Effects of Phosphorylation on Phosphoenolpyruvate Carboxykinase from the C4 Plant Guinea Grass

In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves. To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves. The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms. In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate. The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations. Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plant Urochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass. Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity. Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves. There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation

Phosphoenolpyruvate Carboxykinase Assayed at Physiological Concentrations of Metal Ions Has a High Affinity for CO2

The effect of Mn2+/Mg2+ concentration on the activity of intact, homogeneous phosphoenolpyruvate carboxykinase (PEPCK) from leaves of the C4 grass, Guinea grass (Panicum maximum), have been investigated. Assay conditions were optimized so that PEPCK activity could be measured at concentrations of Mn2+/Mg2+ similar to those found in the cytosol (low micromolar Mn2+ and millimolar Mg2+). PEPCK activity was totally dependent on Mn2+ and was activated at low micromolar concentrations of Mn2+ by millimolar concentrations of Mg2+. Therefore, at physiological concentrations of Mn2+, PEPCK has a requirement for Mg2+. Assay at physiological concentrations of Mn2+/Mg2+ led to a marked decrease in its affinity for ATP and a 13-fold increase in its affinity for CO2. The Km (CO2) was further decreased by assay at physiological ATP to ADP ratios, reaching values as low as 20 μM CO2, comparable with the Km (CO2) of ribulose 1,5-bisphosphate carboxylase-oxygenase. This means that PEPCK will catalyze a reversible reaction and that it could operate as a carboxylase in vivo, a feature that could be particularly important in algal CO2-concentrating systems

A mechanism for morphogen-controlled domain growth

Many developmental systems are organised via the action of graded distributions of morphogens. In the Drosophila wing disc, for example, recent experimental evidence has shown that graded expression of the morphogen Dpp controls cell proliferation and hence disc growth. Our goal is to explore a simple model for regulation of wing growth via the Dpp gradient: we use a system of reaction-diffusion equations to model the dynamics of Dpp and its receptor Tkv, with advection arising as a result of the flow generated by cell proliferation. We analyse the model both numerically and analytically, showing that uniform domain growth across the disc produces an exponentially growing wing disc

Local and global interactions in an evolutionary resource game

Conditions for the emergence of cooperation in a spatial common-pool resource game are studied. This combines in a unique way local and global interactions. A fixed number of harvesters are located on a spatial grid. Harvesters choose among three strategies: defection, cooperation, and enforcement. Individual payoffs are affected by both global factors, namely, aggregate harvest and resource stock level, and local factors, such as the imposition of sanctions on neighbors by enforcers. The evolution of strategies in the population is driven by social learning through imitation, based on local interaction or locally available information. Numerous types of equilibria exist in these settings. An important new finding is that clusters of cooperators and enforcers can survive among large groups of defectors. We discuss how the results contrast with the non-spatial, but otherwise similar, game of Sethi and Somanathan (American Economic Review 86(4):766–789, 1996)

The global burden of cancer attributable to risk factors, 2010–19: a systematic analysis for the Global Burden of Disease Study 2019

BACKGROUND: Understanding the magnitude of cancer burden attributable to potentially modifiable risk factors is crucial for development of effective prevention and mitigation strategies. We analysed results from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 to inform cancer control planning efforts globally. METHODS: The GBD 2019 comparative risk assessment framework was used to estimate cancer burden attributable to behavioural, environmental and occupational, and metabolic risk factors. A total of 82 risk–outcome pairs were included on the basis of the World Cancer Research Fund criteria. Estimated cancer deaths and disability-adjusted life-years (DALYs) in 2019 and change in these measures between 2010 and 2019 are presented. FINDINGS: Globally, in 2019, the risk factors included in this analysis accounted for 4·45 million (95% uncertainty interval 4·01–4·94) deaths and 105 million (95·0–116) DALYs for both sexes combined, representing 44·4% (41·3–48·4) of all cancer deaths and 42·0% (39·1–45·6) of all DALYs. There were 2·88 million (2·60–3·18) risk-attributable cancer deaths in males (50·6% [47·8–54·1] of all male cancer deaths) and 1·58 million (1·36–1·84) risk-attributable cancer deaths in females (36·3% [32·5–41·3] of all female cancer deaths). The leading risk factors at the most detailed level globally for risk-attributable cancer deaths and DALYs in 2019 for both sexes combined were smoking, followed by alcohol use and high BMI. Risk-attributable cancer burden varied by world region and Socio-demographic Index (SDI), with smoking, unsafe sex, and alcohol use being the three leading risk factors for risk-attributable cancer DALYs in low SDI locations in 2019, whereas DALYs in high SDI locations mirrored the top three global risk factor rankings. From 2010 to 2019, global risk-attributable cancer deaths increased by 20·4% (12·6–28·4) and DALYs by 16·8% (8·8–25·0), with the greatest percentage increase in metabolic risks (34·7% [27·9–42·8] and 33·3% [25·8–42·0]). INTERPRETATION: The leading risk factors contributing to global cancer burden in 2019 were behavioural, whereas metabolic risk factors saw the largest increases between 2010 and 2019. Reducing exposure to these modifiable risk factors would decrease cancer mortality and DALY rates worldwide, and policies should be tailored appropriately to local cancer risk factor burden

