by oral cholangiography when stones are of the same density as the contrast media.


Sensory irritation and incapacitation evoked by thermal decomposition products of polymers and comparisons with known sensory irritants. A decrease in respiratory rate in mice during exposure to irritating airborne chemicals has been utilized as a response parameter to characterize the degree of upper respiratory tract irritation (sensory irritation) to the thermal decomposition products of various polymers. These included polystyrene, polyvinyl chloride, flexible polyurethane foam, polytetrafluorethylene, a fiber glass reinforced polyester resin, and Douglas Fir. Each of the materials was thermally decomposed in a low-mass vertical furnace in an air atmosphere at a programmed heating rate of 20 degrees C/min. Mice, in groups of four, were exposed to graded concentrations of the thermal decomposition products of each of the above materials. Dose-response curves were obtained by utilizing the maximum percent decrease in respiratory rate as the response parameter during each exposure. Comparison of these dose-response curves with other sensory irritants such as chlorine, ammonia, hydrogen chloride, sulfur dioxide, and toluene diisocyanate gave an indication of the sensory irrtation potential of the thermal decomposition products of these various polymers versus that of well-known single airborne chemical irritants. Total stress and incapacitation of the organism during exposure to sensory irritants such as from the thermal decomposition products of synthetic polymers is discussed.


Liver resection in children with hepatic neoplasms. In the past ten years, 28 patients with primary tumors of the liver have been treated. There were 11 benign tumors, including four hamartomas, three patients with focal nodular hyperplasia, and two each with congenital cysts and hemangioma. Hamartomas and masses of focal nodular hyperplasia should be excised when possible, but both are benign lesions; therefore life threatening excisions at the porta hepatis should be avoided. Cysts are often resectable, but when occupying all lobes of the liver, they can be successfully managed by marsupialization into the free peritoneal cavity. If resectable, hemangiomas should be removed; when occupying most of the liver as they often do, patients may be subject to platelet trapping or to cardiac failure. In some instances these lesions have been controlled by steroids, radiation therapy or hepatic artery ligation. Of 17 malignant tumors seen, 12 proved to be hepatoblastomas. Nine of the 12 patients underwent liver resection, of whom four are cured, (33%). There were three children with hepatocellular carcinomas and two with embryonal rhabdomyosarcoma. One child from each of these groups is cured by surgical excision. At present the only known cures in children with primary malignant liver neoplasms have been achieved by operative removal.


Disseminated Strongyloides stercoralis infection. Association with ectopic ACTH syndrome and depressed cell-mediated immunity. A patient with disseminated Strongyloides stercoralis was evaluated to elucidate host factors that may have led to the development of this infection. The patient was found to have oat cell carcinoma with hypercortisolism produced by tumor adrenocorticotropic hormone. His serum contained a potent inhibitor of lymphocyte blastogenesis. This patient's high level of endogenous cortisol may have impaired lymphocyte function and thereby facilitated infection with S stercoralis.


Influence of cyclic 3',5'-adenosine monophosphate and adenosine on vascular reactivity. cAMP, 5'-AMP and adenosine in doses of 1, 2 and 5 mg/kg i.v. in rats diminish vascular resistance in the hind limbs in vivo and in isolated limbs perfused with nutrient fluid containing adrenaline (A) (10(-3) M), slowing action of the heart and lowering blood pressure. After administration of cAMP, 5'-AMP and adenosine (5 mg/kg), vasoconstricting action of noradrenaline (NA) and A was depressed, and the vasodilating action of isoprenaline (I) was enhanced. The changes in blood pressure observed after administration of the tested adenosine compounds were not blocked by phentolamine, but after I were blocked by propranolol. cAMP, 5'-AMP and adenosine had no influence on the drop in blood pressure elicited by acetylcholine. The results indicate that cAMP, 5'-AMP and adenosine increase reactivity of beta-receptors of the sympathetic system in the blood vessels and modify the action of catecholamines on blood vessels.


Influence of phenylephrine on levels of cyclic 3',5'-AMP in the heart of rats in vivo. In the course of an investigation into the mode of action of phenylephrine using the radioimmunoisotope method, its influence on levels of cyclic 3'5'-AMP in the heart of rats was studied. Phenylephrine in the dose of 1 microgram/kg/min after five minutes lowered levels of this nucleotide by about 20%. Phenylephrine also inhibited the influence of theophyline, an inhibitor of phosphodiesterase which raises levels of c-AMP. The results suggest that the drop in c-AMP after phenylephrine is connected with lowered activity of adenyl cyclase, but do not exclude the possiblity of an interaction between theophylline and phenylephrine acting on phosphodiesterase, which could be referred to the observed effect.


Investigation of the beta-adrenergic system in regulation of immunologic responses. The influence of pharmacologic stimulation and blocking of the beta-adrenergic system on appearance of PF and RF cells, and intralymphocytic levels of cAMP were investigated. Stimulation of the beta-adrenergic receptor exerted an immunosuppressive effect, reflected by the numbers of PFC and RFC and raised levels of cAMP. Inhibition of the adrenergic beta-receptor stimulated immunologic responses and lowered levels of cAMP in lymphocytes. Confirmation of a specific adrenergic mechanism was provided by the absence of an effect of stimulation after prior blocking of the receptor. The possibility of a nonspecific membrane effect of beta-adrenergic drugs is discussed.


Adrenergic regulation of phosphorolysis and hydrolysis of glycogen in smooth muscle of rabbit stomach in situ. In smooth muscle of rabbit stomachs, activities of hydrolytic and phosphorolytic enzymes of glycogen degradation under the influence of agents stimulating and blocking adrenergic alpha and beta-receptors were studied. The results indicate that beta-adrenergic receptor in smooth muscle activated not only phosphorolytic, but also hydrolytic degradation of glycogen, and the alpha-adrenergic receptor only inhibited phosphorolysis but had no influence on hydrolysis of glycogen.


Amyloid formation in insulinoma.

Amyloid formation in insulinoma. Local amyloid depositions were investigated with electron microscopy in a functioning beta cell adenoma of the pancreas. Beta granule-containing neoplastic cells adjoining small amyloid depositions were shown to have cellular invaginations containing radiating amyloid bundles, indicating the neoplastic cells were involved in the formation of amyloid. Seen in the larger mayloid depositions were attenuated, thin, cellular processes of the neoplastic cells, separating the amyloid stroma into globules. The globular separations of the amyloid correlated well with the light-microscopic globular appearance of amyloid stroma. Possible mechanisms are discussed for the amyloid deposition of insulinoma with relation to amyloidogenesis of other types of amyloidosis.


Intranuclear fibrillary inclusions in influenza pneumonia. Electron microscopical study of the lung tissue from a 75-year-old man who died of influenza pneumonia (A/Victoria/RI/76) demonstrated fibrillary inclusions in the nuclei of many alveolar lining cells, in bronchial epithelial cells, and also in endothelial cells. These inclusions were morphologically different from those previously reported in experimental animals. In view of previous experimental studies indicating the necessity of nuclear participation in the replication of influenza virus, these inclusions may be virus-induced structures. Also, possibly the presence of these inclusions in the nuclei of many endothelial cells might be indicative of endothelial damage of microvessels and may be a pathogenetically important factor in influenza pneumonia.


Electroneurographic evidence of polyneuropathy in chronic liver disease. An electroneurographic study performed on the peripheral nerves of 25 patients with severe cirrhosis following viral hepatitis showed slight slowing (P greater than 0.05) of motor conduction velocity (CV) and significant diminution (P less than 0.001) of sensory CV and mixed sensorimotor-evoked potentials, associated with a significant decrease in the amplitude of sensory evoked potentials. The slowing was about equal in the distal (digital) and in the proximal segments of the same nerve. A mixed axonal degeneration and segmental demyelination is presumed to explain these findings. The CV measurements proved helpful for an early diagnosis of hepatic polyneuropathy showing subjective symptoms in the subclinical stage.


Growth comparisons of avian infectious bronchitis virus strains in organ cultures of chicken tissues. Six strains of avian infectious bronchitis virus (IBV) were used to inoculate explants of a range of 15 chicken tissues and virus growth kinetics observed over a period of 96 hours thereafter. Similar patterns of virus production were given by all 6 strains from explants of any particular tissue such as the nasal turbinates, trachea, lung, air sacs and oviduct. Nevertheless, significant differences in behaviour between strains were noted for different tissues and in the efficiency with which certain tissues produced virus. It is suggested that the method has a potential value in determining the virulence of different strains of IBV by comparing their pathogenesis of chicken tissues in vitro.


Purification of mengovirus by freon extraction and chromatography on protein-coated controlled pore glass. Mengovirus, extracted from infected L-cell cultures with Freon 113 and concentrated from the aqueous phase with polyethyleneglycol, was chromatographed on protein-coated controlled pore glass (CPG). The covalent binding of protein to CPG is described. Further purification and concentration of mengovirus was achieved by isopycnic density gradient centrifugation in solutions of either CsCl or salts of iodinated benzoic acid derivatives. The described procedure is superior to conventional methods for the isolation and purification of large quantities of mengovirus. It yields highly purified virus preparations within a short time and a recovery of more than 50% of the starting infectivity.


Susceptibility of various FMDV strains to urea. Resistance to urea in vitro at 37 degrees C varied for each FMDV strain analysed. The urea marker did not correlate with other markers such as resistance to acid, resistance to acidity or size of plaques under agar on BHK21/13 cells. The resistance to urea of subtypes A24 Cruzeiro, O1 Caseros and C3 Resende varied in accordance with their antigenic potency when administered to swine as a trivalent water-in-oil emulsion type vaccine.


