Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H⁺ (mg Chl)^–1 h^–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal -CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO₃ ^– dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H⁺ (mg Chl)^–1 h^–1) were observed in the presence of detergents (Triton X-100 or n-octyl--D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H⁺ (mg Chl)^–1 h^–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.This revised version was published online in October 2005 with corrections to the Cover Date.     

Comparative studies on the properties of the extrinsic manganese-stabilizing protein from higher plants and of a synthetic peptide of its C-terminus

The present study describes a comparative analysis on the fluorescence properties of the manganese-stabilizing protein (MSP), a synthetic peptide corresponding to its C terminus and a 7:1 (molar ratio) mixture of N-acetyl-tyrosine and N-acetyl-tryptophan, respectively, together with reconstitution experiments of oxygen evolution in MSP-depleted photosystem II (PS II) membrane fragments. It is found: (i) at neutral pH, the fluorescence from Trp(241) is strongly diminished in MSP solutions, whereas it highly dominates the overall emission from the C-terminus peptide; (ii) at alkaline pH, the emission of Tyr and Trp is quenched in both, MSP and C-terminus peptide, with increasing pH but the decline curve is shifted by about two pH units towards the alkaline region in MSP; (iii) a drastically different pattern emerges in the 7:1 mixture where the Trp emission even slightly increases at high pH; (iv) the anisotropy of the fluorescence emission is wavelength-independent (310-395 nm) and indicative of one emitter type (Trp) in the C-terminus peptide and of two emitter types (Tyr, Trp) in MSP; and (v) in MSP-depleted PS II membrane fragments the oxygen evolution is restored (up to 85% of untreated control) by rebinding of MSP but not by the C-terminus peptide, however, the presence of the latter diminishes the restoration effect of MSP. A quenching mechanism of Trp fluorescence by a next neighbored tyrosinate in the peptide chain is proposed and the relevance of the C terminus of MSP briefly discussed.     

Autoradiographic study on [3H]flunitrazepam binding in rat cortex and hippocampus after chronic ethanol treatment

Ethanol alters almost all membrane functions, but it behaves essentially like a benzodiazepine-type GABAergic agonist. The mechanism by which ethanol affects the GABA/benzodiazepine complex is not clear. We studied the possible changes in [³H]flunitrazepam binding induced by chronic ethanol treatment, using light microscopic autoradiography, to try to elucidate the controversy underlying this topic. This technique allows us to measure densities of benzodiazepine receptors in different anatomical brain areas—visual cortex and hippocampus—which seem to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. Autoradiographic studies on the visual cortex and hippocampus from rats chronically treated with ethanol do not show statistically significant differences in the binding of, [³H]flunitrazepam with respect to control animals. Furthermore, we did not find either rostro-caudal or medio-lateral differences, in benzodiazepine receptor densities in each layer of the visual cortex.     

Effect of morphine and abstinence syndrome on [3H]bromoxidine binding to α2-adrenoreceptors in rat brain

At 4 days after the implantation of two subcutaneous 75 mg morphine pellets in the back skin, rats were morphine-dependent. In the three layers studied in the occipital cortex we found that the values of the ₂-adrenergic agonist [³H]bromoxidine binding increased with respect to animals implanted with placebo pellets. Typical behavioral and physiological symptoms of the abstinence syndrome appeared 30 minutes after administration of naloxone, [³H]bromoxidine binding values being similar to those obtained in animals implanted with placebo pellets. The pattern of response of the [³H]bromoxidine binding was similar in the hippocampus and the superficial gray layer of the superior colliculus of the mesencephalon, but the differences were not statistically significant in these areas. This paper concludes that exist brain regional differences in the ₂-adrenoreceptors response under morphine-treatment and possibly under naloxone-induced morphine abstinence syndrome.     

Beyond host-pathogen interactions: microbial defense strategy in the host environment

Many extracellular pathogenic bacteria colonize human or animal bodies through evasion of the host immune system, a process called host-pathogen interaction. What happens when other intruders try to invade the same host and try to establish themselves in the same niche is largely unknown. In one well-studied case, Pseudomonas aeruginosa is known to secrete the protein azurin as a weapon against such invaders as cancers, parasites and viruses. The production of such weapons by pathogenic bacteria could provide important insights into how a pathogen responds in the post-colonization state to impede other intruders for its own survival. Moreover, these molecules might find use in the pharmaceutical industry as next-generation therapeutics.     

Muscular sound and force relationship during isometric contraction in man

The contracting muscle generates a low frequency sound detectable at the belly surface, ranging from 11 to 40 Hz. To study the relationship between the muscular sound and the intensity of the contraction a sound myogram (SMG) was recorded by a contact sensor from the biceps brachii of seven young healthy males performing 4-s isometric contractions from 10% to 100% of the maximal voluntary contraction (MVC), in 10% steps. Simultaneously, the electromyogram (EMG) was recorded as an index of muscle activity. SMG and EMG were integrated by conventional methods (iSMG and iEMG). The relationship between iSMG and iEMG vs MVC% is described by parabolic functions up to 80% and 100% MVC respectively. Beyond 80% MVC the iSMG decreases, being about half of its maximal value at 100% MVC. Our results indicate that the motor unit recruitment and firing rate affect the iSMG and iEMG in the same way up to 80% MVC. From 80% to 100% MVC the high motor units' discharge rate and the muscular stiffness together limit the pressure waves generated by the dimensional changes of the active fibres. The muscular sound seems to reflect the intramuscular visco-elastic characteristics and the motor unit activation pattern of a contracting muscle.     