An Introduction to the Chemistry of heterocyclic Compounds

xvii.;ill.;501 hal.; 30 c

Growth factors and growth factor antagonists

The introduction consists of a brief review of the subject with special reference to antimalarial agents and haematopoietic factors. Part I. Some synthetic Biguanide derivatives. It has been suggested (Curd and Rose, Nature, 1946, 158, 707) that the activity of paludrine as an antimalarial is due to the interference of a dimeric form with a porphyrin enzyme system essential for the growth of the plasmodia. Hawking (Nature, 1947, 159, 409) also showed that paludrine was inactive as an antimalarial in vitro, but was converted into an unidentified biologically active material in vivo. Oxidation to a benziminazole is one possibility, and a series of 2-guanidinobenziminazoles, which are structurally very similar to paludrine, was synthesised for biological examination. The benziminazoles (1 to 15) were all made from the appropriate o-phenylenediamine dihydrochlorides and dicyandiamide, or isopropyl- or n-butyldicyandiamides in aqueous solution, and were usually purified through the picrates or copper derivatives. The hitherto undescribed alkylated dicyandiamides were prepared from sodium dicyanimide and the appropriate amine hydrochlorides in boiling n-butanol. These 2-guanidinobenziminazoles, as their hydrochlorides, were tested against F. gallinaceum in chicks, or P. relictum in canaries, but only two, 7 and 14, had even slight activity. These results showed that 11 could not be Hawking's biologically active metabolite and do not support Curd and Rose's theory. The bimolecular form of 11 is no less like the porphyrin ring system than the postulated paludrine dimer. None of the 2-guanidinobenziminazoles showed large bacteriostatic action against B. coli, or S. aureus broth cultures. Attempts to synthesise the 2-guanidinobenziminazoles in the reverse direction failed. 2-Cyanamino-benziminazole was not formed from o-phenylenediamine and dicyanimide, and when prepared according to Pellizzari (Gazzetta, 1921, 51, I, 140) could not be induced to combine with isopropylamine (experiments by P.C. Spensley). The product of the reaction of o-phenylenediamine with dicyanimide, 2;4-diamino-1:3:5-triazabenzepine (16), is a derivative of a new heterocyclic system. It was stable to dilute acid and alkali, and like its 1-methyl derivative (18) did not react with nitrous acid. A series of these benzepines was prepared from the appropriate diamines and dicyanimide, and one (20) is a possible paludrine metabolite. The compounds had no effect on the growth of B. coli or S. aureus broth cultures. Part II. Alloxazines and isoalloxazines. This was a continuation of earlier work by the author and Prof. F.E. King (J., 1946, 681), on the preparation of alloxazine derivatives for biological testing. Piloty's synthesis of alloxazines (Annalen, 1904, 333, 44) was extended to the isoalloxazine series, and violuric acid was found to react with 3-dimethylamino-β-diethylaminoethylaniline to give the corresponding isoalloxazine (21), isolated as the violurate. The constitution was proved by an independent synthesis from 2-amino-5-dimethylamino-β- diethylaminoethylaniline and alloxan, showing that the violuric acid condensation took place in the 4- position, rather than the 2- position of the benzene ring. The 3-dimethylamino-β-diethylaminoethylaniline was prepared from 3-dimethylamino-p-toluenesulphonanilide by alkylation and hydrolysis. The amine for the alloxan condensation was the reduction product of 2-nitro-5-dimethylamino-β-diethylaminoethylaniline, obtained from β-diethylaminoethylamine and 3:4-dinitrodimethylaniline. The reaction of violuric acid with 3-amino-N-methylaniline was investigated as this could give an alloxazine and/or an isoalloxazine, assuming that this condensation also takes place in a r- position of the benzene ring. A comparison of the ultra-violet absorption spectrum of the product with those of 7-dimethylaminoalloxazine, and 7-dimethylamino-9-methylisoalloxazine, prepared by Piloty's method, showed that it was inhomogeneous. Its sulphuric acid solution was therefore treated with sodium nitrite. The precipitated 7-(N-nitroso-[illegible]-methylamino)-alloxazine