Detection of animal viruses in coastal seawater and sediments. Animal viruses, predominantly enteroviruses, were detected in shallow water at bottom depths and in clastic marine sediments. Viruses accumulated in sandy and slimy deposits of the sea bottom near the shore and could be easily released into water by means of simple mechanical shaking.


Eczema herpeticum. Treatment with methylene blue and light. Kaposi varicelliform eruption due to herpes simplex virus (eczema herpeticum) is a serious disease in infants, with occasional fatal outcome. Photoinactivation therapy consisting of local application of methylene blue followed by light exposure resulted in rapid improvement both clinically and virologically. Methylene blue, at a concentration of 10(-5)M was found to be viricidal to herpesvirus in the light but not in the dark.


Failure of laboratory and radiological studies to predict jejunal mucosal atrophy. Of 58 children with suspected malabsorption, 27 were shown to have subtotal or partial atrophy, and 31 had normal mucosal histology. Oral glucose tolerance tests, faecal fat excretion, D-xylose excretion tests, Prosparol absorption studies, haematological investigations, and radiological examination of the small bowel failed to distinguish these two groups and frequently gave misleading results. It is concluded that in suspected coeliac disease small intestinal biopsy should be performed as a primary investigation.


Nitrogen dioxide and the erythrocyte redox state. Normal human erythrocytes were exposed for two hours at 38 C to an atmosphere of air containing variable concentrations of nitrogen dioxide, in order to detect any primary cytoplasmic effect of NO2 on the calculated oxidation-reduction (redox) ratio ([NAD+]/[NADH]) of a mitochondria-free cell. Substantial increases in the redox ratio were noted only when NO2 concentrations exceeded 15 ppm. In the range of 15 to 500 ppm NO2, the increase in the redox ratio significantly correlated with the NO2 concentration (r=.71; p less than .01). Intracellular to extracellular anion distribution ratios for chloride, lactate, and pyruvate were similar in NO2 and non-NO2 exposed cells, suggesting absence of a substantial hemolytic effect. These data identify a direct cytoplasmic NO2-induced biochemical change that may be mediated by a mechanism other than lipid peroxidation. Alteration of hemoglobin or NAD-NADH-dependent enzyme activity is suggested.


[14 cases of constrictive endocardial fibrosis (or endomyocardial fibrosis]. Constrictive endocardial fibrosis (CEF) or endomyocardial fibrosis (EMF) was studied on 14 files of this rather frequent disease in Ivory Coast. On the basis of the data derived from angiocardiography, catheterization and mechanograms, the clinical signs are interpreted better. Thus one is able to describe with accuracy some clinical forms, the commonest of which being a right heart restriction combined with left-sided signs. It seems important underline how useful the external pulse recordings might be useful for the diagnosis in view of their analogy with the pressure curves. In this disease with a severe course, bold surgical treatments provide some hope. On the contrary, its aetiology remains mysterious. Our best landmark being the link of CEP with tropical ecology, and in particular, in our cases, with the warm and damp forest ecology of some areas.


[Neonatal hypoglycemia due to hyperinsulinism secondary to Langerhans' polyadenomatosis. Recovery after pancreatectomy]. Report of a case of a neonatal hypoglycemia detected at the 11th hour of life, secondary to a Langherans polyadenomatosis in a girl with an hemihypertrophy. Pre- and post-operative tests are reported. In case of neonatal hypoglycemia, the criteria leading to the diagnosis of hyperinsulinism, i.e. the only neonatal hypoglycemia with surgical treatment are reviewed. Except in the case of adenoma, the pancreatectomy should be performed subtotally. The best time for surgery is as soon as the 3rd week, in order to preserve the cerebral development.


[A transplantable myeloid hamster leukemia with high peripheral leukocyte counts and C-particles. II. Histology and cytochemistry (author's transl)]. Results on morphologic and hematologic characterization of a hamster leukemia capable of both cellular and cellfree transmission are described. Solid tumors removed in the course of several passages of leukemia animals, after producing blood smears, and spleen, liver, lymph nodes, bone marrow, thymus, kidney and lung were investigated histologically and histochemically. The morphological picture of the hamster leukemia has not changed during several transplantation generations. In addition to solid tumours, typical leukemic infiltration were detected histologically in spleen, liver, lymph nodes, bone marrow, kidneys and lung. No leukemic proliferation was noticed in the thymus. The final stage of the disease is characterized by an abrupt occurrence of high leukemic cell counts. The demonstration of alkaline phosphatase, but especially of naphtol-AS-D-chloroacetate esterase, in the leukemic cells is interpreted as indicating malignization of cells of the granulocytic line.


Growth inhibition of sarcoma 180 induced by silica and talc. In applying amorphous silica and talc in the site of solid Sarcoma 180 implants, the authors found a suppression and delay of tumor growth which was proveked by peritumoral foreign-body granulomas. Correlating these findings which those experiments which resulted in granulomas of hypersensitivity type, it may be suggested that any kind of peritumoral chronic inflammation enables an anti-tumor effect.


[Alcoholic polyneuropathy. Electrophysiological and clinical findings in 85 patients (author's transl)]. Maximum motor and sensory nerve conduction velocities, amplitudes of muscle action potentials (surface electrodes) and of sensory nerve action potentials, and needle electromyograms were studied in 80 patients with alcoholic neuropathy and in 5 chronic alcoholics without clinical signs of neuropathy. The electrophysiological results were compared to the clinical findings. Neurographic criteria compatible with the diagnosis "alcoholic neuropathy" were defined. Neurographic findings contrary to a diagnosis of alcoholic etiology were demonstrated in 3 patients. Only in a smaller group of patients could a reduction of conduction velocity be found, especially in regions of peripheral nerve entrapment. A decrease in conduction velocity outside of the entrapment sites can be explained in some cases by segmental demyelination in chronic hepatitis or cirrhosis of the liver.


Immunoglobulins and their relative neutralizing efficiency in cattle immunized with infectious bovine rhinotracheitis--parainfluenza-3 (IBR-PI-3) virus vaccine. The localization of anti-IBR and anti-PI-3 activity in the serum and nasal secretory immunoglobulins following intranasal immunization of cattle with a mixed vaccine (IBR-PI-3, MLV, TCO) was studied and was found to reside in the nasal secretory IgA, serum IgM and IgG fractions. The computation of their relative virus neutralizing efficiencies from kinetic data revealed their order of neutralizing efficiencies to be IgM greater than IgA greather than IgG.


[Hepatic amebiasis in an infant].

[Hepatic amebiasis in an infant]. Case report of a 8 month-old North-African infant with liver amebiasis. This disease is not rare in young children and may be observed in all endemic zones of amebiasis. The clinical characteristics, special for the age, and the biological features are reviewed. The diagnosis relies on immunological tests. Treatment is essentially medical, metronidazole often being effective.


[Sensitivity of various primate cells and animal viruses to the antiviral activity of human leukocyte interferon (author's transl)]. U cells (a permanent, human amnion cell line) were protected against infection with Semliki Forest Virus (SFV) by human interferon (HIF) from peripheral leukocytes. Despite the usual genus-specific action of interferons, mouse L929 cultures (a permanent mouse fibroblast cell line) were also protected by HIF. The antiviral action of HIF in six other primate cell cultures was also examined. It is of interest that two lymphoblastoid cell lines, RPMI1788 and Kaplan, were insensitive to HIF. The sensitivity of ten different viruses against HIF in primary African green monkey kidney cell cultures was compared. Among the viruses tested SFV was the most sensitive whereas two strains of vaccinia virus were barely inhibited. In contrast, type 1 herpes simplex virus was relatively sensitive to the action of HIF.


[Hypophyseal gonadotropin secretion in sexual precocity with McCune-Albright's syndrome]. In three young girls with isosexual precocity and fibrous dysplasia of bones, plasma gonadotropins (LH and FSH) response to releasing hormone (LH-RH) has been much under the mean response in true female precocious puberty. These data and those previously reported in the literature suggest that MacCune-Albright's syndrome correlates to an hypothalamo-pituitary dysfunction rather than to a true prematurity.


Structural and growth characteristics of two avian reoviruses. Two virus strains which had been suspected to be the etiological agents of infectious bursitis (Gumboro disease) and of inclusion body hepatitis of chickens were characterized by their morphology, their peptide composition and the segmented genome of their double-stranded RNA to be typical reoviruses. Although the 2 avian strains did not clearly differ in their serological behaviour, the size of some of their RNA segments were not identical. Both strains replicated in tissue cultures prepared from the chorioallantoic membrane of embryonated eggs with growth characteristics of reoviruses.


[Ultrastructure of human hepatocytes in early prenatal ontogenesis]. The structure of hepatic cells was studied in 40 human embryos and fetuses from 4 to 16 weeks of intrauterine development. Depending on the differentiation degree of organells and their amount in the hepatic cell of the embryo and fetus three types of hepatic cells are distinguished: slightly differentiated, transitional and differentiated cells. In the course of early prenatal ontogenesis the amount of slightly differentiated hepatic cells diminishes and the amount of transitional and differentiated forms increases. Glycogen granules are found in the embryo hepatic cells from 7-8 weeks of intrauterine development, and in the course of further embryonic development one can speak of establishment of the protein-forming function of the liver on the basis of increased amount of elements of the endoplasmic network and the presence of numerous ribosomes and polysomes in the cell. The hepatic cells of 14-16 week embryos have well pronounced sinusoid and biliar poles.


[Dark and light neurons in the cortical plate of the forebrain of the swamp turtle]. Electron microscopic study of the neuronal composition of Emys orbicularis cortical zones as compared with the light microscopy data has revealed that the distinctions in the cell cytoplasm ultrastructure with respect to the dendrite branching pattern and the peculiarities of their localization in the cortical plate are suggestive of a possible metabolic heterogeneity and can serve as a foundation of the morpho-functional characteristics of the neurons. A few neuronal types of neurons have been revealed: 1. light neurons distinguished by the cytoplasm matrix devoid of any electron density, a poorly developed endoplasmic reticulum and a few cytoplasmatic organells and inclusions; 2. light neurons having slight osmophility of the cytoplasm matrix, a more developed endoplasmic reticulum with elongated cisternae, a great number of organells including neurofilaments; 3. dark neurons having a high degree of cytoplasm matrix and the nucleus, a large number of the nuclei, cytoplasm, dendrites and axons in contrast to the above described patterns.


[Ultrastructure of experimental virus-induced hemangiopericytomas]. Electron-microscopy studies of malignant low-differentiated hemangiopericytomas, induced by adenovirus of monkeys SA7(C8) and bovine adenovirus type 3 were, carried out. At earlier stages of the development of these tumours in tumourous cells located around capillaries there were observed the ultrastructural characteristics typical of pericytes and cells of human hemangiopericytis: the presence of the basal layer and fibrills as thick as 40 A. As differentiation proceeded, tumorous cells acquired an elongated form, and in some of them there appeared myofilaments typical of smooth-muscle cells, with local thickenings, as well as multitude of pinicytic vacules along the plasmatic membranes of cells. The foregoing confirms the assumption of hemangiopericytomas as a prestage of leio-(angioleio-) myosarcomas. Moreover, the tumour cells revealed the presence of cilia, fibrills as thick as 100A, 75A, annulate lamelae, and myelin-like formations.


[Basal cell adenomas of the salivary glands]. The author presents data on morphology and clinical features of basal-cell adenomas of the salivary gland (10 cases). Singling out this neoplasm into independent onconosological group seems reasonable since basal-cell adenoma not infrequently is erroneously diagnosed as cylindroma or mixed tumour of the salivary gland, which may lead to a wrong clinical prognosis and inadequate therapeutic measures. The clinical course of this tumour is benign. The main morphological feature of the tumour is a monomorphic character of cell elements, their palisade-like distribution over the periphery of individual tumour structures and a clear-cut delimination of the parenchyma from the stroma.


[Basophilic adenoma in combination with a prolapse of adenohypophyseal tissue through the capsule]. During the post-mortem examination of a 15-year patient who had died after the operation connected with incision of commissures a distorted form of the hypophysis was noted. Histological, histochemical and morphometric investigations showed that there was basophilic adenoma of adenohypophysis and prolapse of the tissue (beyond the borders of the adenoma) through a fistula against the background of changes characteristic of chronic diseases in childhood. Oxyphilic shift, moreover, was noted not only in the first zone of the adenohypophysis, but in other zones, as well as with a simultaneous increase in number of basophiles in I--III zones. Prolapse of the tissue, the latter being normal as far as the tumours growth was concerned, through the fistula took place at a considerable distance from the adenoma, which justifies the consideration of this process as a result of hyperplasia not connected with adenoma. Edges of the fistula bordering the prolapse, haemorrhages were to be seen.


Treatment of Cataplexy with Clomipramine. A new antodepressant drug, clomipramine hydrochloride, closely related to imipramine hydrochloride, was used to treat four patients suffering from cataplexy, sleep paralysis, and hypnagogic hallucinations. Attacks of cataplexy were associated with rapid-eye-movement (REM) electroencephalographic patterns. Cloripramine, in doses of 25 to 75 mg/day, completely stopped all attacks of cataplexy, sleep paralysis, and hypnagogic hallucinations within 48 hours of initial therapy. The patients have been free of symptoms for periods of 10 to 21 months. Side effects included impotence in the male patients, but no hematologic, cardiovascular, hepatic, or renal toxic effects were observed. Available evidence suggests that such drugs inhibit those brain stem systems that control the toxic components of REM sleep.


The influence of some fractions of egg yolk on the survival of ram spermatozoa at 5 degrees C. Ram spermatozoa were stored at 5 degrees C in diluents containing various fractions of egg yolk prepared by dialysis, ultrafiltration and ion-exchange chromatography. They survived storage best in the presence of components of egg yolk which were non-dialysable and were not filtered through membranes which retained substances of molecular weight greater than 100 000. The substances isolated in peak B of the ion-exchange chromatogram of whole egg yolk described by Seideman et al. (1969) gave greater protection than those from other fractions from this chromatographic system. These data indicate that the low-density lipoprotein fraction of egg yolk is the most likely source of protection to ram spermatozoa against the effects of storage at 5 degrees C.


Pyridine nucleosidase in bull semen I. Breed and sire differences in enzyme concentration. The concentration of pyridine nucleosidase which cleaves the nicotinamide-ribose bond in NAD and related compounds was assayed using the cyanide addition reaction in semen samples collected by artificial vagina from 246 bulls. Among the 205 sires of recognized dairy breeds the nucleosidase concentration ranged from 0 to 1470 units/ml semen. Of the dairy sires 18% produced semen containing no nucleosidase activity. Among the 41 sires of recognized beef breeds 54% had no nucleosidase and the highest concentration was 630 units/ml semen. Among 20 sires from each of which 5-17 collections were assayed, there were significant differences in average nucleosidase concentration. There was a discontinuous distribution in nucleosidase concentration among the 169 dairy sires producing semen with measurable enzyme activity. Results for 18 dairy sires each with from 4 to 16 sons showed that sires with no seminal nucleosidase produced more sons with no enzyme or lower concentrations of enzyme than sires with higher nucleosidase concentrations. It is suggested that the concentration of the nucleosidase in bull semen is simply inherited.


Mitochondrial changes, ion and water shifts in the cellular injury of ehrlich ascites tumor cells. Cellular injury was produced in Ehlrich ascites tumor cells by an inhibitor of the function of the cell membrane [10(-3M) parachloromercuribenzenesulfonic acid (PCMBS)] or by inhibitors of respiration (10(-4M) Antimycin A), glycolysis (10(-4M), iodoacetic acid, IAA), oxidative phosphorylation (10(-3M) 2,4 dinitrophenol, DNP) or by combinations of these in a medium with or without glucose. The contents of Na+, K+, Ca++, Mg++ and intracellular water in the treated cells and controls were correlated with the percentages of ultrastructural changes in the mitochondria of the cells. A moderate negative correlation (r = -0.66) was observed between K+ content of the cells and the condensation of mitochondria in selected experiments where mitochondrial swelling had not yet started. A moderate positive correlation (r = 0.44) was observed between mitochondrial condensation and Na+ content of the cells in the same experiments. K+ and Mg++ content of cells showed a moderate negative correlation (r = -0.58 and -0.65) with mitochondrial swelling in the material of all experiments and Ca++ content a moderate positive one (r = 0.58), but the Na+ content and intracellular water showed no correlation. The percentages of flocculent densities in mitochondria showed a moderate positive correlation (r = 0.43) with the Ca++ content of the cells and a moderate negative correlation (r = -0.61) with the magnesium content of the cells.


Experimental investigations into the pathogenesis of endocarditis due to shock. Generalized Shwartzman reaction (GSR) was induced in rabbits by two intravenous injections of endotoxin 24 hours apart. Consumption coagulopathy was found in all cases. Histological investigations showed an abacterial endocarditis (thrombotica or verruccosa simplex), comparable to the one seen in humans after shock, in 69% of the treated animals. Biopsies of mitral valve were investigated by electron microscopy. In the early stage of GSR the valvular alterations were characterized by an oedema of the entire valve, an increase in the number of microvesicles in the endothelial cytoplasm, and a marked enlargement of endothelial cell surface by numerous cytoplasmic projections. At a later stage autophagic vacuoles, often showing a destruction of their membranes, and degenerative changes in cytoplasmic organelles occurred in many cells of the valve. On the other hand there were cells in the endothelium, which presented a distinctly increased number of Golgi complexes and of cisternae of rough endoplasmic reticulum. In other regions ruptures of endothelial plasmalemma, as well as hemorrhages into the valvular stroma were observed. Thus predestined loci for thrombogenesis were formed. In conjunction with these structural changes three factors may be involved in the formation of endocarditis due to shock: 1. hypercoagulability in the course of consumption coagulopathy, leading to precipitation of fibrin and platelets in the circulating blood; 2. turbulences of the blood flow, which are produced by the closure of the heart valves, and favour thrombogenesis on valves: 3. direct changes of the endocardium during endotoxemia. Quantitative changes in the different compartments of intracytoplasmic organelles in many cells of the endothelium during the later stage of GSR are discussed.


Malignant lymphoma resembling Burkitt's tumour in rhesus monkeys: (light- and electron microscopic studies). Spontaneous tumours of the haematopoetic system have been only rarely observed in nonhuman primates. We had the opportunity to carry out light and electron microscopic investigations of three lymphomas in immature rhesus monkeys from our colony. All animals had tumours of both retroperitoneum and lymph nodes. The relatively uniform lymphoblastic or lymphoreticular tumour cells as well as the massive occurence of the so-called "starry sky macrophages" were histologically characteristic. Histochemically, the marked pyroninophilia of these tumour cells was evident. The electron nicroscopy revealed the tumour cells as lymphoblasts of about 8 mu diameter with the following features: round or oval, occasionally deeply indented cell nuclei with relatively clear interchromatinic substance; mostly well developed nucleoli; sparsely, rough endoplasmatic reticulum; large numbers of free ribosomes and polyribosomes; a few large, bizzarre mitochondria, often situated at one pole; lipid vacuoles in some cells. These histological, cytological and electron microscopic characteristics correspond to those of the human Burkitt's Lymphoma. This tumour is classified as an undifferentiated malignant lymphoma. Tumours of this type are of particular interest on comparative and epidemiological reasons in light of the existence of morphologically similar experimental, virus induced lymphomas in monkeys.


Thermography and rheumatic diseases.

Thermography and rheumatic diseases. The common factor in most of the rheumatic diseases is an arthritis. Radiometry and thermography have been shown to indicate and measure heat resulting from localised inflammation. In rheumatoid arthritis, juvenile arthritis, osteoarthrosis, gout and ankylosing spondylitis abnormal heat distribution has been recorded over affected joints. Experimental evidence has shown that temperature change reflects the inflammatory state of the joint, and that this may be used to measure the effect of therapy by oral, systemic and local drug therapy, and also surgery, i.e. synovectomy.


Esterase-6 (Es-6) in laboratory mice: hormone-influenced expression and linkage relationship to oligosyndactylism (Os), esterase-1 (Es-1), and esterase-2 (Es-2) in chromosome 8. Allelic differences at an esterase locus designated Es-6 exist between mouse strain C57 BL/6J and a laboratory stock of M.m. molossinus. Strain C57BL/6J has been assigned the allele Es-6a and M. m. molossinus the alternate allele Es-6b. Kidney expression of the electrophoretic esterase band controlled by the Es-6 locus is sex influenced, with increased activity apparently induced by testosterone. A four-point test cross established the gene order Os-Es-1-Es-6-Es-2 within a 10-cM segment on chromosome 8.


Initial expression of the genes for fructose 1,6-diphosphatase, malic enzyme, and aspartate aminotransferase in Japanese quail and chicken--quail hybrid embryos. The initial appearance of a number of enzymes involved in gluconeogenesis was investigated in the early embryogenesis of the Japanese quail (Coturnix coturnix japonica), the domestic chicken (Gallus gallus domesticus), and chicken-quail hybrids. Starch gel electrophoresis and enzyme-specific stains revealed genetic differences between muscle and liver fructose 1,6-diphosphatase (FDPase) as well as malic enzyme (ME) and mitochondrial aspartate aminotransferase (AAT) isozymes of the two species. ME and AAT were present in unfertilized unincubated eggs, indicating maternal storage of these enzymes. The initial expression of the paternally inherited genes in the hybrid occurred before oviposition in the case of ME, and between 12 and 18 hr incubation in the case of AAT. Initial expression of both parental sets of genes for FDPase occurred synchronously between 16 and 24 hr in the hybrid, corresponding to the time of initial appearance of this enzyme in the quail and chicken. Glucose 6-phosphate administration at 0 hr was found to cause no prevention or delay of initial enzyme activation. These results are interpreted in terms of early patterns of enzyme activation regulation and nutrition in the avian embryo.


Purification, properties and amino acid sequence of atypical cytochrome c from two protozoa, Euglena gracilis and Crithidia oncopelti. A basic cytochrome was isolated from the phytomastigophorean protozoan Euglena gracilis and a similar protein from the zoomastigophorean protozoan Crithidia oncopelti. In both cases chromatography on CM-cellulose in first the reduced and then the oxidized form proved to be an efficient means of purification. The two cytochromes can be classed in the cytochrome c family but they have certain atypical features. The alpha peak of the absorption spectrum is shifted towards the red and is asymmetrical. The pyridine ferrohaemochrome has an alpha-peak maximum intermediate between that of c-type cytochromes and proteins containing protohaem IX. The test for free vinyl groups was positive. The amino acid sequences of the two cytochromes were determined. Attention is drawn in the text to those parts of the evidence that are less satisfactory. Both sequences are homologous with the family of cytochrome c, but are unusual in having only one cysteine residue so that the haem is attached through only one thioether bond. Detailed evidence for the amino acid dequences of the two proteins has been deposited as Supplementary Publication SUP 50042 (70 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Wetherby, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5.


A study of the oxidation of butan-1-ol and propan-2-ol by nicotinamide-adenine dinucleotide catalysed by yeast alcohol dehydrogenase. 1. The kinetics of oxidation of butan-1-ol and propan-2-ol by NAD+, catalysed by yeast alcohol dehydrogenase, were studied at 25 degrees C from pH 5.5 to 10, and at pH 7.05 from 14 degrees to 44 degrees C, 2. Under all conditions studied the results are consistent with a mechanism whereby some dissociation of coenzyme from the active enzyme-NAD+-alcohol ternary complexes occurs, and the mechanism is therefore not strictly compulsory order. 3. A primary 2H isotopic effect on the maximum rates of oxidation of [1-2H2]butan-1-ol and [2H7]propan-2-ol was found at 25 degrees C over the pH range 5.5-10. Further, in stopped-flow experiments at pH 7.05 and 25 degrees C, there was no transient formation of NADH in the oxidation of butan-1-ol and propan-2-ol. The principal rate-limiting step in the oxidation of dependence on pH of the maximum rates of oxidation of butan-1-ol and propan-2-ol is consisten with the possibility that histidine and cysteine residues may affect or control catalysis.


A kinetic method to establish the specificity of spin labelling of macromolecules. A method has been devised for establishing the specificity of spin labelling of a macromolecule. Analysis of the rate of incorporation of spin label under different conditions permits the assignment of reacting groups to classes of differing reactivity. The method is illustrated for the enzyme glycogen phosphorylase b.


Effects of anions on a monomeric and a dimeric arginine kinase. 1. Some effects of anions on the rates of phosphoarginine synthesis by monomeric (lobster) and by dimeric (Holothuria forskali) arginine kinases are reported. 2. As with creatine kinase, acetate ions activate both enzymes: Cl- was also found to activate both although this was an inhibitor of creatine kinase. 3. NO3- inhibits the lobster enzyme. Inhibition is of the mixed type with respect to MgATP. Ki greater than Ki' and Ks greater than Ks' indicating that the presence of NO3- promotes the binding of substrate and vice versa. 4. NO3- alone has no effect on the difference spectrum of the lobster enzyme but in the presence of arginine, MgATP, MgADP, MgAMP or MgIDP the difference spectrum is greatly enhanced. A profound effect on the ionization state of tyrosine residues is inferred. 5. With the Holothuria enzyme low concentrations of NO3- activate in a manner that is competitive with arginine. Higher concentrations cause inhibition of the mixed type with respect to arginine in a similar manner to that found with MgATP for the lobster kinase. 6. Of a range of anions tested only NO3- and NO2- enhanced the inhibition of enzyme activity by MgADP, indicating the formation of a pseudo-transition-state dead-end complex, enzyme-arginine-NO3--MgADP. The effect was essentially independent of temperature with the Holothuria enzyme, but with the lobster enzyme was much less marked and temperature dependent. The difference may reflect the different stabilities of the monomer and dimer enzymes, although with neither arginine kinase is the stabilization of the dead-end complex as marked as is found with creatinine kinase.


Glutamine and asparagine as nitrogen donors for reductant-dependent glutamate synthesis in pea roots. Glutamine, in the presence of alpha-oxoglutarate, stimulates nicotinamide nucleotide oxidation by crude extracts of pea roots and leads to a reductant-dependent formation of glutamate. Commercially available asparagine also stimulates nicotinamide nucleotide oxidation in the presence of alpha-oxoglutarate, but the reaction causing the stimulation can occur in the absence of a reductant, is inhibited by transaminase inhibitors, and is additive to the glutamine reaction. The asparagine used was found to be contaminated with aspartate. Repurified asparagine, chromatographically free of aspartate, did not stimulate the rate of nicotinamide nucleotide oxidation, and it is probable that the original stimulation was due to aspartate contamination. It is concluded that pea-root glutamine (amide)-alpha-oxoglutarate aminotransferase (glutamate synthase), in common with the enzyme in leaves, is specific for glutamine as the N donor and alpha-oxoglutarate as the N acceptor. The significance of the enzyme in conjunction with glutamine synthetase in the assimilation of nitrate by roots is discussed.


The distribution and partial characterization of the serum apolipoproteins in the guinea pig. 1. Very-low-density (VLD), low-density (LD) and high-density (HD) lipoproteins were isolated by sequential ultracentrifugation from the serum of male guinea pigs fed on a diet containing 3--4% fat. The apoproteins of these lipoproteins (apo-VLD, apo-LD and apo-HD lipoproteins) were studied after delipidation with organic solvents or extraction with tetramethylurea. 2. The major apolipoprotein of LD lipoprotein isolated by gel filtration was found to closely resemble apolipoprotein B of human serum in its chemical and physical properties. Electrophoresis in sodium dodecyl sulphate-polyacrylamide gel showed that this apoprotein consisted of a number of polypeptides. 3. Tetramethylurea precipitated an apoprotein from guinea-pig serum lipoproteins that is probably the apolipoprotein B-like component. This apoprotein accounted for about 80% of the apo-LD lipoprotein, about 55% of the apo-VLD lipoprotein and about 50% of the apo-HD lipoprotein. 4. The distribution of apolipoproteins soluble in tetramethylurea was determined by densitometric scanning of stained polyacrylamide disc gels. 5. A glycine-rich component of high electrophoretic mobility (band I) and a triplet of soluble apolipoproteins (bands II-IV) were present in both VLD and LD lipoprotein classes. These components constituted a higher proportion of the tetramethylurea-soluble apoproteins of VLD lipoprotein (60--80%) than of LD lipoprotein (40--55%). 6. Small amounts (10--15%) of a component of intermediate mobility, which contained traces of half-cystine, were also present in both VLD and LD lipoproteins. 7. A group of soluble components of basic character (bands VI-X), present as minor components of VLD lipoprotein (10--20%), constituted a major proportion (30--45%) of the soluble apoproteins of LD lipoprotein. Two of these apoproteins were rich in lysine, and two of lower electrophoretic mobility were rich in arginine. 8. The pattern of tetramethylurea-soluble apoproteins in HD lipoprotein was distinguished by the presence of two polypeptides of low electrophoretic mobility as its predominant components. One of these components, band VI, resembled the A-I apolipoprotein of man in both its amino acid profile and in its electrophoretic mobility. The second major component, band VI-B, was rich in lysine and resembled the C-I apolipoprotein of man in amino acid composition. 9. The soluble components of bands I and IX were analogous in physicochemical properties to the R-X1 and R-X2 (high-arginine polypeptide) peptides of human serum lipoproteins respectively.


Kinetic studies on the hydroxylation of p-coumaric acid to caffeic acid by spinach-beet phenolase. 1. A spectrophotometric assay is described that enables the hydroxylation of p-coumaric acid to caffeic acid, catalysed by spinach-beet phenolase, to be followed continuously. 2. Initial-velocity and inhibitor studies indicate that the order of substrate addition is oxygen, p-coumaric acid and electron donor, with an irreversible step separating the binding of each substrate. 3. Caffeic acid is most likely to act as electron donor at the active site; other electron donors, such as ascorbic acid, NADH and dimethyltetrahydropteridine, function mainly to recycle cofactor amounts of caffeic acid. 4. A reaction scheme, consistent with these data, is proposed.


Studies on sex-organ development. Isolation and characterization of an oestrogen receptor from chick Müllerian duct. An oestradiol-binding macromolecule was observed in the left Müllerian duct of the 15-day female chick embryo. The embryonic receptor binds oestradiol with a high affinity and low capacity, having a Kd of 3.2 X 10(-9)M and a maximal number of sites of 5.45 fmol/10(6) cells in the left Müllerian duct. The receptor is protein in nature, as suggested by its susceptibility to proteolysis; in addition, it is organ- and steroid-specific. Judging by glycerol-gradient analysis, the hormone receptors in the cytosol are present in 8S and 4.5S forms, and the 8S form could be dissociated into a 4.5S form in the presence of 0.5M-KCl. A 4.5-6S receptor could be extracted from the nuclei. Under physiological salt conditions, the embryonic receptors bind to DNA-cellulose and can be eluted when the salt concentration is increased to 0.5M-KCl. Determination by isoelectric focusing indicates that the isoelectric point is 5.8 for the 8S and 6.9 for the 4.5S receptor.


Studies on sex-organ development. Ontogeny of cytoplasmic oestrogen receptor in chick Müllerian duct. Oestradiol receptors were observed in the cytoplasm of the chick Müllerian duct at several embryonic stages. The sedimentation coefficients and the dissociation constants of the receptor protein remained unchanged throughout the various stages of development. Specific binding of cytoplasmic receptor to [3H]oestradiol assayed in vitro was shown to be saturable at concentration of 10nM or higher. The number of oeastradiol-binding sites on a per-cell basis increased linearly from day 8 to day 12 of incubation and then levelled off from day 12 to the fourth day after hatching. These results indicate that in the developing embryonic sex organ, the same receptor protein is present throughout prenatal development. The concentration of the oestradiol receptor increases and reaches a constant value, but the capacity for the receptor to interact with the hormone does not change.