Proteomic analysis and protein carbonylation profile in trained and untrained rat muscles

Understanding the relationship between physical exercise, reactive oxygen species and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Unbalanced ROS levels can lead to oxidation of cellular macromolecules and a major class of protein oxidative modification is carbonylation. The aim of this investigation was to study muscle protein expression and carbonylation patterns in trained and untrained animal models. We analyzed two muscles characterized by different metabolisms: tibialis anterior and soleus. Whilst tibialis anterior is mostly composed of fast-twitch fibers, the soleus muscle is mostly composed of slow-twitch fibers. By a proteomic approach we identified 15 protein spots whose expression is influenced by training. Among them in tibialis anterior we observed a down-regulation of several glycolitic enzymes. Concerning carbonylation, we observed the existence of a high basal level of protein carbonylation. Although this level shows some variation among individual animals, several proteins (mostly involved in energy metabolism, muscle contraction, and stress response) appear carbonylated in all animals and in both types of skeletal muscle. Moreover we identified 13 spots whose carbonylation increases after training.     

Combined effects of fatigue and temperature manipulation on skeletal muscle electrical and mechanical characteristics during isometric contraction

Peripheral fatigue and muscle cooling induce similar effects on sarcolemmal propagation properties. The aim of the study was to assess the combined effects of muscle temperature (Tm) manipulation and fatigue on skeletal muscle electrical and mechanical characteristics during isometric contraction. After maximum voluntary contraction (MVC) assessment, 16 participants performed brief and sustained isometric tasks of different intensities in low (Tm(L)), high (Tm(H)) and neutral (Tm(N)) temperature conditions, before and after a fatiguing exercise (6s on/4s off at 50% MVC, to the point of fatigue). During contraction, the surface electromyogram (EMG) and force were recorded from the biceps brachii muscle. The root mean square (RMS) and conduction velocity (CV) were calculated off-line. After the fatiguing exercise: (i) MVC decreased similarly in all Tm conditions (P<0.05), while EMG RMS did not change; and (ii) CV decreased to a further extent in Tm(L) compared to Tm(N) and Tm(H) in all brief and sustained contractions (P<0.05). The larger CV drop in Tm(L) after fatigue suggests that Tm(L) and fatigue have a combined and additional effect on sarcolemmal propagation properties. Despite these changes, force generating capacity was not affected by Tm manipulation. A compensatory mechanism has been proposed to explain this phenomenon.     

Partial persistence of exercise-induced myocardial angiogenesis following 4-week detraining in the rat

Enhanced angiogenesis, or capillary growth, has a prominent role among the various beneficial effects of exercise training on the myocardium. The aim of the present study is to assess if training-induced increases in capillarity and vascularization persist after 4 weeks of detraining. Adult male rats were trained to run on a treadmill for 10 weeks at ∼60% VO_2max, which did not induce cardiac hypertrophy, but increased (P < 0.05) the soleus/body weight ratio, left ventricle capillarity and von Willebrand-positive cell density (n = 6). In another group of animals (n = 6) subjected to training followed by 4-week detraining, the soleus/body weight ratio returned to normal, with only partial reversal of left ventricle capillarity and von Willebrand-positive cell density. Markers of angiogenesis (VEGF, KDR/VEGF-R2 and HIF-1α mRNA, studied by real-time RT-PCR) were upregulated at the end of training, and returned to baseline value after detraining. Electron microscopy highlighted some morphological features in trained hearts (endothelial cell sprouting and bridges and pericyte detachment), suggestive of endothelial cell proliferation and capillary growth that were absent in untrained and detrained hearts. We conclude that the training-induced increase in cardiac capillarity and vascularization are retained for some time upon cessation of the training program even in the absence of angiogenic stimuli.     

Lichen communities in the cathedral of Leon

In the cathedral of León, 36.36% of the sampled surface is covered by up to 39 lichen taxa. Two different communities were found, either on carbonate rocks or on highly nitrified environments. The first is composed of species with crustaceous biotypes, mainly belonging to the generaCaloplaca, Aspicilia andRinodina. The other community is composed of species with foliaceous or lobed crustaceous biotypes of the generaPhyscia, Physconia andPhaeophysica, this latter community being the one producing the higher degree of physical biodeterioration, because of its higher rate of foliaceous species. The species distribution depends directly on nitrophytism, hygrophytism, photophytism and pH index, and to a lesser extent on the variation of the inclination angle, orientation and substratum type.