Detection of virus in water: sensitivity of the tentative standard method for drinking water. The sensitivity of several microporous virus-adsorbent media for reliably detecting low levels of poliovirus from 380 and 1,900 liters of drinking water by use of the tentative standard method was investigated. The virus-adsorbent media tested were (i) nitrocellulose membrane filters, (ii) epoxy-fiber glass-asbestos filters, (iii) yarn-wound fiber glass depth filters, and (iv) epoxy-fiber glass filter tubes. Virus was adsorbed to the filter media at pH 3.5 and eluted with glycine buffer, pH 11.5. The results from 44 samples demonstrated that poliovirus was detected with a 95% reliability at mean virus input levels of 3 to 7 plaque-forming units/380 liters when 1,900 liters of water was sampled. At mean virus input levels of less than 1 to 2 plaque-forming units/380 liters, the detection reliability was 66% in 76 samples when 1,900 liters of water was sampled. No significant difference in virus detection sensitivity was observed among the various virus adsorbent media tested. Overall virus recovery efficiency ranged from 28 to 42%, with a grand average of 35%. Members of the coxsackievirus groups A and B, echovirus, and adenovirus were also detected when 380 and 1,900 liters of water were sampled. These experimental observations attest to the sensitivity of the tentative standard method for detecting low levels of virus in large volumes of drinking water.


Fermentation of glucose-1-phosphate: a screening test for fermentative Bacteroides species. Of 1,504 strains of anaerobic bacteria tested, 544 produced acid from glucose-1-phosphate. Of these, 535 were fermentative strains of Bacteroides; only three fermentative Bacteroides strains were negative. The reaction may be useful for determination of the number of Bacteroides species present in colon content and feces.


Viral infection in wheezy bronchitis and asthma in children. Virus isolation was attempted on 267 out of 360 patients with wheezy bronchitis or asthma admitted to hospital during a 3-year period. Viruses were isolated on 39 occasions, the most common being respiratory syncytial virus and rhinovirus. The peak months for virus isolation were February and August. Virus isolation was significantly more common in readmissions than in first admissions (P less than 0-01). Viruses were isolated in both sexes throughout childhood and though the admission rate fell with increasing age, the isolation rate was unaffected. The possible significance of viral infection as a cause of acute attacks of wheezing in children is discussed.


Restoration of virulence to rat of axenically grown Entamoeba histolytica by cholesterol and hamster liver passage. A virulent strain of Entamoeba histolytica (NIH-200) from a symptomatic human case, cultured in vitro for more than three years, was unable to infect rat caecum or hamster liver. When grown with a mixed bacterial flora, it infected rat caecum but did not produce ulceration. After passaging amoebae growing with bacteria in hamster liver, amoebic hepatic lesions were regularly produced. The amoebae from liver lesions produced ulceration in the caeca of rats. Expoxure to cholesterol of amoebae growing with bacteria or feeding rats orally with cholesterol and injecting these amoebae along with bacteria also led to the production of ulcers in rats. Thus virulent amoebae which have been cultured in vitro for a long time become attenuated but their virulence can be revived.


Interactions of chlorphenesin and divalent metal ions with phosphodiesterase. Chlorphenesin inhibition of the hydrolysis of cyclic AMP by guinea-pig lung phosphodiesterase was reversed by the addition of exogenous magnesium ions. Chlorphenesin and theophylline inhibition of this enzyme was shown to be noncompetitive when the substrate concentration was low. Kinetic studies of the inhibition of beef heart phosphodiesterase by chlorphenesin and theophylline indicated that the substrate concentration was a factor in determining whether inhibition was competitive or noncompetitive. Calcium, cobalt and copper ions were inhibitory to guinea-pig lung phosphodiesterase. The inhibition due to chlorphenesin was partially reversed by low (40 mM or less) concentrations of barium ions; high concentrations of barium ions, or manganese ions, were inhibitory. The concentration of the divalent cation did not affect the type of inhibition that was observed.


Effect of propranolol on the relationship between atrial systolic pressure and type A atrial receptor discharge in cats. The responses of type A atrial receptors to graded infusions of saline and large doses of propranolol were examined in anesthetized cats. Infusion of saline raised the mean atrial pressure, but usually the amplitude of the atrial a wave was reduced. In general the receptor discharge was unaffected. Propranolol reduced the discharge from the control level when it was injected in doses no less than 4 mg/kg. Infusions of saline after propranolol resulted in an increase in the discharge and the increase was related to the amplitude and/or initial pressure of the a wave. In one case the discharge after propranolol was less than that when the atrium was widely slit open. It is concluded that at least part of the effect of the drug is due to a direct depression of the receptor rendering it less sensitive to the stretch provided by atrial contraction. The demonstration of a stimulus-response relationship between the a wave and the receptor discharge at low levels of activity suggests that under normal conditiions the receptor operates on a plateau of maximum activity, thus making a response to small changes in stimulus strength obscure.


Influence of adenosine, cAMP and db-cAMP on responses of the isolated terminal guinea-pig ileum to electrical stimulation. Adenosine, cAMP and db-cAMP were found to inhibit nerve-mediated contractions of the terminal ileum elicited by electrical stimulation (ES). Responses to ES at 30 Hz, which are believed to be of adrenergic origin, were considerably more affected than the responses to ES at 3 Hz, which may be of purinergic orign. The following order of relative inhibitory potencies was established: adenosine greater than cAMP greater than db-cAMP. Adenosine, cAMP and db-cAMP did not affect the contractile responses of the preparation to histamine, potassium and noradrenaline. Therefore, the observed inhibition of nerve-mediated contractions was thought to be due to an action on presynaptic sites, i.e. on the neural network within the intestine. This action was antagonized by an increase in external calcium and by aminophylline, which is known to increase intracellular calcium. The present experiments suggests that the inhibitory action of adenosine, cAMP and db-cAMP results from a reduction of calcium fluxes across the neural membrane and to a subsequent reduction in the release of neurotransmitter(s). The inhibitory effect of cAMP appeared to be nonspecific and was presumably related to adenosine in its molecule.


An alpha-adrenotypic study of the isolated rabbit kidney at normo- and hypothermia. Rabbit kidneys were isolated and perfused at 37degrees and 25degrees C with a balanced salt solution and the effects of an alpha agonist and antagonist were tested in several parameters. Perfusion pressure, resistance, perfusate and urine flow, creatinine and PAH clearance, and Na+ and K+ transport were among the parameters studied. At 25degrees C the vascular responses to norepinephrine (NOR) were potentiated in relation to 37 degrees C. Blockade of the reuptake by cocaine and depletion of catecholamines by reserpine was observed to promote a small potentiation of NOR effects on renal vessels as measured by perfusion pressure. This pharmacological treatment compromised kidney function especially after the first 40 min of perfusion, that is to say that after NOR infusion functional parameters were extremely low. During the infusion of NOR there was a decrease in GFR, urinary flow and CPAH, which was even more intense at 25 degrees C. It was concluded that alpha adrenergic receptors are more responsive at 25 degrees C than at 37 degrees C in kidney vascular receptors. There was no difference in regard to alpha blockade at either temperature tested.


Different membrane mechanisms of action for tiemonium; a comparison with atropine and papaverine. The effects of tiemonium on various membrane events preceding smooth muscle fibre contraction, have been studied in vitro. Classical molecular pharmacological models and methods were used: stimulation of muscarinic, histamine H1 and alpha-adrenergic receptors; induction of contraction by release of membrane phospholipid bound Ca2+ ions, or by intracytoplasmic influx of this cation in depolarized preparations. At each level of investigation, tiemonium was studied comparatively with two reference antispasmodics, atropine and papaverine. Like atropine, tiemonium competitively antagonizes cholinergic stimulation. It also interferes with the contracting effects of BaCl2, as does papaverine. In contrast to papverine, however, tiemonium shows an affinity for histamine H1-receptors, and does not affect alpha-adrenergic receptor stimulation. Tiemonium is therefore a novel antispasmodic which blocks the cholinergic receptor, but being less specific than atropine. This non-specific facet makes it almost as polyvalent as papaverine, in its general inhibition of spasmogenic substances, but its mechanism of action is very different. Whereas papaverine slows intracytoplasmic Ca2+ influx, tiemonium inhibits release, and thus availability, of the same ion. Tiemonium has a totally different mechanism of antispasmodic action from that other substances in the same pharmacological class. The marked competitive interference with acetylcholine and the very weak competitive antagonism of histamine are completed by a membrane stabilizing action. In particular, the latter reinforces the binding of calcium to membrane phospholipids. The absence of any interaction of tiemonium with noradrenaline is compatible with recent theories for noradrenaline's action, which evoke calcium exchanges in an intracytoplasmic compartment. Finally, the results raise the question of the pharmacological suitability of the term "papaverine-like" for describing non-specific antispasmodics.


[Digestive absorption, fixation and excretion of oral disulfiram in the rat]. Absorption, distribution and excretion of disulfiram and of one of its metabolites, diethyldithiocarbamate, were studied in rats orally treated with disulfiram (25 or 250 mg/kg). It was noted that this compound is absorbed in a proportion of 70-90 per cent of the administered dose but that diethyldithiocarbamate can appear already in the gut. After absorption, disulfiram and diethyldithiocarbamate impregnate specifically some tissues such as liver, kidney and muscle; blood and brain contain little or no disulfiram. The largest dose of disulfiram decreases fecal bolus and increases urinary volume favouring perhaps excretion of diethyldithiocarbamate.


Streptozocin-treated Verner-Morrison Syndrome: plasma vasoactive intestinal peptide and tumor responses. A patient with watery diarrhea, hypokalemia, hypochlorhydria, and a non-beta islet cell carcinoma of the pancreas (Verner-Morrison syndrome) was found to have an elevated vasoactive intestinal peptide (VIP) concentration in the plasma as well as in the tumor. Treatment with streptozocin resulted in a dramatic subjective and objective tumor response in this patient. Plasma VIP concentration fell into the normal range after four courses of treatment, diarrhea ceased after the third course of therapy, and measurable tumor mass markedly decreased during that same period of time. The patient remains in clinical remission with no evidence of tumor regrowth 18 months after the beginning of treatment. In this patient, plasma VIP measurements were an excellent marker of tumor activity and correlated well with objective disease measurements and clinical response.


