Respiratory enzymes in muscle: Interaction between environmental temperature, nutrition and growth

1. 1.In young pigs living at 35 or 10°C on a high or low energy intake, respiratory enzyme activities in longissimus dorsi muscle were greater both in the cold and on low intake. The elevated activities in the cold were unlikely to be related entirely to shivering since they were also found in muscle from the diaphragm.

2. 2.In a second study, pigs were kept close to thermal neutrality (26°C) on different levels of food intake and for different periods of time. For all animals, as body weight increased there was a decline in respiratory enzyme activity and the number of dark fibres in skeletal muscle. For those of the same weight, but different age and food intake, there was no difference in enzyme activity or number of dark fibres per unit area.

3. 3.At least part of the difference in respiratory enzyme activities related to energy intake must therefore be due to differences in body size. However, size is not the sole determinant of enzyme activity in skeletal muscle, since in animals of similar size those living at 10°C have greater enzyme activities than those at 35°C.

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Free amino acids from pollens

Pollens from Pinus canariensis, P. nigra, P. pinaster, P. pinea, Castanea sativa, Magnolia grandiflora, Olea sativa cv frantoio, cv itrana, cv pisciottana were examined for their free amino acid composition. A large amount of proline was found in all species; pollens of Olea also contain a large amount of serine.     

Doxorubicin and epirubicin iron-induced generation of free radicalsIn vitro. A comparative study

To ascertain any differences in myocardial injury exerted by the anthracyclines doxorubicin and epirubicin, their ability to generate oxygen free radicals when mixed with Fe(II) was examined in vitro using an oxygen electrode. 5–250 g/ml doxorubicin or epirubicin consumed oxygen when mixed with 50 or 100 mol/1 Fe(II). Addition of 75 mol/1 cytochrome C showed that of the consumed oxygen, approximately 80% entered the monovalent pathway of oxygen reduction. The strong inhibitory effect of 250 mg/1 catalase indicates that most of the superoxide radicals generated are further reduced to hydrogen peroxide by both anthracyclines. Addition of metal chelators DTPA (100/mol/1), or DDTC (50 mol/1) did not affect oxygen consumption, whereas EDTA (100/mol/1) or desferrioxamine (100 mol/1) with anthracyclines and Fe(II) rather stimulated oxygen consumption. It is concluded that there are no significant differences in the amount or proportion of generated oxygen free radicals between doxorubicin and epirubicin when mixed with Fe(II) in a cell-free system in vitro. Thus, the ability of the anthracyclines, in conjunction with iron alone, to generate radicals does not explain the differences of the drugs in causing myocardial injury.     

Pheophytin-mediated energy storage of photosystem II particles detected by photoacoustic spectroscopy

The photoacoustic (PA) characteristics (energy storage and heat dissipation) of photosystem II (PSII) core-enriched particles from barley were studied (i) in conditions where there was electron flow, i.e., in the presence of a combination of the electron acceptor K₃ Fe (CN)₆, referred to as FeCN, and the electron donor diphenylcarbazide (DPC), and (ii) in conditions where electron flow was suppressed, i.e., in the absence of FeCN and DPC. The experimental data show that a decrease of heat dissipation with a minimum at 540 nm can be interpreted as energy storage resulting from the presence of pheophytin (Pheo) in the PSII particles. On account of the capability of the PA method to measure the energy absorbed by the chromophores which is converted to heat, it is suggested that the PA detection of Pheo present in the PSII complex will permit to clarify the function of processes involving non-radiative relaxation of excited states in P680-Pheo-Q_A interactions.Abbreviations -Car -Carotene - Chl Chlorophyll - DPC Diphenylcarbazide - EPR Electron Paramagnetic Resonance - FeCN potassium ferricyanide - HEPES N-2-hydroxyethylenepiperazine-N-2-ethanesulfonate - P680 reaction center of PSII - PA Photoacoustic - Pheo pheophytin - PSI photosystem I - PSII photosystem II - Q_A primary electron acceptor of PSII     

Waterlogging responses in dune,swale and marsh populations of Spartina patens under field conditions