[Ultrastructural organization of the mammalian pineal body during postnatal ontogenesis]. Ultrastructural organization of the components composing the mammal pineal gland is analyzed on the basis of literature data and personal observations. Variations in the form, size and ultrastructural organization of the cellular elements in the pineal gland--pinealocytes, interstitial and glial cells--are discussed. The problem of dark and light pinealocytes is considered as a part of a general biological problem on the presence of dark and light cells as a reflection of the law on "intermittent activity of functional structures". An attention is paid to the organization of the microcirculatory bed and pericapillary space to the peculiarities of the secretory process in the organ. Dynamic changes in the cytologic organization of pinealocytes and other components of the organ in the postnatal ontogenesis are presented in detales.


[Mechanism of formation of the psammoma bodies in serous adenocarcinomas of the ovaries]. Histological and electron microscopic examinations of 7 serous adenocarcinomas of the ovaries permitted to follow all the stages of psammous body formation starting from the submicroscopic level. The formation of psammous bodies was shown to be connected with a relatively differentiated epithelium of the adenocarcinomas. Initially, microcrystals of oxyapatites are deposed on accumulations of protein substances in ergastoplasm cysterns and on mitochondrial cristae. With further accumulation of these crystals relatively large intracytoplasmic inclusions of oxyapatites are formed which after confluence fill the cell which results in its death. In this way a small psammous body becomes free-lying.


[Heterologous mesodermal tumor of the uterus of the chondrolipomyxosarcoma type]. A case of heterologous mesodermal tumor of the uterus of the chondrolipomyxosarcoma type in a woman of 70 is described. This observation was peculiar in that the tumor was benign for 25 years. Malignancy and extensive metastases into bones developed during the last year of life.


[Epithelioid sarcoma].

[Epithelioid sarcoma]. The analysis of data from literature and of the author's own 3 observations suggests that epithelioid sarcoma of Enzinger should be separated from soft tissue tumors of obscure genesis. It has a peculiar morphological structure, affects predominantly distal parts of the extremities in the area of tendons and joint capsules, and has some features of the clinical course and prognosis.


[Neural pathway of Powassan virus spread in the central nervous system of white mice]. Electron microscopic investigation of the brains and lumbar spinal cords of adult albino mice infected with Powassan virus was carried out. Virus particles were found within all parts of neurons (perikarya, dendrites, axon), as well as within synaptic apparatus and intercellular gaps of the central nervous tissue. The possibility of the virus spread both throughout the cytoplasm of nerve cells and their processes and the extracellular spaces of the brain was confirmed. Localization of virions within neurons, synapses and myelinated fibers of the spinal cord after intracerebral inoculation suggests that virus spread in the CNS can occur through the CNS parenchyma and also through the nervous conduction pathways. The possible mechanisms of virus dissemination in the CNS of albino mice with experimental Powassan virus encephalomyelitis are discussed.


Bilateral retinal detachment following cytomegalovirus retinitis. Bilateral retinal detachments developed in a renal allograft patient several months after the onset of cytomegalovirus retinitis. Laser photocoagulation was used to limit the posterior extent of one detachment until the detachment was surgically repaired. The thinned, atrophic retina that results from the necrotizing retinitis makes localization of retinal holes difficult and, in this case, contributed to the initial impression that these detachments were nonrhegmatogenous in origin.


Malignant fibrous histiocytoma of the maxillary sinus. Malignant fibrous histiocytomas are an unusual neoplasm in the head and neck, and only six cases involving the maxillary sinus have been reported. The combination of an anaplastic storiform "fibroblastic" stroma with xanthomatous tumor giant cells histologically identifies this tumor. The high incidence of local recurrence of this neoplasm necessitates wide surgical resection. Regional lymph node and distant metastases, although not previously reported in maxillary sinus fibrous histiocytomas, occurred in this case.


Recovery of eighth nerve function after cerebellopontine angle surgery. Retrocochlear lesions cause typical patterns of hearing loss as detailed in special audiometric tests. Such losses, however, may be temporary deficits. Indeed, return to near normalcy following severely depressed auditory function is documented in this article. Conservative surgery on lesions influencing eighth nerve function should be considered. This is not to say that neoplasia can be partially removed, but rather that when tumors can be resected while preserving the cochlear nerve, this should be done inasmuch as good hearing levels may be restored.


Proliferation of cells and intracellular c-AMP content: comparative investigations of autologous lymphatic cells from palatine tonsils, nasopharyngeal tonsils, and blood. The c-AMP content and thymidine incorporation were determined in tonsillogenic, adenoid, and peripheral lymphatic cells. A significant c-AMP decrease with simultaneous increase of the thymidine incorporation compared to the blood lymphocytes was noted in the tonsillogenic and adenoid cells. The negative regulative effect of c-AMP on cell proliferation and the function of tonsils at this processes are discussed.


Effect of waterborne fluoride on fluoride concentration and solubility of dental enamel. Enamel F level and depth of acid etch were measured in 166 teeth from 8 communities with water F ranging from .07-5.0 ppm. Enamel F decreased progressively from the surface inward and was positively correlated with water F. Depth of acid etch was inversely related to enamel F and water F. Relationships between water F, enamel F and enamel solubility were most consistent at water F levels up to 1.0-2.0 ppm.


An adenovirus survey of poultry flocks during the growing and laying periods. Chicken adenoviruses were isolated from asymptomatic chickens on each of 7 farms tested; a majority of them induced cytopathology by the second serial passage. Although adenoviruses were isolated from chickens ranging in age from 8 to 34 weeks, the highest isolation rate was from those 8 to 14 weeks. Eight serotypes (1, 2, 4, 5, 6, 7, 8, and 9) were identified in a relatively small geographic area; serotypes 1, 4, 7, and 9 were isolated most frequently. Multiple serotypes were found on 6 of 7 farms, with 1 farm having 4 serotypes identified. Repeat isolations of the same serotypes were noted on 2 farms. The pattern of virus isolations was generally related to age but varied according to farm. Although new serotypes kept appearing, even after birds came into lay, no clinical problems were associated with them. The incidence of precipitin reactors tended to be low (10-40%) in immature birds but reached 80% or more after sexual maturity. The "antiserum pool" modification of the VN test was found to be accurate and less cumbersome than the conventional procedure for typing isolants.


Studies of flocks with high mortality from lymphoid leukosis. Two pairs of commercial white-egg parent flocks were selected for study, because one of each pair was observed to be dying with lymphoid leukosis at a high rate. The proportion of each flock producing eggs with lymphoid leukosis virus in the albumen was studied. In one pair the rate of birds shedding was no different in the high- and low-mortality flocks. The total rate of shedding was lower in the other pair, but the rate was higher in the high-mortality flock. These data indicate that the rate of virus infection is not always proportional to the development of lymphoid leukosis, and that other environmental factors may play a role. Direct complement-fixation tests on the albumens which were positive for lymphoid leukosis virus showed that group-specific antigen could be detected in 83%. Therefore, the direct complement-fixation test can be used on albumens as a rough estimate of shedding rate.


Experimental infection of chickens with hemorrhagic enteritis virus. Chickens were experimentally infected with hemorrhagic enteritis virus, causing lesions similar to those observed in turkeys. Lesions were induced by intraperitoneal and oral infection. Virus particles with the morphology of adenovirus were demonstrated in the spleens of infected chickens. Splenic extracts from infected chickens caused lesions of hemorrhagic enteritis in turkeys. Recovery from the infection in chickens was followed by the production of antibodies to HEV, demonstrated by agar-gel precipitin test.


Serologic and pathogenicity studies of avian adenovirus isolated from chickens with inclusion body hepatitis. A virus initially thought to be inclusion body hepatitis virus (IBHV), Tipton strain, was classified as an avian adenovirus (AAV) and shown to be antigenically related to 2 serotypes of AAV, 764 and YR36. The virus was antigenically unrelated to AAV serotype TR-22, which included IBHV, Tipton strain. Inoculating specific-pathogen-free chickens with the virus produced hepatitis with basophilic and eosinophilic staining intranuclear inclusion bodies.


Spontaneously occurring lymphoproliferative disease in ducks. A naturally occurring lymphoproliferative disease was observed in a domestic flock of Muscovy ducks. Gross lesions were observed only in one of three ducks necropsied (in the small intestine). Microscopic lesions consisted of mononuclear cell infiltrates composed primarily of reticuloendothelial cells, mature lymphocytes, and plasma cells in livers, spleens, small intestines, peripheral nerves, and brains. Antibodies to reticuloendotheliosis virus were detected in sera of four ducks, whereas all six ducks examined lacked antibodies ot Marek's disease virus.


A clinical evaluation of chickens inoculated with several avian adenovirus isolants. Clinical-chemistry values were determined in young specific-pathogen-free single-comb White Leghorn chickens, both uninoculated and inoculated intra-abdominally at one day old with one of several avian adenovirus isolants. Clinical values for plasma proteins and enzymes indicative of liver function were determined at 3, 7, 10, 14, and 21 days postinoculation. Plasma glutamic oxalacetic transaminase and glutamic pyruvic transaminase activities were elevated in the virus-inoculated birds, along with decreased  albumin levels suggesting liver necrosis, Subsequent histopathologic evaluations confirmed hepatic necrosis in the virus-inoculated groups, demonstrating the potential value of clinical-chemistry assay techniques for diagnosing subclinical disease associated with avian adenoviruses.


An improved method for extracting cell-free herpesviruses of Marek's disease and turkeys from infected cell cultures. Sonic extraction of cell-free Marek's disease herpesvirus (MDHV) and turkey herpesvirus (HVT) from infected cell cultures was improved by incorporating sorbitol in the suspending media. Yields of cell-free virus of virulent MDHV were significantly increased with 10% sorbitol added to SPGA-EDTA buffer. Avirulent strains of MDHV and HVT were respectively readily extracted with SPGA-EDTA and SPGA as the suspending medium, and extraction of their cell-free viruses was moderately improved by adding sorbitol to the suspending medium.


Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide-adenine dinucleotide-binding studies. 1. Two methods of preparing pig heart soluble malate dehydrogenase are described. A slow method yields an enzyme composed of three electrophoretically separable subforms. The more rapid method reproducibly gives a high yield of an enzyme that consists predominantly of the least acid subform. 2. The A(1%) (1cm) of the protein was redetermined as 15 at 280nm. By using this value the enzyme molecule was found to contain two independent and indistinguishable NADH-binding sites in titrations with NADH. 3. No evidence was found for the dissociation of the enzyme in the concentration range 0.02-7.2mum. 4. l-Malate (0.1m) tightened the binding of NADH to both pig and ox heart enzyme (2-fold), but, in contrast with the report by Mueggler, Dahlquist & Wolfe [(1975) Biochemistry14, 3490-3497], did not cause co-operative interactions between the binding sites. 5. Fructose 1,6-bisphosphate had no effect on the binding of NADH to the pig heart enzyme, but with the ox heart enzyme the NADH is slowly oxidized. This slow oxidation explains the ;sigmoidal' binding curves obtained when NADH was added to ox heart soluble malate dehydrogenase in the presence of fructose 1,6-bisphosphate [Cassman (1973) Biochem. Biophys. Res. Commun.53, 666-672] without the postulate of site-site interactions. 6. It is concluded that neither l-malate nor fructose 1,6-bisphosphate could in vivo modulate the activity of soluble malate dehydrogenase and alter the rates of transport of NADH between the cytosol and the mitochondrion. 7. Details of the preparation of soluble malate dehydrogenase have been deposited as Supplementary Publication SUP 50080 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1978) 169, 5.


Purification and properties of the S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B. The S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B, was separated from other alkylsulphohydrolases and purified to homogeneity. Under the experimental conditions used the enzyme completely hydrolysed d-octan-2-yl sulphate (d-1-methylheptyl sulphate), but showed no activity towards the corresponding l-isomer. Additional evidence has been obtained to indicate that it is probably optically stereospecific for d-secondary alkyl sulphate esters with the ester sulphate group at C-2 and with a chain length of at least seven carbon atoms. Enzyme activity towards racemic samples of heptan-2-yl sulphate (1-methylhexyl sulphate), octan-2-yl sulphate and decan-2-yl sulphate (1-methylnonyl sulphate) increased with increasing chain length. l-Octan-2-yl sulphate is a competitive inhibitor of the enzyme, as are certain primary alkyl sulphates and primary alkanesulphonates. Inhibition by each of the last two types of compounds is characteristic of the behaviour of an homologous series. Inhibition increases with increasing chain length and plots of log K(i) values against the number of carbon atoms in each alkyl chain show the expected linear relationship. A crude preparation of the S2 secondary alkylsulphohydrolase was used to show that this particular enzyme hydrolyses l-octan-2-yl sulphate, but is probably inactive towards the corresponding d-isomer. The similarity of the S1 and S2 enzymes to the CS2 and CS1 enzymes respectively of Comamonas terrigena was established, and some comments have been made on the possible roles of these and other alkylsulphohydrolases in the biodegradation of detergents.


Comparison of guinea-pig serum lipoproteins after iodination by two different methods. 1. Guinea-pig low-density lipoproteins were isolated by ultracentrifugation and iodinated either by the IC1 method or by the chloramine-T procedure. 2. The efficiency of labelling by both methods was essentially the same. 3. When the two products were compared by ultracentrifugation, gel chromatography and immunodiffusion analysis, no significant difference in their properties was detected. 4. When they were compared by gradient-gel electrophoresis, aggregates were found in the product of the IC1 method, but not in the lipoprotein iodinated by the chloramine-T process. 5. Both products were metabolized by the guinea pig with essentially the same fractional catabolic rate. 6. The fractional catabolic rate of lipoprotein iodinated by the chloramine-T method was not significantly different from that of lipoprotein biologically labelled in the protein moiety with [75Se]selenomethionine. 7. It is concluded that the products of both methods of iodination are almost equally acceptable, provided that the optimum conditions for the chloramine-T reaction are carefully established.


Differential activation of type-I and type-II adenosine 3':5'-cyclic monophosphate-dependent protein kinases in liver of glucagon-treated rats. The protein-bound cyclic AMP and the activity of cytosolic protein kinases in the presence and absence of cyclic AMP were determined in rat liver up to 2h after injection of glucagon. On the basis of the different salt-sensitivities of the activated cyclic AMP-dependent proteinkinases I and II, an activation of protein kinase II restricted to the high cyclic AMP concentrations present in the first 30 min after hormone injection was found. Essentially the same result was obtained by chromatographic analysis on DEAE-cellulose of liver cytosol from untreated rats and from rats killed at 2 and 60 min after glucagon injection. Protein kinase II activation was only detected at 2 min after injection. In contrast, the cyclic AMP-dependent protein kinase I was found to be nearly totally activated at 2 min and to be still almost as active at 60 min after the hormone stimulus, whereas the amount of bound cyclic AMP and the activation of total cytosolic protein kinases had fallen to two-thirds of their maximal values during this time period. A third cyclic AMP-independent protein kinase, which co-chromatographed with protein kinase type II, could be clearly distinguished from the two cyclic AMP-dependent kinases by use of the heat-stable inhibitor from bovine muscle, which totally inhibited the cyclic AMP-dependent enzymes, but stimulated the cyclic AMP-independent protein kinase.


Typing field isolates of infectious bronchitis by the plaque-reduction test. A technique is described for typing field isolates of infectious bronchitis virus by plaque-reduction assay of the serum antibody response of chickens experimentally infected with such isolates. Also reported are use of the technique with field isolates and a comparison of results obtained in plaque-reduction assay and in tracheal-ring organ-culture assay.


Comparative studies on the 25-hydroxylations of cholecalciferol and 1 alpha-hydroxycholecalfierol in perfused rat liver. The 25-hydroxylations of [(3)H]cholecalciferol and 1alpha-hydroxy[(3)H]cholecalciferol in perfused rat liver were compared. Results showed that about twice as much 1alpha(OH)D(3) (1alpha-hydroxycholecalciferol) was incorporated into the liver as cholecalciferol. 25-Hydroxy[(3)H]cholecalciferol and 1alpha-25-dihydroxy[(3)H]cholecalciferol were not incorporated significantly. Livers isolated from vitamin D-deficient rats formed the 25-hydroxy derivatives of cholecalciferol and 1alpha(OH)D(3) respectively linearly with time for at least 120min. The rate of 1alpha,25(OH)(2)D(3) (1alpha,25-dihydroxycholecalciferol) production increased exactly 10-fold on successive 10-fold increases in the dose of 1alpha(OH)D(3), suggesting that hepatic 25-hydroxylation of 1alpha(OH)D(3) is not under metabolic control. On the other hand, the rate of conversion of cholecalciferol into 25(OH)D(3) (25-hydroxycholecalciferol) did not increase linearly with increase in the amount of cholecalciferol in the perfusate. The 25-hydroxylation of cholecalciferol seemed to proceed at a similar rate to that of 1alpha(OH)D(3) at doses of less than 1nmol, but with doses of more than 2.5nmol, the conversion of cholecalciferol into 25(OH)D(3) became much less efficient, though the linear relation between the amounts of substrate and product was maintained. A reciprocal plot of data on the 25-hydroxylation of cholecalciferol gave two K(m) values of about 5.6nm and 1.0mum, whereas that for the 25-hydroxylation of 1alpha(OH)D(3) gave a single K(m) value of about 2.0mum. These results suggest that there are two modes of 25-hydroxylation of cholecalciferol in the liver, which seem to be closely related to the mechanism of control of 25(OH)D(3) production by the liver.


Response to several avian respiratory viruses as affected by infectious bursal disease virus. After being inoculated with the infectious bursal disease virus (IBDV) at one day of age, specific-pathogen-free chickens were inoculated with either Newcastle disease virus (NDV), infectious bronchitis virus (IBV), or infectious laryngotracheitis virus (ILTV). Their immunity was challenged 2-3 weeks later with homologous virus, and antibody titers were determined for ILTV and IBV. Several studies were made to determine the effects of IBDV on the development of persistent or chronic virus infections. Chickens susceptible to IBDV were exposed to IBDV at one day and inoculated at two weeks of age with either NDV, IBV, or ILTV. Two, three, four, and five weeks postinoculation with the respiratory viruses, tracheal swabbings were collected for virus isolations. The birds were challenged with homologous virus at five weeks postinoculation. The results show that early (1-day) infections with IBDV affect the response of young birds to several avian respiratory viruses, as indicated by lowered resistance to challenge and humoral antibody levels for birds inoculated with ILTV and NDV. Birds infected with IBV and IBDV withstood IBV challenge but were much more prone to persistent infections than were birds not exposed to IBDV. High death rates and decreased weights in all IBDV-inoculated groups demonstrated the damaging effects of early IBDV infection.


Calcium metabolism in rat hepatocytes.

Calcium metabolism in rat hepatocytes. 1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.


Reaction of formiminoglutamate with liver glutamate dehydrogenase. 1. Kinetic aspects of the reaction between crystalline bovine liver glutamate dehydrogenase and formiminoglutamate were investigated to establish the conditions under which the latter may interfere with the assay of glutamate by using glutamate dehydrogenase and to explain why formiminoglutamate accumulates in vivo after histidine loading, although it can react with glutamate dehydrogenase. The Km and Vmax. values were compared with those of the enzyme reacting with glutamate. At pH 7.4 Km for formiminoglutamate was much higher and Vmax. much lower than the values for glutamate. 2. The equilibrium constant at pH 7.0 was 0.017 micrometer with formiminoglutamate, i.e. about one two-hundredths that with glutamate. 3. In vivo the interaction between glutamate dehydrogenase and formiminoglutamate is minimal even when the concentration of the latter in the liver is greatly raised, as in cobalamine or folate deficiency after histidine loading. 4. At pH 9.3, i.e. under the conditions for the assay of glutamate by glutamate dehydrogenase, formiminoglutamate reacts readily with the enzyme.