[Gluconeogenetic capacity of suckling piglets and young pigs]. No marked gluconeogenetic performance was recordable from nursed piglets aged, between one and five days, in response to ACTH nor glucocorticosteroid application. Store pigs, aged twelve weeks, however, exhibited glucose rises of 34 per cent one hour after injection or 55 per cent three hours from ACTH application. The point was made, in an attempt to elucidate the above findings, that in newborn piglets, few days after birth, the gluconeogenetic capacity is insufficient because of functional immaturity of the liver. The behaviours of several metabolites in the liver and muscles of store pigs were examined under differentiated model conditions to check up and verify that view.


The influence of central chemical sympathectomy and reserpine on peripheral effects of noradrenaline and cyclic AMP dibutyrate injected into the cerebral ventricles. Chemical sympathectomy of the central nervous system by injection of 6-hydroxy-dopamine (2 X 250 mug) or 6-hydroxydopa (90 mug) intensified some of the peripheral effects of noradrenaline and cyclic AMP dibutyrate injected into the cerebral ventricles. Reserpine (5 mg/kg) injected intraperitoneally weakened the peripheral reactions to noradrenaline injected intraventricularly. The results of the experiments indicate that peripheral reactions to intraventricularly injected noradrenaline depend on changes in the content of endogenous narodrenaline in the brain and on the mechanisms leading to these changes.


AKR mouse leukemia cells in tissue culture. I. Establishment of leukemic cell line AKR 87 and its morphologic and biologic characteristics. A line of AKR leukemic mouse cells cultured in vitro, designated AKR 87, was established and characterized. Two types of cells were distinguished in this line fibroblasts and lymphoblasts. The lymphoblast-like cells resembled the original tumor cells spontaneous leukemia of AKR mice. In the XC test, cells of this line reacted specifically with XC cells, forming syncytia characteristic of cells infected with mouse leukemia viruses. Ultrathin sections of cells of this line examined on the electron microscope showed presence of type C viral particles.


AKR mouse leukemic cells in tissue culture. II. Immunologic studies on the AKR 87 cell line. The continuous AKR 87 cell line was derived from the thymus and spleen of AKR mice with spontaneous leukemia. Previous studies showed that the line has retained the original morphologic features of leukemic cells, reacts positively in the XC test, and produces virus particles of type C morphology. The present paper presents an immunologic characterization of cells of the AKR 87 line and of the virus produced by them, in comparison with the cells of spontaneous leukemia of AKR mice, TGV line productively infected with Gross virus, and normal mouse tissues. Results of the hemagglutination test, immunoprecipitation in agar gel, cytotoxic test and immunofluorescence indicate that the AKR 87 line produces virus antigen of the Gross type.


Production of interferon in human diploid cell cultures. Eleven human diploid cell lines were compared for their ability to produce interferon in response to Newcastle Disease Virus. Sensitivity of the cultures of MM, Sindbis VSV and VR viruses was also studied. Human cells derived from various tissue of a single embryo responded variably to interferon inducer. Using selected viruses interferon production was studied in two systems, in cultures of leukocytes and in human embryonic cells.


[Association of a bifocal nephroblastoma with Beckwith's syndrome]. A nephroblastoma occurred in the evolution of a case of Beckwith's syndrome. This rare association is not fortuitous. The bifocal character of the tumor is noted for the first time in the Beckwith's syndrome; however, it has already been observed in other conditions, which are known to promote the development of nephroblastomas. Wilm's tumor is only one of the tumors which may be associated with Beckwith's syndrome. These morbid associations and certain histological features bring together some material for the understanding of the nature of the histo-genetic relationship between different aspects of constitutional pathology and tumors.


Chronic hypoxia and chemodectomas in bovines at high altitudes. A severe degree of hyperplasia of the chief cells occurs in bovine carotid bodies at high altitudes, compared to sea level. As a result, the carotid body from an animal at high altitudes is significantly heavier and larger than the carotid body from an animal at sea level (P less than .001). In eight of 20 (40%) animals at high altitudes, the hyperplastic reaction had progressed to form chemodectomas. The findings suggest that neoplasia can result from chronic stimulation by a biologically essential environmental factor (atmospheric PO2) that acts pharmacologically on the target tissues (chief cells of the carotid body).


Morphogenesis of avian infectious bronchitis virus in primary chick kidney cells. Primary chick kidney cells were infected with avian infectious bronchitis virus (IBV) and examined by electron microscopy. Virus particles entered the cells by viropexis and distinction could be made between engulfment by cell processes (phagocytosis) and entry by micropinocytosis in coated transport vesicles. Virus maturation occurred by budding into either the cisternae of the endoplasmic reticulum or cytoplasmic vacuoles, and evidence was obtained to suggest that the viral surface projections could be attached during the budding process. Late in infection large numbers of virus particles were present, mainly in cytoplasmic vacuoles, and the majority were released by cell lysis. Release by fusion of vacuoles with the plasma membrane was also observed, and individual virions could be transported from the endoplasmic reticulum to the surface within coated vesicles.


Individual translational efficiencies of SV40 and cellular mRNAs. The technique of selective inhibition of peptide chain initiation by growth medium hypertonicity was adapted for cell monolayer cultures and applied to a study of protein synthesis in SV40 infected BSC-1 cells. The translational efficiences for individual peptide chain initiation sites on SV40 mRNAs were determined and compared to those for cellular mRNA species under conditions of a reduced rate of peptide chain initiation. The SV40 mRNAs show different translational efficiences. The results indicate that the synthesis of the SV40 proteins VP1, 2 and 3 are initiated independently and with different rates on viral mRNA(s). It is proposed, therefore, that VP2 is not a precursor for VP3.


Antiviral activity of carbopol, a cross-linked polycarboxylate. Carbopol, a polymer of acrylic acid cross-linked with allylsucrose, was found to impart resistance to virus infection in mice which received an intraperitoneal injection of the polycarboxylate 1 or 4 days before either intravenous vaccinia virus challenge or intranasal herpes simplex virus challenge. An interferon-like substance was detected in the blood stream 20-40 hours after the intraperitoneal injection of carbopol. The antiviral activity of this cross-linked polycarboxylate could be due to activation of peritoneal macrophages and/or interferon production.


In vivo antiviral activity of D-glucosamine. Intraperitoneal treatments with D-glucosamine, an inhibitor of the glycosylation of the viral envelope, decreased the growth rate of tumors induced in quails or in chicks by Rous sarcoma virus and increased the survival of mice inoculated with human influenza virus.


Comparison of the humoral and cellular immune response after immunization with live, UV inactivated herpes simplex virus and a subunit vaccine and efficacy of these immunizations. Antibody and cell-mediated immune responses were measured in rabbits immunized with live, UV inactivated herpes simplex virus or with a subunit vaccine containing envelope proteins. All the types of immunization procedures induced the production of antibody as well as a specific cellular immunity. Furthermore, the subunit vaccine was as effective as the immunization with live or UV inactivated virus to prevent death upon challenge with live HSV. Live HSV induced a transient unresponsiveness of both B and T cells to in vitro stimulation with various mitogens.


Inhibitors of foot-and-mouth disease virus. Temperature-dependence of the effect of guanidine on virus growth. In suspended secondary calf kidney cells infected with foot-and-mouth disease virus (FMDV) the temperature range for optimal virus growth is shifted down by 3 to 5 degrees C in the presence of 1--2 mM guanidine. For some virus strains this shift is so effective that at infraoptimal temperatures virus yield in guanidine-treated cells exceeds that of the corresponding control by more than one log10. On the contrary, at supraoptimal temperatures inhibition of virus growth by the drug is strongly enhanced. At a concentration of 1 to 2 mM guanidine virus yield reduction or enhancement is based on a decrease in increase, respectively, of the number of virus producing cells (infective centers; I.C.), while virus yield per I.C. is less affected. Besides this "thermomimetic" effect virus production is inhibited by guanidine depending on the concentration of this substance. A mutant of FMDV strain O1L, resistant to 4.2 guanidine, did not differ from the original virus in its antigenic behaviour in the passive immunohemolysis test.


Electron microscope observations on the entry of avian infectious bronchitis virus into susceptible cells. Infectious bronchitis virus was observed to enter cells of chicken chorioallantoic membrane by viropexis. There was no support for the suggestion that entry took place by fusion of viral and plasma membranes. The results of electron microscopy showed that virus attachment occurred both at 4 degrees and at 37 degrees C. Viropexis was not observed until the preparations were warmed. Similar results were obtained using chicken kidney cells. Quantitative data obtained from a plaque counting system employing chicken kidney cells indicated that attachment was the same at both temperatures and that some virus particles were taken up at 4 degrees C. Virus uptake was triggered by attachment of the virus to the cell membrane and the subsequent process of virus entry visualised by E. M. appeared to proceed without the involvement of lysosomal enzymes. No intracellular virus was located by electron microscopy in warmed preparations when virus was treated with specific antiserum, either before or after adsorption to the cells.


Comparative studies on the structural proteins of T3 and T7 phages. A comparison of the coat protein patterns of the wild types of the related phages T3 and T7 on SDS polyacrylamide gel electrophoresis was carried out. After infection of the nonpermissive host with T7 amber mutants in genes 7, 11, 12, 13 or 17 and T3 amber mutants in genes 11, 12, 13 and 17 respectively, noninfectious, DNA containing particles were produced. The protein pattern, as well as electron microscopy of defective particles of T3 and T7 led to the conclusion that the proteins specified by genes 11, 12, 13 and 17 are tail proteins whereas the proteins coded by genes 7, 8, 10, 14, 15 and 16 enter the head structure. The "serum blocking protein" (gene 17 product) seems to play a different role in the assembly of T3 and T7 tails, since T3 particles defective in gene 17 did not show any tail structure connected with the head whereas T7 particles defective in gene 17 had a tail which looked different from that of the wildtype. Treatment of wildtype particles with ammonium nitrate or sodium pyrophosphate led to morphologically abberrant forms which had partially or completely lost the hexagonal head structure. After treatment with ammonium nitrate ball-like structures were obtained, both for T3 and T7. However, in the case of T3 these abberrant forms contained the proteins specified by genes 7, 8 and 10 whereas those derived from T7 contained only the proteins specified by genes 8 and 10. Sodium pyrophosphate treatment of T3 and T7 wildtype particles led to a release of the phage tails. The isolated tails were examined by electron microscopy thus revealing for the first time a detailed substructure of the T3 and T7 phage tails. In order to find out more about the tail proteins, absorption experiments with isolated bacterial cell walls were carried out.


