Purification and Synthesis under Anaerobic Conditions of Rice Arginine Decarboxylase

Arginine decarboxylase (ADC; EC 4.1.1.19 [EC] ) which catalyzes thesynthesis of putrescine, is involved in the responses of plantsto stress. The enzyme was purified 1,561-fold from rice coleoptilesby steps that included ammonium sulfate fractionation, gel filtration,ion-exchange chromatography and chromatofocusing. The purifiedenzyme had a pI of 5.3, a molecular mass of 176 kDa and appearedto be composed of three subunits of 63 kDa. A polyclonal antibodywas raised in a rabbit and the IgG fraction was purified fromserum. On Western blots the antibody recognized the ADC fromboth rice and E. coli. Immunoprecipitation with the ADC-specificantibodies allowed detection of radiolabelled ADC in extractsfrom aerobically and anaerobically grown rice seedlings thathad been supplied with a mixture of ¹⁴C-amino acids. This resultis discussed in relation to the role of ADC under anaerobicconditions. (Received April 28, 1994; Accepted September 27, 1994)     

Genetic diversity and molecular fingerprinting of Prunus cerasus var. austera from central Italy

In central Italy, Prunus cerasus var. austera is cultivated as small stands or scattered trees in marginal areas for the production of jam and wine. Thanks to the healthy attributes of its products and its ability to grow in different environmental conditions, this variety has gained new interest in the development of marginal areas. We assessed the level of the genetic variability of P. cerasus var. austera germplasm from central Italy and identified a ‘core collection’ representative of the present genetic diversity. A total of 161 trees, morphologically identified as var. austera, and one tree, identified as var. caproniana were collected and genotyped by 14 SSRs. Two individuals provided by a commercial plant nursery, one of P. cerasus var. caproniana and one of P. cerasus var. austera, were used as control. Thirteen SSRs presented private alleles in austera. Seven individuals morphologically identified as austera revealed private alleles specific to caproniana. The PCoA and Bayesian clustering analysis showed a main genetic group including var. austera, while a second group included all the caproniana-like genotypes. A core collection of 31 trees (46% of austera genotypes) was selected. This study can be considered as a starting point for future investigations on this variety.     

Polyamines and Anaerobic Elongation of Rice Coleoptile

The role of polyamines in the anaerobic elongation of rice (Oryzasativa L.) coleoptiles was studied. The reduced growth of ricecoleoptiles under anoxic conditions was accompanied by a massiveaccumulation of free putrescine. Putrescine was synthesizedfrom arginine in a reaction catalyzed by arginine decarboxylase(ADC). The anoxic titer of putrescine was closely correlatedwith elongation of coleoptiles. In experiments in which putrescineand inhibitors [-difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO)] of the synthesis of polyamines were exogenously supplied,we demonstrated an absolute requirement for putrescine, synthesizedby ADC, for anaerobic elongation of coleoptiles. The presenceof exogenous putrescine (alone or in combination with DFMA)increased the rate of anaerobic elongation of coleoptile by30–40%. (Received December 1, 1988; Accepted June 19, 1989)     

Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: Cholesterylphosphoryldimethylethanolamine

Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca²⁺. It has a K_i of about 30 m. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca²⁺-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.Abbreviation CPD cholesterylphosphoryldimethylethanolamine     

Accumulation and Interconversion of Amino Acids in Rice Roots under Anoxia

In excised rice roots, anaerobic degradation of proteins gaverise to an increase of free-amino acids. Anoxic accumulationof alanine, -aminobutyric acid and proline was the consequenceof the interconversion of glutamate, aspartate and amides. Theshift in the composition of the amino acid pool appears to becaused by changes in keto acid levels. The role of reactionsinvolved in amino acid interconversion and the physiologicalsignificance of these interconversions are considered and discussed. (Received November 25, 1988; Accepted June 9, 1988)     

An extended x-ray absorption fine structure study of the high-affinity cation-binding site in the purple membrane.

The structure of the high-affinity cation-binding site of bacteriorhodopsin was studied using extended x-ray absorption fine structure techniques. The results obtained for Mn2+ in aqueous solution and for the complex BR-Mn2+ (1:1 molar ratio) show great similarities, suggesting that Mn2+, when bound to this site, is coordinated with six atoms of oxygen, forming an octahedral disposition. The interatomic distance between the atoms of oxygen and the Mn2+ was found to be 2.17 A for the complex BR-Mn2+, similar to Mn2+ in solution (2.15 A). In addition, the absence of any other peak at greater distances in the Fourier-transformed spectrum indicates that neither phosphorus nor sulphur atoms are present in the second coordination shell. This suggests that this binding site is located in the protein, discarding the proximity of lipid polar headgroups.     

Effect of infusing branched-chain amino acid during incremental exercise with reduced muscle glycogen content

The aim of this study was to investigate whether, when muscle glycogen is reduced, a pre-exercise infusion of branched-chain amino acids (BCAA) modifies exercise performance or the metabolic and respiratory responses to incremental exercise. Six moderately trained volunteers took part in the following protocol on two occasions. On day 1, at 9 a.m. in the postabsorptive state, they performed a graded incremental exercise (increases of 35 W every 4 min) to exhaustion (Ex-1). A meal of 1,000 kcal (4,200 kJ; 60% protein, 40% fat) was consumed at 12 p.m. No food was then allowed until the end of the experiment (20–21 h later). A 90-min period of exercise at alternating high and moderate intensities, designed to deplete muscle glycogen, was performed between 6 p.m. and 7.30 p.m. The morning after (day 2), the subjects randomly received either a mixed solution of BCAA (260 mg × kg^–1 × h^–1 for 70 min), or saline. They then repeated the graded incremental exercise to exhaustion (Ex-2). Metabolic and respiratory measurements suggested a muscle glycogen-depleted state had been achieved. No significant differences were observed in total work performed, maximal oxygen uptake or plasma ammonia, alanine, and blood pyruvate concentrations in the two treatments. After BCAA infusion, higher blood lactate concentrations were observed at maximal power output in comparison with those during saline [BCAA 4.97 (SEM 0.41) mmol × l^–1, Saline 3.88 (SEM 0.47) mmol × l^–1,P < 0.05]. In summary, in conditions of reduced muscle glycogen content, after a short period of fasting, BCAA infusion had no significant effect on the total work that could be performed during a graded incremental exercise.     

Protection of Cells against Oxidative Stress by Nanomolar Levels of Hydroxyflavones Indicates a New Type of Intracellular Antioxidant Mechanism

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.     

Computational modelling of the binding of arachidonic acid to the human monooxygenase CYP2J2

An experimentally determined structure for human CYP2J2—a member of the cytochrome P450 family with significant and diverse roles across a number of tissues—does not yet exist. Our understanding of how CYP2J2 accommodates its cognate substrates and how it might be inhibited by other ligands thus relies on our ability to computationally predict such interactions using modelling techniques. In this study we present a computational investigation of the binding of arachidonic acid (AA) to CYP2J2 using homology modelling, induced fit docking (IFD) and molecular dynamics (MD) simulations. Our study reveals a catalytically competent binding mode for AA that is distinct from a recently published study that followed a different computational pipeline. Our proposed binding mode for AA is supported by crystal structures of complexes of related enzymes to inhibitors, and evolutionary conservation of a residue whose role appears essential for placing AA in the right site for catalysis.

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Graphical Abstract Arachidonic acid docked in the active site of CYP2J2 assumes a catalytically competent binding mode stabilised by hydrogen bonds to Arg117