Cyclic AMP-dependent histone-specific nucleoplasmic protein kinase from rat liver. A nucleoplasmic histone kinase activity was isolated from livers of adult rats and purified 39-fold compared with whole nuclei by ultracentrifugation of the nuclear extract and Sephadex G-200 gel filtration in the presence of cyclic AMP. Analysis by polyacrylamide-gel electrophoresis as well as by gel filtration indicates a mol.wt. of approx. 60,000 for the catalytic subunit and 130000-150000 for the cyclic AMP-binding activity. The purified enzyme displays a 20-fold greater preference for histone fractions 1 and 2b than for any non-histone substrate, including alpha-casein. Endogenous protein in the preparation is not appreciably phosphorylated. The unfractioned enzyme is stimulated significantly by cyclic GMP, cyclic IMP and dibutyryl cyclic AMP as well as by cyclic AMP. The catalytic reaction requires Mg2+ (Km 1.9mM) and ATP (Km 15.4 micron). Half-maximal activity of the enzyme is observed with histone 2b at 12micron and histone 1 at a higher substrate concentration. The pH optima are 6.1 and 6.5 with histones 2b and 1 respectively. This nuclear protein kinase appears to be distinct from other nuclear enzymes that have been reported, on the basis of histone specificity, univalent-salt-sensitivity, pH optima and nuclear location. However, the enzyme possesses many properties similar to those of the cytoplasmic kinases, including cyclic AMP-dependence, Mg2+ and ATP affinities and pH optima. It differs from cytoplasmic protein kinase type I, the major form in the liver, with respect to bivalent-cation effects and response to the heat-stable protein kinase inhibitor protein isolated from ox heart.


Problems encountered in measuring the activity of phosphatidate phosphohydrolase. The measurement of phosphate release from phosphatidate overestimates the microsomal activity of phosphatidate phosphohydrolase from rat liver, since phosphate is also produced via the glycerol phosphate that results from the deacylation of phosphatidate. The determination of phosphate production can be a reliable assay for the soluble phosphatidate phosphohydrolase in rat liver, because the glycerol phosphate formed is not hydrolysed under the conditions used.


Stopped-flow spectrophotometric studies on the reaction of turkey liver xanthine dehydrogenase with reducing substrates. The kinetics of reduction of turkey liver xanthine dehydrogenase by substrates were investigated by stopped-flow spectrophotometry. The results may be explained in terms of the known redox potentials of the various centres in the enzyme [Barber, Bray, Cammack & Coughlan (1977) Biochem. J. 163, 279-289]. They are, morover, consistent with the scheme [Olson, Ballou, Palmer & Massey (1974) J. Biol. Chem. 249, 4363-4382] in which reduction occurs in three consecutive steps, one molecule of substrate reacting with the active site at each step. First-order rate constants believed to correspond respectively to the combined first and second steps and to the third step in the reduction by excess of xanthine and of NADH were determined. The rates of reaction with these substrates in the combined first and second steps are independent of the degree of enzyme functionality.


Filtration of chylomicrons by the liver may influence cholesterol metabolism and atherosclerosis. A fenestrated endothelial lining of sinusoids in rat liver has been shown to separate chylomicrons of different sizes following their injection into the portal vein. This sieving may have physiological importance, since during low dietary fat intake some intestinal lipoproteins are probably small enough to contact liver cells, but during high dietary fat loads most chylomicrons are too large to pass through the filter and must first be degraded to smaller remnants. The liver plays a central role in cholesterol metabolism since it catabolises dietary cholesterol which inhibits synthesis of cholesterol to be circulated as liver-derived very low density lipoproteins (VLDL) and low density lipoproteins. The sieving of chylomicrons, remnants and other lipoproteins by the sinusoidal endothelium of the liver may thus play an important role in lipid transport, affecting the balance of various lipoprotein moieties which in turn may affect the relationship of dietary lipids to various hyperlipidaemias and atherosclerosis.


Multipeaking of the compound electromyographic response to single nerve stimulus: its occurrence and prevention in an experimental model. The compound electromyographic response to a single nerve stimulus was examined in the cat tibialis anterior muscle preparation, to determine the cause of and to find a method of preventing multipeaking of the waveform. Bundles of the sciatic nerve were separated and stimulated while the response was detected by unipolar electrodes and analysed with an instrument specifically designed for this purpose. It was found that the most cephalad bundle of the sciatic nerve at the sciatic notch innervated the tibialis anterior muscle and that restriction of stimulation to this bundle most readily rendered the response single-peaked. It is concluded that nerve-branching is a common cause, and therefore a straightforward alternative to "repetitive response of the muscle" as the explanation, of multipeaking of the neurally-elicited compound electromyogram. A method of eliciting and recording the compound electromyogram in the cat tibialis anterior model is described.


Biochemical and morphological comparison of two tumour-cell-aggregation factors from rat ascites hepatoma cells. Two tumour-cell-aggregation factors, derived from rat ascites hepatoma cells, had different antigenicity; one was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. Their activities were both lost by digestion with trypsin, but remained unchanged by oxidation with periodate, suggesting the role of the protein portions in their molecules. The potency of the unabsorbed factor was inhibited specifically by alpha-methyl-D-mannoside or D-mannose, while that of the absorbed factor was inhibited specifically by N-acetyl-D-glucosamine, suggesting that these carbohydrates may be concerned with the respective receptor structures at the tumour-cell surface. The unabsorbed factor induced not only cell aggregation (as shown in the form of simple apposition) but also cell adhesiveness characterized by development of intermediate junctions, desmosomes and tight junctions, while the absorbed factor produced only simple apposition, suggesting their functional difference.


Enhancing and inhibiting effects of spleen cells from tumour-bearing mice on growth of virus-induced primary sarcoma. The effects of adoptive transfer of spleen cells from tumour-bearing mice on the growth of Moloney murine sarcoma virus (M-MuSV)-induced primary tumours in BALB/c mice were studied. The effects were in 2 directions, depending on the time of lymphocyte inoculation; when spleen cells from tumuor-bearing mice 12 days after M-MuSV inoculation were inoculated into recipient mice before M-MuSV inoculation, the appearance of tumours was significantly delayed and their incidence was reduced, whereas when these lymphocytes were inoculated after the development of tumours in recipient mice, tumour growth was significantly enhanced. The data indicated that these inhibiting and enhancing effects were mediated mainly by T cells. The mechanisms of the parodoxical effects were investigated.


[Persistency of serum antibodies against Entamoeba histolytica]. With the purpose to determine the duration of serum antibodies against E. histolytica by means of the contraimmunoelectrophoresis technique, 31 patients with the diagnosis of intestinal or liver invasive amebiasis were included in this study. In 92.3% of the 13 patients subjected to transversal study, no specific antibodies were found from 10 to 24 months after the occurrence of amebic disease. In 64.7% of 17 children with liver amebiasis who were studied longitudinally, no serum antibodies were found between 4 to 10 months after the disease. The possible factors involved in the varaibility of the duration of serum antibodies against E. histolytica are discussed.


Neutrophil and eosinophil granulocytes in bacterial infection: sequential studies of cellular and serum levels of granule proteins. The intraneutrophilic concentrations of lactoferrin, myeloperoxidase, collagenase and chymotrypsin-like cationic proteins were measured sequentially during acute bacterial infection. The serum levels of lactoferrin and myeloperoxidase were also followed as well as the 'eosinophil' cationic protein as a marker for eosinophil leucocytes. During the early course of infection there was a profound but reversible decrease of intraneutrophilic lactoferrin. The levels of cellular collagenase and chymotrypsin-like cationic proteins also tended to decrease reversibly during day 2-8 in most cases; myeloperoxidase levels were normal except for two cases. Serum myeloperoxidase and lactoferrin correlated with blood neutrophil counts. In spite of the absence of peripheral eosinophils the 'eosinophil' cationic proteins of serum were increased on the first day of infection, which may reflect increased eosinophil turnover.


Synthesis and characterization of 2-nitro-5-aziodobenzoylglycyloxytocin, an oxytocin photoaffinity label. The oxytocin analogue, 2-nitro-5-azidobenzoylglycyloxytocin (NAB-Gly-oxytocin), has been synthesized and purified. The analogue is a full agonist for the stimulation of osmotic water flow in the toad urinary bladder (one-half maximal activity at 3.2 X 10(-6)M). It also enhances [14C]urea permeability in this tissue. Repetitive photolysis in the presence of NAB-Gly-oxytocin (8 X 10(-6)M) results in a progressive permanent inhibition of oxytocin stimulated urea permeability but does not alter hormone induced 3H2O movement. The inhibition is dependent on the photogeneration of the aryl nitrene intermediate and is relieved by protecting the hormone receptor with excess oxytocin (10(-6)M) during the photolysis. These results suggest that the photodependent permanent inhibition of the response to oxytocin in the toad bladder is due to covalent incorporation of the photoaffinity label, NAB-Gly-oxytocin, into the hormone receptor.


Small-angle X-ray scattering and differential scanning calorimetry studies on reversibly modified human-serum low density lipoproteins. Small-angle x-ray scattering diagrams of human serum low density lipoprotein (LDL) were recorded at several temperatures in solutions of different freezing points. It was found that modifications of the x-ray patterns observed on cooling the lipoprotein samples below 0 degrees C are due to reversible alterations of the LDL surface structure induced by the freezing process (independent of temperature). With both intact and partially dehydrated LDL, differential scanning calorimetry (DSC) carried out in the body temperature range revealed a heat absorption characteristic of the transition from a liquid crystal to an isotropic liquid phase of cholesteryl esters within the lipoproteins (Deckelbaum, R. J., Shipley, R. J., Small, P. M., Lees, R. S., & George, P. K. (1975) Science 190, 392). However, small-angle x-ray scattering diagrams recorded with the same LDL sample before and after the partial removal of water were found to be very different: the scattering curve for intact LDL showed a strong band centered at (36 A)-1 which disappeared upon drying and reappeared upon restoring the water. Our results suggest that the presence of this signal strongly depends on the molecular structure of the lipoprotein surface.


Synthesis of mouse mammary tumor virus ribonucleic acid in isolated nuclei from cultured mammary tumor cells. Glucocorticoid hormone treatment of GR cells, a cultured line derived from mouse mammary tumor tissue, selectively stimulates the rate of transcription of integrated proviral genes specifying mammary tumor virus (MTV). We have incubated isolated nuclei from these cells under conditions in which all three endogenous RNA polymerases appear to be active. RNA synthesized in vitro is distinguished from preexisting nuclear RNA by labeling the in vitro products with [3H]CTP, and the level of MTV RNA synthesis is measured by molecular hybridization with unlabeled viral DNA. Synthesis requires the addition of nucleoside triphosphates, and is inhibited by actinomycin D. Pretreatment of GR cells with dexamethasone, a synthetic glucocorticoid, has no significant effect on the amount of total RNA synthesis in isolated nuclei. In contrast, synthesis of MTR RNA is stimulated 10-20-fold in nuclei from dexamethasone-treated cells relative to untreated control nuclei; the sensitivity of in vitro viral RNA synthesis to inhibition by alpha-amanitin suggests that it is carried out exclusively by RNA polymerase II. The fraction of total RNA synthesis which is viral specific (about 0.2-0.4% in nuclei from dexamethasone-treated cells and 0.01-0.03% in controls) is similar to that detected in pulse labeled RNA from whole GR cells in culture. Our procedures for labeling and hybridization of RNA appear to avoid artifacts recently noted in other in vitro transcription systems.


Kinetic studies on the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes. Steady-state kinetic techniques have been used to investigate each of the reactions catalyzed by the bifunctional enzyme, chorismate mutase-prephenate dehydrogenase, from Aerobacter aerogenes. The results of steady-state velocity studies in the absence of products, as well as product and dead-end inhibition studies, suggest that the prephenate dehydrogenase reaction conforms to a rapid equilibrium random mechanism which involes the formation of two dead-end complexes, viz, enzyme-NADH-prephenate and enzyme-NAD+-hydroxyphenylpyruvate. Chorismate functions as an activator of the dehydrogenase while both prephenate and hydroxyphenylpyruvate acted as competitive inhibitors in the mutase reaction. By contrast. bpth NAD+ and NADH function as activators of the mutase. Values of the kinetic parameters associated with the mutase and dehydrogenase reactions have been determined and the results discussed in terms of possible relationships between the catalytic sites for the two reactions. The data appear to be consistent with the enzyme having either a single site at which both reactions occur or two separate sites which possess similar kinetic properties.


Sodium transport by isolated bullfrog small intestine. Effect of prostaglandin E1. Addition of 446 micron prostaglandin E1 (PGE1) to the serosal medium of isolated short-circuited bullfrog small intestine elicited small increases transmural potential difference and short-circuit current while addition of PGE1 to the mucosal medium caused no change in the electrical parameters. Addition of 100 micron indomethacin to the mucosal medium inhibited both potential difference and short-circuit current with a resultant increase in steady-state tissue resistance. In the presence of mucosal 100 micron indomethacin, serosal 60 micron PGE1 markedly stimulated transmural potential difference and short-circuit current with a resultant decrease in steady-state tissue resistance. Serosal arachidonic acid (330 micron) stimulated transmural potential difference and short-circuit current and this effect was abolished by the addition of 100 micron indomethacin to the mucosal medium. Serosal 60 micron PGE1 only stimulated the M (mucosa) leads to S (serosa) unidirectional flux of sodium. These results strongly suggest that the PGE1 action is mediated either via a series of metabolic reactions which possibly increase the permeability of the mucosal membrane to sodium or via direct stimulation of rheogenic sodium pump activity.


Correlation between the thermotropic behavior of sphingomyelin liposomes and sphingomyelin hydrolysis by sphingomyelinase of Staphylococcus aureus. The hydrolysis of D-erythro beef brain sphingomyelin and D,L-erythro-N-palmitoylsphingomyelin dispersed as multilamellar liposomes by sphingomyelinase of Staphylococcus aureus is correlated with the thermotropic behavior of the sphingomyelins. In both cases maximal enzymatic hydrolysis was achieved at the beginning of the gel to liquid crystalline phase transition (30 degrees C for beef brain sphingomyelin and 41 degrees C for N-palmitoylsphingosine-phosphorylcholine) with much lower activity both below and above these temperatures. The enzymatic activity was depressed in the presence of cholesterol in the bilayer which also depressed the phase-transition. The profile of the enzymatic activity is explained by the uniqueness of the lipid molecules arrangement at the phase transition.


Magnetic resonance studies of eukaryotic cells. III. Spin labeled fatty acids in the plasma membrane. XC Sarcoma, Vero and Aedes aegypti plasma membranes have been studied in viable cells and in purified membrane of XC Sarcoma cells by the spin label method. The temperature dependence of the order parameter of fatty acid spin labels is found to be linear in all three cells and membrane and shows no evidence of a lipid phase transition. The order parameter of the fatty acid labels substituted at the 5-position is shown to increase as a function of the cholesterol: phospholipid molar ratio in cells that have been studied to date. Cells attached to their growing surface are studied for the first time by electron paramagnetic resonance spectroscopy (EPR). The resulting spectra are anisotropic due to the non-spherical shape of the cells and show that these labels orient preferentially perpendicular to the cell surface. The viscosity of the extracted XC cell membrane is estimated to be 2.5 P from rotational correlation time measurements of the spin label 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO).


Purification and characterization of a long chain, fatty-acid-binding protein supplying the mitochondrial beta-oxidative system in the heart. A fatty-acid-binding protein with a molecular weight of approximately 12 000 was purified from rat heart and the binding investigated by electron spin resonance. The stearic acid bound to the protein was found to be transferred to the mitochondrial beta-oxidative system, suggesting a role as transcytoplasmic fatty acid carrier for this protein. For the first time a physiological cytoplasmic protein was used as a carrier supplying the mitochondrial beta-oxidative system. A new mechanism of action is proposed to explain the control exerted by this type of protein in some membrane-linked enzymatic processes.


[Paramagnetic centers and photochemical reactions of photosystem 2 subchloroplast fragments]. ESR signals of highly purified subchloroplast fragments containing the Photosystem 2 (PS 2) were obtained. Reversibility of the ESR II signal in darkness in the presence of dichlorophenolindophenol (DCPIP) was observed. Reduction of DCPIP with chloroplast fragments in the absence of exogenous donors takes place in light without simultaneous liberation of O2. Electron transfer may be blocked by the displacement of manganese with Mg2+ and Ba2+ bivalent cations. Cd2+ cations partially suppress the electron transfer catalysed by PS 2. The inhibiting effect of Cd2+ is removed in the presence of exogenous Mn2+ or hydroxylamine.


[Light-dependent reactions of Mn2+ and P700+ in chloroplasts during flash illumination]. The changes of the light-induced ESR signal I and exogeneous Mn2+ ions in chloroplasts of Vicia faba plants have been studied under short flash excitation and weak background far red illumination. It is shown that Mn2+ ions reactivate the noncyclic electron transport in chloroplast previously inactivated with high tris concentrations under pulse illumination. Simultaneously with P700+ reduction the ESR signal of free Mn2+ ions decays stoichometrically to P700+ reduction.


Reactivity of antiinflammatory and superoxide dismutase active Cu(II)-salicylates. The activity of chelated Cu(II) with four different aspirin-like drugs in various superoxide dismutase assays was examined. Prior to these studies the oxidation state of the involved copper was measured by x-ray photoelectron spectrometry and was found to be +II throughout. All copper complexes were able to suppress the xanthine-xanthine oxidase mediated reduction of both cytochrome c and nitroblue tetrazolium as well as the formazan formation by KO2 in a specific manner. The hydroxylation of benzo-[alpha]-pyrene as well as the demethylation of 7-ethoxycoumarin using induced hepatic rat microsomes could be successfully inhibited by the employed Cu(II) chelates. Cu(II)-acetylsalicylate was the most active copper complex. Our findings support the proposal that Cu(II) chelates are the active forms of aspirin-like antiinflammatory agents.


Coordination chemistry of molybdenum and tungsten--VIII. Oxomolybdenum(V) complexes of 8-hydroxyquinoline and relevance of EPR spectra to binding sites in flavoenzymes. MoOCl3(THF)2 (THF = tetrahydrofuran) reacts with 8-hydroxyquinoline (QH) to form [MoOCl3(QH)2], which contains neutral monodentate ligands, [MoOCl(Q)2], and anionic bidentate ligands, and the dimeric [Mo2O3(Q)4], which contains anionic bidentate ligands and both terminal and bridging oxo donors. In dichloromethane [MoOCl3(QH)2] dissolves to give three species, and epr measurements identify these as unchanged [MoOCl3(QH)2], [MoOCl(Q)2] and a third species characterised by a value of 1.979. No g value of this magnitude has previously been obtained for molybdenum(V) complexes which do not contain sulphur donors, and the significance of epr measurements as an indication of the nature of molybdenum coordination in flavoenzymes must be questioned. These complexes have also been characterised by vibrational and electronic spectral measurements.


[Cytochrome concentration in the liver at different stages of adaptation to cold]. Concentration of cytochroms in the liver of adult male rats has been studied during the acclimation to cold. One group of rats was kept at about 24 degrees, another at 2--4 degrees. After a month of acclimation to cold the cytochroms concentration in the liver increased, whereas the mitochondrial concentration of cytochroms remained unchanged. It is possible to distinguish three steps of cytochroms concentration changes; 1. on the 9th day of acclimation to cold cytochroms concentration increased 1.5--2 times; 2. by the 14th day the cytochroms concentration decreased (to the control level and lower); 3. after about one month of acclimation to cold the cytochroms concentration increased again. In the skeletal muscle it increased on the 14th (but not on the 9th) day.


[Inhibitors of exonuclease A5].

[Inhibitors of exonuclease A5]. Over 30 compounds resembling to or being structural elements of the minimal substrate of exonuclease A5 were tested for their ability to inhibit the reaction catalyzed by this nuclease. The compounds containing less than two phosphate groups were shown to possess a low inhibitory activity, if any. CDP, double-stranded DNA and nucleoside-3',5'-diphosphates (pNp) proved to be effective exonuclease A5 inhibitors. Pyrophosphate stimulated the reaction in the case of low molecular weight substrates only. For the inhibitory activity of pNp to occur, the intactness of the nucleoside moiety as a whole was shown to be necessary, the activity level depending on the structure of both the base and the sugar components. A competitive mechanism of the inhibitory action was demonstrated for pTp, pdCp, pdGp, pdAp and pUp and the Ki values were determined. The affinity for the inhibitor decreased in the following order: pdCp greater than or equal to pTp greater than pdGp greater than pdAp. Ki for pdCp and pTp were found to be approximately 3.10(-6) M. The investigation of the inhibition mechanism as well the determination of Ki were accomplished with the help of homogenous low molecular weight substrates--ApApA and the phosphoamide MeOPheNH(pdA)2. These were chosen after kinetic parameters determination of the hydrolysis of 22 exonuclease A5 substrates, predominatly of the RpNpN type. On the basis of data obtained the specificity of exonuclease A5 is also discussed. Possible usefulness of immobilized competitive inhibitors of the pNp type not only for single nuclease isolation but for the separation of a mixture of different nucleases is considered. This possibility is based on the almost universal inhibitory effect of pNp on different nucleases and at the same time on their different affinity for the enzymes. In particular, this approach might be useful for the elimination of exonucleases and some other nucleolytic enzymes from the preparations of endonucleases-restrictases.


Stochastic ESR analysis of rat liver and hepatoma mitochondrial lipids. A commonly used model for the interaction of the motional narrowing of ESR lines is shown to be qualitatively misleading. An analysis of lipid extracts of mitochondrial preparations labeled with 12-nitroxide stearic acid produced linear plots of the logarithm of the correlation time versus the reciprocal of the absolute temperature when analyzed with stochastic computer simulations. However, when the data were analyzed with isotropic Lorentzian line shape approximations, nonlinear plots were obtained.


[Comparative study of the photopotential and EPR signal arising after photooxidation of metal substituted pheophytins]. Ethanolic solutions of Zn-, Cd-Phe a and b and Chl a and b with p-benzoquinone as electron acceptor have been studied at pH 4. In all systems positive photopotentials and singlet ESR lines of the pigment cation radicals are observed. A conclusion is made that the photopotential is only partly attributable to an electrode reaction of the cation radicals, to a greater extent arising due to unknown, non-radical particles.


[Study of the responses of tumor and normal tissues to exposure to ionizing radiation by the EPR method. I. Comparison of the low temperature EPR spectra of irradiated tumor and normal tissues]. ESR spectra of liver samples of normal mice and liver tumour samples of mice (line C3HA) injected hepatomo 22-a irradiated in the dose of 1 Mrad at --196 degrees C were studied. The ESR spectra of normal and tumour tissues irradiated at --196 degrees C are shown to differ. Only the ESR spectra of tumour and liver of the tumour host is characterized by an asymmetric signal with deltaH=6 Oe and g=2.0005 ("tumour" signal). The intensity of the "tumour" signal depends on the developmental stage of hepatoma. It is changed after the animals are injected with ionol.


Role of microtubules in granulocyte adherence. The adherence of human polymorphonuclear leukocytes (PMN) to nylon fibers is inhibited in a dose-dependent fashion by exposure in vitro of these cells to either colchicine or VM-26, both of which agents prevent microtubule assembly. Mean adherence of human PMN was 48% +/- 2%, following treatment with 10(-5) M colchicine or 10(-4) M VM-26 it was reduced to 31% +/- 2% and 7%, respectively. Peritoneal PMN obtained from mice and mink with Chediak-Higashi syndrome (CHS) thought to have a microtubule-membrane disorder affecting the PMN had a mean adherence of 29% +/- 3% and 40% +/- 8% compared to control values of 46% +/- 5% and 73% +/- 8%, respectively, from the mice and mink. Both ascorbic acid and bethanechol, shown previously to enhance microtubule assembly in humans with CHS, normalized granulocyte adherence in PMN obtained from mice with CHS. Cyclic nucleotide levels were not altered by treatment of human PMN with colchicine, nor did they differ between normal and CHS animals. Thus it appears that the state of microtubule assembly may directly affect the properties of the PMN plasma membrane without requiring alterations of cyclic nucleotides as an intermediary.


Involvement of the periaqueductal grey matter and spinal 5-hydroxytryptaminergic pathways in morphine analgesia: effcts of lesions and 5-hydroxytryptamine depletion. 1 Electrolytic lesions of the periaqueductal grey matter (PAG) were made in rats. The analgesia produced by intraperitoneal injection of morphine (10 and 20 mg/kg), tested by the tail flick and hot plate methods, was substantially reduced in the lesioned rats. Baseline pain thresholds were unaffected by the lesions.2 The lesion effects were not due to damage to the dorsal raphé nucleus. The extent of histologically determined damage to the dorsal raphé and the resulting decrease in striatal 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations did not correlate with the reduction in morphine analgesia produced by the lesion. Furthermore, microinjections of 5, 6-dihydroxytryptamine (5,6-DHT) into the dorsal raphé nucleus produced a similar fall in 5-HIAA levels but had no effect on morphine analgesia.3 Selective destruction of the periventricular catecholamine system produced by microinjection of 6-hydroxydopamine (6-OHDA) caused a slight decrease in morphine analgesia, thus raising the possibility that catecholamines may be involved in the action of morphine in the PAG.4 5,7-Dihydroxytryptamine-induced lesions of the spinal cord 5-hydroxytryptaminergic pathways reduced cord 5-HT concentration by 70% and markedly attenuated morphine analgesia as determined by the tail flick test.5 These experiments provide additional evidence that the PAG is a major site of action of opiates in producing analgesia. Furthermore, they have demonstrated the probable involvement of spinal 5-hydroxytryptaminergic pathways in the mediation of opiate analgesic effects.


Effects of prostaglandin E2 and a prostaglandin endoperoxide analogue on neuroeffector transmission in the rat anococcygeus msucle. 1 Investigations were made into the effects of prostaglandin E(2) (PGE(2)) and a prostaglandin endoperoxide analogue (Upjohn compound U-46619) on the responses of the rat anococcygeus muscle to field stimulation of the intrinsic sympathetic nerves, and to exogenous noradrenaline. The effects of PGE(2) on responses to stimulation of intrinsic inhibitory nerves were also studied.2 PGE(2) (5.6 x 10(-8) or 2.8 x 10(-6) mol/l) decreased motor (sympathetic) responses to field stimulation at all frequencies tested (2 to 24 Hz). The prostaglandin also reduced the inhibitory responses to field stimulation, seen when the tone of the preparation had been raised and its sympathetic innervation had been blocked by guanethidine. However, these inhibitory responses were also reduced by other spasmogens (carbachol and 5-hydroxytryptamine) which, like PGE(2), further increased the tone of guanethidine-treated preparations.3 At a concentration of 5.6 x 10(-8) mol/l, PGE(2) had no effect on responses to noradrenaline, whereas at a fifty-fold higher concentration the prostaglandin potentiated these.4 Unlike PGE(2), U-46619 (5.6 x 10(-8) mol/l) greatly potentiated motor responses to field stimulation, at frequencies from 0.75 to 24 Hz. This effect did not represent a specific facilitation of sympathetic neurotransmission, as responses to carbachol and 5-hydroxytryptamine, as well as to noradrenaline, were also potentiated.5 The results are discussed in relation to the effects of prostaglandins and prostaglandin endoperoxides on neuroeffector transmission in other sympathetically innervated tissues. It is concluded that PGE(2) inhibits sympathetic neurotransmission in the rat anococcygenus muscle by a prejunctional action, whereas the predominant effect of U-46619 is direct excitation of the muscle. The effect of PGE(2) on inhibitory responses to field stimulation may represent an interference with inhibitory neuroeffector transmission in this tissue, or may simply be a consequence of the spasmogenic action of the prostaglandin.