Summary Soil waterlogging responses were examined in three Spartina patens populations along a steep flooding gradient in coastal Louisiana. Root anatomy and physiological indicators of anaerobic metabolism were examined to identify and compare flooding responses in dune, swale and marsh populations, while soil physicochemical factors were measured to characterize the three habitats. Soil waterlogging increased along the gradient from dune to marsh habitats and was accompanied by increases in root porosity (aerenchyma). Aerenchyma in marsh roots was apparently insufficient to provide enough oxygen for aerobic respiratory demand, as indicated by high root alcohol dehydrogenase activities and low energy charge ratios. Patterns of root metabolic indicators suggest that dune and swale roots generally respired aerobically, while anaerobic metabolism was important in marsh roots. However, in each population, relatively greater soil waterloging was accompanied by differences in enzyme activities leading to malate accumulation. In dune and swale roots under these circumstances, depressed adenylate energy charge ratios may have been the result of an absence of increased ethanol fermentation. These trends suggest that: 1) Aerenchyma formation was an important, albeit incomplete, long-term adaptation to the prevalent degree of soil waterlogging. 2) All populations adjusted root metabolism in response to a relative (short-term) increase in soil waterlogging.     

Reversible effects of moderately elevated temperature on the distribution of excitation energy between the two photosystems of photosynthesis in intact avocado leaves

Initial (F_o), maximum (F_m) and steady-state (F_s) levels of modulated chlorophyll fluorescence were measured in intact avocado leaves (Persea americana Mill.) during state 1-state 2 transitions using a combination of modulated and non-modulated lights with synchronized detection. Under normal temperature conditions (20°C), transition from state 2 to state 1 was associated with a substantial increase (about 20%) in F_m and F_o whereas the F_m/F_o ratio remained constant, reflecting increased absorption cross-section of PS II. On the contrary, at moderately elevated temperature (35°C), these fluorescence changes were very limited, indicating marked inhibition of the state regulation. The fraction of light distributed to PS II () was calculated from the F_o, F_m and F_s levels for both types of leaves. In control leaves, varied from 48% (in state 2) to values as high as 58% (in state 1). In contrast, mild heat treatment resulted in values close to 50% in both states, indicating the inability of heated leaves to reach extreme state 1. The results suggested that avocado leaves under moderately elevated temperature conditions are blocked in a state close to state 2. This effect was shown to occur in a non-injurious temperature range (as shown by the preservation of the (photoacoustically monitored) oxygen evolution activity) and to be rapidly reversed upon lowering of the temperature. Thermally induced development of state 2 (independent on the light spectral quality) could possibly be a protective mechanism to avoid photodamage of the heat-labile PS II by high light intensities which usually accompany heat stress in the field.     

Estimation of uncertainties in X-ray refinement results by use of perturbed structures

The uncertainties in the refined parameters for a 1.5-A X-ray structure of carbon-monoxy (FeII) myoglobin are estimated by combining energy minimization with least-squares refinement against the X-ray data. The energy minimizations, done without reference to the X-ray data, provide perturbed structures which are used to restart conventional X-ray refinement. The resulting refined structures have the same, or better, R-factor and stereochemical parameters as the original X-ray structure, but deviate from it by 0.13 A rms for the backbone atoms and 0.31 A rms for the sidechain atoms. Atoms interacting with a disordered sidechain, Arg 45 CD3, are observed to have larger positional uncertainties. The uncertainty in the B-factors, within the isotropic harmonic motion approximation, is estimated to be 15%. The resulting X-ray structures are more consistent with the energy parameters used in simulations.     

Subcellular distribution,molecular dynamics and catabolism of plasmalogens in myocardium

Recent studies have implicated accelerated sarcolemmal phospholipid catabolism as a mediator of the lethal sequelae of atherosclerotic heart disease. We have demonstrated that plasmalogens are the predominant phospholipid constituents of canine myocardium and that plasmalogens are hydrolyzed by a novel calcium independent plasmalogen selective phospholipase A2. Since the activities of phospholipases are modulated by the molecular dynamics and interfacial characteristics of their phospholipid substrates, we compared the molecular dynamics of plasmenylcholine and phosphatidylcholine vesicles by electron spin resonance spectroscopy and deuterium magnetic resonance spectroscopy. Plasmenylcholine vesicles have separate and distinct molecular dynamics in comparisons to their phosphatidylcholine counterparts as ascertained by substantial decreases in the angular fluctuations and motional velocities of probes attached to their sn-2 aliphatic constituents. Furthermore, since free radical oxidation of myocardial lipid constituents occurs during myocardial ischemia and reperfusion, we demonstrated that ¹O₂ mediated oxidation of plasmenylcholine resulted in the generation of several products which have chromatographic characteristics and molecular masses corresponding to 2-acyl lysophosphatide derivatives. Taken together, these studies underscore the biologic significance of the predominance of sarcolemmal plasmalogens present in mammalian myocardium and suggest that their catabolism by plasmalogen selective phospholipases and/or oxidative processes may contribute to the lethal sequelae of myocardial ischemia.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol