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Spermadhesins are a family of seminal plasma proteins composed of a single CUB domain, which appear to be involved in various aspects of the fertilization process in pigs. PSP-I and PSP-II, the most abundant porcine spermadhesins, occur in seminal plasma as noncovalent heterodimers devoid of heparin-binding capability. Of note is the stability of this dimer, which is significantly affected by physiologically relevant conditions such as Zn2+ ions. Here, we show that PSP-I and PSP-II when separated appear to conserve the overall fold of the CUB domain observed in the crystal structure of the PSP-I/PSP-II heterodimer, as concluded from gel filtration, analytical ultracentrifugation, differential scanning calorimetry, and circular dichroism analyses. However, Zn2+ concentrations in the range of those found in boar seminal plasma induce the unfolding and self-association of PSP-I, apparently as a consequence of the exposure of hydrophobic core residues, whereas they have no effect on PSP-II. Remarkably, Zn2+-denatured and self-associated (but not structured monomeric) PSP-I is retained on a heparin column, resembling the behavior of free PSP-I and homologous spermadhesins of the heparin-binding fraction of boar seminal plasma, which also exhibit different aggregation states. Thus, the modulation of the structural organization and heparin-binding ability of PSP-I by Zn2+ might be a physiological phenomenon in seminal plasma.

Spermadhesins are a family of 12-16 kDa proteins with a single CUB domain. PSP-I and PSP-II, the most abundant boar spermadhesins, are present in seminal plasma as a noncovalent heterodimer. Dimerization markedly affects the binding ability of the subunits. Notably, heparin and mannose 6-phosphate binding abilities of PSP-II are abolished, indicating that the corresponding binding sites may be located at (or near) the dimer interface. Pursuing the hypothesis that cryptic binding sites in PSP-I/PSP-II may be exposed in specific physiological environments, we examined the influence of Zn2+ and acidic pH on the heterodimer stability. According to near-UV CD spectra, the core native fold is preserved in the presence of physiological concentrations of Zn2+, a cation unusually abundant in boar seminal plasma. However, the thermostability of the heterodimer decreases significantly, as observed by CD and differential scanning calorimetry. The effect is Zn2+-specific and is reversed by EDTA. Destabilization is also observed at acidic pH. Gel filtration analysis using radioiodinated PSP-I/PSP-II reveals that dissociation of the heterodimer at low (nanomolar) protein concentrations is promoted by both Zn2+ and acidic pH. Although the integrity of the heterodimer in seminal plasma seems to be guaranteed by its high concentration, dissociation may be facilitated in the female genital tract because of dilution of the protein in the intraluminal fluids of the cervix and the uterus, and the acidic fluid of the uterotubal junction. Such a mechanism may be relevant in the regulation of uterine immune reactions.

The influence of nitrogen and phosphorus pulses on Chaetomorpha linum (Muller) Kutzing growth and photosynthesis was studied in laboratory experiments. Photosynthesis and growth of C. linum from Tancada lagoon seems limited by both nitrogen and phosphorus, as indicated by the high rate (4.7–11.6 mg O 2 g −1 dry weight h −1 ) of light-saturated photosynthesis ( P m ) and growth rates observed under nitrogen plus phosphorus enrichment in relation to enrichment by nitrogen alone (2.9–7.6 mg O 2 g −1 dry weight h −1 ). Significant increase in nitrogen and phosphorus content as percentage of dry weight was observed in C. linum fertilized with a single nutrient or with nitrogen plus phosphorus. In Tancada lagoon, when availability of nitrogen to primary producers is by pulses, an increase of nitrate concentration in the water column (from 6 to 100 μM) has a greater effect on growth of C. linum (growth rate: 0.13 day −1 ) than an increase in ammonium concentration (from 20 to 100 μM and growth rate: 0.11 day −1 ). For a given thallus nitrogen content (0.6–1.4% N), both P m and the photosynthetic efficiency ( α ) normalized to dry weight were correlated ( r 2 = 0.73, p α and P m may be a general characteristic of thin-structured opportunistic algae in more variable estuarine environments.

In this study the dynamics of leaf fall, decomposition and nutrient dynamics of Phragmites australis was investigated beginning in the standing position and after leaves fall into the water. The study was conducted in a pond at the end of a temporary Mediterranean stream. The results showed that the decomposition rate was higher in the standing position (0.0098d -1 ) than after submergence of the detritus (0.0034-0.0042d -1 ). A significant variation in leaf nutrient content was observed during the standing dead phase, increasing during the first days and decreasing afterwards. After submergence, significant effects of macroinvertebrates, mainly gastropods, were observed on the nutrient content of the detritus after 60 days. The overall decomposition rate, considering the 184 days from the beginning of the experiment with emerged leaves, in the standing position, to the end of measurements with submerged leaves, was 0.0076d -1 . This rate is higher than values reported in the literature for P. australis leaves in submerged conditions, suggesting that decomposition takes place mostly in the standing position.

The spatial variability of plant organic matter processing was studied experimentally in a shallow coastal lagoon (Tancada lagoon, average depth: 37 cm, area: 1.8 km 2 ) in the Ebro River Delta (NE Spain). To determine theeffect of hydrology and sediment characteristics on plant organic matter processing, leaves of Phragmites australis at the end of its vegetative cycle and whole plants of Ruppia cirrhosa (PETAGNA) GRANDE, just abscised, were enclosed in litter bags. Two different mesh sizes (100 μm and 2 mm) were used to study the effect of macroinvertebrates on decomposition. The bags were placed in the water column and approximately 15 cm above the sediment at 6 different locations in the lagoon. The experiment was performed twice, in autumn-winter and spring-summer. The effect of macroinvertebrates on decomposition rate was not significant in Tancada lagoon. Breakdown rates showed spatial differences only in spring-summer. In the autumn-winter experiment, the effect of strong wind masked the effects of environmental variables and hydrology on decomposition rate. In the spring-summer experiment, characterised by high stability of the water column, dissolved inorganic nitrogen (DIN) concentration in the water column and organic matter in the sediment were the main factors determining the variability of organic matter processing. A positive relationship was calculated between P. australis decomposition rate and dissolved inorganic nitrogen in spring-summer (r 2 = 0.92, p < 0.001).

We have characterized the stability and folding behavior of the isolated extrinsic PsbQ protein of photosystem II (PSII) from a higher plant, Spinacia oleracea, using intrinsic protein fluorescence emission and near- and far-UV circular dichroism (CD) spectroscopy in combination with differential scanning calorimetry (DSC). Experimental results reveal that both chemical denaturation using guanidine hydrochloride (GdnHCl) and thermal unfolding of PsbQ proceed as a two-state reversible process. The denaturation free-energy changes (GD) at 20 C extrapolated from GdnHCl (4.0 ± 0.6 kcal mol-1) or thermal unfolding (4.4 ± 0.8 kcal mol-1) are very close. Moreover, the far-UV CD spectra of the denatured PsbQ registered at 90 C in the absence and presence of 6.0 M GdnHCl superimpose, leading us to conclude that both denatured states of PsbQ are structurally and energetically similar. The thermal unfolding of PsbQ has been also characterized by CD and DSC over a wide pH range. The stability of PsbQ is at its maximum at pH comprised between 5 and 8, being wider than the optimal pH for oxygen evolution in the lumen of thylakoid membranes. In addition, no significant structural changes were detected in PsbQ between 50 and 55 C in the pH range of 3-8, suggesting that PsbQ behaves as a soluble and stable particle in the lumen when it detaches from PSII under physiological stress conditions such as high temperature (45-50 C) or low pH (

11 p.-7 fig.-3 tab., This work was supported by Dirección General de Investigación Científica y Técnica Grants BIO2000-1307, BMC2000-1002, BIO2003-01952, BMC2003-00074, and by a Comunidad Autónoma de Madrid Grant of the Program for Strategic Groups.

We have analyzed the in vitro disassembly of the capsid of the minute virus of mice, and the stability of capsid chimeras carrying heterologous epitope insertions. Upon heating in a physiological buffer, empty capsids formed by 60 copies of protein VP2 underwent first a reversible conformational change with a small enthalpy change detected by fluorescence. This change was associated with, but not limited to, externalization of the VP2 N terminus. Irreversible capsid dissociation as detected by changes in fluorescence, hemagglutination activity, and electrophoretic mobility occurred at much higher temperatures. Differential scanning calorimetry in the same conditions indicated that the dissociation/denaturation transition involved a high enthalpy change and proceeded through one or more intermediates. In contrast, in the presence of 1.5 M guanidinium chloride, heat-induced disassembly fitted a two-state irreversible process. Both thermally and chemically induced dissociation/denaturation yielded a form that had lost a part of the tertiary structure, but still retained the native secondary structure. Data from chemical dissociation indicates this form may correspond to a molten globule-like monomeric state of the capsid protein. All five antigenic peptide insertions attempted in exposed loops, despite being perhaps among the least disruptive, led to defects in folding/assembly of the capsid and, in most cases, to reduced capsid stability against thermal dissociation. The results with one of the simplest viral capsids reveal a complex pathway for disassembly, and a reduction in capsid assembly and stability upon insertion of peptides, even within the most exposed capsid loops.

1. Coastal ecology is mostly regulated by hydro-meteorological factors and human disturbances. The spatial and temporal scales of variation of the processes associated with these factors may not correlate. 2. Monitoring of water and ecosystem quality in the coastal zone must be performed at spatial and temporal scales that adequately address the objectives. They range between two extreme states, i.e. high water turnover due to surface water exchanges and evaporation driven turnover, and can cover a range of ecological situations depending on hydrological variables. 3. The spatial scale for monitoring coastal aquatic ecosystems may be better related to water masses circulation than to physiographic features. Biologically mediated processes require monitoring taking account of meteorological, seasonal and interannual time scales. 4. Variables to be monitored must be in accordance with objectives. They may range from intra-compartment variables (e.g. demography of a biological population) to inter-zone relationships (e.g. budgets of nutrient exchange with the sea). Larger and longer scales, such as including buffer zones around lagoons and the continental and sea basins, and the consideration of interdecadal and secular time scales are required for wider monitoring perspectives. 5. Monitoring the impacts of global changes (both direct, such as urbanization and eutrophication of the coastal zone, and indirect, through relative sea-level rise due to climate change) requires a wider conceptual scale of approach to coastal zone ecology and even longer monitoring programmes. Copyright © 2004 John Wiley & Sons, Ltd.

11 p.-5 fig.-2 tab., Pneumococcal bacteriophage-encoded lysins are modular choline binding proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) against streptococcal infections. Here we present the crystal structures of the free and choline bound states of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1. While the catalytic module displays an irregular (β/α)5β3 barrel, the cell wall-anchoring module is formed by six similar choline binding repeats (ChBrs), arranged into two different structural regions: a left-handed superhelical domain configuring two choline binding sites, and a β sheet domain that contributes in bringing together the whole structure. Crystallographic and site-directed mutagenesis studies allow us to propose a general catalytic mechanism for the whole glycoside hydrolase family 25. Our work provides the first complete structure of a member of the large family of choline binding proteins and reveals that ChBrs are versatile elements able to tune the evolution and specificity of the pneumococcal surface proteins., This work was supported by grants BIO2000-1307 and BIO2002-02887 from Dirección General de Investigación and by grant of Contrato-Programa de Grupos Estratégicos (BMC2000-1002) de la Comunidad Autónoma de Madrid.

The litter-bag technique was used to study the effect of seasonality (spring–summer versus autumn–winter) and nutrient availability on leaf decomposition in two Mediterranean watercourses: the Ebro river, which is eutrophic, and the Senia stream. To establish the effect of macroinvertebrates on litter breakdown, we used air-dried leaves from the two dominant species of riparian vegetation (Populus alba and Populus nigra) along each watercourse and litter bags of two mesh sizes (100 μm and 5 mm). Macroinvertebrates were collected from the bags and ash free dry weight and carbon, nitrogen and phosphorus contents of the plant detritus remaining were measured. Litter decomposition rates, in days and degree-days, were estimated using a simple exponential model. A significant effect of macroinvertebrates and seasonality on litter decay was observed only in the Ebro river. Decomposition rates were faster in this river, P. alba showing the highest rates in spring–summer (0.022–0.038 day−1). The macroinvertebrates collected from the bags were mostly collector-gatherers of the genus Chironomus. Their density increased over time and was greater in the experiments performed in spring–summer (two to ten fold higher). Higher temperatures together with increased macroinvertebrate densities led to augmented decomposition rates in the Ebro river but no effect was observed in the Senia stream. Our data show that leaf decay is both quantitatively and qualitatively affected by inorganic nutrient levels and temperature in the ambient environment, regardless of initial C:N and C:P ratios.

The effect of dissolved nutrients on growth, nutrient content and uptake rates of Chaetomorpha linum in a Mediterranean coastal lagoon (Tancada, Ebro delta, NE Spain) was studied in laboratory experiments. Water was enriched with distinct forms of nitrogen, such as nitrate or ammonium and phosphorus. Enrichment with N, P or with both nutrients resulted in a significant increase in the tissue content of these nutrients. N-enrichment was followed by an increase in chlorophyll content after 4 days of treatment, although the difference was only significant when nitrate was added without P. P-enrichment had no significant effect on chlorophyll content. In all the treatments an increase in biomass was obseved after 10 days. This increase was higher in the N+P treatments. In all the treatments the uptake rate was significantly higher when nutrients were added than in control jars. The uptake rate of N, as ammonium, and P were significantly higher when they were added alone while that of N as nitrate was higher in the N+P treatment. In the P-enriched cultures, the final P-content of macroalgal tissues was ten-fold that of the initial tissue concentrations, thereby indicating luxury P-uptake. Moreover, at the end of the incubation the N:P ratio increased to 80, showing that P rather than N was the limiting factor for C. linum in the Tancada lagoon. The relatively high availability of N is related to the N inputs from rice fields that surround the lagoon and to P binding in sediments. A partir de experimentos en laboratorio se ha estudiado la influencia de la disponibilidad de nutrientes disueltos en el crecimiento, contenido en nutrientes y sus tasas de absorción en la especie de macroalga Chaetomorpha linum procedente de una laguna costera típicamente mediterránea (La Tancada, Delta del Ebro, NE España). Se fertilizó con diferentes formas de nitrógeno, nitrato o amonio y fósforo. Se ha observado un incremento significativo en el contenido de N y P en los tejidos vegetales tanto en las fertilizaciones con P o N como con N+P. La fertilización con N dio como resultado un aumento de la concentración de clorofila a los 4 días de iniciado el experimento aunque sólo se han observado diferencias significativas en el tratamiento con nitrato. La fertilización con P no produjo efecto significativo sobre la concentración en clorofila. En todos los tratamientos se ha observado un aumento de biomasa al cabo de 10 días y este incremento fue mayor cuando se fertilizaba con N+P. La tasa de absorción ha sido siempre más elevada en los microcosmos fertilizados que en los controles sin fertilizar. Las tasas de absorción de amonio y P fueron significativamente más elevadas en los tratamientos simples, con sólo amonio o P, sin embargo en el caso del nitrato la tasa de absorción fue mayor cuando se fertilizaba con N+P. Se ha observado un aumento en 10 veces el contenido inicial en los cultivos enriquecidos con P lo cual sería indicador de un consumo extra de fósforo, lo que unido al valor bajo inicial de N:P de 80 sería indicador de limitación del crecimiento por P en la laguna de la Tancada. La relativa disponibilidad alta de N con respecto al P podría estar relacionada con la entrada de N disuelto de los arrozales y la inmovilización de P en el sedimento.

The distribution biomass and photosynthesis of three species of rooted macrophytes, Ruppia cirrhosa (Petagna) Grande, Potamogeton pectinatus L. and Zostera noltii Hornem and a floating macroalga Chaetomorpha linum Kutz, were studied in Buda lagoon (River Ebro delta, NE Spain) during spring (May), summer (July) and autumn (October) 1995. Buda lagoon was characterized by a marked gradient of conductivity due to freshwater discharges from rice fields from June to October and by seawater input due to the regression of the delta of the River Ebro during the last 10 years. A typical spatial distribution was observed: monospecific stands of P. pectinatus developed near freshwater inputs in the inner part of the lagoon and mixed stands of R. cirrhosa and P. pectinatus developed in transitional zones between freshwater and seawater influence. The part of the lagoon where the influence of seawater was highest was covered by dense mixed stands of Z. noltii , R. cirrhosa and C. linum . Maximum biomass and production of P. pectinatus were reached in July (biomass of 501 gDW m −2 , and maximum photosynthetic rates, Pm, of 14 mgO 2 g −1 DW h −1 ). Maximum biomass of R. cirrhosa and Z. noltii (456·5 and 250 gDW m −2 respectively) and photosynthetic rate of R. cirrhosa (23·9 mgO 2 g −1 DW h −1 ) occurred in May, whereas no significant differences in production were detected between May and July in Z. noltii . Maximum C. linum production was reached in October (5·4 mgO 2 g −1 DW h −1 ). In October P. pectinatus coverage and production decreased, which was related to high turbidity and density of benthivorous fish due to freshwater inflows. Implications of lower freshwater inflow and higher seawater intrusion in the spatial distribution of aquatic macrophytes in this coastal lagoon are discussed.

The main objective of this work was to devise a method to measure the production and turnover rate of a macrophyte with thin leaves such as Ruppia cirrhosa (Petagna) Grande. Net production was calculated from values of biomass, decomposition rates and mortality obtained in a Mediterranean coastal lagoon (Tancada lagoon, Ebro Delta, NE Spain). Overall, the biomass of rhizomes and roots was constant during the annual cycle, but stem biomass varied substantially. Above-ground biomass peaked in September with 532 g AFDW m −2 and reached a minimum in April with 30 g AFDW m −2 . The net production was calculated to be 361.1 g C m −2 per year, which is the highest value for R. cirrhosa reported in the literature, and a turnover rate of 1.34. The net production obtained by this method is within the estimates based on harvesting techniques (303–545 g C m −2 per year).

39 p.-10 fig.-3 tab., Natural amino acid substitution by single-site nucleotide polymorphism can become a valuable tool for structure-activity correlations, especially if evidence for association to disease parameters exists. Focusing on the F19Y change in human galectin-8, connected clinically to rheumatoid arthritis, we here initiate the study of consequences of a single-site substitution in the carbohydrate recognition domain of this family of cellular effectors. We apply a strategically combined set of structural and cell biological techniques for comparing properties of the wild type and variant proteins. The overall hydrodynamic behavior of the full-length protein and of the separate N-domain is not noticeably altered, but displacements in the F0 β-strand of the β-sandwich fold in the N-domain are induced, as evidenced by protein crystallography. Analysis of thermal stability by circular dichroism spectroscopy revealed perceptible differences for the full-length proteins, pointing to an impact of the substitution beyond the N-domain. In addition, small differences in thermodynamic parameters of carbohydrate binding are detected. On the level of two types of tumor cells, characteristics of binding appeared rather similar. In further comparison of influence on proliferation, the variant proved to be more active as growth regulator in the six tested lines of neuroblastoma, erythroleukemia and colon adenocarcinoma. The seemingly subtle structural change identified here thus has functional implications in vitro, encouraging further analysis in autoimmune regulation and, in a broad context, work with other natural single-site variants, using the documented combined strategy., This work was generously supported by grants BFU2009-10052, BFU2011-24615, BFU2012-36825 and CSD2009-00088 from the Spanish Ministry of Science and Innovation, the CIBER of Respiratory Diseases (CIBERES), an initiative from ISCIII, the Regional Government of Madrid (S2010/BMD-2353 and S2010/BMD-2457) and funding from the European Union’s Seventh Framework Program FP7/2007-2013 under REA grant agreements no. 260600 (GlycoHIT) and 317297 (GLYCOPHARM), F.M.R. by an ESF/CSIC funded JAE-Doc Contract.

The litter bag technique was used to study leaf decomposition in the large forested eutrophic (160.9 μmol l -1 DIN, 1.7 μmol l -1 SRP) Ebro river and in the forested Senia (12.8 μmol l -1 DIN, 0.7 μmol l -1 RSP), Air dried leaves (3g) of Phragmites australis were used in both watercourses while Populus alba leaves were studied in the Ebro river only, and Populus nigra leaves and a submerged rooted macrophyte (stems and leaves), Potamogeton coloratus, were studied only in the Senia stream. Litter bags of 100 μm and 1 mm mesh size were collected on days 0, 3, 15, 30, 90 and 120 and 5 mm mesh bags were sampled once after 30 days. Macroinvertebrates inside the leaf bags were retrieved and ash free dry weight and carbon and nitrogen contents of the remaining detritus were determined. Litter decomposition rates, k, were estimated using a simple exponential model. No significant differences in decomposition rates were observed between 100 μm and I mm mesh bags. In 100 μm litter bags, decomposition of P. nigra leaves from the Senia stream (0.0104 day -1 ) was faster than that of P. alba leaves from the Ebro river (0.0049 day -1 ) (ANCOVA, p < 10 -5 ). The decomposition rate of P. australis was higher in the Ebro river (0.009 ± 0.002 day -1 ) than in the Senia stream (0.0056 ± 0.0004 day -1 ) for the first 30 days of decomposition, but no differences were observed from day 30 onwards. The number of macroinvertebrates increased over time and were mainly collectors-gatherers of the genus Chironomus. Percentage of detritus nitrogen increased in litter that had initially high C/N ratios (28.7 in P. alba and 64.7 in P. nigra) and decreased when initial C/N ratios were low (12.6 and 14.6 for P. australis from the Senia and Ebro river respectively).

A number of experimental freshwater wetlands (150 m long x 75 m wide) with different ages since they were abandoned as rice fields, were used to analyze the prospects of multipurpose wetland restoration for such degraded areas. Nitrogen and phosphorus removal rate of the wetlands were determined monthly during the flooding season to estimate their efficiency as filters to remove nutrients from agricultural sewage. The number of wetland birds was recorded regularly to identify their habitat preferences. Both the temporal dynamics and changes in the spatial pattern of land use cover during the last 20 years were determined from aerial photographs and field analysis. All the wetlands appeared to be very efficient in the removal of nitrogen and phosphorus exported from rice fields. Usually 50-98% of the nitrogen and less than 50% of the soluble phosphorus were removed by the wetlands at any stage of restoration. Wetland birds preferred wetlands with intermediate plant cover for resting and sleeping activities better than rice fields and either very open wetlands or very dense ones with tall vegetation. Apart from the improvement in water quality and the restoration of natural habitats, restoration of wetland belts around lagoons will increase spatial heterogeneity and diversity of the landscape.

This study examines the effect of pH changes on photosynthetic characteristics and the role of dissolved inorganic carbon (DIC) in determining the dominance of three species of macroalgae Chaetomorpha linum (O.F. Muller) Kutzing, Gracilaria verrucosa (Hudson) Papenfuss and Ulva sp. in a Mediterranean coastal lagoon. Fluctuations of pH were measured in the lagoon in summer. Water column CO 2 and HCO 3 − concentrations inside the algal mat showed significant diurnal fluctuations, from a morning peak to an afternoon low, decreasing 96 and 40%, respectively. The response of photosynthesis to increased pH was examined in laboratory conditions in spring (May) and summer (July). The photosynthetic rate declined rapidly at pH above 8.5 and below 6.5. G. verrucosa responded differently in spring and summer showing acclimation to higher pH in summer than in spring. In Ulva sp. incubations, we observed optimum photosynthesis between pH 6 and 7.5. The decrease in photosynthetic rate below pH 6 was lower (12.30%) than above pH 8 (81.03%). This difference may be related to the origin of the macroalgae, suggesting acclimation to the original pH of the environment. Results from instantaneous photosynthesis measurements indicate that low DIC-availability limits the photosynthetic capacity of G. verrucosa,, C. linum and Ulva sp. in spring and of C. linum at high irradiances in summer. Our results also show that G. verrucosa has a higher efficiency at low CO 2 concentrations than C. linum and Ulva sp. It is suggested that G. verrucosa may be better adapted to maintain higher photosynthetic rates than other macroalgae in conditions of tissue N sufficiency and low water DIC concentrations which are typical of shallow coastal environments in summer.

During the last 10 years, a drastic change in the structure of the community of primary producers has been observed in Tancada Lagoon (Ebro Delta, NE Spain). This consisted of a decrease in the abundance of submerged rooted macrophyte cover and a spring and summer increase in floating macroalgae. Two spatial patterns have been observed. In the west part of the lagoon, Chaetomorpha linum Kutzing, dominated during winter and decreased progressively in spring when Cladophora sp. reached its maximum development. In the east part of the lagoon, higher macroalgal diversity was observed, together with lower cover in winter and early spring. Cladophora sp., Gracilaria verrucosa Papenfuss and Chondria tenuissima Agardh, increased cover and biomass in summer. Maximum photosynthetic production was observed in spring for G. verrucosa (10·9 mg O 2 g −1 DW h −1 ) and C. tenuissima (19·0 mg O 2 g −1 DW h −1 ) in contrast with Cladophora sp. (15·9 mg O 2 g −1 DW h −1 ) and Chaetomorpha linum (7·2 mg O 2 g −1 DW h −1 ) which reached maximum production in summer. Increased conductivity from reduced freshwater inflow, and higher water temperatures during periods of lagoon isolation, mainly in summer, were the main physical factors associated with an increase in floating macroalgal biomass across the lagoon. Reduced nitrogen availability and temperature-related changes in carbon availability during summer were related to a decrease in abundance of C. linum and increases in G. verrucosa and Cladophora sp.

Two-dimensional NMR spectroscopy has been used to monitor the exchange of backbone amide protons in ribonuclease A (RNase A) and its subtilisin-cleaved form, ribonuclease S (RNase S). Exchange measurements at two different pH values (5.4 and 6.0) show that the exchange process occurs according to the conditions of the EX2 limit. Differential scanning calorimetry measurements have been carried out in 2H2O under conditions analogous to those used in the NMR experiments in order to determine the values of DeltaCp, DeltaHu and Tm, corresponding to the thermal denaturation of both proteins. For the amide protons of a large number of residues in RNase A, the free energies at 25 degreesC for exchange competent unfolding processes are much lower than the calorimetric denaturation free energies, thus showing that exchange occurs through local fluctuations in the native state. For 20 other protons, the cleavage reaction had approximately the same effect on the exchange rate constants than on the equilibrium constant for unfolding, indicating that those protons exchange by global unfolding. There is a good agreement between the residues to which these protons belong and those involved in the putative folding nucleation site identified by quench-flow NMR studies. The unfolding free energies of the slowest exchanging protons, DeltaGex, as evaluated from exchange data, are much larger than the calorimetric free energies of unfolding, DeltaGu. Given the agreement between DeltaDeltaGex(A-S), the difference in free energy from exchange for a given proton of the two proteins, and DeltaDeltaGu(A-S), the difference in the calorimetric free energy of the two proteins, the discrepancy indicates that the intrinsic exchange rates in the unfolded state of those protons cannot be approximated by those measured in short unstructured peptides and, consequently, exchange for those protons in RNase A and S must occur through a rather structured denatured state.

The relationship between primary production and irradiance was analyzed over an annual cycle in the submerged macrophytes, Chara hispida and Potamogeton pectinatus, of two mediterranean stream ponds. The effect of temperature on photosynthesis was also studied. The population of P. pectinatus reached maximum production values of 19.67 mgO2 g−1 AFDW h−1 in December at 20°C. In summer and owing to the development of floating filamentous algae, P. pectinatus growth decreased. This can be observed in a decrease in production and chlorophyll content. At the end of autumn a `self-pruning' mechanism in P. pectinatus caused increase of light penetration and new shoots were observed. C. hispida showed maximum production values of 10.86 mg O2 g−1 AFDW h−1 in May. In summer, in agreement with temperature increase C. hispida fructifies and decays, increasing P. pectinatus growth and cover in the same pond. Photoinhibition was observed in C. hispida in October, in agreement with a low radiation adaptation owing to an unusual decrease in light penetration in the pond, caused by inputs of suspended material from a fire suffered in the watershed at the end of summer. Variations in temperature had no effect on Pm/R ratio in P. pectinatus and decreased sharply from young to old tissue. The effect of temperature on the Pm/R ratio in C. hispida was significant. P. pectinatus showed great adaptation capacity to temperature fluctuations and light and this adaptation is higher than in C. hispida, which is affected by temperature increase and light penetration reduction.

J. Jesús Casas et al., Terrestrial plant litter is important in sustaining stream food webs in forested headwaters. Leaf litter quality often decreases when native species are replaced by introduced species, and a lower quality of leaf litter inputs may alter litter decomposition at sites afforested with non-native species. However, since detritivore composition and resource use plasticity may depend on the prevalent litter inputs, the extent of the alteration in decomposition can vary between streams. We tested 2 hypotheses using 2 native and 3 introduced species of tree differing in quality in 4 Iberian regions with contrasting vegetational traits: 1) decomposition rates of all plant species would be higher in regions where streams normally receive litter inputs of lower rather than higher quality; 2) a higher resource-use plasticity of detritivores in regions vegetated with plants of lower litter quality will cause a greater evenness in decomposition rates among plant species compared to regions where streams normally receive higher-quality plant litter inputs. Results showed a highly consistent interspecific ranking of decomposition rates across regions driven by litter quality, and a significant regional effect. Hypothesis 1 was supported: decomposition rates of the five litter types were generally higher in streams from regions vegetated with species producing leaf litter of low quality, possibly due to the profusion of caddisfly shredders in their communities. Hypothesis 2 was not supported: the relative differences in decomposition rates among leaf litter species remained essentially unaltered across regions. Our results suggest that, even in regions where detritivores can be comparatively efficient using resources of low quality, caution is needed particularly when afforestation programs introduce plant species of lower litter quality than the native species. © 2013 Elsevier B.V., This research was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) projects IMPARIOS (CGL2007-66664-C04) and TEMPERIOS (CGL2010-22129-C04).JJC and MM contributed to this manuscript during tenure of the grants RIBARID (CGL2012-39635) and CARBONET (CGL2011-30474-C02-02) (MINECO), respectively.

Actinoporins are water-soluble proteins with the ability to form pores upon insertion into biological membranes. They constitute a family of proteins with high degree of sequence identities but different hemolytic activities, suggesting that minor conformational arrangements result in major functional changes. A good example of this situation is the sea anemone Stichodactyla helianthus which produces two very similar actinoporins, sticholysins I (StnI) and II (StnII), but of very different hemolytic efficiency. Within this idea, given that the high resolution three-dimensional structure of StnII is already known, we have now solved that one corresponding to StnI in order to analyze the influence of particular residues on the conformation and activity of these proteins. In addition, random mutagenesis has been also used to produce five less hemolytic variants of StnI. All these mutations map to functionally relevant regions because they are probably involved in conformational changes associated with pore formation, which take place after membrane binding, and involve long-distance rearrangements of the polypeptide chain of actinoporins., This work was supported by the Spanish Ministerio de Ciencia e Innovación through projects CTQ2008-00080/BQU, CTQ2011-22514, and BFU2009-10185, and one FPU fellowship granted to S.G.-L.

Phage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny.Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from-14.93 to-3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity against Streptococcus pneumoniae (including multiresistant strains), Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-μg dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected with S. pneumoniae or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules. Copyright © 2013, American Society for Microbiology. All Rights Reserved., This research was funded by grants from the Ministerio de Ciencia e Innovación (MICINN) to P. García (SAF2009-10824) and E. García (SAF2012-39444-C02-01) and from the MICINN (BFU2009-10052 and BF42012-36825) and the Consejería de Educación de la Comunidad de Madrid (S2010/BMD/2457) to M. Menéndez. Additional funding was provided by the CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII). Roberto Díez-Martínez was the recipient of a fellowship from the MICINN (FPI program).We declare a competing financial interest. We are coinventors on a Spanish patent application (no. P201330777) covering the results contained in this article. Any potential income generated by exploitation of the patent rights and received by our employers, the CSIC, and CIBERES shall be shared with us according to Spanish law

37 p-.8 fig.-3 tab., The comparatively small number of members of the family of adhesion/growth-regulatory galectins in chicken predestines this system as an attractive model to study divergence of these lectins after gene duplications. Expression profiling of the three homodimeric (proto-type) chicken galectins (CG-1A, CG-1B and CG-2) has raised evidence for distinct functionalities, explaining the interest in a detailed crystallographic analysis of CG-2. As revealed here, marked differences are found in the ligand-binding site and in the contact pattern within the homodimer interface, underlying a characteristic orientation of the two subunits. Notably, a distinctive trimer of dimers that is unique in all galectin crystal structures reported to date forms the core unit of the crystallographic assembly. Combination with spectroscopic and thermodynamic measurements, and comparisons with CG-1A and CG-1B, identifies differential changes in circular-dichroism spectra in the presence of lactose, reflecting the far-reaching impact of the ligand on hydrodynamic behavior, and inter-galectin differences in both the entropy and the enthalpy of binding. This structural information is a salient step to complete the analysis of the full set of galectins from this model organism., This work has been kindly supported by grants BFU2009-10052, BFU2011-24615, BFU2012-36825 and CSD2009-00088 from the Spanish Ministry of Science and Innovation, the CIBER of Respiratory Diseases (CIBERES), an initiative from ISCIII, the Regional Government of Madrid (S2010/BMD-2353), and funding from the European Union’s Seventh Framework Program FP7/2007-2013 under REA grant agreements no. 260600 (“GlycoHIT”) and 317297 (“GLYCOPHARM”).

The secondary and tertiary structures of the choline-dependent major pneumococcal autolysin LytA amidase and of its COOH-terminal domain, C-LytA, have been investigated by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Deconvolution analysis shows that the far-UV CD spectrum of both proteins is governed by chiral contributions, ascribed to aromatic residue clusters contained in the COOH-terminal module. The secondary structure of LytA, determined from the FTIR spectral features of the amide I′ band, results in 19% of α-helix and tight loops, 47% of β-sheets, 23% of turns, and 11% of irregular structures. Similar values are obtained for C-LytA. The addition of choline significantly modifies the far- and near-UV CD spectra of LytA and C-LytA. These changes are attributed to alterations in the environment of their aromatic clusters, since the FTIR spectra indicate that the secondary structure is essentially unaffected. CD choline titration curves at different wavelengths show the existence of two types of binding sites/subunit. Data analysis assuming protein dimerization upon saturation of the high affinity sites reveals positive cooperativity between the low affinity sites. Thermal denaturation of both proteins occurs with the formation of unfolding intermediates and the presence of residual secondary structure in the final denatured state. The irreversibility of the thermal denaturation of LytA and C-LytA results from the collapse of the polypeptide chain into intermolecular extended structures. At saturating concentrations, choline prevents the formation of these structures in the isolated COOH-terminal module.

The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel beta-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgroup of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5 degrees C and 78.6 degrees C, respectively. The calorimetric enthalpy changes (delta Hcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that beta-structure and turns are the major structural element of both PSP-I/PSP-II (53% of beta-sheet, 21% of turns) and aSFP (44% of beta-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, alpha-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-beta-structure proteins, and would support a beta-barrel topology like that predicted for the CUB domain. Other beta-structure folds, such as the Greek-key pattern characteristic of many carbohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms.

Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes., This research was supported by funds from SAF2013-49179-C2-1-R JPND_CD_FP-688- 059 (AC14/00037 ISCIII) to MCV, SIMR to KS, SAF2013-49179-C2-2-R JPND_CD_FP-688-059 (AC14/00037 ISCIII) to DVL, BFU2012-36825, S2011/BMD-2457 (Comunidad de Madrid)

11 p.-4 tab.-7 fig., Endolysins comprise a novel class of selective antibacterials refractory to develop resistances. The Cpl-7 endolysin, encoded by the Streptococcus pneumoniae bacteriophage Cp-7, consists of a catalytic module (CM) with muramidase activity and a cell wall-binding module (CWBM) made of three fully conserved CW_7 repeats essential for activity. Firstly identified in the Cpl-7 endolysin, CW_7 motifs are also present in a great variety of cell wall hydrolases encoded, among others, by human and live-stock pathogens. However, the nature of CW_7 receptors on the bacterial envelope remains unknown. In the present study, the structural stability of Cpl-7 and the target recognized by CW_7 repeats, relevant for exploitation of Cpl-7 as antimicrobial, have been analyzed, and transitions from the CM and the CWBM assigned, using circular dichroism and differential scanning calorimetry. Cpl-7 stability is maximum around 6.0–6.5, near the optimal pH for activity. Above pH 8.0 the CM becomes extremely unstable, probably due to deprotonation of the N-terminal amino-group, whereas the CWBM is rather insensitive to pH variation and its structural stabilization by GlcNAc-MurNAc-L-Ala-D-isoGln points to the cell wall muropeptide as the cell wall target recognized by the CW_7 repeats. Denaturation data also revealed that Cpl-7 is organized into two essentially independent folding units, which will facilitate the recombination of the CM and the CWBM with other catalytic domains and/or cell wall-binding motifs to yield new tailored chimeric lysins with higher bactericidal activities or new pathogen specificities., This work was supported by grants SAF2009-10824, and BFU2009-10052 from Ministerio de Ciencia e Innovacio´n (MICINN), and BIPPEP2 from Comunidad Autónoma de Madrid (S2010/BMD-2457). Additional funding was provided by the CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the ISCIII. P. Rico-Lastres was supported by a fellowship from MICINN.

Amyloidogenic neurodegenerative diseases are incurable conditions with high social impact that are typically caused by specific, largely disordered proteins. However, the underlying molecular mechanism remains elusive to established techniques. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these >neurotoxic proteins> triggers the pathogenic cascade. We use force spectroscopy and a novel methodology for unequivocal single-molecule identification to demonstrate a rich conformational polymorphism in the monomer of four representative neurotoxic proteins. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the critical monomeric β-conformational change, neurotoxicity, and neurodegeneration. Hence, we postulate that specific mechanostable conformers are the cause of these diseases, representing important new early-diagnostic and therapeutic targets. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt, or reverse multiple neurodegenerative diseases. © 2012 Hervás et al.

1 pags. -- 56th Annual Meeting of the Biophysical-Society, FEB 25-29, 2012, San Diego, CA, Amyloidogenic neurodegenerative diseases are incurable conditions caused by specific largely disordered proteins. However, the underlying molecular mechanism remains elusive. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these ‘‘neurotoxic proteins’’ triggers the pathogenic cascade. Using force spectroscopy with unequivocal singlemolecule identification we demonstrate a rich conformational polymorphism at their monomer level. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the monomeric b-conformational change and neurodegeneration. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt or reverse multiple neurodegenerative diseases

Leaf litter decomposition is a crucial process providing matter and energy to communities inhabiting headwater streams. This process could be affected by many man-made landscape transformations and its response can vary depending on the climate setting. In this study, we test the hypothesis that the presence of small headwater reservoirs decreases litter decomposition downstream, as reported for temperate Oceanic climatic regions, and that this effect is more accentuated in the Mediterranean. The effect of small dams on the decomposition of alder (Alnus glutinosa) leaves was studied in four headwater streams in Catalonia (NE Spain). The presence of a dam affected litter decomposition rates in three of the four streams studied, and this depended on reservoir typology. In those with seasonal surface release, decomposition rates were slower downstream from the dams, but in the case of a continuous hypolimnetic release, it was faster, with higher DIN and temperature and abundance of shredders. Alder litter decomposition rates were twice those reported for Oceanic climatic conditions. In Mediterranean headwaters, the effect of small dams will even be more evident at an annual scale due to the diminished flow rates in summer and this effect will be more pronounced than in the more Oceanic. © 2012 Springer Science+Business Media B.V., This study was funded by the Spanish Ministry of Education and Science (projects CGL2007-462 66664-C04 and CGL2011-30474-C02).

Gemini surfactants have two polar head groups and two hydrocarbon tails. Compared with conventional surfactants, geminis have much lower (οM vs. mM) critical micelle concentrations and possess slower (ms vs οs) monomer micelle kinetics. The structure of the gemini surfactants studied is [HOCH 2CH 2-, CH 3-, CH 3(CH 2) 15-N +-(CH 2) s-N +-(CH 2) 15CH 3,-CH 3,-CH 2CH 2OH]·2Br - where s=4, 5, or 6. Our objective is to reveal the effect of these cationic gemini surfactants on the structure and stability of two model proteins: Ribonuclease A (RNase A) and Hen Egg White Lysozyme (HEWL). 2D 1H NMR and Circular Dichroism (CD) spectroscopies show that the conformation of RNase A and HEWL is unaffected at low to neutral pH where these proteins are positively charged, although hydrogen exchange shows that RNase A's conformational stability is slightly lowered. At alkaline pH, where these proteins lose their net positive charge, fluorescence and CD spectroscopies and ITC experiments show that they do interact with gemini surfactants, and multiple protein•gemini complexes are observed. Based on the results, we conclude that these cationic gemini surfactants neither interact strongly with nor severely destabilize these well folded proteins in physiological conditions, and we advance that they can serve as useful membrane mimetics for studying the interactions between membrane components and positively charged proteins. © 2011 Elsevier Inc.

The recognition of protein surfaces by designed ligands has become an attractive approach in drug discovery. However, the variable nature and irregular behavior of protein surfaces defy this new area of research. The easy to understand "lock-and-key" model is far from being the ideal paradigm in biomolecular interactions and, hence, any new finding on how proteins and ligands behave in recognition events paves a step of the way. Herein, we illustrate a clear example on how an increase in flexibility of both protein and ligand can result in an increase in the stability of the macromolecular complex. The biophysical study of the interaction between a designed flexible tetraguanidinium-calix[4]arene and the tetramerization domain of protein p53 (p53TD) and its natural mutant p53TD-R337H shows how the floppy mutant domain interacts more tightly with the ligand than the well-packed wild-type protein. Moreover, the flexible calixarene ligand interacts with higher affinity to both wild-type and mutated protein domains than a conformationally rigid calixarene analog previously reported. These findings underscore the crucial role of flexibility in molecular recognition processes, for both small ligands and large biomolecular surfaces.

11 páginas, 8 figuras -- PAGS nrs. 5445-5455, Thioglycosides offer the advantage over O-glycosides to be resistant to hydrolysis. Based on initial evidence of this recognition ability for glycosyldisulfides by screening dynamic combinatorial libraries, we have now systematically studied dithiodigalactoside on a plant toxin (Viscum album agglutinin) and five human lectins (adhesion/growth-regulatory galectins with medical relevance e.g. in tumor progression and spread). Inhibition assays with surface-presented neoglycoprotein and in solution monitored by saturation transfer difference NMR spectroscopy, flanked by epitope mapping, as well as isothermal titration calorimetry revealed binding properties to VAA (K(a): 1560 +/- 20 M (1)). They were reflected by the structural model and the affinity on the level of toxin-exposed cells. In comparison, galectins were considerably less reactive, with intrafamily grading down to very minor reactivity for tandem-repeat-type galectins, as quantitated by radioassays for both domains of galectin-4. Model building indicated contact formation to be restricted to only one galactose moiety, in contrast to thiodigalactoside. The tested glycosyldisulfide exhibits selectivity between the plant toxin and the tested human lectins, and also between these proteins. Therefore, glycosyldisulfides have potential as chemical platform for inhibitor design., This work has been generously supported by grants CTQ2009-08536, SAF2008-00945, BFU2006-10288 and BFU2009-10052 from the Spanish Ministry, an EC Marie Curie Research Training Network grant (contract no. MRTN-CT-2005-019561), the EC research program GlycoHIT (contract ID 260600), the Verein zur Forderung des biologisch-technologischen Fortschritts in der Medizin e. V. and the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII). We thank Drs B. Friday and S. Namirha for inspiring discussions as well as M. V. Lopez-Moyano for excellent technical assistance.

The main purpose of our work was to elucidate factors responsible for the geographical differences in leaf-litter decomposition rates in Spanish oligotrophic headwater streams. Decomposition experiments with alder (Alnus glutinosa) leaf litter were carried out in 22 headwater streams in 4 different climatic regions across the Iberian Peninsula (Cornisa Cantbrica, Cordillera Litoral Catalana, Sierra de Guadarrama, and Sierra Nevada). Streams that were similar in size, flowed mainly over siliceous substrate in catchments with scarce human settlements and activities, and fell within a range of low nutrient concentrations were chosen in each region. Breakdown rates were regionally variable and were low (0.1090.198% ash-free dry mass [AFDM]/degree day [dd]) in the Cornisa Cantbrica, the most mesic and Atlantic region, and high (0.3020.639% AFDM/dd) in Sierra de Guadarrama, one of the coldest and most inland areas. Temperature was not the determining factor affecting differences in breakdown rates among regions, and breakdown rates were not related to concentrations of dissolved nutrients. However, microbial reproductive activity (sporulation rates) was significantly correlated with dissolved P concentration. Breakdown rates were explained better by presence and feeding activities of detritivores than by decomposer activity. Incorporation of breakdown rates in assessment schemes of stream ecological status will be difficult because leaf processing does not respond unequivocally to environmental factors when climatic regions are considered. Thus, regional adjustments of baseline standards in reference conditions will be required. © 2011 by The North American Benthological Society., This study was funded by the Spanish Ministry of Education and Science (project CGL2007-66664-C04), by the University of the Basque Country (Research grant GIU05/38), and by the Basque Government (Research grants IT-422-07 and IT-302-10)

Agricultural runoff and urban activities can increase the inputs of nitrogen (N) and phosphorus (P), into headwater streams, leading to eutrophication and thus substantially affecting the structure and functions of benthic communities. A high P concentration in water stimulates the activity of heterotrophic microorganisms associated with leaf litter and, hence, influences decomposition rates and the availability of detrital resources for macroinvertebrates. Litter breakdown of alder (Alnus glutinosa) leaves enclosed in coarse mesh bags was studied in five low-order Mediterranean streams with different trophic status defined by their soluble reactive phosphorus (SRP) concentrations. Decomposition rates differed significantly between these streams and increased with the eutrophication gradient, but these differences were not always related to the availability of P in water. Leaf mass loss was directly correlated with shredder density, but macroinvertebrate density and diversity were not related to P availability in water, and ammonium concentration had a negative effect on macroinvertebrate diversity and shredder relative abundance. A significantly positive effect of nitrate concentration in water on aquatic hyphomycete sporulation rates was observed, but there also was a negative effect of % ammonium on dissolved inorganic nitrogen (DIN). The predominantly available ionic form of DIN could thus affect the structure of the aquatic hyphomycete community. These results suggest that the response of litter decomposition to eutrophication in forested headwater streams is strongly influenced by local stream characteristics and by the nature of nutrient pollution. © 2011 Springer Science+Business Media B.V., This study was funded by the Spanish Ministry of Education and Science (project CGL2007-462 66664-C04)

Classical ‘proton sponges’ related to 1,8-bis(dimethylamino) naphthalene (DMAN) and new ones derived from the replacement of one or both dimethylamino groups by iminophosphorane groups are compared (i) in the solid state using crystallographic data, (ii) in solution in deuteriochloroform and [2H6]dimethyl sulfoxide by 1H and 13C NMR spectroscopy, (iii) in water and ethanol–water mixtures by determining their pKa′ s. The conclusion is that the new sponges are stronger than the classical ones but, since they decompose when the acid proton is removed are of no practical value as chemical reagents.

21 p.- 7 fig.- 4 tab.-2 fig. supl.-1 tab. supl., Bacteriophage endolysins include a group of new antibacterials reluctant to development of resistance. We present here the first structural study of the Cpl-7 endolysin, encoded by pneumococcal bacteriophage Cp-7. It contains an N-terminal catalytic module (CM) belonging to the GH25 family of glycosyl hydrolases and a C-terminal region encompassing three identical repeats of 42 amino acids (CW_7 repeats). These repeats are unrelated to choline-targeting motifs present in other cell wall hydrolases produced by Streptococcus pneumoniae and its bacteriophages, and are responsible for the protein attachment to the cell wall. By combining different biophysical techniques and molecular modeling, a three-dimensional model of the overall protein structure is proposed, consistent with circular dichroism and sequence-based secondary structure prediction, small angle x-ray scattering data, and Cpl-7 hydrodynamic behavior. Cpl-7 is an ∼115-Å long molecule with two well differentiated regions, corresponding to the CM and the cell wall binding region (CWBR), arranged in a lateral disposition. The CM displays the (βα)5β3 barrel topology characteristic of the GH25 family, and the impact of sequence differences with the CM of the Cpl-1 lysozyme in substrate binding is discussed. The CWBR is organized in three tandemly assembled three-helical bundles whose dispositions remind us of a super-helical structure. Its approximate dimensions are 60 × 20 × 20 Å and presents a concave face that might constitute the functional region involved in bacterial surface recognition. The distribution of CW_7 repeats in the sequences deposited in the Entrez Database have been examined, and the results drastically expanded the antimicrobial potential of the Cpl-7 endolysin, This work was supported by Grants BFU2006-10288, SAF2009-10824, BIO2007-61336, and BFU2009-10052 from Ministerio de Ciencia e Innovación, Grant BIPPED-CM from the Comunidad de Madrid, the CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the ISCIII, Glycodynamics Network Grant FP6-UE MCTN-CT-2005-019561, and European Community beam time Proposal 48086

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Human tandem-repeat-type galectin-9 is a potent adhesion/growth-regulatory effector via lectin capacity of its N- and C-terminal domains. This bioactivity prompted further crystallographic study of the N-domain, combined with analysis in solution. Binding of lactose markedly increased the N-domain's resistance to thermal denaturation. Crystallography revealed its intimate contact profile, besides detecting an extension of the beta-sandwich fold by an antiparallel beta-strand F0 aligned to the C-terminal F1 strand. Ligand accommodation in its low-energy conformation leads to a movement of Arg87's side chain. As consequence, the ligand's glucose moiety and Arg87 become hydrogen bonded. The resulting predictions for spatial parameters in solution were verified by determining (a) the pattern of magnetization transfer from the protein to protons of lactose and Forssman disaccharide by NMR spectroscopy and (b) the ellipticity changes at wavelengths characteristic for Trp/Tyr residues in near-UV CD spectroscopy. Whereas solid-phase assays confirmed a previously noted tendency for homo- and heterotypic aggregation, gel filtration and ultracentrifugation disclosed monomeric status in solution, in line with crystallographic data. Using cell mutants with defects in glycosylation, this lectin domain was shown to preferentially bind N-glycans without alpha2,3-sialylation. Since proximal promoter sequences were delineated to diverge markedly among galectin genes and resulting differences in expression profiles were exemplarily documented immunohistochemically, the intrafamily diversification appears to have assigned this protein to a characteristic expression and activity profile among galectins. Our data thus take the crystallographic information to the level of the lectin in solution and in tissues by a strategic combination of spectroscopic and cell/histochemical assays.

Experimental determinations have been carried out of the thermodynamic basicities and acidities in the gas phase and in aqueous solution of pyrazoles substituted at positions 3 and 5 with methyl and trifluoromethyl groups. Corresponding theoretical calculations at the STO-3G level have also been done. The results are highly consistent both between relative gas-phase properties and theoretical calculations (including STO-3G calculations of anions) and between gas-phase and aqueous solution values. The trifluoromethyl group is found to considerably decrease the basicity (more in position 3 than in position 5) and increase the acidity (more in position 5 than in position 3)

6 pags, 6 figs, Protein p53 is a transcription factor crucial for cell cycle and genome integrity. It is able to induce both cell arrest when DNA is damaged and the expression of DNA repair machinery. When the damage is irreversible, it triggers apoptosis. Indeed, the protein, which is a homotetramer, is mutated in most human cancers. For instance, the inherited mutation p53-R337H results in destabilization of the tetramer and, consequently, leads to an organism prone to tumor setup. We describe herein a rational designed molecule capable of holding together the four monomers of the mutated p53-R337H protein, recovering the tetramer integrity as in the wild-type structure. Two ligand molecules, based on a conical calix[4]arene with four cationic guanidiniomethyl groups at the wider edge (upper rim) and hydrophobic loops at the narrower edge (lower rim), fit nicely and cooperatively into the hydrophobic clefts between two of the monomers at each side of the protein and keep the tetrameric structure, like molecular templates, by both ion-pair and hydrophobic interactions. We found a good agreement between the structure of the complex and the nature of the interactions involved by a combination of theory (molecular dynamics) and experiments (circular dichroism, differential scanning calorimetry and 1H saturation transfer difference NMR). © 2008 by The National Academy of Sciences of the USA., S.G. thanks B. Claasen for her help on the STD experiments design and T. Tarrago for biological support. Some of the simulations were run at the Barcelona Supercomputing Center-Mare Nostrum. This work was supported by the Spanish Ministry of Science and Education (MEC) Projects BIO2005-00295, CTQ2005-06909-C02-02/BQU, and CTQ2005-08989-C02/BQU, Consolider Ingenio 2010 Grant CSD2006-0003, and the Institute of Chemical Research of Catalonia (ICIQ) Foundation. S.G., V.M., and E.S. received predoctoral fellowships from Generalitat de Catalunya-Agencia de Gestio d'Ajuts Universitaris i de Recerca, MEC, and the ICIQ Foundation.

18 páginas, 14 figuras, 2 tablas -- PAGS nros. 5086-5103, The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic inactivation of the drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4′) from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4′) displays a wide tolerance to conformational variations in the drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Ψ orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4′) against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4′) as aminoglycoside receptors, Financial support from DGES (grants CTQ2004-04494/BQU, CTQ2007-67403/BQU, and CTQ2004-03523/BQU) is acknowledged. J.R. and F.C. thank the Ministerio de Educación y Ciencia for Juan de la Cierva and Ramon y Cajal contracts, respectively. We also thank CESGA (Santiago de Compostela) for computer support. T.V. thanks CSIC for a JAE contract

11 páginas. 7 figuras, 4 tablas, 1 esquema --pags NROS. 28618-28628, The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 °C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 °C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 ± 0.5 sites/monomer) behave as equivalent (Kd = 2.7 ± 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 °C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 °C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile., The integrity of the cell wall is essential for bacterial survival, and peptidoglycan-metabolizing enzymes have to work closely together to prevent the rupture of the cell wall and the cell lysis during cell growth and division (1). In particular, the potentially suicidal activity of autolytic enzymes should be strictly controlled (2), and tight binding to the cell envelope (3) seems to work as a mechanism for the regulation and control of many murein hydrolases from different bacterial species (4). In Streptococcus pneumoniae, the presence of phosphorylcholine residues on lipoteichoic and teichoic acids (5), a characteristic shared by other related microorganisms (6), determines the activity of murein hydrolases. In addition to the catalytic module (CM),3 these enzymes comprise a choline-binding module (CBM) that specifically recognizes and binds the choline residues on the cell wall (4). Moreover, it has been suggested that phosphorylcholine residues would be acting as a selective pressure factor to preserve the cell wall recognition module in all of the murolytic enzymes encoded by pneumococcus so far characterized. The CBM, usually located at the C terminus of choline-binding proteins, is made up of tandemly arranged homologous repeats of about 20 amino acids (Pfam accession code PF01473) (7). Each repeat forms a β-hairpin followed by a loop and a coiled region that tend to fold into a left-handed β-solenoid (8-10). Choline binds at the interface of two consecutive β-hairpins, in a cavity lined by three aromatic side chains (8-10), and the multivalent interacting surface presented by the CBM can provide a strong binding to the pneumococcal envelope. The same type of modular organization is found in the endolysins produced by most pneumococcal bacteriophages and suggests a genetic interchange during evolution between the host and the parasites (7)., Only two autolytic enzymes (autolysins) have been unequivocally identified so far in S. pneumoniae: the well known LytA amidase and the LytC lysozyme (11). LytC is an autolysin designed to remodel the cell wall, with maximum activity at 30 °C. This feature suggests that LytC could be more active in habitats like the upper, well ventilated respiratory tract (11). In fact, LytC plays a role in the colonization of the rat nasopharynx (12), where it could also contribute to DNA release in competent cells (13). LytC is directed to the outer surface by a leader peptide (33 residues), and it remains tightly bound to the cell wall in a mature, active form that comprises a CBM made of 11 (p1-p11) repeating units (264 residues) and a CM belonging to the GH-25 family of glycosyl hydrolases (204 residues). The unprocessed form is also detected in the cytoplasm (11). The slow hydrolysis of pneumococcal cultures carried out by LytC contrasts with the fast, uncontrolled lysis of the host cell performed by Cpl-1, the lysozyme encoded by the pneumococcal phage Cp-1, in order to release the infective particles after the virion replication (11). Cpl-1 and LytC are built of homologous modules (Fig. 1) although assembled in opposite locations (7). In Cpl-1, whose three-dimensional structure has been solved (9), the catalytic module is localized at the N terminus (188 residues), and the CBM (139 residues) is made up of six repeating units (p1-p6) and a short terminal tail (7). However, only two choline-binding sites, located at the interfaces of the three first repeats (p1-p2 and p2-p3), seem to be functional, according to the crystal structure of Cpl-1 (9). In contrast, molecular modeling of LytC reveals a higher number of potential sites for choline. In addition, the alternation of shorter (17 residues) and longer (21-23 residues) repeats (see Fig. 1) in LytC leads to an uneven distribution of choline-binding sites along the CBM surface (14), previously unseen in other CBMs of known structure (8-10). We have performed a comparative study of LytC and Cpl-1 lysozymes using different approaches. The differences found in structure, stability, conformational plasticity, choline-binding avidity, and complex dynamics contribute to determine the lytic profiles of these two lysozymes. Overall, LytC and Cpl-1 represent a good illustrative example of how natural selection can tailor at the genetic level different solutions using homologous building blocks and module shuffling, This work was supported by Dirección General de Investigación Cientifica y Técnica Grants BIO2003-01952, BFU2006-10288, and SAF2006-00390. Additional funding was provided by the COMBACT program (S-BIO-0260/2006) of the Comunidad de Madrid, the CIBER of Respiratory Diseases (CIBERES), an initiative of the ISCIII, and the Glycodynamics Network (FP6-UE and MCTN-CT-2005-019561). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Pneumococcal bacteriophage-encoded lysins are modular proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) in treatment of streptococcal infections. The first x-ray crystal structures of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1, in complex with three bacterial cell wall peptidoglycan (PG) analogues are reported herein. The Cpl-1 structure is folded in two well defined modules, one responsible for anchoring to the pneumococcal cell wall and the other, a catalytic module, that hydrolyzes the PG. Conformational rearrangement of Tyr-127 is a critical event in molecular recognition of a stretch of five saccharide rings of the polymeric peptidoglycan (cell wall). The PG is bound at a stretch of the surface that is defined as the peptidoglycan-binding sites 1 and 2, the juncture of which catalysis takes place. The peptidoglycan- binding site 1 binds to a stretch of three saccharides of the peptidoglycan in a conformation essentially identical to that of the peptidoglycan in solution. In contrast, binding of two peptidoglycan saccharides at the peptidoglycan-binding site 2 introduces a kink into the solution structure of the peptidoglycan, en route to catalytic turnover. These findings provide the first structural evidence on recognition of the peptidoglycan and shed light on the discrete events of cell wall degradation by Cpl-1., This work was supported by Grants BFU2005-01645, BIO2003-01952, and BFU2006-10288 from Dirección General de Investigación by 08.2/0030.1/ 2003 from the Comunidad de Madrid, by “Factoria de Cristalización,” CONSOLIDER INGENIO-2010, and by a grant from the National Institutes of Health.