Action of guanethidine on rabbit atrial membranes. 1 Intracellular potentials were recorded in driven left atria from reserpine-treated rabbits. Guanethidine 2 X 10(-5) M slightly increased Vmax and shortened the total duration (TD) of the action potential (AP) without causing hyperpolarization. For the first 30 min after 4 X 10(-4) M, Vmax increased without hyperpolarization and AP height increased slightly. Thereafter, Vmax and height decreased with a slight and gradual depolarization. This depolarization was irreversible. TD was increased after 15 minutes. Guanethidine 2 X 10(-3) M initially decreased Vmax and height before causing depolarization. 2. Pretreatment with tetrodotoxin (TTX) 1.6 X 10(-7) M prevented or reversed the initial increases in Vmax, height and TD induced by guanethidine (4 X 10(-4) M). 3 TTX 3.1 to 6.2 X 10(-6) M, added 15 or 30 min after guanethidine 4 X 10(-4) M, delayed or prevented depolarization by guanethidine. 4 Ouabain 10(-5) M incubated for 20 and 90 min greatly inhibited Na+, K+-adenosine triphosphatase and K+-phosphatase activities; guanethidine was without effect. 5 Guanethidine probably increases resting sodium permeability after the promotion of increases in sodium permeability during the AP. High doses of the drug decrease sodium permeability during the AP.


Reversal of the action of amino acid antagonists by barbiturates and other hypnotic drugs. 1 The effects of pentobarbitone (PB) and other sedative/hypnotic drugs have been examined in relation to gamma-aminobutyric acid (GABA) in vitro on the superfused isolated superior cervical ganglion of the rat and in vivo on single units in the brain stem of the anaesthetized rat.2 PB, and other barbiturates, depolarized the ganglion in a dose-dependent manner (threshold concentration 100-300 muM, cf. GABA depolarization threshold 1 muM). The depolarization was reduced in the presence of the selective GABA antagonist (+)-bicuculline methochloride (Bic). Other non-barbiturate sedatives e.g. chlordiazepoxide, amitriptyline, promethazine at concentrations up to 2mM produced no depolarization.3 PB, tested at concentrations up to 80 muM, produced variable effects on the dose-response curve to GABA. On most occasions a slight potentiation occurred in responses to low concentrations of GABA (below 10 muM) coupled with a depression in the responses to concentrations of GABA greater than 10 muM.4 Superfusion with PB in the presence of Bic reversed the depression in the response to GABA produced by Bic. This reversal phenomenon occurred at concentrations of PB too low to depolarize the ganglion and was dependent not only on the concentration of PB but also on that of Bic.5 The reversal potency within an homologous series of barbiturates increased with the size of the alkyl substituent (R2) at C5 on the barbiturate ring. The most potent occurred when the substituent contained 5 carbon atoms (pentobarbitone and amylobarbitone); above this, activity decreased.6 PB reversed the effects of the other GABA antagonists, tetramethylenedisulphotetramine and isopropyl bicyclophosphate and also the non-selective antagonism produced by strychnine. A concomitant reduction by strychnine of responses to the cholinomimetic, carbachol, was not reversed by PB.7 Non-barbiturate sedative/hypnotics also reversed the GABA antagonism produced by Bic. The benzodiazepines were effective at lower concentrations than PB (chlordiazepoxide threshold concentration 0.5 muM, cf. PB 5 muM), however, they only produced a partial reversal even at concentrations much higher than the maximally effective concentration of PB.8 The Bic reversal effect of chloridazepoxide (and other benzodiazepines) lasted many hours after removal from the superfusion solution. By contrast the effect of PB lasted only 15-30 min after its removal.9 Chlordiazepoxide (30 muM) applied in the absence of Bic did not affect the response to GABA but did reduce the depression produced by the subsequent application of Bic even though the chlordiazepoxide had been removed 40 min earlier.10 In the rat brain stem in vivo PB, applied iontophoretically in amounts which neither decreased the spontaneous neuronal firing rate nor affected the response to GABA or glycine, reversed the GABA antagonism induced by iontophoretic application of Bic (in all 23 neurones tested). PB also reversed the antagonism produced by strychnine of responses to glycine although this was less readily observed (5 out of 14 neurones tested).11 Iontophoretic application of other barbiturates and chlordiazepoxide also reversed the effect of Bic. Chlordiazepoxide only produced a partial reversal, as in the isolated ganglion, and no reversal could be demonstrated with flurazepam.12 Intravenous administration of thiopentone (1.3 mg/kg) pentobarbitone (0.4-5.5 mg/kg) hexobarbitone (0.4-0.8 mg/kg) and clonazepam (0.1-0.2 mg/kg) also reversed the effect of iontophoretically applied Bic. The reversal by clonazepam was of much longer duration than that produced by the barbiturates.13 It is suggested that the reversal exhibited by PB and the other hypnotics may be explained by assuming that the amino acids and their antagonists bind to the membrane at separate sites. If the reversal agent has particular affinity only for the antagonist binding site then it may displace the antagonist without affecting the receptor.


Species of differences in postganglionic motor transmission to the retractor penis muscle. 1 Graded motor responses were elicited in isolated, desheathed, thin strips of dog, horse, pig and sheep retractor penis (RP) muscles by field stimulation with trains of 0.2 ms pulses at 10 hertz. These twitches were shown to be neurogenic in all four species, by their prompt extinction in tetrodotoxin.2 alpha-Adrenoceptor blocking drugs abolished the contractile response to noradrenaline and to tyramine in all four species.3 Motor transmission was wholly adrenergic in the horse as in the dog RP because phentolamine rapidly abolished the electrically induced twitches in both these species; but in the pig and in the sheep RP a large proportion of the motor transmission was unaffected by phentolamine given in many times the concentration required to abolish matching noradrenaline-induced contractions.4 Because of the occurrence of periodic spasms in sheep preparations, further investigation of the phentolamine-resistant transmission was confined to the pig RP. Its responses were shown to be entirely postganglionic in origin because they were unaffected by pentolinium.5 In the pig RP a considerable proportion of the phentolamine-resistant motor transmission persisted after combined blockade of alpha- and beta-adrenoceptors by phenoxybenzamine plus propranolol and was more resistant to guanethidine and bretylium than the motor transmission to the dog RP; it was not extinguished after reserpine treatment.6 The pig RP is contracted by histamine but is rather insensitive to acetylcholine, 5-hydroxytryptamine and adenosine-5'-triphosphate. The motor transmission remained unaffected after responses to these substances were blocked by the following antagonists, given alone or in combination: mepyramine, burimamide, atropine, (+)-tubocurarine, methysergide and 2-2'-pyridylisatogen tosylate.


Effects of magnesium on contractile responses induced by electrical transmural stimulation and noradrenaline in rabbit thoracic aorta. 1 In rabbit isolated thoracic aortae, effects of magnesium ions on the contraction and (3)H-efflux in response to electrical transmural stimulation and on the contractile responses induced by noradrenaline and KCl were investigated.2 Addition of magnesium (1.2, 3.6, 12.0 and 24.0 mM) to the bathing solution inhibited the electrically induced contractions in a dose-related manner; the inhibition was complete with a concentration of 24.0 mM.3 The increase in (3)H-efflux induced by electrical transmural stimulation was inhibited by the addition of magnesium to the superfusing fluid, but a complete block was not obtained even in high concentrations of magnesium.4 Magnesium ions (1.2, 3.6, 12.0 and 24.0 mM) inhibited the contractile responses induced by low concentrations of noradrenaline (2 x 10(-8)M) and KCl (2 x 10(-2)M). However, the responses induced by higher concentrations of noradrenaline (5 x 10(-7) and 10(-5)M) and KCl (3 and 4 x 10(-2)M) were enhanced by low concentrations of magnesium.5 Magnesium ions affect both presynaptic and effector sites in rabbit thoracic aortae but in a different manner; magnesium manifests only an inhibitory effect on noradrenaline release from the adrenergic nerves, but dual effects on reactivity of vascular smooth muscle, depending on concentrations of magnesium and stimulants; it is suggested that the change in vascular reactivity is more important than the reduction in transmitter release when magnesium inhibits the response to nerve stimulation.


The neuronal origin of prostaglandin released from the rabbit portal vein in response to electrical stimulation. Transmural electrical stimulation of the isolated portal vein of the rabbit was accompanied by the release of a prostaglandin-like substance (PLS). Thin layer chromatography coupled with bioassay indicated that this substance was probably prostaglandin E2 (PGE2). 2 Indomethacin potentiated the response of the portal vein to electrical stimulation at 2 Hz and abolished the release of the PLS. 3 There was no significant change in the amount of PLS released from the portal vein in response to electrical stimulation at 2 Hz when the contractile response of the portal vein was prevented by pretreatment with phentolamine or guanthidine. 4 In vitro denervation of the portal vein with 6-hydroxydopamine or the omission of Ca2+ from the bathing solution caused a significant reduction in the amount of PLS released from the portal vein in response to electrical stimulation at 2 hertz. 5 It is concluded that electrical stimulation of the isolated portal vein of the rabbit is accompanied by the release of a PLS, probably PGE2, from a neuronal source. The synthesis and release of the PLS is Ca2+ -dependent.


Pharmacological and electrophysiological studies of morphine and enkephalin on rat supraspinal neurones and cat spinal neurones. 1 The actions of morphine, methionine and leucine enkephalin, administered electrophoretically, were studied on supraspinal neurones in the cortex and brainstem of the rat anaesthetized with urethane and on spinal Renshaw cells and dorsal horn interneurones in the cat anaesthetized with pentobarbitone.2 The majority of Renshaw cells and cortical and brainstem neurones were excited by all three compounds although some supraspinal neurones were depressed.3 Naloxone reversibly antagonized both excitatory and depressant actions of morphine and enkephalin. Acetylcholine-induced excitation but not amino acid-induced excitation was also antagonized by naloxone.4 Neither morphine nor the enkephalins had any naloxone-reversible action on dorsal horn neurones when ejected from conventional multibarrelled electrodes. However, morphine but not enkephalin, administered into the substantia gelatinosa region of the spinal cord selectively reduced responses to noxious stimuli of neurones in deeper laminae. Naloxone administered into the same region antagonized this action of morphine.5 Intravenous morphine also antagonized responses of dorsal horn neurones to noxious stimuli and subsequent intravenous naloxone reversed this effect.6 It was concluded that the excitatory and inhibitory effects of morphine and enkephalin on central neurones may be mediated by actions on different opiate receptors and that depression of noxious responses of dorsal horn neurones may be relevant to the analgesic action of morphine.