Biological and electron microscopic studies on the phenotypic mixing of the thermolabile mutant of vesicular stomatitis virus, tl-17, with avian RNA tumor viruses. A thermolabile as well as thermosensitive mutant of vesicular stomatitis virus, VSV-tl-17, could be thermostabilized by phenotypic mixing with avian RNA tumor viruses (ATV). Biological, immunological and morphological studies revealed that this effect is due to a replacement of the thermolabile projections of the VSV by the avian tumor viral projections. The arrangement of projections of VSV and ATV on phenotypically mixed viria was studied by electron microscopy. A-type particles were detected in chick embryo cells which were doubly infected by ATV and VSV. Their intracytoplasmic appearance seemed to be the result of a disturbed maturation of ATV due to the relative deficiency of envelope proteins which were depleted by VSV maturation in the course of phenotypic mixing.


The pathogenesis of vesicular stomatitis virus, serotype Indiana, in Aedes aegypti mosquitoes, after imbibition of a viremic blood meal. This study showed that Vesicular Stomatitis Virus (Indiana) in most instances was not capable of replicating in Aedes aegypti when imbibed by the mosquitoes on a viremic host. Rapid inactivation of the virus was observed in some cases within 24 hours after imbibition. Attempts to demonstrate virus inactivation by midgut contents in vitro were not successful.


Mucolipidosis IV. Clinical, ultrastructural, histochemical, and chemical studies of a case, including a brain biopsy. A 7-year-old Ashkenazi Jewish boy with normal early development started to regress at 8 months of age and made no further developmental progress. Corneal clouding was noted at age 10 months. Corneal and conjunctival biopsy at 14 months, cerebral biopsy at 24 months, and fibroblast cultures at 32 months showed lysosomal inclusions, suggesting the storage of lipid-like and mucopolysaccharide-like material. In the brain, dense fluorescent inclusions resembled those in ceroid-lipofuscinosis. Total ganglioside content of white matter was raised, but the pattern was normal. The level of nonlipid hexosamine in the brain was normal. The cornea and conjunctiva contained electronlucent vacuoles resembling those in the mucopolysaccharidoses. Cornea, brain, and lymphocytes contained concentric membranous lamellar structures reminiscent of those in the gangliosidoses. The clinical picture and ultrastructural findings support the impression that this case belongs to a new variant of the mucolipidoses, mucolipidosis IV.


Axonal degeneration in beriberi neuropathy. Ultrastructural and teased-fiber studies were carried out on the sural nerves of nine patients with beriberi neuropathy Axonal degeneration was the most prominent feature, and large myelinated fibers were more affected than unmyelinated ones. An unusual change with accumulation of flattened sacs or tubuli was recognized in the axoplasm of myelinated fibers of untreated patients. Active regeneration was extensive in patients receiving vitamin B. Segmental demyelination, remyelination, and early onion bulbs were scarece in patients with a long and relapsing course before treatment.


Peripheral nerve involvement in Hunter syndrome (mucopolysaccharidosis II). A 20-year-old man with confirmed Hunter syndrome had entrapments of median and ulnar nerves. Sural nerve biopsy specimen revealed clear, lamellated, and granular inclusions in Schwann cells, fibroblasts, and perineurial cells. The clear inclusions are believed to represent storage of mucopolysaccharide, and the zebra bodies, ganglioside. Focal alterations in myelin were found, associated with moderately dense granular inclusions. Following carpal tunnel release operations, the patient experienced dramatic releif of symptoms.


Granular cell myoblastoma of the orbit. Report of a case. A light and electron microscopical study of a case of granular cell myoblastoma of the orbit is described. Its possible histogenesis is discussed and, from the study of this case and of the literature, it is believed that the most likely histogenesis is from a primitive mesenchymal cell resembling a fibroblast.


The placement of viruses in the aetiology of cancer. The role of viruses as causative agents of cancer in man has been examined for more than 50 years. These studies have recently been intensified by employing newly developed, more sensitive techniques in virology and immunology. In spite of proof that viruses cause cancer in animals and that about one-quarter of the known 600 viruses possess oncogenic potential, definite proof of their aetiology in human cancer remains elusive even though cancer research is a highly respectable and costly pursuit. Nevertheless, the probabilities are high that human cancer viruses have been and will be identified and that their induced diseases can be prevented or controlled. It is accepted that some benign tumours of man (such as warts) are induced by a virus infection. This article examines the current situation and highlights the difficulties, hopes and usefulness of research in this field.


Why sleep?

Why sleep? Alert clinicians realize that much more data are fragmentedly available than has coherently been assimilated for practical application to the understanding of sleep--its vital function, its vicissitudes and its values. Now may be the time to add to sleep research, a dimension of field trips away from the labs. Cross cultural data might also be correlated, bringing social anthropologists and sleep physiologists together for didactic exchange.


[The quantitative cytoarchitecture of Morris hepatoma 66 (author's transl)]. To obtain a statistic comparable picturg oncogenesis, tissue of a middle-fast growing Morris-hepatoma 66 was transplanted into the femoral musculature. 35 days later the animals were killed by decapitation and hepatoma tissue was taken. The morphometric analysis based on the technique described by Weibel (1969). In the Morris-hepatoma 66 hepatocyte and nucleus show a much smaller single volume in comparison to normal liver cell. Smaller is also the number of nucleoli per nucleus. The decreased numerical density of mitochondria in liver tissue and their smaller content of mitochondrial grana suggest a reduction of the oxidative metabolism. The enlargement of membrane surface of the rough endoplasmatic reticulum in liver tissue is accompanied by a cisternal collapse. The smooth endoplastmatic reticulum is rudimentary in the hepatoma cells. The number of peroxisomes is equal in hepatoma and normal liver. Only half as large is, however, their volume density in liver tissue and so also their single volume.


[Congenital mesoblastic nephroma - a semimalignant fibroleiomyomatous kidney tumor of the newborn (author's transl)]. The congenital mesoblastic nephroma is a distinct tumor entity, which should be clearly distinguished from Wilmus-tumor. The pure mesenchymal tumor is usually present at birth and palpated as a mass in the kidney. Macroscopically the tumor reveals a striking resemblance with an uterine fibroid. Histologically the tumor tissue ist characterized by 1. interlacing bundels of spindle cells with uniform cell nuclei and regular mitotic figures, 2. collagen fibres between the tumor cells, 3. an angiomatous marginal zone, no tumor capsule, 4. hematopoetic foci and dysplastic glomeruli and tubuli in areas where normal kidney parenchyma mixes with tumor tissue, 5. small myxomatous areas within in the tumor, 6. no invasion of blood vessels or pelvis. Prognosis of the congenital mesoblastic nephroma is much better than in Wilms-tumor. Metastases have not been described so far. If, however, the tumor tissue is incompletly removed during operation, the neoplasm may recur and prove fatal. Ultrastructural and DNA cytophotometric studies suggests a low grade malignancy rather than a truely benign behaviour of this tumor.


[Diagnosis and differential diagnosis of rinderpest]. Starting from the clinical symptoms and the pathological-anatomical changes hints are given on the diagnosis of the rinderpest and how to distinguish it from other diseases. The paper discusses the differential diagnosis of the rinderpest with respect to mucosal disease, malignent catarrhal fever, Nairobi sheep disease, salmonellosis, pasteurellosis, and coccidiosis.


Particulate' hexokinase activity in rat intestine. The comparative contributions of mitochondria and brush-border membranes. The activity of hexokinase was examined in brush-border membranes purified from rat intestine. Compared with homogenates, purified membranes exhibited a 20-fold increase in sucrase specific activity and a 15-fold decrease in hexokinase specific activity, which argues against the structural localization of hexokinase within the brush-border membrane.


Carboxymethylation of methionine residues in bovine pituitary luteinizing hormone and its subunits. Effects on the binding activity with receptor sites and interactions between subunits. The reaction of iodoacetic acid with bovine lutropin (luteinizing hormone) at pH 3.0 was specific for methionine residues; it was slow and reached its equilibrium after 12 h at 37 degrees C. The number of modified methionine residues increased proportionately with the amount of the alkylating reagent in the reaction mixture. In the presence of a 20-fold molar excess of iodoacetic acid with respect to methionine, essentially all methionine residues in both subunits of bovine lutropin were carboxymethylated. Studies of various recombinations of modified and native alpha and beta subunits showed that methionine residues in bovine lutropin were not essential for interactions between subunits. Various recombinants were characterized by polyacrylamide-gel electrophoresis and gel filtration of Sephadex G-100. Immunological cross-reactivity by radioimmunoassay of the recombinants of modified alpha and beta subunits was relatively similar to that of the native subunits. However, the biological activity measured by receptor-site binding of the recombinants of alpha and beta chains with a total of three alkylated methionine residues was less than 5% of the activity of native lutropin. It is noteworthy that recombinants of a modified subunit and a native counterpart subunit regenerated 20-30 % of biological activity. These findings suggested that at least 1-2 methionine residues in each subunit are involved in the hormone-receptor interaction for bovine lutropin.


Amino acid sequence of cytochrome c from Tetrahymena pyriformis Phenoset A. The cytochrome c of Tetrahymena pyriformis GL (Phenoset A) had an isoelectric point of 6.5 and by sequence the following composition: Asp(7) Asn(2) Thr(4) Ser(8) Glu(6) Gln(2) Pro(7) Gly(13) Ala(13) Val(7) Met(2) Ile(5) Leu(6) Tyr(2) Phe(5) Lys(11) His(3) Trp(1) Arg(3) Cys(2) (total 109 residues). The peptides derived from the protein afforded complete overlap, so a complete sequence could be determined without reference of homologous proteins. Alignment with other mitochondrial cytochromes c required two internal deletions totalling three residues and an N-terminal region two residues longer than, and a C-terminal region one residue shorter than, the previously known limits. The sequence was the most divergent of the known mitochondrial cytochromes c, suggesting a distant relationship of ciliates to other eukaryotes. Details of the sequence data have been deposited as Supplementary Publication no. SUP 50068 (37 pages) at The British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153,5.


Identification of cysteine as the reactive group in pyruvate kinase alkylated by 5-chloro-4-oxopentanoic acid. 4-Hydroxypentanoic acid alanine thioether was synthesized and characterized by n.m.r. spectroscopy. This derivative corresponded to the modified amino acid obtained by allowing 5-chloro-4-oxo[3,5-3H]pentanoic acid to react with rabbit muscle pyruvate kinase. Performic acid oxidation of 4-oxo[3,5-3H]pentanoic acid alanine thioether in pyruvate kinase gave [3H]succinate (67%) and [3H]carboxymethylcysteine (33%) as expected. Evidence is presented to show that NaBH4 reduction followed by periodate oxidation and analysis of radioactive formaldehyde production may provide a convenient method for distinguishing between thiol and amino alkylation by halogenomethyl ketone compounds. Peptide 'mapping' confirms that the modification by 5-chloro-4-oxopentanoic acid occurs primarily at one region of pyruvate kinase.


The action of snake venom, phospholipase A and trypsin on purified myelin in vitro. 1. Purified myelin was incubated with snake venom or phospholipase A in the presence of or absence of trypsin at 37 degrees C, pH7.4, for different times. 2. Analysis of the myelin pellet obtained after centrifugation of the myelin sample incubated with snake venom or phospholipase A alone showed conversion of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine into their corresponding lyso compounds. No significant loss of myelin protein was observed in these samples. 3. A marked digestion of basic proteins and proteolipid protein was observed from the myelin pellet when trypsin was present in the incubation mixture. 4. The digestion of basic protein and particularly of proteolipid from myelin suggest that phospholipases may make protein more exposed to proteolytic enzyme for its digestion. 5. The relevance of the co-operative effect of phospholipases and proteinases as a model system of the mechanism of myelin breakdown in degenerative brain diseases is discussed.


Circular-dichroism and electron-microscopy studies of human subcomponent C1q before and after limited proteolysis by pepsin. 1. A fragment of human subcomponent C1q was prepared by limited proteolysis with pepsin at 37 degrees C for 20 h, and at pH4.4, followed by gel filtration on Sephadex G-200. This fragment was shown to contain all the collagen-like features known to be present in the intact molecule [Reid (1976) Biochem. J. 155, 5-17]. 2. Circular-dichroism studies showed the presence of positive bands at 230 and 223 nm in the intact subcomponent C1q and pepsin fragment respectively, compared with a positive band at 220 nm obtained for lathyritic rat skin collagen. These bands were abolished by collagenase treatment, which suggested that there may some collagen-like triple-helical structure in subcomponent C1q and that this structure resides in the pepsin-resistant portion of the molecule. However, the 230 and 223 nm bands had a substantially lower magnitude than that obtained for the unaggregated single fibres of totally triple-helical collagen. 3. Thermal-transition temperatures obtained for subcomponent C1q, the pepsin fragment and the reduced and alkylated pepsin fragment were 48 degrees, 48 degrees and 39 degrees C respectively, compared with a value of 38 degrees C obtained for lathyritic rat skin collagen. 4. Only the unreduced pepsin fragment regained significant amounts (up to 60%) of collagen-like structure, after heat denaturation and cooling, as estimated by circular-dichroism measurements. 5. Electron-microscopy studies of subcomponent C1q and the collagen-like pepsin-resistant fragment of subcomponent C1q showed that the six peripheral globular regions of the molecule were fragmented by pepsin leaving the six collagen-like connecting strands and fibril-like central portion intact.


Location of nucleotide pyrophosphatase and alkaline phosphodiesterase activities on the lymphocyte surface membrane. 1. Isolated mouse spleen lymphocytes hydrolysed UDP-galactose added to the medium. Nucleotide pyrophosphatase activity that accounted for this hydrolysis was enriched to a similar extent as alkaline phosphodiesterase and 5'-nucleotidase in a lymphocyte plasma-membrane fraction. 2. The cell surfaces of mouse spleen and thymus lymphocytes were iodinated with 125I by using the lactoperoxidase-catalysis method. Detergent extracts of the cells were mixed with a purified anti-(mouse liver plasma-membrane nucleotide pyrophosphatase) antiserum and the immunoprecipitates analysed by polyacrylamide-gel electrophoresis. Only one major radioactive component, similar in size (apparent mol.wt 110000-130000) to the liver enzyme, was observed. 3. Electrophoresis of an iodinated spleen plasma-membrane fraction indicated peaks of radioactivity, including one of apparent mol.wt 110000-130000. 4. When detergent extracts of spleen lymphocytes were passed through a Sepharose-bead column containing covalently attached anti-(nucleotide pyrophosphatase) antiserum, the nucleotide pyrophosphatase activity was retained by the beads, whereas protein and leucine naphthylamidase activity were eluted. 5. The results indicate that nucleotide pyrophosphatase and alkaline phosphodiesterase activities are due to the location of the same or similar enzymes at the outer aspect of the lymphocyte plasma membrane. Some possible functions of enzymes at this location are discussed.


Purification and characterization of the catalytic subunit of adenosine 3':5'-cyclic monophosphate-dependent protein kinase from bovine liver. 1. The catalytic subunit of bovine liver cyclic AMP-dependent protein kinase (EC2.7.1.37) was purified essentially by the method of Reimann & Corbin [(1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1384]. 2. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation-velocity centrifugation and sedimentation-equilibrium centrifugation showed that the catalytic subunit was monodisperse. Polyacrylamide-gel isoelectric-focusing electrophoresis revealed the presence of at least three isoenzyme forms of catalytic subunit activity with slightly different pI values (6.72, 7.04 and 7.35). 3. Physical properties of the catalytic subunit were determined by several different methods. It had mol.wt. 39000-42000, Stokes radium 2.73-3.08 nm, so20.w 3.14S, f/fo 1.19-1.23 and, assuming a prolate ellipsoid, axial ration 4-5. 4. Amino acid analysis was performed on the catalytic subunit. It had one cysteine residue/molecule which was essential for activity. Inhibition by thiol-specific reagents was partially prevented by the presence of ATP-Mg2+. 5. The circular-dichroic spectrum showed the catalytic subunit contained 29% alpha-helical form, 18% beta-form and 53% aperiodic form. Near-u.v. circular dichroism showed the presence of aromatic residues whose equivalent molar ellipticity was greatly altered by the addition of ATP-Mg2+. 6. Kinetic experiments showed that the catalytic subunit had an apparent Km for ATP of 7 muM. 5'-Adenylyl imidodiphosphate inhibitied competitively with ATP with a Ki of 60 muM. The kinetic plot for histone (Sigma, type II-A) was biphasic showing 'high'-and 'low'-Km segments. Under assay conditions the specific activity of the catalytic subunit was 3 X 10(6) units/mg of protein. Of various metal ions tested, the catalytic subunit was most active with Mg2+.7. When assayed with histone (Sigma, type II-A) as substrate, the activity of the catalytic subunit was increased by non-ionic detergents or urea. No such activation was observed with casein as substrate.


Interaction of pigeon-liver nicotinamide-adenine dinucleotide kinase with cibacron blue F3GA. The interaction of pigeon liver NAD kinase with Cibacron Blue F3GA was investigated. By using steady-state rate measurements, spectrophotometric titration and chromatography of the enzyme on immobilized dye, it was shown that binding occurs at two nucleotide sites with different affinities, and also at a site distinct from the substrate-binding region.


The influence of theophylline on the motility and the cyclic 3',5'-AMP content in the pregnant and non-pregnant rat uterus in vitro. The tension developed by muscle strips obtained from pregnant and non-pregnant rat uteri and uterine cyclic 3',5'-AMP was measured in the presence of increasing amounts of theophylline. In the pregnant uterus the initial tension was elevated by 112% and the tissue level of cAMP by 59% as compared to the normal organ. 1 mM theophylline relaxed the normal uterus by 81% and the pregnant uterus by 36% without any change in the cAMP concentrations. Higher amounts of theophylline (3-10 mM) able to relax the pregnant uterus completely also increased uterine cAMP in a dose dependent manner. It seems likely that the effect of high amounts of theophylline is mediated by cAMP, which, however, is not involved in the action of low (therapeutic) doses of the drug.


Lymphocyte response to virus antigens in systemic lupus erythematosus. The cell-mediated immune response of lymphocytes to rubella, measles, parainfluenza types 1, 2, and 3, varicella-zoster and herpes virus type 1 virus antigens was evaluated in 15 SLE patients and 15 matched controls by incorporating 3H-thymidine in whole blood cultures as a measure of blastic transformation. SLE patients were less responsive than normal individuals to six of eight virus antigens tested. Culture of washed SLE cells in AB plasma did not reverse the hyporesponsiveness. The results indicated that a functional impairment of the circulating lymphocytes appeared to be responsible for the in vitro hyporesponsiveness of SLE patients to virus antigens.


Stimulation of anti-tumour immunity in guinea-pigs by methanol extraction residue of BCG. The immunoprophylactic effects of the methanol extraction residue (MER) of BCG were investigated in Strain 2 guinea-pigs injected with cells of the transplantable, diethylnitrosamine-induced, Line 10 hepatocarcinoma. Pretreatment with MER at times ranging from 18 to 182 days prior to tumour implantation protected approximately 40% of guinea-pigs from progressive neoplastic disease. In addition, MER-treated animals developed specific cell-mediated anti-tumour immunity both more rapidly and at higher levels than did non-MER-treated tumour-bearing controls. It was not possible, however, to prognosticate from the results of such laboratory studies to the outcome of immunoprophylaxis.


Hepatitis B surface antigen produced by a human hepatoma cell line. The human hepatoma cell line, PLC/PRF/5, was shown to produce hepatitis B surface antigen (HBsAg). Immunologically reactive material was present in the supernatant tissue culture medium in significant amounts, and was associated with spherical particles approximately 20 nm in diameter. The rate of antigen production by the cells was estimated at 500 ng/day/10(6) cells by reference to a purified HBsAg standard. All immunological activity was neutralized by specific antibody and the subtype was ad. The studies reported here broaden the scope of investigations on both the in vitro production of HBsAg and the association between this antigen and primary liver cancer.


Electron spin resonance study of changes during development of solid Yoshida tumour. I: Ascorbyl radical. The ascorbyl radical concentration has been observed, by means of electron spin resonance spectroscopy, in the blood and spleen of female Wistar rats carrying a Yoshida tumour. The ascorbyl radical concentration of the tumour tissue itself was studied as the tumour was developing, and as it was regressing after treatment with methylene dimethane sulphonate. Changes in the concentration of this radical may be related to host tumour reactions.


Electron spin resonance study of changes during development of solid yoshida tumour. II. Paramagnetic metal ions. Electron spin resonance (ESR) spectroscopy was used to examine changes in the concentration of paramagnetic metal ions in Yoshida tumours carried by female Wistar rats. Blood, spleen and lymph nodes from these animals were also examined by ESR. A decrease in the concentration of a paramagnetic species associated with mitochondrial activity, and marked increases in those thought to be associated with inflammatory or immune reactions and cell lysis, were observed in the tumours within one day of implantation. During development of the tumour, and during its regression after treatment with methylene dimethane sulphonate (MDMS), further changes were observed in the concentration of the species. These were dependent on the region of the tumour examined. In blood, development of the tumour produced an increase in ceruloplasmin and a decrease in iron-transferrin. An increase in spleen weight, as the tumour developed, was accompanied by a small decrease in the concentration of species with g-values of 6-0 and 4-3, which was reversed on regression of the treated tumour. The presence of metastases in the regional lymph nodes produced distinguishable changes in the ESR spectra.


Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol. Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDPdiglyceride):L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase) is bound tightly to the ribosomes in crude extracts of Escherichia coli. After separation of the enzyme from the ribosomes by the method of Raetz and Kennedy (Raetz, C.R.H., and Kennedy, E.P. (1974), J. Biol. Chem. 249, 5038), we have purified the enzyme to 97% of homogenekty. The major portion of the overall 5500-fold purification was attained by substrate-specific elution from phosphocellulose using CDP-diglyceride in the presence of detergent. The purified enzyme migrated as a single band with an apparent minimum molecular weight of 54 000 when subjected to electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. The purified enzyme catalyzed exchange reactions between cytidine 5'- monophosphate (CMP) and CDP-diglyceride and between serine and phosphatidylserine. The enzyme also catalyzed the hydrolysis of CDP-diglyceride to form CMP and phosphatidic acid. dCDP-diglyceride was equivalent to CDP-diglyceride in all reactions catalyzed by the enzyme. In addition, the purified enzyme catalyzed the formation of phosphatidylglycerol or phosphatidylglycerophosphate at a very slow rate when serine was replaced as substrate by glycerol or sn-glycero-3-phosphate, respectively. These results suggest catalysis occurs via a ping-pong mechanism through the formation of a phosphatidyl-enzyme intermediate.


Kinetics of Novikoff cytoplasmic messenger RNA methylation. Methylation patterns of Novikoff cytoplasmic mRNA were determined as a function of labeling time with L-[methyl-3H]methionine. The 5'-terminal m7G could be released from whole mRNA by treatment with nucleotide pyrophosphatase. Subsequent alkaline phosphatase treatment of this mRNA, followed by KOH digestion, yielded N'mpNp and N'mpNp from cap 1 (m7GpppN'mpN) and cap 2 (m7GpppN'mpN''mpN), respectively. Our results indicate that the relative amounts of labeled cap structures do change with time and that the amount of internal N6-methyladenosine decreases, relative to 5'-cap structures, as the cytoplasmic mRNAs age and the average size decreases. The formation of cap-2 structures by the addition of second 2'-O-methyl group at position N''m appears to be cytoplasmic event. Thus, after very short labeling times, greater than 80% of the labeled methyl groups in cap 2 are found in this position. These results, along with earlier data obtained on L-cell heterogeneous nuclear RNA methylation, are consistent with a model in which the nucleus is the cellular site of three mRNA methylation events producing 5'-terminal m7G, the first 2'-O-methylnucleoside (N'm) found in cap-1 structures and internal N6-methyladenosine. Subsequently, these nuclear methylations are followed by the cytoplasmic methylation at N''m. Analysis of the methynucleoside composition of cap-1 structures, along with comparable "core" structures (m7GpppN'm) generated from cap-2 by removal of N''m, indicates that at any single labeling time the methylnucleoside composition of a given cap-1 and the cap-2 "core" structure is remarkably similar. On the other hand, comparisons of the methylnucleoside composition of the cap structures at different labeling times indicate an increase in Cm in the first 2'-O-methylnucleoside (N'm) with time.


Copper and the oxidation of hemoglobin: a comparison of horse and human hemoglobins. Oxidation studies of hemoglobin by Cu(II) indicate that for horse hemoglobin, up to a Cu(II)/heme molar ratio of 0.5, all of the Cu(II) added is used to rapidly oxidize the heme. On the other hand, most of the Cu(II) added to human hemoglobin at low Cu(II)/heme molar ratios is unable to oxidize the heme. Only at Cu(II)/heme molar ratios greater than 0.5 does the amount of oxidation per added Cu(II) approach that of horse hemoglobin. At the same time, binding studies indicate that human hemoglobin has an additional binding site involving one copper for every two hemes, which has a higher copper affinity than the single horse hemoglobin binding site. The Cu(II) oxidation of human hemoglobin is explained utilizing this additional binding site by a mechanism where a transfer of electrons cannot occur between the heme and the Cu(II) bound to the high affinity human binding site. The electron transfer must involve the Cu(II) bound to the lower affinity human hemoglobin binding site, which is similar to the only horse hemoglobin site. The involvement of beta-2 histidine in the binding of this additional copper is indicated by a comparison of the amino acid sequences of various hemoglobins which possess the additional site, with the amino acid sequences of hemoglobins which do not possess the additional site. Zn(II), Hg(II), and N-ethylmaleimide (NEM) are found to decrease the Cu(II) oxidation of hemoglobin. The sulfhydryl reagents, Hg(II) and NEM, produce a very dramatic decrease in the rate of oxidation, which can only be explained by an effect on the rate for the actual transfer of electrons between the Cu(II) and the Fe(II). The effect of Zn(II) is much smaller and can, for the most part, be explained by the increased oxygen affinity, which affects the ligand dissociation process that must precede the electron transfer process.


On the inhibitory activity of 4-vinyl analogues of pyridoxal: enzyme and cell culture studies. Analogues of pyridoxal and of pyridoxal phosphate in which the 4-CHO group is replaced with CH = CH2 were synthesized and were found to be potent inhibitors of pyridoxal kinase and pyridoxine phosphate oxidase of rat liver. They also inhibited the growth of mouse Sarcoma 180 and mammary adenocarcinoma TA3 in cell culture. Saturation of the vinyl double bond, replacement of the 5-CH2OH with methyl, methylation of the phenolic hydroxyl, or conversion to the N-oxide resulted in diminution or loss of all these activities. Similarly, the introduction of a beta-methyl group into the vinyl analogues of pyridoxal reduced all these inhibitory activities. The 4-vinyl anatogue of pyridoxal was shown to be a substrate of pyridoxal kinase and the product a potent inhibitor of pyridoxine oxidase, competing with pyridoxal phosphate. The affinity of this phosphorylated pyridoxal analogue to some apoenzymes varied greatly, indicating striking differences among the cofactor binding sites of these enzymes. The growth inhibitory effects of these analogues on cells in culture correlated well with their effects on pyridoxal kinase and pyridoxine phosphate oxidase in cell-free systems.


Inhibition of estrogen-induced increases in uterine guanosine 3',5'-cyclic monophosphate levels by inhibitors of protein and RNA synthesis. Uterine guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels are elevated significantly from 2 to 12 h after a single injection of estradiol-17 beta or diethylstilbestrol to mature, ovariectomized, or immature rats. The accumulation of cyclic GMP is greater in endometrial- than myometrial-enriched uterine tissue. The estrogen-induced increase in cyclic GMP can be prevented by administration of the protein synthesis inhibitors, cycloheximide and puromycin, or by relatively large doses of the RNA synthesis inhibitor, actinomycin D, but not by the muscarinic antagonist, atropine. The requirement for a protein with a relatively rapid rate of turnover is suggested by the demonstration that cycloheximide, when administered after estrogen, can within a 3-h period restore the estrogen-elevated levels of cyclic GMP to those of the non-estrogen-treated tissue.


EPR and optical spectroscopic properties of the electron carrier intermediate between the reaction center bacteriochlorophylls and the primary acceptor in Chromatium vinosum. 1. A reaction center-cytochrome c complex has been isolated from Chromatium vinosum which is capable of normal photochemistry and light-activated rapid cytochrome c553 and c555 oxidation, but which has no antenna bacteriochlorophyll. As is found in whole cells, ferrocytochrome c553 is oxidized irreversibly in milliseconds by light at 7 K. 2. Room temperature redox potentiometry in combination with EPR analysis at 7 K, of cytochrome c553 and the reaction center bacteriochlorophyll dimer (BChl)2 absorbing at 883 nm yields identical results to those previously reported using optical analytical techniques at 77 K. It shows directly that two cytochrome c553 hemes are equivalent with respect to the light induced (BChl)2+. At 7 K, only one heme can be rapidly oxidized in the light, commensurate with the electron capacity of the primary acceptor (quinone-iron) being unity. 3. Prior chemical reduction of the quinone-iron followed by illumination at 200K, however, leads to the slow (t1/2 approximately equal to 30 s) oxidation of one cytochrome c553 heme, with what appears to be concommitant reduction of one of the two bacteriophytins (BPh) of the reaction center as shown by bleaching of the 760 nm band, a broad absorbance increase at approx. 650 nm and a bleaching at 543 nm. The 800 nm absorbing bacteriochlorophyll is also involved since there is also bleaching at 595 and 800 nm; at the latter wave-length the remaining unbleached band appears to shift significantly to the blue. No redox changes in the 883 absorbing bacteriochlorophyll dimer are seen during or after illumination under these conditions. The reduced part of the state represents what is considered to be the reduced form of the electron carrier (I) which acts as an intermediate between the bacteriochlorophyll dimer and quinone-iron. The state (oxidized c553/reduced I) relaxes in the dark at 200K in t1/2 approx. 20 min but below 77 K it is trapped on a days time scale. 4. EPR analysis of the state trapped as described above reveals that one heme equivalent of cytochrome becomes oxidized for the generation of the state, a result in agreement with the optical data. Two prominent signals are associated with the trapped state in the g = 2 region, which can be easily resolved with temperature and microwave power saturation: one has a line width of 15 g and is centered at g = 2.003; the other, which is the major signal, is also a radical centered at g = 2.003 but is split by 60 G and behaves as though it were an organic free-radical spin-coupled with another paramagnetic center absorbing at higher magnetic field values; this high field partner could be the iron-quinone of the primary acceptor. The identity of two signals associated with I-. is consistent with the idea that the reduced intermediary carrier is not simply BPh-. but also involves a second radical, perhaps the 800 nm bacteriochlorophylls in the reduced state...


Metabolism of pyridoxine in the liver of vitamin B-6-deficient rats. The metabolism of [6-3H]pyridoxine - HCl was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding ad libitum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 muCi of [6-3H] pyridoxine - HCl and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H+) columns and the percent radioactivity for each vitamin compound was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days. In vitamin B-6 deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5'-P and pyridoxal 5'-P reached 36 and 44% levels, respectively. Pyridoxine 5'-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5'-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5-P was falling, pyridoxamine 5'-P was linearly synthesized reaching a platuau low level (3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5'-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [3H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an extended label retention period, (b) a rapid pyridoxal 5'-P synthesis, and (c) a continuous synthesis (and accumulation) of pyridoxamine 5'-P which is not utilized or further metabolized.


Phospholipid metabolism and lysosomal enzyme secretion by leukocytes. Effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP. The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.


Adenosine 3',5'cyclic monophosphate in adipose tissue of diabetic rats. Normal male rats were made chronically diabetic by injection of alloxan or acutely diabetic by injection of anti-insulin serum. The concentration of cyclic AMP in epididymal adipose tissue was increased approximately 2 1/2-fold 24 h after alloxan administration and up to 7-fold 72 h post-alloxan. Treatment of alloxan-diabetic rats with insulin for 4 h completely suppressed lipolysis but only partially suppressed cyclic AMP levels; 6 h following insulin treatment cyclic AMP levels were normal. When segments of the epididymal fat bodies were incubated in vitro the high cyclic AMP levels were not maintained but instead decreased spontaneously. Addition of insulin to the incubation media decreased lipolysis in tissues of diabetic rats to levels measured in tissues of normal rats and accelerated the decline in cyclic AMP levels but did not return cyclic AMP levels to normal. Rats rendered acutely insulin deficient by injection of anti-insulin serum showed increased plasma glucose and free fatty acid levels and increased adipose tissue free fatty acid, and cyclic AMP levels 30 min following injection of the antiserum. Plasma glucagon levels increased but not until 2 h following anti-insulin serum, thereby excluding the possibility that an increment in plasma glucagon is the primary stimulus for the acceleration of lipolysis in diabetes. These data are consistent with the view that control of adipose tissue cyclic AMP levels in situ is an important physiologic action of insulin.


Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex. Suspensions of renal cortical tubules were incubated with 33Pi and exposed to parathyroid hormone (40 mlg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [gamma-32P]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 150 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.


Insulin degradation. XVIII. On the regulation of glutathione-insulin transhydrogenase in the hyperglycemic obese (ob/ob) mouse. The occurrence of insulin-degrading activity in the liver of the obese hyperglycemic mouse (ob/ob) and its litter mate has been studied. The trichloroacetic acid-soluble product formed from insulin upon incubation with liver homogenate was identified as the A chain of insulin. In Ouchterlony double-diffusion experiments with antibody to purified rat liver glutathione-insulin transhydrogenase, mouse liver homogenate and the microsomal fraction each gave a single precipitation band of identity with the purified rat liver enzyme. These results indicate that the insulin-degrading activity present in the mouse liver is, in fact, glutathione-insulin transhydrogenase. Subcellular distribution studies of glutathione-insulin transhydrogenase and marker enzymes indicate that the transhydrogenase is located primarily in the microsomal fraction of mouse liver homogenate. The ob/ob mouse, which is a genetic mutant characterized by obesity, hyperinsulinism and resistance to the hypoglycemic action of insulin, contains hepatic glutathione-insulin transhydrogenase activity (per mg microsomal protein) markedly higher (40--60%) than its lean litter mates. However, a major portion of the increased hepatic enzyme in the ob/ob mouse occurs in a latent state; the increased amount of enzyme either is unavailable or is nonfunctional, although the ob/ob mouse still contains more of the functional form than the lean mouse. Thus, the results are consistent with the suggestion that the hepatic glutathione-insulin transhydrogenase is probably under a feedback control by circulating insulin.


Independent modulation of hepatic protein kinase activities. The effects of hormonal status on protein kinase activity was examined in homogenates of rat liver. Protein kinase activity was evaluated from incorporation of 32P from [gamma-32P]ATP into protamine or histone as receptor substrates. Protamine phosphorylation in the presence or absence of cyclic AMP exceeded histone phosphorylation by at least a factor or two. Hypophysectomy markedly increased protamine phosphorylation in the presence or absence of saturating amounts of cyclic AMP. In contrast, hypophysectomy only slightly increased cyclic AMP independent phosphorylation of histone. These results could not be amounted for by differences in ATPase or protein phosphase activities. Cortisone (2 mg/day x 3) decreased total protein kinase activity in livers of hypophysectomized rats when protamine was substrate, but had no effect on the total activity toward histone. Growth hormone (100 mug/day x 3) significantly increased histone, but not protamine phosphorylation in livers of hypophysectomized rats. Administration of 5 mug of triiodothyonine/day to hypophysectomized rats also markedly increased the phosphorylation of histone, but not protamine when saturating amounts of cyclic AMP were present. These results support the hypothesis that liver may contain more than one type of protein kinase activity and that the different protein kinase activities can be separately affected by hormones. Such control distal to cyclic AMP might allow selective modulation of cyclic AMP-dependent processes in cells which carry out more than one such process.


The development of phosphomonesterases in the testes of prepuberal chicks. 1. The biochemical development and histochemical localisation of phosphomonoesterases in the testes of prepuberal chicks have been studied. 2. Maximum acid phosphatase activity was observed at 12 weeks with a decrease in enzyme activity after this age, whereas alkaline phosphatase activity fluctuated with age. 3. Acid phosphatase activity in chicks was similar to that of the cockerel in being tartarate-insensitive. 4. There was a low level of significant correlation between acid phosphatase activity and testes weight. 5. Both alkaline and acid phosphatase activities were observed in the basement membrane of the seminiferous tubules, and acid phosphatase activity also in the various spermatogenic elements. 6. The results suggest that acid phosphatase is more involved in spermatogenesis, and more widely distributed than alkaline phosphatase in testicular tissue during testicular development.


Disaggregation of hydroxyapatite crystals. A system has been developed to measure quantitatively the disaggregation of hydroxyapatite crystals. Disaggregation was induced by pyrophosphate, ethane-1-hydroxy-1,1-diphosphonate, dichloromethylene diphosphonate, haparin and citrate. Hyaluronic acid stimulated aggregation at low concentrations and disaggregation at high concentrations. Lactate had no effect. The possible role disaggregation might play in the resorption of calcified tissues in vivo id discussed.


Absorption and circular dichroism spectra of different forms of mushroom tyrosinase. The circular dichroism spectrum of resting mushroom tyrosinase between 800 and 400 nm showed two bands at 755, and 653 nm. The CD spectrum of resting tyrosinase between 400 and 250 nm showed oxygen-sensitive changes at 350 nm upon treatment of tyrosinase with hydroxylamine or hydrogen peroxide. These were similar to changes observed on regeneration of aged hemocyanin by similar procedures. A structural relationship between the active sites of hydroxylamine- or hydrogen peroxide-treated tyrosinase and hemocyanin is suggested by these observations, confirming inferences based upon other studies (Jolly, Jr., R.L., Evans, L.H., Makino, N. and Mason, H.S. (1974) J. Biol. Chem. 249, 335-345 and Schoot Uiterkamp, A.J.M. and Mason, H.S. (1973) Proc. Natl. Acad, Sci. U.S. 70, 993-996).


Low temperature magnetic susceptibility of a human Co(II) carbonic anhydrase B sulphonamide complex. The magnetic susceptibility of the acetazolamide complex of human Co(II) carbonic anhydrase B was measured between 1.4 and 160 K. From the temperature dependence of the paramagnetic contribution of Co(II) a g-value of 2.23 +/- 0.02 and a zero-field splitting of (33 +/- 1) cm-1 were estimated. The effective number of Bohr magnetons as T leads to 0 is 3.35 in excellent agreement with the number (3.38) calculated from the apparent g'-values of the EPR spectrum at 16 K. Extrapolation to high temperatures gave an effective number of Bohr magnetons of 4.32.


A study of the enzymic dephosphorylation of beta-casein and a derived phosphopeptide. beta-Casein, and the phosphate containing peptide derived from it by tryptic digestion, have been dephosphorylated by the action of two phosphatases. Escherichia coli alkaline phosphatase (EC 3.1.3.1) has been shown to remove the phosphates from these substrates in two distinct stages. Substrate molecules retaining three of the original phosphoseryl residues accumulate during the reaction and are resistant to further dephosphorylation at low enzyme concentrations. In contrast bovine spleen phosphoprotein phosphatase (EC 3.1.3.16) achieves complete dephosphorylation of these substrates sequentially without any of the intervening species showing resistance to the action of the enzyme. The phosphopeptide has been partially dephosphorylated by the action of the two phosphatases and the resultant peptides containing three phosphoseryl residues compared in their reactivity toward the E. coli alkaline phosphatase. The results obtained are discussed in relation to the mode of action of the two enzymes.


The effect of phospholipase C on DNA synthesis, morphology and phospholipid content of isolated HeLa cell nuclei. Isolated HeLa cell nuclei have been treated with purified phospholipase C (Bacillus cereus) and sphingomyelinase (Staphylococcus aureus). The phospholipids of untreated nuclei consisted of about 67% phosphatidylcholine, 23% phosphatidylethanolamine, 7% sphingomyelin, 2% phosphatidylserine and 1% phosphatidylinositol. Phospholipase C degraded 80-90% of the total phospholipids of the nuclei. Such nuclei seemed ultrastructurally intact, and had an average diameter and a protein loss during incubation which were not significantly different from those of controls. Their rate of DNA synthesis was only slightly reduced after treatment with phospholipase C alone and slightly more reduced when phospholipase C was used in combination with sphingomyelinase. This suggests that the polar head-groups of the nuclear phospholipids are of very limited importance in DNA synthesis. Since it has been reported that phospholipase C treatment releases nascent DNA from a membrane complex, the absence of a concommitant reduction in DNA synthesis may suggest that this complex is not necessary for the replication of DNA. Phospholipase C did not significantly influence the stability of the DNA product and gave only a slight inhibition of cytosol and nuclear DNA polymerases when tested with exogenous template.


Selenalysine and protein synthesis.

Selenalysine and protein synthesis. Selenalysine is a lysine analog having the gamma-methylene group substituted by a selenium atom. It has been demonstrated that selenalysine is activated and transferred to tRNAlys by either Escherichia coli or rat liver aminoacyl-tRNA synthetases, and inhibits lysine incorporation into polypeptides in protein-synthesizing systems from E. coli, rat liver or rabbit reticulocytes. All tests were performed in comparison with thialysine, a lysine analog having the gamma-methylene group substituted by a sulfur atom. In all the reactions studied, both thialysine and selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine shows a slightly lower activity as lysine inhibitor.


Characterization of the 3'-terminal region of the large molecular weight RNA subunits from normal and transformation-defective Rous sarcoma virus. In avian sarcoma viruses a function required to transform cells is frequently lost giving rise to non-transforming virus. The genetic information for this function is localized near the 3' end of the genome. Short 3'-terminal poly(A)-linked RNA fragments from the genomes of a transforming Rous sarcoma virus and its non-transforming derivative were isolated and gave rise to identical fingerprint patterns, suggesting that internal deletion rather than terminal elimination leads to non-transforming virus.


Comparison of DNA facilitators in the uptake and intracellular fate of infectious herpes simplex virus type 2 DNA. The comparative efficiencies of polyornithine, CaCl2 and DEAE-dextran in enhancing the infectivity of exogenous herpes simplex virus (HSV) type 2 DNA were examined. CaCl2 was 12-times more effective in promoting genetic expression of viral DNA than DEAE-dextran, while polyornithine did not mediate any HSV DNA infectivity. A comparison of sedimentation profiles of DNA extracted from cells inoculated with viral DNA in the presence of each facilitator revealed that input 56 S HSV DNA underwent marked alteration with time. There was also extensive processing of a 20 S DNA species. The data indicated that the biological efficiency of each facilitator was related to the amount of 20 S DNA remaining at the final time periods. One possible explanation for the efficiency of CaCl2 as a facilitator was that it allowed for the reutilization of the 20 S DNA species during HSV replication. The persistence of the 20 S peak in cells treated with DEAE-dextran was a measure of the decreased efficacy of this facilitator. Finally, the absence of a 56 S peak and the greatly elevated levels of 20 S DNA at final time points in polyornithine-treated cells accounted for the failure of this compound to promote HSV DNA infectivity.


The effects of glucose and cyclic GMP on RNA synthesis and nuclear morphology in starved rats. Feeding rats in diet high in glucose has been demonstrated to inhibit the induction of many enzymes, block the action of glucocorticoids, and, in general, appears to result in decreased cyclic AMP activity. We found that glucose feeding depresses both messenger RNA (mRNA) and non-mRNA synthesis. Electron microscopic examination of the nucleus revealed that glucose feeding decreases the granular component of liver cell nucleoli. It only slightly decreases liver cyclic AMP levels, but produces a sixfold elevation in levels of the cyclic AMP antagonist, cyclic GMP. Administration of bromocyclic GMP, like glucose feeding, depresses mRNA synthesis, but does not simulate the effect of the carbohydrate on nuclear morphology. In addition, glucose feeding halves liver inorganic phosphate and triples ATP levels. Phosphorylation of nuclear proteins, however, remains unaltered. Despite the antagonism between glucose feeding and glucocorticoid activity, the former compound did not change the binding of dexamethasone to liver nuclei.


Characterization of a protein kinase-phosphoprotein system in free cytoplasmic ribonucleoprotein particles of plasma cell tumours. A protein kinase, associated with free cytoplasmic ribonucleoprotein particles (free dRNP) has been purified from mouse plasma cell tumours. This protein kinase is able to phosphorylate in vitro endogenous protein from free dRNP. Some characteristics of this protein kinase have been studied. This protein kinase behaves as being cyclic AMP independent. The properties of this protein kinase were compared with other protein kinases: soluble, ribosome-bound, and nuclear protein kinases. Although there are minor differences it is very similar to a ribosome-associated protein kinase from the plasma cell tumours.


Plasma membrane studies on drug-sensitive and -resistant cell lines. II. Ouabain sensitivity of (Na+ +K+)-stimulated Mg2+-ATPase. Mutant cell lines have been selected from the murine plasmocytoma MOPC 173 for their resistance to ouabain, dibutyryl cyclic AMP, theophyllin and concanavalin A. We have compared three wild-type cell lines with their seven resistant counterparts. All resistant mutants exhibited a (Na+ + K+)-stimulated Mg2+-ATPase resistance to ouabain inhibition when measured in microsomes. The homogeneity of ouabain binding sites has been found in most of the cell lines; however, two different populations of sites have been detected in one wild-type and in one resistant cell lines. These results led us to hypothetise the (Na+ + K+)-ATPase-ouabain interaction being modulated by a non-specific membrane structure.


Comparison of the epinephrine-mediated activation of adenylate cyclase in plasma membranes from liver and ascites hepatomas of rats. (1) The apparent [3H]epinephrine binding parameters of plasma membranes from rat liver and ascites hepatomas such as AH-7974, AH-371A and AH-130, as measured by equilibrium dialysis and/or Millipore filtration, were almost similar to each other. The epinephrine binding sites in the plasma membranes were heterogenous (alpha, beta-receptors and non specific sites), but the pattern of these binding sites in the liver membranes appeared almost similar to that in the hepatoma membranes. 2. The beta-receptor seemed to be specifically involved in the epinephrine-mediated activation of adenylate cyclase of the liver membranes. In spite of the presence of almost similar beta-receptors and adenylate cyclase, the adenylate cyclase of hepatoma membranes was found to be less sensitive to the epinephrine-mediated activation. 3. GTP alone was found to activate adenylate cyclase of liver and hepatoma membranes to some extents when the concentration of ATP was lower (0.3 mM). When GTP was added with epinephrine, a marked, synergistic activation of adenylate cyclase was observed in liver plasma membranes, but not in hepatoma ones. 4. The synergistic activation of adenylate cyclase by epinephrine plus GTP showed a characteristic kinetic feature, reaching a maximal peak within 1 min or so after mixing. 5. Binding of [3H]epinephrine to liver membranes proceeded monophasically in the absence of GTP, while it proceeded biphasically in the presence of GTP, showing the retardation of binding at some earlier stages. GTP added at the time of binding equilibrium induced the temporary release of bound epinephrine from the beta-receptors. The GTP-induced temporary release of bound epinephrine, occurring within 4-5 min after the addition of GTP, was less marked in the hepatoma membranes as compared with the liver membranes. 6. Possible impairment of the GTP-dependent coupling mechanism in the receptor-adenylate cyclase system of hepatoma plasma membranes was suggested.


Correlated x-ray diffraction and freeze-fracture studies on membrane model systems. Perturbations induced by freeze-fracture preparative procedures. Lipid-water and protein-lipid-water phases have been examined by X-ray methods before and after freezing. Frozen samples have been subsequently fractured and replicated, thus permitting an evaluation of the nature of structural perturbations in samples examined by freeze-fracture electron microscopy. Important results are summarized: (1) Freezing low water content (approx. less than 25%) phases causes perturbations in the packing of hydrocarbon chains. The results suggest that freezing liquied paraffin chains produces a condensed "glass-like" packing. (2) Additional perturbations occur in high water content samples. After freezing, much smaller lamellar repeat distances, intense ice reflections, and extensive perturbation of fracture faces are consistant with the expulsion of water from between lamellae. Presence of glycerol generally relieves these perturbations but in some cases introduces additional lattice disorder. (3) Surprisingly, cooling by a stream of cold N2 gas (-140 degrees C) produces qualitatively the same results as rapid cooling in liquid Freon-22 (-160 degrees C). (4) Complex perturbations occur in phases containing integral membrane proteins. Interesting results have been obtained with cytochrome b5-lecithin lamellar associations which display both smooth and rough fracture faces without clearly defined particles.


Glycolipids in model membranes. Spin label and freeze-etch studies. 1. Several types of glycolipid are examined in lipid bilayer model membranes as part of a program to clarify their fuction in living cells. 2. Data obtained with three spin labelled derivatives of galactosyl ceramide is reported showing a fatty acid fluidity gradient similar to that obtained with phospholipid spin labels. Some possible structural implications of the observed differences are considered. 3. Results obtained using Freeze-Etch electron microscopy and hemagglutination inhibition are given showing beef brain gangliosides in lipid vesicles to be effective receptors for influenza virus.


Use of phospholipid-clay complexes for determining vibrational spectra of membrane related systems. For determining the infrared and Raman spectra of membrane related systems, a method is developed to incorporate phospholipid bilayer assemblies in a clay matrix to form ultra-thin, self-supporting films. These films, containing stabilized bilayers arranged between the silicate layers of hectorite, are in the shape of discs which measure about 2 cm in diameter and 25 microns thick and require approximately 2 mg of phospholipid for preparation. Although several spectral regions below 1100 cm-1 are masked by the host clay, both head group and acyl chain vibrations may be conveniently observed and monitored for phospholipid conformational changes.


Effect of the hydrophile-lipophile balance of non-ionic detergents (Triton X-series) on the solubilization of biological membranes and their integral b-type cytochromes. The solubilization of four integral membrane proteins (i.e. cytochrome b-561 of the chromaffin granule membrane, cytochrome b5 of the endoplasmic reticulum and the mitochondrial b-type cytochrome(s) as well as cytochrome c oxidase) has been studied at 0 degrees C using the non-ionic detergents of the Triton X-series having the common hydrophobic 4(1,1,3,3-tetramethylbutyl)phenoxy (t-octyl-phenoxy) group and a variable average number (n) of polar ethylene oxide units added. Following a pre-extraction of peripheral membrane and matrix proteins with low and high salt concentration and a weak non-ionic detergent (Tween 20, average hydrophile-lipophile balance (HLB) = 16.7), the amount of heme proteins solubilized by subsequent Triton X-solutions was measured. With the detergents tested the degree of solubilization decreased in the sequence cytochrome b-561 greater than cytochrome b5 greater than mitochondrial cytochrome(s) b and parallelled the effect of the detergents on light scattering and the phospholipid to protein ratio of the three membranes. For all the b-cytochromes, the solubilizing power of the detergent increased with decreasing average length of the polar ethylene oxide chain and the hydrophile-lipophile balance as long as clouding did not occur (e.g. Triton X-114,n = 7.5 and HLB = 12.4). Thus, the greatest difference in the degree os solubilization of the three cytochromes was observed with Triton X-405 (n = 40 and HLB = 17.9). All the cytochromes were most efficiently solubilized (i.e. approx. 90%) by Triton X-100 (n = 9.5 and HLB = 13.5).


Interactions of concanavalin A with chick embryo fibroblasts transformed by Rous sarcoma virus. Study with an RSV mutant thermosensitive for transformation. The interactions between concanavalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 degrees C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 degrees C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 degrees C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 degrees C. Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 degrees C.


Cell growth and quabain-sensitive 86Rg+ uptake and (Na++K+)-ATPase activity in 3T3 and SV40 transformed 3T3 fibroblasts. The uptake of ouabain-sensitive 86Rb+ uptake measured at 5 min and the uptake measured at 60 min was 4.5- and 2.7-fold greater respectively for SV40 transformed 3T3 cells compared to 3T3 cells during the late log phase of growth. This uptake, however, varied markedly with cell growth. Ouabain-sensitive 86Rb+ uptake was found to be a sensitive indicator of protein synthesis as measured by total protein content. Cessation of cell growth as measured by total protein content was associated with a decline in ouabain-sensitive 86Rb+ uptake in both cell types. This increase ouabain-sensitive cation transport was reflected in increased levels of (Na++K)-ATPase activity for SV40 3T3 cells, which showed a 2.5-fold increase V but the same Km as 3T3 cells. These results are compared with the results of related work. Possible mechanisms for these effects are discussed and how changes in cation transport might be related to alterations in cell growth.


In vitro synthesis of simian virus 40 DNA. I. Synthesis by a soluble extract from infected CV1 cells. Simian Virus 40 (SV40) DNA replication was studied in vitro using cell free extracts prepared from SV40 infected CV1 cells. The cells were fractionated into a soluble cytoplasmic fraction and nuclei. The nuclei were lysed with high salt and used to prepare a soluble nuclear fraction. Both fractions displayed DNA polymerase activity as measured with activated calf thymus DNA. However, only the cytoplasmic fraction was active when SV40 DNA comonent I molecules were used as template. Under these conditions, the cytoplasmic extract was shown to catalyse the SV40 DNA dependent, in vitro incorporation of the four deoxyribonucleotides into DNA molecules which had, at both neutral and alkaline pH, the same sedimentation behavior as authentic SV40 DNA component I and component II molecules. Optimal Mg++ concentration was 5-8 mM. Incorporation of label into DNA component I molecules showed an initial lag of about 15 min., after which it was linear with time for up to 5 hrs at 32 degrees. Incorporation into DNA component II molecules proceeded without obvious lag and reached a plateau after approximately 2 hrs of incubation. It is concluded that the cytoplasmic extract supports the in vitro synthesis of SV40 DNA and that DNA component II molecules appear to be a precursor to DNA component I molecules in the reaction. Labeling of viral DNA molecules was highly dependent on ATP and on an ATP generating system. In the absence of ATP and of the energy generating system, incorporation occurred but both template and newly synthesized DNA molecules were extensively degraded.


In vitro synthesis of simian virus 40 DNA. II. Evidence for a repair mechanism. The technique of density labeling of DNA by BrdU was used to characterize the material synthesized in vitro by cytoplasmic extracts of SV40 infected cells incubated in the presence of simian virus 40 (SV40) DNA component I molecules (Girard et al, Biochimie, this volume). In a first experiment, the template was labeled beforehand in vivo using [14C]-BrdU, and the in vitro incubation was carried out in the presence of [3H]-dGTP and [3H]-dTTP. In a second experiment, the template was labeled in vivo with 32P, and the in vitro incubation was in the presence of [3H]-dGTP and BrdUTP. After digestion with the restriction endonuclease Hind II + III, the fragments from the end products of the reaction were analyzed by density gradient centrifugation, at pH 7 and pH 13. In both experiments the DNA product molecules had the same density as the resepctive DNA templates. Cellular enzymes seem to be responsible for this in vitro synthesis of DNA, since cytoplasmic extracts from uninfected cells were almost as active as those from SV40 infected cells. The system was proved efficient in the conversion of "open circular" molecules (component II DNA molecules) to covalently closed circular DNA molecules (relaxed component I molecules). The use of DNA complexed with histones did not impart viral specificity to the system. It is concluded that the cytoplasmic extract is only capable of supporting the repair synthesis of added viral DNA.


[Phospholipid exchange between mitochondria and microsomes of rat hepatoma 27]. The phospholipid exchange in vitro between mitochondria and microsomes from rat liver and rat hepatoma 27 was investigated. On incubation with a postmicrosomal protein fraction the phospholipid exchange between subcellular fractions of the tumor was found to proceed much faster and less specific than between mitochondria and microsomes from normal liver. These results indicate that the earlier demonstrated lipid dedifferentiation of tumor cell membranes may be connected with an altered transmembrane phospholipid exchange in vivo.


[Structure of the active center of the catalytic subunit of histone kinase]. A number of unknown ATP analogues is isolated when studying the structure of the active site of catalytic histonekinase subunit. Adenosine-5'-chloromethanepyrophosphonate adenosine-5'-(beta-bromoethanepyrophosphonate) and adenosine-5'-(p-fluorosulphonylphenylphosphate) were isolated under the reaction of chloromethanephosphonic acid, beta-bromoethanephosphonic acid and n-phenolsulphofluoride respectively with AMP imidazolide. Adenosine-5'-(beta-chloroethylphosphate) was obtained from AMP morpholide and ethylenechorohydrine. Adenosine-5'-chloracetylaminomethanephosphonate and adenosine-5'-(p-fluorosulphonylbenzoylaminomethanephosphonate) were obtained in the reaction of chloroacetyc anhydride and n-fluorosulphonylbenzoylchloride. Adenosine-5'-(p-aminophenylphosphate) is synthesized under the reduction of AMP mononitrophenyl ester. The treatment of the former with chloroacetyc anhydride produced adenosine-5'-(p-chloroacetylaminophenylphosphate. Interaction of ATP analogues obtained and also of early synthesized adenosine-5'-chloromethanephosphonate and adenosine-5'-(beta-bromoethanephosphonate) with homogenous catalytic histonekinase subunit is studied. The decrease in the reaction rate of Hi histone phosphorylation is found to take place. pH optimum of the enzyme inactivation with adenosine-5'-chloromethanepyrophosphonate and adenosine-5'-(beta-chloroethylphosphate) and the protective effect of the substrate (ATP) indicate covalent blocking imidazole ring in the active site. The date obtained suggest that the functional group of the active site of catalytic histonekinase subunit is histidine imidazole ring located close to terminal ATP phosphate.


[Effect of detergents and proteolytic enzymes on membrane-bound phosphohydrolases in Escherichia coli cells with repressed and depressed biosynthesis of these enzymes]. Solubilization of protein membranes by detergents and protein liberation from the membranes induced by proteolytic enzymes results in a change of activity of membrane-bound phosphohydrolases--alkaline phosphatase and polyphosphatase. The activity of enzymes under conditions of repressed and derepressed biosynthesis of phosphohydrolases changes differently, thus indicating their different membrane environment in the two types of membranes. Some data were obtained on the localization of alkaline phosphatase in a hydrophobic region, possibly in lipid bilayer and polyphosphatase in the surface layers of the membrane.


Sleep analysis during drug-free weekends in chronic schizophrenic patients. The authors made a polygraphic registration of the night sleep in a sample of 14 chronic schizophrenic patients who for several months (mean 8 months) have been on a stable, relatively low maintenance dosage of neuroleptics administered according to the drug-free weekend schedule (two consecutive drug-free days at the weekend). During this treatment none of them showed a relapse or deterioration (BPRS, CGI, and NOSIE rating scales were applied periodically). Their only complaint was of sleep deterioration during the drugfree weekend nights, especially during the second night. The polygraphic night-sleep pattern of each patient was studied during two consecutive weeks. No difference was found between the adaptation night on medication and the consecutive night on medication during the first week, and between the adaptation and readaptation nights on medication during two consecutive weeks. There was no difference in any sleep parameters between the nights on medication and the first drug-free nights. There was a signifcant difference in the total sleep time between the nights on medications and the second drug-free nights. No difference was found in any other sleep parameters in nights analysed as a blocks and in the distribution of NREM and REM stages in the first vs. the second half of the night when B3 was compared with A2. The practical implication is, that to avoid any change in nocturnal behavior it is preferable to withdraw the medication on two nonconsecutive days in the week. The evaluation of both daily and nocturnal behavior seems to be a useful tool in evaluating the first sign of the drug-withdrawal syndrome.