was collected and the filtrate gave an olive green azo-dye with β-naphthol in dilute alkali. From the respective yields of these products the ratio of alloxazine to isoalloxazine in the mixture was approximately 10:60. The first stage of the Piloty reaction is probably the attack of the 4-carbonyl of the violuric acid on an amino group of the diamine. If the first stage were the attack of the oximino group in violuric acid on the benzene nucleus the initial product would be an anil of a type that does not cyclise to an alloxazine derivative (Tischler, Wellman and Ladenburg, J. Amer. Chem. Soc., 1945, 67, 2165). Part III. The synthesis of some Benziminazoles. The reaction of phenylacetiminomethylether with o-phenylenediamine N to give 2-benzylbenziminazole was investigated. The free iminoether reacted with the diamine only at elevated temperatures to give poor yields of the benziminazole, but the reaction was acid catalysed. In the presence of one, or two equivalents of acid good yields of the benziminazole were obtained, but if three equivalents were used the yield dropped. This was also found with phenylacetiminothiobenzylether, and the reaction mechanism has been interpreted as an attack of the iminoether cation on an uncharged aromatic amino group. The reaction was extended to various mono-N-alkylated o-phenylenediamines with good results. Three benziminazoles (22–24) were prepared, for antimalarial testing, from the appropriate diamines by the Phillips method; 22 and 23 were inactive against P. gallinaceum in chicks, and the results for 24 are awaited. Acetiminothiobenzylether hydrochloride with 2-amino-5-chloro-γ-diethylaminopropylaniline also gave 23, but only small quantities of 22 were isolated from the reaction of acetiminomethylether hydrochloride with 2-amino-4-chloro-γ-diethylaminopropylaniline under various conditions. The main product of the reaction was provisionally regarded as 2-(N-acetamidino)-4-chloro-γ-diethylaminopropylaniline. In support of the idea that the electrophilic chlorine atom para to the secondary amino group in the amine was reducing benziminazole formation the corresponding 2-amino-4-methoxy-γ-diethylaminopropylaniline gave a much larger yield of 1-(γ-diethylaminopropyl)-2-methyl-4-methoxy-benziminazole with acetiminomethylether hydrochloride than the chloro derivative. Part IV. The synthesis of Pteroylglutamic acid and Related Compounds. The benziminazole analogue (25) of ethyl pteroate was prepared from chloroacet-2-nitroanilide, which reacted with ethyl p-aminobenzoate to give 4-carbethoxyphenylglycine-2-nitroanilide. On hydrogenation this gave the corresponding amine which cyclised to ethyl N-(2-benziminazole)-4-aminobenzoate (25) under acid conditions. Others completed work in this series, and details are in press (King, Spensley, and Nimmo-Smith, Nature, 1948). N-(2-quinoxaline)-4-aminobenzoic acid (26) was prepared from 2-chloroquinoxaline and p-aminobenzoic acid. It had little effect on the growth of S. lactis R. The pteroylglutamic analogue (28) was prepared by the hydrolysis of ethyl N4-(2-quinoxaline)-4-aminobenzoyl-l-glutamate (27), the condensate of 2-chloroquinoxaline and ethyl p-aminobenzoyl-l-glutamate. Its inhibitory action on the growth of S. lactis R was prevented by pteroylglutamic acid, and quantitative biological results are awaited. Many attempts to synthesise N-(quinoxaline-2-methyl)-4-aminobenzoic acid (29) and some of its derivatives were made. It was not found possible to prepare 2-bromomethyl-quinoxaline by any route examined, but quinoxaline-2-aldehyde reacted with p-aminobenzoic acid, and its ethyl ester, to give the corresponding anils. Nothing homogeneous could be isolated from the reduction of N-(quinoxaline-2-methylene)-4-aminobenzoic acid, but a poor yield of ethyl N-(quinoxaline-2-methyl)-4-aminobenzoate (30) was obtained from the reduction of the corresponding ester. One attempt at hydrolysis failed. Methylglyoxal on bromination gave [illegible], which reacted with 2:4:5-triamino-6-hydroxypyrimidine and p-aminobenzoyl-l-glutamic acid to give a product containing about 5% of pteroylglutamic acid. [For the figures to accompany this abstract, please consult the PDF]</p