Enhanced ability of skeletal muscle containing cyclocreatine phosphate to sustain ATP levels during ischemia following beta-adrenergic stimulation

Breast muscle of young chicks fed chow diets containing the creatine analog 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine) accumulated up to 40 mumol/g wet weight of the synthetic phosphagen 1-carboxymethyl-2-imino-3-phosphonoimidazolidine (cyclocreatine-P2-). ATP levels were sustained at high values substantially longer in breast muscle of cyclocreatine-fed chicks, compared to control-fed chicks, during total ischemia initiated 2 h after injection of both groups with the beta-adrenergic agonist isoproterenol (5 mg/kg subcutaneous). For example, in chicks fed 0.5% cyclocreatine for 10-19 days ATP levels in isoproterenol-stimulated breast muscles after 1 h of ischemia at 37 degrees C were 6.1 mumol/g, compared to 1.9 mumol/g for the control-fed group, and after 2 h of ischemia were 3.5 mumol/g compared to 0.6 mumol/g for controls. Creatine-P reserves in isoproterenol-stimulated breast muscles of all dietary groups were essentially exhausted within the first hour of ischemia. In contrast, breast muscle of chicks fed either 1 or 0.5% cyclocreatine still contained 28 and 19 mumol/g of cyclocreatine-P, respectively, after 1 h of ischemia; after 2 h of ischemia, the respective cyclocreatine-P values were 20 and 13 mumol/g. Isoproterenol-stimulated chick breast muscle provides the first skeletal muscle model system for studying the molecular mechanisms by which dietary cyclocreatine helps sustain ATP levels during ischemia. Although adaptive factors are also involved, it is suggested that a significant portion of the ATP-sustaining activity of dietary cyclocreatine in ischemic breast muscle can be attributed to the unique thermodynamic properties of the accumulated cyclocreatine-P. These properties enable cyclocreatine-P to continue to thermodynamically buffer the adenylate system and transport high energy phosphate throughout the long muscle fibers at cytosolic pH values and phosphorylation potentials well below the range where the creatine-P system can function effectively. Synergism between glycolysis and this long-acting synthetic phosphagen might well help delay depletion of ATP levels in skeletal muscles during ischemia. Cyclocreatine feeding provides a unique experimental tool for quantitative evaluation of the proposed protective role of ATP against irreversible cellular damage in skeletal and cardiac muscles during ischemic episodes.     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.     

Lipid composition of thylakoid membranes of cadmium treated wheat seedlings.

Cadmium (200 ppm) applied through the rooting medium to 30-day-old wheat plants decreased chlorophyll content, net CO2 exchanges and PSII activity by 34, 54 and 43% respectively. Thylakoid total lipids, total glycolipids, total phospholipids and total neutral lipids decreased by 22, 23, 12 and 25%, respectively, under cadmium treatment. Thylakoid membrane glycolipids had three major constituents, viz. monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulphoquinovosyl diacylglycerol. Monogalactosyl diacylglycerol and digalactosyl diacylglycerol contents decreased by 32 and 27%, respectively, under cadmium. Cadmium application also decreased the concentration of phosphatidyl glycerol and phosphatidyl choline to the extent of about 57 and 31%, respectively. On the other hand, phosphatidic acid and free fatty acids content showed an increase. These compositional changes in thylakoid membranes might be responsible for reduced PSII activity and rate of photosynthesis as observed under cadmium treatment.     

Chemical modification of spinach plastocyanin using 4-chloro-3,5-dinitrobenzoic acid: characterization of four singly-modified forms

Chemical modification of plastocyanin was carried out using 4-chloro-3,5-dinitrobenzoic acid, which has the effect of replacing positive charges on amino groups with negatively charged carboxyl groups. Four singly-modified forms were obtained which were separated using anion exchange FPLC. The four forms were modified at the N-terminal valine and at lysines 54, 71 and 77. The rates of reaction with mammalian cytochrome c were increased for all four modified plastocyanins. In contrast, the rates of reaction with cytochrome f were inhibited for the forms modified at residues 1, 54 and 77, whereas no effect was observed for the form modified at residue 71. Modification had no effect on either the midpoint redox potential or the reaction with K3Fe(CN)6. These results are consistent with a model in which charged residues on plastocyanin located at or near the binding site for cytochrome f recognize the positively-charged binding site on cytochrome f. In contrast, charged residues located at points on plastocyanin distant from the cytochrome f binding site recognize the net negative charge on the cytochrome f molecule. Based on these considerations, Glu-68 may be within the interaction sphere of cytochrome f, suggesting that cytochrome f may donate electrons to plastocyanin at either Tyr-83 or His-87.     

Trichome-borne and artificially applied acylsugars of wild tomato deter feeding and oviposition of the leafminer Liriomyza trifolii

Oviposition and adult feeding of the leafminer Liriomyza trifollii (Burgess) (Diptera, Agromyzidae) on Lycopersicon pennellii (Corr.) D'Arcy and its F₁ hybrid with Lycopersicon esculentum (Mill.) was significantly less than that on the cultivated tomato, L. esculentum. The resistance of L. pennellii and the F₁ was reduced following rinsing of foliage with ethanol. Resistant attributes of L. pennellii were transferred to L. esculentum through appression of L. pennellii foliage to L. esculentum leaflets. Application of purified 2,3,4-tri-O-acylglucoses (the principal component of type IV glandular trichome exudate of L. pennellii) to L. esculentum significantly decreased feeding and oviposition on L. esculentum leaflets by 61–99%. Therefore the principal mechanism of resistance to this leafminer by L. pennellii is the secretion of these acylglucoses. Dose response analysis of acylglucoses applied to L. esculentum shows that dosages as low as 10% those found on L. pennellii provide large reductions (91%) in leaf punctures and mines.     

Choline transport in Fusarium graminearum A 3 5

Fusarium graminearum A 3/5 possesses a high affinity system (Km = 32 +/- 8 microM; mean +/- SE) for uptake of choline, which was shown to be energy-dependent and constitutive. The maximum rate of choline uptake by this system was repressed by ammonia and glucose, showing a three-fold increase in maximum activity after nitrogen (2 h) or carbon (4 h) starvation. The system was highly specific for choline with only dimethylethanolamine (Ki = 198 +/- 29 microM), betaine aldehyde (Ki = 95 +/- 14 microM) and chlorocholine (Ki = 352 +/- 40 microM) acting as competitive inhibitors. Hemicholinium-3 acted as a mixed (non-competitive) inhibitor (KIES = 1.9 +/- 0.6 microM; KIE = 3.6 +/- 1.9 microM).     

Hypoxanthine phosphoribosyltransferase activity in tissues and hypoxanthine concentrations in plasma and CSF of the horse in comparison with other species

1. Plasma hypoxanthine and xanthine concentrations are very low in the horse and low in rat, mouse and greyhound compared to concentrations in beagles, man, sheep and rabbit. 2. Activities in erythrocytes of the main enzyme metabolizing hypoxanthine, hypoxanthine phosphori-bosyltransferase, show a similar pattern (Tax et al., 1976, Comp. Biochem. Physiol. 54B, 209-212); thus low activities have been found where plasma concentrations were low. 3. Hypoxanthine phosphoribosyltransferase activities in horse tissue other than erythrocytes are similar to those in man and rabbit with high activities in brain; this enzyme may therefore be functionally important in equine brain.