Glucocorticoids and the regulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in the rat. The effect glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP)(EC4.1.1.32) in rat liver and kidney in vivo was studied immunochemically. The glucocorticoid analogue triamcinolone (9alpha-fluoro-11beta, 21-dihydroxy-16alpha,17alpha-isopropylidenedioxypregna-1,4-diene-3,20-dione) increased the synthesis rate of the kidney enzyme in starved animals. Both triamcinolone and cortisol decreased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP) in fed and starved rats, but were without effect on the degradation rate of the enzyme. This effect of triamcinolone in liver was reversed by injection of dibutyryl cyclic AMP. However, in diabetic animals glucocorticoids increased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP). Triamcinolone administration to starved rats in vivo is shown to cause an increase in the portal blood concentrations of insulin and glucose. Since the physiological de-inducer of liver phosphoenolpyruvate carboxykinase (GTP) is insulin, this is the probable cause of the decrease in the synthesis rate of the hepatic enzyme noted when glucocorticoids are administered to non-diabetic animals.


Progesterone synthesis of pig corpus luteum tissue during superfusion. A modified superfusion technique is described with which it was demonstrated that the action of gonadotrophin on progesterone secretion by pig corpus luteum tissue is twofold, in that it first stimulates the rapid release of progesterone (either performed or partially synthesized), which is followed by prolonged synthesis of the steroid de novo from acetate.


[The mechanism of action of bencyclane on smooth musculature]. Bencyclane (N-[3-(1-benzyl-cycloheptyloxy)-propyl]-N,N-dimethyl-amine-hydrogenfumarate, Fludilat), inhibits phosphodiesterase(PDE)-activity in vitro similarly to several other smooth muscle relaxants. Compared with papaverine this inhibitory effect of bencyclane on PDE is weak despite of its strong relaxant effect on smooth muscle, which is about equal to that of papaverine. 14C-Bencyclane is accumulated 8-fold in the smooth muscle tissue of bovine coronary arteries, indicating that relaxation is caused by 8-fold higher concentrations in the tissue than in the organ bath. A comparison of (corrected) ED50-values for relaxation with Ki-values for PDE-inhibition obtained with several PDE-inhibitors, including bencyclane, yields a significant correlation between both parameters. Since in subsequent studies in isolated tracheal muscle strips bencyclane at maximum relaxing concentrations did not increase cAMP, which was in contrast to the actions of papaverine or aminophylline, it is likely that bencyclane-induced smooth muscle relaxation is unrelated to inhibition of PDE or cAMP. In the same dose range in which bencyclane relaxes smooth muscles it exerts a non-specific antiadrenergic inhibitory effect, possibly due to its local anesthetic action at the cell membrane. It is also possible that the myocardial inhibitory effect of bencyclane is caused by a direct Ca++-antagonistic mechanism (Fleckenstein et al. 1971).


Inhibition of platelet cyclic 3',5'-adenosine-monophosphate phosphodiesterases by pentifylline. Pentifylline (1-hexyl-3,7-dimethylxanthine equals SK 7, the main active principle in Cosaldon) inhibits the soluble and the particulate cyclic AMP phosphodiesterases (PDE I and PDE II) from bovine platelets. I50 PDE I equals 2.0 x 10(-3)M; I50 PDE II equals 8.1 x 10(-4)M. The type of inhibition of both PDE's was non-competitive.


[The mode of action of bisacodyl on the smooth muscle of the small and the large intestine of the guinea pig]. 4,4'-Diacetoxy-diphenyl-(pyridyl-2)-methan (Bisacodyl, Dulcolax, 12--240 mug/ml) was shown to initiate dose-dependently contracile responses in the guinea pig isolated terminal ileum and taenia coli (a preparation of pure longitudinal muscle fibers). In contrast to muscle contractions induced by 1 mug/ml acetylcholine (ACh) and 1 mug/ml histamine, respectively bis-acodyl-induced contractile responses were antagonized neither by the anticholinergic agent atropine (6 mug/ml) nor by the antihistaminic compound pheniramine (6 mug/ml). On the other hand, bisacodyl inhibited contractile responses induced by ACh or histamine in a dose-dependent manner. The possibility that bisacodyl-induced contractions were due to a fall in cyclic 3,5-AMP level was excluded by estimation of endogenous cyclo-AMP. Since exogenous cyclic 3,5-AMP (which should possess calcium-antagonistic properties in smooth muscle) as well as verapamil (a calcium inhibitor) inhibited bisacodyl-induced contractions, a site of action on the calcium-dependent contractile system of the smooth muscle cell was discussed. Furthermore, bisacodyl should diminish the sensibility of the contractile system to other contractile compounds (ACh, histamine).


Enhancement of phagocytosis by Compound 48/80 and its influence upon Ca++-dependent ATP-ase. A new activity of p-methoxy-phenylethyl-methyl-amine condensed by formaldehyde (Cpd 48/80) has been established: enhancement of phagocytosis. This property seems to be correlated to its ability to activate lysosomes. Enhancement of phagocytosis is discussed in relation to the virus penetration process. Furthermore it was found that Cpd 48/80 and to a smaller degree the dimer inhibit the Ca++-dependent ATP-ase (E. C. 3.6.1.3.) from human red blood cells. The enzyme from bovine myosin is not inhibited by the dimer, but somewhat more by poly-THIQ (7-methoxy-tetrahydroisoquinoline condensed by formaldehyde) than by Cpd 48/80. These properties are discussed in relation to a general hypothesis of virus-induced giant cell formation. Finally it has been shown that the drug does not influence the PHA-stimulation (phytohemagglutinin) of lymphocytes, indicating selectivity of its activity.


Actions of various methylxanthines and papaverine on the synthesis of corticosterone in vitro. Actions of various methylxanthines (theophylline, theobromine, caffeine) and papaverine, i.e. drugs which are known to inhibit phosphodiesterase (PDE), were studied on the basal and stimulated synthesis of corticosterone in vitro by using rat adrenal slices. When slices were incubated with methylxanthines, the synthesis of corticosterone was slightly increased. The order of potency, expressed as the efficacy (intrinsic activity) was: theophylline greater than caffeine greather than theobromine. Papaverine did not stimulate the synthesis. The synthesis stimulated by ACTH or the dibutyryl derivative of c-AMP (DBA) was reduced by all of the inhibitors of phosphodiesterase. The molar concentration of the inhibitors which reduced the stimulated steroidogenesis by 50% was lowest for papaverine, higher for theobromine and theophylline and highest for caffeine. Papaverine was active in concentrations of about 10(-5)M, while the methylxanthines were effective in concentrations of 10(-3)--10(-2)M. When the PDE-inhibitors were added to the incubate simultaneously with the stimulant (ACTH or DBA), there was a delay of 60 min before the synthesis was completely blocked. When the stimulant was added 30 min before the administration of an inhibitor, the inhibitory action was no more evident. The inhibitory action of theophylline, but not that of papaverine, was reversed by washing adrenal slices free from the inhibitor, before adding the stimulant. The type of inhibition for papaverine as well as for methylxanthines in antagonizing both the ACTH- and DBA-stimulated synthesis of corticosterone was not competitive but of a mixed type.


Response of plasma lipid fractions to the administration of exogenous phospholipids. Plasma lipidic fractions (total lipids, free and total cholesterol, triglycerides, total phospholipids and low density lipoproteins) of 16 hyperlipemic subjects have been followed up over a 4-week period. Intravenous injection of exogenous phospholipids prepared from pig heart resulted in a significant decrease of all the examined lipidic fractions. The possible mechanims of exogenous phospholipids action is discussed.


[Controlled application of cynarin in the treatment of hyperlipemic syndrome. Observations in 60 cases]. In a controlled trial 2 groups of 30 patients presenting various dislipidemic pictures were treated for 50 days with either 3,5-dihydroxy-1,4-bis-(3,4-dihydroxy-cinnamoyloxy)-cyclohexan-carbonic acid-(1) (cynarin, Listrocol) or placebo. Cynarin provied to be able to induce a significant reduction of the hypercholesteremia, the level of pre-beta-lipoproteins, the beta/alpha-lipoprotein ratio and patients' body weight. The administration of cynaria is, therefore, deemed useful in the prophylaxis and therapy of dislipidemic pictures of the arteriosclerotic disease.


The relation of immune response to pathogenesis, vaccination and epidemiology in virus induced leukaemia. The antigenic systems of oncornaviruses and particularly feline leukaemia virus (FeLV) are reviewed briefly. The use of immunological methods in studying the epidemiology of the disease is described. The incidence of FeLV infection as judged by a serological survey is at least 100 times greater than that of leukaemia in the cat population. Horizontal transmission, due to virus replication in respiratory and alimentary epithelial cells, is common. A method of producing high titres of antibody against membrane antigens of virus infected cells is described; the use of such vaccination is discussed in relation to several epidemiological facets of feline leukaemia virus infection. Leukaemia viruses are well known to cause immunodepression to heterologous antigens. The hypothesis is advanced that depression of the humoral antibody response to leukaemia virus antigens and cell membrane antigens may be an early event allowing establishment and replication of virus in haemic and the lymphatic tissues. Subsequent depression of cell mediated immunity through direct action of thymic cells is known to take place in the cat system. This may allow further spread of the virus with replication in epithelial cells which are not susceptible to cytotoxic action. Thus the primary events leading to leukaemogenesis may be an interplay between immunostimulation and immunodepression.


Marek's disease--the disease and its prevention by vaccination. Marek's disease (MD) is a common lymphoproliferative disease of the domestic chicken caused by a cell associated herpesvirus. Vaccines used for the prophylaxis of MD have been derived from non-pathogenic field Marek's disease virus (MDV), pathogenic MDV and the herpesvirus of turkeys (HVT). Vaccines derived from MDV have been developed by attenuation of virus strains which produce the acute form of MD and virus strains which produce the classical form. They have also been developed by suitable modification, where necessary, of non-pathogenic MDV. All MDV derived vaccines have to be used in the cell associated form. The turkey herpesvirus vaccine can be used in either the cell associated or cell free lyophilized form. All these types of vaccine appear to be safe and to provide significant levels of protection under field conditions.