Stimulation of presynaptic beta-adrenoceptors enhances [3H]-noradrenaline release druing nerve stimulation in the perfused cat spleen. 1 The effects of isoprenaline, propranolol and phosphodiesterase inhibitors on (3)H-transmitter overflow elicited by low frequency nerve stimulation were determined in the isolated perfused spleen of the cat.2 (-)-Isoprenaline (0.14, 1.4, and 14 nM) produced a concentration-dependent increase in [(3)H]-transmitter overflow evoked by nerve stimulation at 1 Hz and was more effective at 1 Hz than at 2 hertz.3 A concentration of propranolol (0.1 muM), devoid of neurone blocking activity, blocked this effect of (-)-isoprenaline. These results are compatible with the presence of beta-adrenoceptors in the noradrenergic nerve endings of the cat spleen.4 (+)-Isoprenaline (140 nM) failed to increase the release of radioactivity induced by nerve stimulation, indicating that the beta-adrenoceptor mediating the facilitation of transmitter release was stereospecific.5 The increase in (3)H-transmitter overflow induced by nerve stimulation during exposure to the phosphodiesterase inhibitor, papaverine (27 muM) was more pronounced than that obtained in the presence of 3-isobutyl-1-methyl xanthine (IBMX) 0.5 mM. The facilitation in transmitter release induced by papaverine was not correlated with the granular effect produced by this drug.6 In the presence of papaverine, the concentration-effect curve for (-)-isoprenaline on transmitter release was shifted to the left and its maximum was increased. In addition, propranolol significantly reduced the enhancement in noradrenaline release obtained by exposure to papaverine under conditions in which the granular effect produced by the phosphodiesterase inhibitor was even greater than in the absence of the beta-blocker.7 It is concluded that activation of presynaptic beta-adrenoceptors in the perfused cat spleen leads to an enhancement in transmitter release which appears to be linked to an increase in cyclic adenosine 3',5'-monophosphate levels in noradrenergic nerve endings.


Hepatocellular carcinoma associated with oral contraceptives. A primary hepatic tumour occurred in a 36-year-old woman who had been taking oral contraceptives for 6 years; she was treated by partial hepatectomy. The neoplasm had the characteristics of a well-differentiated hepatocellular carcinoma and the regional lymph nodes contained similar tumour.


The effect of age and lung pathology on cytochrome a,a3 redox levels in rat cerebral cortex. The steady-state reduction/oxidation (redox) ratio of cytochrome a,a3 in the non-stressed or 'resting' cerebral cortex was compared in 'healthy' mature and aged rats and in animals with varying degrees of lung pathology present. By using noninvasive dual wavelength spectrophotometry, cytochrome a,a3 was found to be approximately 30% reduced under 'resting' conditions in both mature and 'aged' brain. Although no significant age-related or strain differences were apparent, the 'resting' redox level of cytochrome a,a3 was markedly affected by the presence of lung pathology. The redox ratio was lower in animals where lung lesion involvement was not extensive, and higher (indicative or cellular hypoxia) in animals having both extensive acute and chronic lung pathology. These studies demonstate that, regardless of age or lung pathology, the cortical cytochrome a,a3 redox state is labile to changes in the amount of inspired oxygen, a condition differing from that of isolated mitochondria. These results indicate that dysfunction of the mitochondrial respiratory chain is not a direct or primary consequence of chronlogical aging in the 'resting' brain.


Formation of synapses between cells of a neuroblastoma X glioma hybrid clone and mouse myotubes. Synapses form between cells of a neuroblastoma X glioma hybrid clone and cultured mouse skeletal myotubes. The synapses are cholinergic, and the acetylcholine release mechanism is dependent on calcium ions. The transmitter output of the synapses is low, with considerable variability in the latency and amplitude of the postsynaptic responses to presynaptic action potentials. The fine structure of physiologically identified functional junctions was examined electron microscopically. Small (50 nm) clear vesicles were seen presynaptically and there were areas with a wide (approx. 50 nm) gap containing basement membrane-like material between the pre- and postsynaptic cells. In addition, in some regions there was a densely staining material lining the muscle membrane and some suggestion of infolding of the muscle membrane. In none of the cases, however, have areas been found where small, clear vesicles cluster around pre- and postsynaptic membrane densities. Thus, functional synapses can occur in the absence of the highly organized synaptic structure seen at mature synapses.


Epileptic properties of leucine- and methionine-enkephalin: comparison with morphine and reversibility by naloxone. Morphologically similar epileptic seizures were recorded from the cortex of rats after injections into the lateral ventricle of 100 microgram of leucine-enkephalin, methionine-enkephalin, and morphine. Seizures were either greatly attenuated or blocked completely by prior systemic administration of naloxone (10 mg/kg). These findings suggest that such seizures result from an interaction of these compounds with opiate receptors in the brain. The epileptogenic potency of the enkephalins was illustrated by the observation that seizures and other pathological manifestations could still be elicited by doses as low as 10 microgram. Leucine-enkephalin was seen to have greater epiliptic potency than methionine-enkephalin. At doses of 1 microgram both enkephalins typically evoked cortical spindles resembling those seen in drowsy animals. Enkephalin-induced analgesia was seen in only one animal at the 100 microgram dose. Results obtained with repeated injections of morphine suggest that the epileptogenic effect of opiates may be subject to either tolerance or potentiation, depending on the prior occurrence of seizures. A synthesis of the present findings with several other lines of evidence suggests both that endogenous enkephalins play some role in normal mechanisms of reward, and that, when regulatory processes are disturbed, they may contribute as well to the elaboration of certain epileptic phenomena.


Isolation and partial characterization of rat CNS axolemma enriched fractions. Axolemma-enriched fractions were prepared from rat brain by osmotic shock of a purified preparation of myelinated axons and subsequent separation of myelin, two axolemma-enriched fractions and myelin-free axons by density gradient centrifugation. Compared with the starting whole homogenate, the fractions were enriched in specific activity of Na+K+ ATPase, acetylcholinesterase, 5'nucleotidase as well as 2',3'-cyclic nucleotide 3'phosphohydrolase. Compared with myelin, the axolemmal fractions are greatly enriched in high molecular weight proteins. The 1.0/1.2 fraction has a predominant peak of fucose-labeled glycoprotein with a molecular weight between that of the myelin associated glycoprotein and the Wolfgram protein which is absent from the myelin glycoprotein profile. Polyacrylamide gel electrophoresis showed that the protein profile of myelin isolated by this procedure was similar to that of myelin isolated by other procedures and that the myelin specific basic and proteolipid proteins were virtually absent in the axolemma-enriched fractions. Both axolemma fractions were enriched in higher MW proteins, some of which resembled proteins in the myelin protein profile. Both axolemma-enriched fractions specifically bind between 2 and 3 pmoles of [3H]tetrodotoxin per mg protein. The axolemma-enriched fractions incorporated [3H]leucine and [14C]fucose exclusively into high molecular weight proteins and glycoproteins. In contrast myelin concomitantly isolated with the axolemma-enriched fractions had a significant amount of [3H]leucine labeled protein in myelin proteolipid and basic proteins. In addition to the myelin associated g-ycoprotein the [14C]fucose labeled a glycoprotein of slightly larger apparent molecular weight than proteolipid protein was found in the myelin fraction while the comparable labeled glycoprotein was absent in the axolemma-enriched fractions. The possible extent of contamination of these fractions by myelin or myelin subfractions and relationship of these axolemma-enriched fractions to other axolemma preparations are discussed.


The influx of pyrophosphate ions into calvaria in vitro. The flux of 32P-labeled inorganic pyrophosphate (PPi) into bone was studied in vitro using Ussing chamber techniques and calvaria from newborn rat pups. Because insignificant hydrolysis and backflux of PPi took place under experimental conditions, it was possible to study the penetration of bone membranes by PP1 unambiguously. At physiological concentrations, the influx was found to be linearly concentration dependent and to follow first order kinetics, apparently a simple diffusion process. In magnitude, the intrinsic constant for influx was found to be approximately one-half that of inorganic phosphate ions under comparable conditions.


Radiogenic free radicals as molecular probes in bone. Exposure of bone mineral to X-rays generates free radicals. These are usually very labile, but can be stabilized at liquid nitrogen temperatures for study by electron spin resonance spectroscopy. The free radicals thus detected in the present study included one with resonances arising from an electron excess center and 2 species with electron-deficit centers: a phosphate anion radical and a radical associated with carbonate. Each of these radicals seemed to be located chiefly at the mineral surface and was sensitive to the surface environment. Presence of an organic phase, as in whole bone, modified free radical production in a manner that suggests interference with the formation of electron deficit centers. Comparison with other synthetic minerals suggests that precipitated carbonate-apatites are good models for bone mineral.


Differential behavioral effects of ACTH 4-10 and [D-Phe7] ACTH 4-10. Intraventricular administration of an access of ACTH 4-10 does not interfere with the excessive grooming behavior of rats, elicited by intraventricular administration of [D-Phe7] ACTH 4-10. In an avoidance extinction paradigm, the two ACTH analogs have opposite effects. ACTH 4-10 counteracts the facilitation of extinction seen after [D-Phe7] ACTH 4-10, only under conditions that treatment with ACTH 4-10 alone results in retardation of that extinction. The data are discussed in terms of a multiple interaction of these peptides with brain function.