Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. M?ssbauer and EPR characterization of the metal centers

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and M?ssbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.     

Effect of a Single Dose of β,β''-Iminodipropionitrile In Vivo on the Properties of Neurofilaments In Vitro: Comparison with the Effect of Iminodipropionitrile Added Directly to Neurofilaments In Vitro

The biochemical properties of neurofilaments isolated from control and iminodipropionitrile-treated rats were compared with regard to autophosphorylation capacity, hydrolysis of ATP, and the formation of a viscous gel between filaments. Both preparations exhibited a similar polypeptide composition, and no covalent cross-linking between neurofilament subunits was induced by iminodipropionitrile in vivo. An ATPase activity, systematically present in all preparations, was unaffected by the administration of iminodipropionitrile to the rats. Conversely, the autophosphorylation of neurofilament subunits in vitro was significantly higher in preparations from iminodipropionitrile-treated rats than from control animals, with a marked increase of the phosphorylation of a high molecular weight neurofilament-associated protein. Iminodipropionitrile provoked a higher gelation capacity of neurofilaments as measured in vitro, with a lower critical concentration for the preparation from treated animals. A similar increased interaction was obtained with millimolar concentrations of iminodipropionitrile added to bovine neurofilaments in vitro, involving likely neurofilament-associated molecules, because the effect of the drug was lost after their extraction by 0.8 M KCl. These results support the hypothesis that iminodipropionitrile interferes with the neurofilament networks through a preferential interaction with the neurofilament-associated proteins, resulting in a change in their properties and consequently in an increased capacity of interaction between the polymers.     

On the mechanism of hydrogen peroxide formation during precipitation of hemoglobin with perchloric acid

During acid precipitation of hemoglobin, hydrogen peroxide is formed. The oxidant is generated from hemoglobin-bound as well as from physically dissolved oxygen, provided divalent hemoglobin-iron is present. The extent of hydrogen peroxide formation seems to be dependent on the precipitating reagent. From experiments on GSH oxidation during precipitation of red cells and from a literature survey formation of at least one second reactive oxygen species has to be assumed. Different reaction pathways for generating these oxidants during acid precipitation are discussed: Hydroperoxide which is probably liberated by proton catalysis may contribute to the observed oxidation reactions. At pH approximately 1 it rapidly disproportionates yielding oxygen and hydrogen peroxide, which might produce reactive oxidants with hemoglobin intermediates formed during precipitation. In addition a new hypothesis is presented explaining the ability of N-ethylmaleimide to inhibit GSH oxidation during precipitation of red cells. Accordingly, the SH-reagent rapidly penetrates the cell membrane and reacts with GSH before access of protons prevents alkylation.     

GROWTH PATTERN,REPRODUCTION, AND SELF-THINNING IN SEAWEEDS1

In unispecific plant stands, the logarithm of mean individual weight (w) depends on the logarithm of density (d) by the -3/4 power law (a slope of -1.5 and an intercept ranging from 2.3 to 5.0). The analysis of the w and d relationships in whole cohorts of two seaweed species from the Strait of Gibraltar shows deviations from the canonical equation. The kelp Phyllariopsis purpurascens (C. Agardh) Henry et South (Phaeophyta) growing at a 30-m depth has the lowest intercept value (0.6) recorded for any plant species and a slope not significantly different from -1.5. The slope value is in accordance with those found in species whose growth is not stopped by reproduction. Irradiance under a single layer of blades was lower than the photosynthetic light compensation point, and this could be due to overdispersion of the shoots and, in consequence, to the low intercept value of the self-thinning equation. The w to d relationships in Asparagopsis armata Harvey (Rhodophyta) show two different components: no dependence between these two variables (slope not significantly different from 0) at densities < 500 shoots·m^?2, and a slope more negative (-2.1) than proposed by the -3/2 power law at densities > 500 shoots·m^?2. The pattern at high densities could be due to intraspecific competition for light, whereas the slope ～0 at low densities could be related to inhibition of growth by reproduction (cystocarp and carpospore production). Therefore, rather than being considered exceptional, we suggest that a gradient of variability could be expected in the dependence of w on d when specific growth patterns and reproduction are considered.     

Small-scale association measures in epibenthic communities as a clue for allelochemical interactions

The small-scale associations in a rocky subtidal community in the northwestern Mediterranean were studied by a development of the continuous line transect method. This method allowed the overall measurement of non-randomness in interspecific contacts and the assignment of an association index to each species-pair, whose, significance was tested by Monte Carlo procedures. At the same time, the continuous recording allowed the study of the weakening of the interactions with increasing distances. Our purpose was to uncover evidence for allelochemical mechanisms of space occupation and maintenance. A strong non-randomness was found in the interspecific associations. This was mostly due to the interactions of the poecilosclerid sponge Crambe crambe (Schmidt) with its neighbours, especially its negative associations with other sponge species. The strength of the relationships fell drastically over the first few centimeters from the contact borders of the different species. The results pointed strongly to an allelochemical mechanism. The extracts of this sponge featured high bioactivity in laboratory assays, and field experiments demonstrated that the sponge can inhibit the growth of species in the community studied. Standard sampling techniques would have overlooked the spatial structure present in the data. The study emphasizes the need for both contact data and distance data in order to identify the underlying processes reliably. The line transect method provides both types of information easily and allows testing of models and identification of organisms likely to use chemical defenses in space competition. Its use as a preliminary step in studies of chemical ecology might help to detect presumptive allelochemical processes prior to experimental work on the potentially active species.     

Colonization and deterioration processes in Roman mortars by cyanobacteria,algae and lichens

The mortars covering some walls of the Roman city of Baelo Claudia (Cadiz, Spain) support an abundant colonization of cyanobacteria, algae and lichens. The distribution of these organisms is closely related to microclimatic parameters. Furthermore, the development, specific composition and biomass of algal cryptoendolithic communities are related to the wall orientation. The effect of these communities on mortar deterioration is discussed.     

On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement

Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO(2) was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell(-1) h(-1) at 10% air saturation to 4.5 pmol cell(-1) h(-1) at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. (c) 1996 John Wiley & Sons, Inc.     

A New Type of Manchette Structure in Spermatids of Culex tigripes(Culicidae)

Abstract In young spermatids of Culex tigripes, microtubules organize a manchette which surrounds the nucleus. When the nucleus elongates, 1–5 expansions appear on the wall of the microtubules. They grow and branch out while the nucleus elongates and chromatin condenses. Expansions are straight or curved in shape. They have the same thickness as the microtubule walls, but they rarely constitute links between microtubules. The manchette disappears naturally at the end of spermiogenesis. The action of colchicine on spermatids leads to the complete disappearance of the microtubules and expansions, and inhibits the lengthening of the nucleus.     

Starvation-Induced Stress Resistance in Lactococcus lactis subsp. lactis IL1403

Carbohydrate-starved cultures of Lactococcus lactis subsp. lactis IL1403 showed enhanced resistance to heat, ethanol, acid, osmotic, and oxidative stresses. This cross-protection seems to be established progressively during the transitional growth phase, with maximum resistance occurring when cells enter the stationary phase. Chloramphenicol or rifamycin treatment does not abolish the development of a tolerant cell state but, on the contrary, seems to provoke this response in L. lactis subsp. lactis.