On the lack of response to catecholamine stimulation by the adenyl cyclase system in psoriatic lesions. When epidermis from the uninvolved skin of psoriatic patients was incubated for 5 min in Hank's medium containing adrenaline and theophylline, the cyclic AMP level consistently increased 20-30 times over the level observed when adrenaline was not added to the medium. On the other hand, when epidermis from the involved skin of psoriatic patients was incubated under the same experimental conditions, the cyclic AMP level increased only 2-5 times. Even when theophylline, and inhibitor of specific cyclic AMP-phosphodiesterase, was omitted from the medium, a clearly demonstrable difference in sensitivity to adrenaline was evident in normal appearing and lesional psoriatic epidermis. These results indicate a faulty adenyl cyclase system in the involved epidermis of psoriatic lesions rather than a defective degradation process by the specific phosphodiesterase. Since the Km for adrenaline activation of adenyl cyclase was approximately the same in both the uninvolved and the involved epidermis and since the cyclic AMP increase by adrenaline was abolished by the addition of propranolol, the basic nature of the beta-receptor (specifically the binding affinity to adrenaline) in the involved epidermis does not appear to be defective. On the other hand, the finding that the Vmax for adrenaline activation is 10-20 times higher in the uninvolved than in the involved epidermis suggests that the poor response in the involved epidermis may be due to fewer available binding sites for adrenaline in the psoriatic lesion.


Chloroperoxidase: P-450 type absorption in the absence of sulfhydryl groups. The oxidation state of the two half-cystine residues in the native ferric form of chloroperoxidase and in the reduced ferrous chloroperoxidase has been examined in order to evaluate the role of sulfhydryl groups as determinants of P-450 type spectra. Mössbauer and optical spectroscopy studies indicate that the ferrous forms of P-450cam and chloroperoxidase have very similar or identical heme environments. Model studies have suggested that sulfhydryl groups may function as axial ligands for developing P-450 character. However, chemical studies involving both sulfhydryl reagents and amperometric titrations show that neither the ferric nor the chemically produced ferrous forms of chloroperoxidase contain a sulfhydryl group. These results rule out the hypothesis that sulfhydryl groups are unique components for P-450 absorption characteristics. The optical and electron paramagnetic resonance (EPR) spectra of the nitric oxide complex of chloroperoxidase have been obtained and compared to those of myoglobin, hemoglobin, and cytochrome c and horseradish peroxidase. The EPR spectrum of the NO-ferrous chloroperoxidase complex, which is similar to that of cytochrome P-450cam, does not show the extra nitrogen hyperfine structure which appears to be characteristic of those hemoproteins which have a nitrogen atom as an axial heme ligand.


Infrared linear dichroism investigations of deoxyribonucleic acid complexes with poly(L-arginine) and poly(L-lysine). Complexes between DNAs from various sources and poly(L-lysine) and poly(L-arginine) were studied by means of infrared linear dichroism. The measurements of dichroic ratios allowed us to determine the orientation of the phosphate group of DNA in the complexes with basic polypeptides. At high relative humidities (higher than 90%, B form), the bisector of the less than OPO in the complexes forms an angle with respect to the helical axis which has a value lower by about 4 degrees than in the corresponding DNA sample. This change of orientation of the phosphate group of DNA indicates a modification of the B form upon binding of polylysine or polyarginine. The structural transitions B leads to A and B leads to C measured as a function of relative humidities were not affected by formation of complexes with both basic polypeptides. Similar results were obtained for complexes prepared by direct mixing or by salt gradient dialysis. The presence of A and C forms was observed in complexes of DNA with poly(L-lysine) and poly(L-arginine) at lower relative humidity. Thus, the conformational flexibility of DNA in complexes with polylysine and polyarginine is not changed despite a substantial increase in the Tm (melting temperature). These results are considered as a model for the understanding of interactions between DNA and histones particularly of the binding of the N-terminal fragment, lysine or arginine rich.


Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose. SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.


8-Substituted derivatives of adenosine 3',5'-cyclic phosphate require an unsubstituted 2'-hydroxyl group in the ribo configuration for biological activity. Derivatives of adenosine 3',5'-cyclic phosphate (cAMP) with modifications in both the 2' and the 8 positions were synthesized and their enzymic activities as activators of cAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterases were determined. Three types of derivatives were investigated: 8-substituted derivatives of O2'-Bt-cAMP, 8-substituted derivatives of 9-beta-D-arabinofuranosyladenine 3',5'-cyclic phosphate (ara-cAMP), and 8-substituted derivatives of 8,2'-anhydro-9-beta-D-arabinofuranosyladenine 3,'5'-cyclic phosphate (8,2'-anhydro-cAMP). The 8-substituted O2'-Bt-cAMP derivatives were synthesized by acylation of the preformed 8-substituted cAMP (8-HS-cAMP, 8-MeS-cAMP, and 8-PhCH2S-cAMP). 8-Br-O2'-tosyl-cAMP was sued as an intermediate for the preparation of 8,2'-anhydro-cAMP derivatives (8-HO-, 8-SH-, 8-H2N-, and 8-H3 CHN derivatives of 8,2'-anhydro-cAMP). 8-Substituted ara-cAMP derivatives were obtained by ring opening of 8-HO-8,2'-anhydro-cAMP with H+/H2O, NH3/MeOH, or MeONa/MeOH (to yield the 8-HO-, 8-H2N-, and 8-MeO-ara-cAMP derivatives). All of these doubly modified derivatives of cAMP are less than one-hundredth as active as cAMP at activating protein kinase and did not serve as substrates for the phosphodiesterase. These data show that the general inactivity of 2' derivatives of cAMP with kinase was not overcome by addition of an 8-substituent, even though many 8-substituted derivatives of cAMP activate the kinase more efficiently than does cAMP itself. In addition they show that while 2'-modification were tolerated by the phosphodiesterase, addition of an 8-substituent countermanded the allowable 2'-modification. The 8-substituted derivates of 02'-Bt-cAMP were found in general to be slightly better inhibitors of phosphodiesterase than the parent compounds containing no o2'-Bt substitution. As a group, the 8-substituted ara-cAMP derivatives were poorer inhibitors of phosphodiesterase than 8-substituted cAMP derivatives while the 8,2'-anhydro-cAMP derivatives were much poorer inhibitors than the 8-substituted ara-cAMP derivatives.


Conformational and functional studies of chemically modified cytochrome c: nitrated and iodinated cytochromes c. The purification of iodinated (E. B. McGowan and E. Stellwagen (1970), Biochemistry 9, 3074) and of nitrated (M. Sokolovsky et al. (1970), Biochemistry 9, 5113) cytochromes c resulted in the recovery from the former preparation of diiododityrosyl-cytochrome c (DIDT-) with modification of Tyr-67 and Tyr-74, and, from the latter, a mononitromonotyrosyl-cytochrome c (MNMT-), with modification of Tyr-67, and mononitrodityrosyl-cytochrome c (MNDT-), with the added modification of Tyr-48. The three purified preparations were conformationally characterized using pH-spectroscopy, circular dichroism, thermal denaturation, reducibility with ascorbate, autoxidation with molecular oxygen, and binding with CO. These results are related to the two aspects of biological function, reducibility, measured by NADH-cytochrome c reductase, and oxidizability, with cytochrome c oxidase, as well as to structure-function relationships in the protein. MNMT-cytochrome c was found to be, structurally and conformationally, a single isomer, reducible with ascorbate, with a small, but definite affinity for both oxidation with molecular oxygen and binding of CO. Conformationally, in both valence states of the metal atom, it represents a molecular form with native-like conformation with small but definite perturbations in the immediate vicinity of the heme group, reflected by the destabilization of the Met-80-S-Fe linkage. MNMT-ferricytochrome c exhibits a pK of 6.2 for the transformation of the low-spin, native-like spectral form II containing the 695-nm band to form lacking lacking the 695-nm band. The isomerization at pK = 6.2, when analyzed in terms of the isomerization of the native protein with a pK of 9.2 and the nature of the group involved, indicates that Tyr-67 is not involved in the isomerization of the modified preparation, and possibly not in the native protein as well. In terms of biological function, the partial derangement of redecibility (24%) and the unaltered oxidizability point to the functional significance of Tyr-67, and provide another example of selectivity between the two aspects of physiological functional function, in agreement with the two-function, two-path operational model of the protein. The MNDT- and DIDT-ferricytochromes c exhibited physicochemical properties indicative of gross derangement of both the conformation of the protein as well as of the coordination configuration of the metal atom. The complete inability to accept an electron from NADH-cytochrome c reductase in both cases, and the retention of 50% of the oxidizability property of DIDT-cytochrome c, were interpreted to be the result of conformational derangement, rather than the added modification of Tyr-48 or of Tyr-74.


Conformational flexibility of luteinizing hormone-releasing hormone in aqueous solution. A carbon-13 spin-lattice relaxation time study. The carbon-13 nuclear magnetic resonance (13C NMR) spectra of luteinizing hormone-releasing hormone (LH-RH) and lower homologous peptides have been assigned in aqueous solutions at various pH values. 13C spin-lattice relaxation times (T1) have been measured for all proton-bearing carbons at 25.2 and 67.9 MHz. From the T1 data the rates of overall molecular motion and intramolecular motion of side chains have been estimated. LH-RH is a flexible molecule in solution, having segmental motion along the backbone as well as in the nonaromatic side chains.


Electrophoretic mobilities of RNA tumor viruses. Studies by Doppler-shifted light scattering spectroscopy. We have used laser beat frequency light scattering spectroscopy to measure, at several pH values, the electrophoretic mobilities of purified avian myeloblastosis (AMV), murine leukemia (MuLV), murine mammary tumor (MuMTV), and feline leukemia (FeLV) viruses. The mobilities of these viruses are similar at pH greater than or equal to7 (-2.7 to -3.2 X 10(-4) (cm/sec)/(V/cm). The isoelectric points of MuLV and AMV are apparently less than pH 3, whereas for FeLV the data could be interpreted to indicate an isoelectric point between 3 and 5. Using a Debye-Hückel model to describe the interaction between electrolytes and virus, we show that our values for the mobility of MuMTV, obtained in ionic strength 0.005, are consistent with the values of Sarkar et al. ((1973), Cancer Res. 33, 2283), obtained in ionic strength of 0.10. This model is then used to calculate surface charge densities. In terms of the density of charged groups, the RNA tumor virus envelope is not very different from the erythrocyte membrane.


Isolation and characterization of the testosterone-estradiol-binding globulin from human plasma. Use of a novel affinity column. This report concerns the purification and characterization of the testosterone-estradiol-binding globulin (TeBG) from human plasma. Cohn fraction IV was submitted sequentially to ammonium sulfate preciptation, affinity chromatography, gel filtration, and isoelectric focusing. The final product was homogeneous in polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. Its activity was demonstrated by the finding of slightly more than one binding site/mole for dihydrotestosterone. Association constants (M-1) at 4 and 37degreesC were ascertained for three steroids: dihydrotestosterone; 2.4 x 10(9) and 0.99 x 10(9); testosterone, 1.1 x 10(9) and 0.35 x 10(9); estradiol, 0.60 x 10(9) and 0.22 x 10(9). TeBG is a glycoprotein having a molecular weight of 94000 and both the amino acid and carbohydrate content are presented along with other physical properties.


Manganese (II) And spin-labeled uridine 5'-diphosphate binding to bovine galactosyltransferase. The kinetically observed Mn(II) activation as well as inhibition has been clarified for bovine galactosyltransferase. An electron spin resonance (ESR) titration of MnCl2 with galactosyltransferase alone at pH 8.0 clearly shows the existence of at least two metal ion binding sites with microscopic dissociation constants of 0.84 +/- 0.1 and 9.0 +/- 1.0 mM, respectively. The second site corresponds with either published kinetic constant for Mn(II) of 8.5 mM (inhibition) or 3.40 mM (activation). The contribution of the binary complex Mn(II)-UDPGal is of lesser significance, as concluded by its ESR measured Kdiss of 14.5 +/- 1.1 mM at pH 8.0. A spin-labeled inhibitor analog of UDPgalactose, UDP-4-O-(2,2,6,6-tetramethyl-4-piperidinyl-1-oxy), or UDP-R, was synthesized as a competitive inhibitor for UDPGal. It was shown from inhibition kinetics to be almost as potent an inhibitor as UDPGlu. The Ki values at pH 8.0 in the N-acetyllactosamine and lactose reactions were 0.38 +/- 0.04 and 0.63 +/- 0.06 mM, respectively, as compared with 0.10 +/- 0.01 and 0.094 +/- 0.009 mM for UDPGlu. An ESR titration of UDP-R with galactosyltransferase at pH 8.0 yielded direct physical dissociation constants of 0.40 +/- 0.07 and 0.53 +/- 0.08 mM in the absence and presence of alpha-lactalbumin, respectively. No other substrates (glucose of N-acetylglucosamine) nor Mn(II) were present.


Hydrolysis of phosphonate esters catalyzed by 5'-nucleotide phosphodiesterase. 4-Nitrophenyl and 2-napthyl monoesters of phenylphosphonic acid have been synthesized, and an enzyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commerical calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NH4) 2SO4, and gel filtration. Detergent-solubilized enzyme from fresh bovine intestine was purified after (NH4)2SO4 fractionation by the same technique. The purified enzyme is homogeneous by polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, contains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homogeneous intestinal enzyme, an efficient catalyst of phosphonate ester hydoolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification was the ability of the enzyme to hydrolyze 5'-ATP to 5'-AMP and PPi, NAD+ to 5'-AMP and NMN, TpT to 5'-TMP and thymidine, pApApApA to 5'-AMP, and only the single-stranded portion of tRNA from the 3'-OH end. Snake venom 5'-nucleotide phosphodiesterase also hydrolyzes phosphonate esters, but 3'-nucleotide phosphodiesterase of spleen and cyclic 3',5'-AMP phosphodiesterase do not. Thus, types of phosphodiesterases can be conveniently distinguished by their ability to hydrolyze phosphonate esters. As substrates for 5'-nucleotide phosphodiesterases, phosphonate esters are preferable to the more conventional esters of nucleotides and bis(4-nitrophenyl) phosphate because of their superior stability and ease of synthesis. Furthermore, the rate of hydrolysis of phosphonate esters under saturating conditions is greater than that of the conventional substrates. At substrate concentrations of 1 mM the rates of hydrolysis of phosphonate esters and of nucleotide esters are comparable and both superior to that of bis(4-nitrophenyl) phosphate.


31 P nuclear magnetic resonance studies of the interaction of pyridine nucleotide coenzymes with dehydrogenases. 31P nuclear magnetic resonance spectra of the pyrophosphate group in NAD+ and NADH were recorded in the presence of beef heart lactate dehydrogenase and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. At high lactate dehydrogenase concentrations (60 mg/ml), two NADH resonances are observed: a slowly exchanging peak which is shifted to 1.9 ppm downfield (relative to free NADH) and a rapidly exchanging peak with a downfield shift of 0.5-0.6 ppm. At lover concentrations (15 mg/ml) only the rapidly exchanging peak is observed thus indicating that the peak observed at-1.9 ppm is due to coenzyme bound to an aggregated enzyme species. With NAD+, rapid exchange and downfield shifts are observed at both enzyme and concentrations, with shifts of about 1.5 ppm and 0.6 ppm at 60 and 15 mg/ml, respectively. In the presence of glyceraldehydephosphate dehydrogenase, the results are independent of enzyme concentration, and slow exchange and upfield shifts of 0.4-0.6 ppm occur with each coenzyme. These data indicate that the environment of the pyrophosphate group of oxidized and reduced coenzyme is the same for a given dehydrogenase, but is different in one enzyme from the other. The resonances observed with glyceraldehydephosphate dehydrogenase are broader than those observed with lactate dehydrogenase. This is indicative of either shorter relaxation times with the former enzyme, or the presence of multiple, unresolved resonances.


Polypeptide composition of cell membranes from chick embryo fibroblasts transformed by rous sarcoma virus. Chick embryo fibroblasts were transformed by the Bryan high-titer strain of Rous sarcoma virus (RSV-BH), or a mutant (RSV-BH-Ta) inducing temperature-dependent transformation. Surface membranes from normal and transformed cells were isolated as membrane vesicles by differential centrifugation, and as cell ghosts after ZnCl2 treatment and separation in an aqueous two-phase system. These preparations were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or phenol/urea/acetic acid. In general a greater resolution of individual bands was found in gels containing phenol/urea/acetic acid, which separates polypeptides on the bases of size and charge. Electrophoresis of preparations from nontransformed cells showed that two polypeptides (molecular weights 200 000 and 250 000) found in cell ghosts were missing in membrane vesicles. In cell ghosts, transformation by RSV-BH resulted in a significant decrease of the 250 000 molecular weight complex. Also a polypeptide (molecular weight 73 000) prominent in membrane vesicles from nontransformed cells was decreased in transformed cells. Surfaces from cells transformed by RSV-BH-Ta at 37 degrees C presented patterns similar to those for RSV-BH infected cells. Shifting these cells to 41 degrees C resulted in an increase in the 250 000 molecular weight complex, although the amount of this protein(s) never reached that found in noninfected cells. Inhibitors of RNA and protein synthesis failed to block the morphological changes occurring in RSV-BH-Ta cells after temperature shifts from 41 degrees C to 37 degrees C or vice-versa. The same inhibitors caused a reduction in the levels of the 250 000 molecular weight complex at both temperatures. These data indicate that these large membrane-associated polypeptides play little or no role in the morphological changes associated with transformation and its reversal.


The interaction of nitric oxide with ascorbate oxidase. 1. The reaction of nitric oxide with oxidized and reduced ascorbate oxidase (L-ascorbate: oxygen oxidoreductase, EC 1.10.3.3) has been investigated by optical absorption measurements and electron paramagnetic resonance, and the results are compared with those of ceruloplasmin. 2. Upon anaerobic incubation of oxidized ascorbate oxidase with nitric oxide a decrease of the absorbance at 610 nm is found, which is due to an electron transfer from nitric oxide to Type-1 copper. 3. In the presence of nitric oxide the EPR absorbance of ascorbate oxidase decreases and shows predominatly a signal with characteristics of Type-2 copper (g parallel = 2.248; A parallel = 188 G), whereas the type-1 copper signal has vanished. 4. Comparison of the intensities of the EPR signals before and after NO-treatment points to the presence of one Type-2 and three Type-1 copper atoms per molecule of ascorbate oxidase. 5. It is shown that the changes in the optical and the EPR spectrum of ascorbate oxidase induced by nitric oxide are reversible. No difference in enzymic activity is found between the native enzyme and the NO-treated enzyme after removal of nitric oxide.


Regulation of platelet phosphorylase.

Regulation of platelet phosphorylase. A sensitive fluorimetric enzyme assay was developed for study of activation of glycogen phosphorylase (EC 2.4.1.1) in intact platelets and in platelet extracts. Activity was calculated as AMP independent (activity in the absence of AMP), total (activity in the presence of 1 mM AMP), and AMP dependent (difference between AMP independent and total). The following observations were made with intact rat platelets. (1) Stimulation of platelets with thrombin caused a 7-fold increase in total activity, with increases in both AMP-dependent and AMP-independent activities. Maximum activation was obtained within 10 s after addition of thrombin. (2) The divalent cation ionophore A23187 caused a similar, though less pronounced, activation of phosphorylase. (3) Acceleration of glycogenolysis by inhibition of respiration with cyanide caused similar changes in phosphorylase activity but with the maximum effect observed only after 45 s. (4) Dibutyryl cyclic AMP had two effects; it partially activated phosphorylase and blocked further activation by thrombin, but not A23187. Similar effects were observed with human platelets, but low resting levels of phosphorylase activity could not be maintained so that changes were not as large as with rat platelets. Experiments with extracts of rat platelets gave the following results. (1) Phosphorylase activity in many extracts of non-stimulated platelets could be increased by incubation with Mg2+-ATP and Ca2+; ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) partially inhibited. (2) In some extracts there was essentially no activation by incubation with Mg2+-ATP and Ca2+, but addition of cyclic AMP GAVE PARTIAL ACTIVATIon while addition of rabbit muscle phosphorylase kinase gave full activation. (3) Incubation of extracts of thrombin-stimulated platelets caused conversion of AMP-dependent to AMP-indeptndent activity. It is concluded that platelet phosphorylase exists in an inactive and two active forms. Conversion of the inactive to the active forms and of the AMP-dependent to the AMP-independent form is catalyzed by a kinase(s) that requires Ca2+ for full activity and is activated through a cyclic AMP-mediated process. The major change following physiological stimulation is an increase in both active forms, with little change in their ratio.


Aspartokinase isoenzymes of the fruiting myxobacterium Myxoccus xanthus. Two isoenzymes of aspartokinase can be found in extracts of the differentiating bacterium Myxococcus xanthus. Aspartokinase I is repressed by L-lysine and feedback is inhibited by meso-diaminopimelate and by low concentrations of L-lysine. However, the inhibition by L-lysine is no longer observed at high concentration of this amino acid. Aspartokinase II is repressed and feedback inhibited specifically by L-threonine. Both enzymes are stimulated significantly by L-methionine and L-isoleucine; the effect is greater with aspartokinase I. The role of these enzymes in relation to growth conditions of the organism is discussed and a correlation with life cycle activity is indicated.


Multiple thermal discontinuities in glucose-6-phosphatase activity. The temperature dependence of glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase EC 3.1.3.9) was studied in rat liver and kidney microsomal fractions. Arrhenius plots were non-linear and showed four distinct discontinuities in enzyme activity over the temperature range 2-41 degrees C. The discontinuities occurred at approx. 39, 30, 20 and 12 degrees C in the liver and were similar to this in the kidney. Changes in the energy of activation for the enzyme were noted at approx. 20 degrees C in both tissues. The multiple discontinuities in glucose-6-phosphatase activity are viewed as a reflection of complex reorganization and/or change in physical state of the membrane components, primarily lipid.


Cyclic nucleotide phosphodiesterase of retinal photoreceptors. Partial purification and some properties of the enzyme. 1. A cyclic nucleotide phosphodiesterase (EC 3.1.4.16) has been partially purified from bovine rod outer segments. The enzyme preparation obtained has a very high specific activity towards cyclic GMP and is still able to hydrolyze cyclic AMP. Upon polyacrylamide gel electrophoresis, one major and three minor protein bands are seen, the enzyme activity being associated with the major band. The enzyme eluted from the gels still hydrolyzes both cyclic nucleotides. At all substrate concentrations tested, cyclic GMP was hydrolyzed at a faster rate. The enzyme eluted from the gel columns migrated as a single band upon electrophoresis in 0.1% sodium dodecyl sulfate-polyacrylamide gels corresponding to a molecular weight of 105 000. 2. A complex kinetic pattern was observed for cyclic GMP hydrolysis: the plot of velocity vs substrate concentration was hyperbolic at low and sigmoidal at higher concentrations. By contrast, simple kinetics were observed for cyclic AMP hydrolysis yielding an apparent Km of 0.1 mM. The unusual kinetics may be implicated in the regulation of cyclic GMP levels in rod outer segments. 3. Cyclic AMP stimulated the hydrolysis of cyclic GMP at low and inhibited it at higher concentrations. Addition of Mg2+ appeared to be necessary for optimum activity. The activity measured in the absence of exogenous Mg2+ was abolished by EDTA.


Cyclic nucleotide phosphodiesterases associated with bovine retinal outer-segment fragments. ATP-dependent cyclic GMP phosphodiesterase activity (EC 3.1.4.16) associated with bovine retinal outer-segment fragment preparations was stimulated an order of magnitude by light, confirming the results of Miki et al. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3820-3824 at Yale for the frog system. In contrast to the results of the Yale group, however, light stimulation was not observed for cyclic AMP as substrate. A direct relationship of bovine rhodopsin bleaching to phosphodiesterase activation differs from a previous report by the Yale group that full activation of the frog enzyme was achieved by bleaching of a maximum of 2% rhodopsin. Phosphodiesterase activity could be qualitatively removed from the fresh outer-segment preparations with isotonic sucrose which apparently did not disrupt the plasmalemma or discs. Activity recovered from the washing was not light sensitive. Two Km values were determined for cyclic AMP, 5 and 0.05 mM; for cyclic GMP a Km of 0.22 mM was found. All Km values were determined in the presence of 1 mM ATP in the dark. Sonication of fresh outer segments or storing at -20 degrees C abolished the light response. However, storage at -76 degrees C fully preserved it.


Classification of fructose-1,6-bisphosphate aldolases based on 18O retention in the cleavage reaction. Oxygen (18) was used as a mechanistic probe in the investigation of several different sources of fructose 1,6-bisphosphate aldolases (EC 4.1.2.13) which, due to differences in some physical and chemical properties, could not be clearly put in either Class I or Class II. Aldolases may be identified as belonging to a particular class on the basis of the amount of 180 retained in the dihydroxyacetone phosphate produced in the cleavage of [2-Oxygen (18)] fructose 1,6-biphosphate. The mechanism of Class I aldolases involves an obligatory exchange of the C-2 oxygen atom of fructose 1,6-bisphosphate, leading to the absence of 180 in the product. For Class II aldolases, the C-2 oxygen atom is retained in the aldol cleavage reaction. Aldolases from spinach and L. casei base intermediate. Aldosase from C. perfringens was found to be Class II, suggesting a metal-chelate intermediate. Results with Euglena aldolase confirmed that this organism contained both types of aldolases with approximately 78% Class II. The data show that despite a wide variety of physical and chemical properties, there are important mechanistic similarities within each class of enzyme and significant differences between the two classes. The determination of 180 retention in the product of the cleavage reaction using [2-180] fructose 1,6-biphosphate is an accurate means of classifying these enzymes since it is a measure of a property which is directly related to the mechanisms of the reactions.


Formation of adenosine 3',5'-monophosphate from adenosine in mouse thymocytes. The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3':5'-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E1, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E1 and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly within 1 min and was maximal by 10 to 20 min with approx. 2 and 10 muM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E1, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [14C]adenine exhibited dramatic increases in cyclic [14C]AMP 10 min after addition of adenosine or prostaglandin E1 which corresponded to simultaneously determined increases in total cyclic AMP. Using [14C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.


On the mechanism of action of cholera toxin on isolated rat adrenocortical cells. Comparison with the effects of adrenocorticotropin on steroidogenesis and cyclic AMP output. The effects of cholera toxin on isolated rat adrenocortical cells have been investigated. Both steroid and cyclic AMP output from adrenal cells were increased by the toxin in a dose dependent fashion. The concentration of toxin for half maximal stimulation for both of these responses was about 40 ng/ml. Maximal steroidogenesis and cyclic AMP output was obtained with similar concentrations of the toxin. A correlation was observed between the low amounts of cyclic AMP produced in response to all doses of cholera toxin and to physiologically significant concentrations of adrenocorticotropin (ACTH) (less than 0.1 munit/ml; i.e. submaximal for steroidogenesis in this system). This was in direct contrast to the much higher levels of cyclic AMP generated by concentrations of ACTH greater than 1 munits/ml. Time course studies demonstrated a time-lag between toxin addition and steroid response of at least 40 min. Binding of cholera toxin to adrenal cells was rapid and was 90% complete within 15 min at both 37 and 0 degrees C. These data indicate that most of the delay in response to cholera toxin is due to processes subsequent to the initial binding interaction. Following the initial delay the subsequent maximal rate of steroidogenesis brought about by cholera toxin was very similar to that obtained with a concentration of ACTH that was maximal for steroidogenesis. Significant increases in cyclic AMP levels were detected about 20 min before increased steroidogenesis was apparent. Possible explanations for this result are considered. The results presented indicate great potential use for cholera toxin in the study of adrenal steroidogenic control mechanisms, particularly at the level of receptor mechanisms and the role of cyclic AMP.


Prostaglandin E1 induced changes in cyclic AMP levels in WI-38 human fibroblasts. Two different effects of cell density. WI-38 lung diploid fibroblasts respond to protaglandin E1 with increased levels of cyclic adenosine 3':5'-monophosphate. This increase is affected by cell density in two ways: (a) The initial rate of accumulation of intracellular cyclic AMP increases with increasing cell density. (b) However, the elevated levels of cyclic AMP are more stably maintained in lower-density cells, and this stability decreases with increasing cell density. Cyclic AMP phosphodiesterase activities, as well as the efflux of intracellular cyclic AMP into the medium are similar at all cell densities.


Effect of calcium(ion) uptake by rat adrenal mitochondria on pregnenolone formation and spectral properties of cytochrome P-450. The effect of calcium on pregnenolone formation from endogenous precursors has been studied in mitochondria from rat decapsulated and capsular adrenal glands. In the presence of succinate, addition of calcium chloride in the concentration range 20-150 muM caused an inhibition of pregnenolone formation in both decapsulated and capsular adrenal mitochondria. 11beta-hydroxylation of added deoxycosticosterone in decapsulated adrenal mitochondria was also inhibited. Under these conditions, calcium inhibited the reduction of adrenodoxin, a component of the cytochrome P-450 reductase system, presumably because uptake of calcium by the mitochondria competes with energy-linked transhydrogenase for high-energy intermediates. For this reason, incubations were carried out in the presence of succinate plus isocitrate plus NADP+. Under these conditions, calcium chloride in the concentration range 120-875 muM caused a 2-4-fold stimulation of pregnenolone formation, but had no effect on corticosterone formation from added deoxycorticosterone. The effect of calcium on the optical spectra of cytochrome P-450 has also been examined in mitochondria from decapsulated and capsular rat adrenals. In the presence of succinate, calcium induced a spectral change resembling a type I difference spectrum of cytochrome P-450. Thus it appears that uptake of calcium by adrenal mitochondria can stimulate pregnenolone formation by increasing the interaction of mitochondrial cytochrome P-450 with endogenous substrate.


An adenosine 3',5'-monophosphate-requiring mutant of the luminous bacteria Beneckea harveyi. We have isolated a mutant of the luminous bacterium Beneckea harveyi, which requires exogenous adenosine 3',5'-monophosphate (cyclic AMP) to synthesize luciferase and emit light. The mutant was pleiotropic, lacking not only the ability to luminesce, but also the capacities to form flagella and the ability to utilize a variety of carbohydrates for growth. All these deficiencies could be corrected by added cyclic AMP. The cyclic AMP-induced de novo synthesis of luciferase was possible only after autoinduction had occurred. The induction time by cyclic AMP ranged between 6 and 10 min at 27 degrees C.


Phosphonopyruvic acid: A probable precursor of phosphonic acids in cell-free preparation of Tetrahymena. 1. In cell-free preparations of Tetrahymena, doubly labelled [32P]phosphoenol-[3-14C]pyruvate gives rise to 2-aminoethylphosphonate and 2-amino-3-phosphonopropionate, labelled with the two isotopes in the same ratio as the starting compound. The result is consistent with an intra-molecular rearrangement of phosphoenolpyruvate in the biosynthetic sequence of carbon-phosphorus bond formation. 2. Incubation of [32P]phosphoenolpyruvate with the same preparation, followed by treatment with 2,4-dinitrophenylhydrazine, yielded labelled hydrazones. When these were subjected to hydrogenolysis, the radioactivity was recovered in 2-aminoethylphosphonate and 2-amino-3-phosphonopropionate, suggesting that 2-phosphonoacetaldehyde and 3-phosphonopyruvic acid were probable precursors of the aminoalkylphosphonic acids. 3. Radioactivity from 2-amino-3-phosphono-[3-14C]propionic acid was incorporated into 2-aminoethylphosphonic acid, but incorporation of the radioactivity into lipids was negligible.


Induced changes in the electron paramagnetic resonance spectra of mammalian catalases. The EPR spectra of bovine liver catalase, rat liver catalase and human erythrocyte catalase have been measured at 9.0 degrees K. In N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) and Tris buffers at pH 7.0, the liver catalases show EPR spectra typical of rhombically distorted high spin ferric heme with major lines at g = 6.50, 5.35, 1.98. A number of extra lines are also seen; these are weak or absent in human erythrocyte catalase. The effect of the addition of formate, nitrite, acetate, fluoride, azide, hypophosphite and of inactivation with 3-amino-1,2,4-triazole on the degree of rhombic distortion has been studied. There is a good correlation between the low temperature EPR and room temperature optical changes for the binding of formic acid in HEPES and Tris. There is no evidence from EPR spectra for the presence of heme-heme interactions in the binding of formic acid to human erythrocyte catalase. The properties of catalase are altered in phosphate and in distilled water. This is a consequence of the low temperature of measurement.


