Specific effects of chloride on the photocycle of E194Q and E204Q mutants of bacteriorhodopsin as measured by FTIR spectroscopy

Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.     

Acute stress-induced tissue injury in mice: differences between emotional and social stress

Emotional stress affects cellular integrity in many tissues including the heart. Much less is known about the effects of social stress. We studied the effect of emotional (immobilization with or without cold exposure) or social (intermale confrontation) stress in mice. Tissue injury was measured by means of the release of enzyme activities to blood plasma: lactate dehydrogenase (LDH), creatine kinase (CK), aspartate transaminase (AST), and alanine transaminase (ALT). Tape-immobilization increased all these activities in the plasma. AST-ALT ratio was also increased in these animals. Electrophoretic analysis of CK isoenzymes showed the appearance of CK-MB. These results indicate that the heart was injured in immobilized mice. Analysis of LDH isoenzymes and measurement of alpha-hydroxybutyrate dehydrogenase (HBDH) activity suggests that other tissues, in addition to the heart, contribute to the increase in plasma LDH activity. Restraint in small cylinders increased plasma LDH, CK, AST, and ALT activities, but to lower levels than in tape immobilization. Because the decrease in liver glycogen and the increase in plasma epidermal growth factor (EGF) were also smaller in restraint than in the tape-immobilization model of emotional stress, we conclude that the former is a less intense stressor than the latter. Cold exposure during the restraint period altered the early responses to stress (it enhanced liver glycogen decrease, but abolished the increase in plasma EGF concentration). Cold exposure during restraint enhanced heart injury, as revealed by the greater increase in CK and AST activities. Intermale confrontation progressively decreased liver glycogen content. Plasma EGF concentration increased (to near 100 nM from a resting value of 0.1 nM) until 60 minutes, and decreased thereafter. Confrontation also affected cellular integrity in some tissues, as indicated by the rise in plasma LDH activity. However, in this type of stress, the heart appeared to be specifically protected because there was no increase in plasma CK activity, and both AST and ALT increased, but the AST-ALT ratio remained constant. Habituation to restraint (1 h/d, 4 days) made mice resistant to restraint-induced tissue injury as indicated by the lack of an increase in plasma LDH, CK, AST, or ALT activities. Similar general protection against homotypic stress-induced injury was observed in mice habituated to intermale confrontation.     

Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization

The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4′,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of the Cytophaga-Flavobacterium cluster were most abundant in all layers, followed by the sulfate-reducing bacteria.     

Role of Histidine Residues in Agonist and Antagonist Binding Sites of A1 Adenosine Receptor

Abstract: The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-N⁶-[adenine-2,8-³H, ethyl-2-³H]phenylisopropyladenosine ([³H]R-PIA) and antagonist 8-cyclopentyl-1,3-[³H]-dipropylxanthine ([³H]DPCPX). Two ionizable groups, of pK 7.0 and pK 7.4, are involved in the [³H]R-PIA associations with high- and low-affinity states of the receptor, and another group, of pK 6.0, is involved in the association with the low-affinity state. No ionizable group is involved in the dissociation process for the high-affinity state, whereas two ionizable groups, of pK 6.0 and 6.5, are involved in the low-affinity state. For [³H]DPCPX, three ionizable groups (pK 6.0, 7.4, and 8.0) are involved in the association process and only one group, (pK 6.0), is involved in the dissociation step. The apparent pK values obtained agree with histidine residues. We thus studied the effect of diethylpyrocarbonate (DEP), which reacts irreversibly with histidine residues, on agonist and antagonist binding to A₁ adenosine receptors from pig brain cortical membranes. DEP treatment of membrane reduced the affinity (K_D) and the total binding (R) of the agonist and the antagonist. Membrane preincubation with unlabeled ligand (R-PIA or DPCPX) prevented the effect of DEP modification observed when the same ligand, but with label, is added to the same membranes, but did not prevent the DEP modification on different, labeled ligand. The pattern of protective action of R-PIA, DPCPX, adenosine, and guanylylimidodiphosphate in DEP treatment and the displacement curves of radiolabeled agonist and antagonist by both unlabeled ligands indicated that the interaction site for agonist and antagonist binding is the same, although the complete mechanisms for recognition and binding differ.     

Phosphorus equivalence of a Consensus phytase produced by Hansenula polymorpha in diets for young turkeys

An experiment was conducted to determine the efficacy of a so-called consensus phytase preparation produced by Hansenula polymorpha on growth, tibia and toe ash and P retention of young turkeys. A total of 192 female turkeys (BUT 9 strain) were placed into 96 batteries at two per cage and assigned to one of eight diets: A negative control containing 2.5 g non-phytate P per kg feed (T-1); T-1 plus 125, 250, 500, 1000 or 10 000 phytase units (U) per kg feed, respectively, (T-2 to T-6); T-1 plus 0.5 or 1.0 g Pi/kg feed as dicalcium phosphate dihydrate (DCP), respectively, (T-7 and 8). The experiment lasted 32 d, and excreta were collected and weighed between 26 and 29 d of age. Feed was also weighed during this period in order to calculate P retention. Performance was calculated for the 0 to 32 d period. At the end of the experiment one bird per pen was killed for tibia and toe ash content determination, except for T-2, T-3, and T-7. Body weight, FCR, toe and tibia ash, and P retention responded to phytase or Pi supplementation. Using linear or quadratic models for comparing performance of the treatments containing supplemental Pi with phytase treatments, an equivalence between phytase and Pi was calculated. Body weight, toe ash, tibia ash and P retention showed a significant response to phytase supplementation. The values of equivalence for body weight, toe ash, tibia ash and P retention were 251, 597, 391 and 390 U to 1 g Pi/kg feed. At 10 000 U/kg feed there was a significant response in terms of weight gain and P retention, indicating that turkeys respond to levels greater than 1 000 U/kg feed.     

Characterisation of exposure to airborne fungi: measurement of ergosterol

In order to gain a clearer understanding of the level of fungal air contamination in indoor environments, we have adapted and tested a method to evaluate fungal biomass. Liquid phase chromatography (HPLC) of ergosterol, a component of the cell membrane of microscopic fungi, was employed. This method permits the detection and identification of ergosterol molecules at a concentration of 40 microg/ml (n=33, sigma=5). By combining this assay with a rotating cup collection apparatus, it was possible to measure fungal flora levels with a limit of quantification of 0.4 ng/m3 or a theoretical value of 150 spores per cubic meter (m3). Measurements of ergosterol levels performed on different sites showed that this method reflected the different situations of exposure of occupants to airborne fungal flora.     

The role of the AU-rich elements of mRNAs in controlling translation

Adenosine- and uridine-rich elements (AREs) located in 3'-untranslated regions are the best-known determinants of RNA instability. These elements have also been shown to control translation in certain mRNAs, including mRNAs for prominent pro-inflammatory and tumor growth-related proteins, and physiological anti-inflammatory processes that target ARE-controlled translation of mRNAs coding for pro-inflammatory proteins have been described. A major research effort is now being made to understand the mechanisms by which the translation of these mRNAs is controlled and the signalling pathways involved. This review focuses on the role of ARE-containing gene translation in inflammation, and the disease models that have improved our understanding of ARE-mediated translational control.     

KCNQ1/KCNE1 channels during germ-cell differentiation in the rat: expression associated with testis pathologies

KCNQ1/KCNE1 channels are responsible for the Jervell-Lange-Nielsen cardiac syndrome, which is also characterized by congenital deafness. KCNQ1/KCNE1 is crucial for K+ transport in the inner ear. We show that KCNQ1 and KCNE1 are associated in testis and that their expression is closely regulated during development. Both genes were expressed in undifferentiated germ cells in 21-day-old rats and mostly confined to basal immature germ cells in adulthood. Leydig and Sertoli cells were negative. KCNQ1 and KCNE1 were also studied in various germ-cell pathologies. First, in spontaneous unilateral rat testis atrophy, hematoxylin-eosin analysis revealed massive germ-cell aplasia with only Sertoli cells and groups of interstitial Leydig cells. In these samples, KCNQ1 and KCNE1 were not expressed. In human seminoma samples characterized by a proliferation of undifferentiated germ cells, KCNQ1/KCNE1 protein levels were higher than in healthy samples. Our results demonstrate that the expression of KCNQ1 and KCNE1 is associated with early stages of spermatogenesis and with the presence of undifferentiated healthy or neoplastic germ cells. The presence of a K+ rich-fluid in the seminiferous tubule suggests that KCNQ1/KCNE1 is involved in K+ transport, probably during germ-cell development.     

The Introduced Green Alga Caulerpa Taxifolia Continues to Spread in the Mediterranean

The tropical green alga Caulerpa taxifolia in the Mediterranean has spread steadily since its introduction in 1984. At the end of 2000, approximately 131km² of benthos had been colonized in 103 independent areas along 191km of coastline in six countries (Spain, France, Monaco, Italy, Croatia and Tunisia). Large regions neighboring the invaded areas appear favorable to further colonization, and there is thus no reason to believe that spreading will slow down in the years to come.     

Distribution and trophic ecology of chaetognaths in the western Mediterranean in relation to an inshore-offshore gradient

This study examines the distribution patterns and feeding ecologyof chaetognaths in the Catalan Sea in relation to mesoscalefeatures along an inshore–offshore gradient. The studywas conducted during two different periods of the year: latespring of 1995 and late summer of 1996. The two periods differedin hydrographic conditions and mesoscale processes, which affectedthe distribution patterns of the different species of chaetognathsfound. The diet of the chaetognaths was mainly composed of copepodsand differed between species. Prey size was not always stronglyrelated to chaetognath size and for certain species, there wasan overlap in prey size spectrum. Trophic niche breadth (ona ratio scale) appeared to be constant with growth. Ingestionrates and predation pressure by chaetognaths did not followa clear trend related to the mesoscale features in the area,such as the presence of a density front. The impact of chaetognathson copepod standing stock appeared to be extremely low (<1%),but it became more relevant when the species and prey size specificityof the chaetognaths was taken into account.