Electron paramagnetic resonance spectra of catalase in mammalian tissues. The relatively small number of paramagnetic species and the high concentration of catalase in mammalian liver and blood make it possible to directly study this enzyme in frozen whole tissue. The EPR spectra of catalase are dependent on the heme environment and in human blood only catalase A, gxy = 6.48, 5.36 is observed whereas in liver a second spectrum, catalase B, gxy = 6.80, 5.07 can also be seen. Using rapid freeze techniques it has been shown that in rat liver catalase A corresponds to the in vivo steady state and that after death this is largely converted into catalase B. Data from the perfusion of rat livers with oxygenated and deoxygenated blood and dextran solutions together with results from in vitro studies of catalase are interpreted as indicating that catalase B results from the interaction of catalase with an organic acid, most probably formic acid, that the acid is a peroxidative substrate for catalase in vivo and that peroxidation of the acid is not the major role for catalase in rat liver. Catalase binding with other small molecules in intact liver has been demonstrated by perfusion with nitrite-containing dextrans and by intraperitoneal injection of 3-amino-1,2,4-triazole.


Magnetic studies of the four-iron high-potential, non-heme protein from Chromatium vinosum. Extensive EPR studies on high-potential, iron-sulfur protein from Chromatium vinosum indicate that the singular spectrum of this four-iron, non-heme protein consists of a superposition of three distinct signals; namely, two principal signals of equal weight, one reflecting axial and the other rhombic symmetry, and a third nearly isotropic minority component. In addition, magnetic susceptibility experiments on two oxidation states of the protein from 4.2 to approx. 260 degrees K indicate antiferromagnetic exchange coupling between iron atoms. Possible origins of the complex EPR signals are discussed, and a preferred model that is consistent with EPR, magnetic susceptibility, NMR, X-ray, and Mössbauer data is presented.


Characterization of the respiratory nitrate reductase of Klebsiella aerogenes as a molybdenum-containing iron-sulfur enzyme. 1. In respiratory nitrate reductase I of Klebsiella aerogenes, 0.24 atom of molybdenum, eight iron-sulfur groups and four tightly bound, non-heme iron atoms per molecule of enzyme (Mr 260 000) are found. 2. EPR spectra at 83 degrees K of oxidized and reduced nitrate reductase I show complex lines at g = 2.02 and g = 1.98, which are more intense in the reduced than in the oxidized enzyme. The resonances, the shape and intensity of which are rather temperature insensitive, are attributed to two species of paramagnetic molybdenum. In dithionite-reduced enzyme all these lines are saturated at the same microwave power of 15 mW. This is not the case in oxidized enzyme, where the resonance at g = 2.02 is hard to saturate. Addition of nitrate to dithionite-reduced reductase I decreases the intensity of the EPR lines to about that of oxidized enzyme. The participation of molybdenum in the electron transfer process has been discussed. 3. At 18 degrees K the oxidized enzyme exhibits an axial-symmetrical signal with g parallel = 2.10 and g = 2.03, and a signal with unknown symmetry at g = 2.015. Upon reduction by dithionite, a ferredoxin type of signal is observed with g values at 2.05, 1.95 and 1.88, while the g = 2.015 signal disappears. Reoxidation by nitrate causes a concomitant disappearance of the ferredoxin type of signal and reappearance of the g = 2.015 signal; hence iron-sulfur centres participate in the transfer of electrons to nitrate. 4. Nitrate reductase II, containing only two (Mr 117 000 and 57 000) of the three subunits found in nitrate reductase I and lacking the tightly bound iron, does not exhibit the axial-symmetrical signal (g = 2.10 and 2.03). Thus, it suggested that this signal in nitrate reductase I stems from an iron centre in the low-molecular weight subunit (Mr 52 000). 5. Inhibition studies confirm the participation of metals in the transfer of electrons from reduced benzylviologen to nitrate and show that the binding sites for these substrates are different.


Preparation of lutropin with acetyl or acetimidinyl substituents on the amino groups of the beta-subunit. The free amino groups in ovine lutropin beta subunit were acylated with acetic anhydride and methyl acetimidinate-HCl to produce the corresponding acetyl and acetimidinyl ovine lutropin beta derivative. These two derivatives recombined with ovine lutropin alpha as well as native ovine lutropin beta, but produced lutropin derivatives which were 33-50% less active than the ovine lutropin alpha + ovine lutropin beta in biological assays.


D-galactose accumulation in rabbit ileum. Effects of theophylline on serosal permeability. The effects of theophylline and dibutyryl cyclic AMP, on in vitro unidirectional galactose fluxes across the mucosal and serosal borders of rabbit ileum have been studied. 1. When Ringer [galactose] = 2mM, theophylline and dibutyryl cyclic AMP reduce both mucosal-serosal and serosal-mucosal galactose flux by approx. 50%. The K1 for theophylline inhibition of flux in both directions is 2 mM. 1 mM dibutyryl cyclic AMP elicits a maximal inhibitory response. Concurrent with the inhibition in transmural galactose fluxes, theophylline and dibutyryl cyclic AMP increase the tissue accumulation of [galactose] and the specific-activity ratio R of 3H : 14C-labelled galactose coming from the mucosal and serosal solutions respectively. It is deduced that theophylline and dibutyryl cyclic AMP are without effect on the mucosal unidirectional permeability to galactose but cause a symmetrical reduction in serosal entry and exit permeability. 2. Reduction in the asymmetry of the mucosal border to galactose by reducing Ringer [Na], raising Ringer [galctose] or adding ouabain reduces the theophylline-dependent increase in galactose accumulation. 3. Hypertonicity in the serosal solution increases the permeability of the serosal border to galactose and reduces tissue galactose accumulation. Serosal hypertonicity partially reverses the theophylline-depedent effects on galactose transport. Replacing Ringer chloride by sulphate abolishes the theophylline-dependent effects on galactose transport. 4. It is considered that the theophylline-dependent increase in galactose accumulation results from the reduction in serosal permeability. This is shown to be a quantitatively consistent inference. 5. Further support for the view that the asymmetric transport of galactose in rabbit ileum results from convective-diffusion is presented.


A comparison of the interfacial interactions of the apoprotein from high density lipoprotein and beta-casein with phospholipids. The conformations adopted by beta-casein and the total apoprotein from serum high density lipoprotein when spread at the air-water interface are compared; the monolayer data are consistent with the apoprotein being alpha-helical and the beta-casein being disordered with segments distributed in loops and trains. The penetration of these hydrophobic proteins into phosphatidylcholine monolayers in different physical states was investigated. More protein can penetrate into monolayers when they are in the liquid-expanded state; for penetration at constant total surface area the lateral compressibility of the lipid is an important factor. The charge and conformation of the polar group of the phospholipid does not have a major influence on the interaction. The mixed films of lipid and protein have a mosaic structure; probably the beta-casein is in a compressed state whereas the apoprotein is extended as alpha-helices in the plane of the interface. The chain-length depedences of the interaction of the apoprotein with phosphatidylcholine monolayers and bilayers are different; when the apoprotein binds to bilayers of shorter-chain phosphatidylcholines it alters the shape of the lipid-water interface whereas with monolayers the interface remains planar throughout.


Partial purification and characterization of DNA polymerases from a Rous sarcoma virus-transformed rat cell line. A new DNA polymerase was partially purified from cell-free extracts of a continuous rat cell-line (XC). The XC cells had been transformed by the Prague strain of Rous sarcoma virus but did not produce infectious virus. The molecular weight of the DNA polymerase is 70,000, as estimated by glycerol gradient centrifugation and by Sephadex gel filtration. This enzyme can be distinguished from the other cellular DNA polymerases by its elution pattern on DNA-cellulose column chromatography, its molecular weight, and its primer-template specificity. The enzyme has some characteristics of the murine leukemia virus reverse transcriptase. It is partially inhibited by immunoglobulin G purified from rabbit antiserum prepared against Rauscher leukemia virus reverse transcriptase, but is not inhibited by IgG from rat antiserum prepared against avian myeloblastosis virus reverse transcriptase. However, the XC cell enzyme can be distinguished from the murine leukemia virus reverse transcriptase by its inefficiency in copying an oligo(dG)12-poly(rC)primer-template.


Nuclear phosphoprotein kinase activities in normal and neoplastic tissues. Nuclear phosphoprotein kinases from normal rat liver and transplantable neoplasms were fractionated and compared. A phosphoprotein kinase fraction activated by Mn2+ was found to be present only in the neoplasms. This nuclear protein kinase phosphorylated nuclear proteins represented by one major and several minor bands as determined by polyacrylamide gel electrophoresis (M approximately 50,000).


Uptake of [3H] thymidine and cell DNA synthesis during the early multiplication phase of herpesvirus hominis in BHK cells. Experiments about the interaction of herpes viruses with BHK-cells during the first 6 h after infection concerning uptake and incorporation of dThd have been reported. During adsorption and penetration, the inhibition of uptake and of incorporation of [3H] dThd is sensitive to heat, but not to ultraviolet irradiation or cycloheximide. The eclipse is characterized by a strongly increased uptake of [3H] dThd and by inhibition of cell DNA synthesis. Both are sensitive to ultraviolet irradiation of the particles and cycloheximid treatment of the cells. It is concluded that the events during adsorption and penetration are dependent on the particles themselves, whereas the events during the eclipse depend on the activity of the viral genome. The implications of the findings are discussed.


Relation of cell type and cell density to the degree of post-transcriptional modification of tRNALys and tRNAPhe. An examination of the reversed-phase chromatographic profiles of tRNALys and tRNAPhe from SV40-transformed BALB/3T3 cells grown to different cell densities, untransformed BALB/3T3 cells grown to confluency and BALB/c mouse liver indicates that with increasing cell density in culture the degree of the peroxy-Y modification in tRNAPhe and an undetermined modification in tRNALys become more like that of differentiated tissue (liver). Because precursor/product relationships appear to exist among the unmodified and modified forms of the isoaccepting species for each of these tRNAs, the present findings support the view that the often reported differences in tRNA isoaccepting spectra result primarily from differences in post-transcriptional modifications, rather than from different tRNA transcripts.


Synthesis of viral proteins by the avian myeloblastosis viral core component. Avian Myeloblastosis Viral (AMV) core component was isolated and shown to synthesize AMV proteins in vitro. This reaction was linearly dependent on viral core concentration, proceeded linearly with time, and was inhibited by puromycin and aurintricarboxylic acid. The proteins synthesized in vitro co-electrophoresed and co-chromatographed with known proteins, and were immunoprecipitated by total and monospecific antibodies to known AMV proteins.