Incorporation of either molybdenum or tungsten into formate dehydrogenase from<Emphasis Type="Italic"> Desulfovibrio alaskensis</Emphasis> NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria

We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126±2 kDa composed of two subunits, =93±3 kDa and =32±2 kDa, which contains 6±1 Fe/molecule, 0.4±0.1 Mo/molecule, 0.3±0.1 W/molecule, and 1.3±0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5–40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d¹ configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme.Abbreviations AOR aldehyde oxidoreductase - FDH formate dehydrogenase - NAP periplasmic nitrate reductase - SRB sulfate-reducing bacteria     

A survey of the year 2003 literature on applications of isothermal titration calorimetry

Over the last decade isothermal titration calorimetry (ITC) has developed from a specialist method which was largely restricted in its use to dedicated experts, to a major, commercially available tool in the arsenal directed at understanding molecular interactions. The number of those proficient in this field has multiplied dramatically, as has the range of experiments to which this method has been applied. This has led to an overwhelming amount of new data and novel applications to be assessed. With the increasing number of publications in this field comes a need to highlight works of interest and impact. In this overview of the literature we have attempted to draw attention to papers and issues for which both the experienced calorimetrist and the interested dilettante hopefully will share our enthusiasm.     

A novel mammalian display system for the selection of protein-protein interactions by decoy receptor engagement

The emerging field of proteomics has created a need for new high-throughput methodologies for the analysis of gene products. An attractive approach is to develop systems that allow for clonal selection of interacting protein pairs from large molecular libraries. In this study, we have characterized a novel approach for identification and selection of protein-protein interactions, denoted SPIRE (selection of protein interactions by receptor engagement), which is based on a mammalian expression system. We have demonstrated proof of concept by creating a general plasma membrane bound decoy receptor, by displaying a protein or a peptide genetically fused to a trunctated version of the CD40 molecule. When this decoy receptor is engaged by a ligand to the displayed protein/peptide, the receptor expressing cell is rescued from apoptosis. To design a high-throughput system with a highly parallel capacity, we utilized the B cell line WEHI-231, as carrier of the decoy receptor. One specific peptide-displaying cell could be identified and amplified, based on a specific receptor engagement, in a background of 12 500 wild-type cells after four selections. This demonstrates that the approach may serve as a tool in post-genomic research for identifying protein-protein interactions, without prior knowledge of either component.     

Diffusion ordered spectroscopy as a complement to size exclusion chromatography in oligosaccharide analysis

A series of N-acetyl-chitooligosaccharides (GlcNAc)(1-6) have been studied by a nuclear magnetic resonance (NMR) method, diffusion ordered spectroscopy (DOSY). DOSY has also been applied to two additional synthetic related oligosaccharides [GlcNH(2)-(GlcNAc)(4) and GlcNH(2)-(GlcNAc)(2)-GlcNAcSO(3)Na]. A plot of the log of the determined diffusion coefficients (logD) of (GlcNAc)(n) versus the log of molecular weight was linear (6 points, R(2) = 0.995). The molecular weights of the two synthetic chitin derivatives could be estimated to within 10% error. The processed NMR data of all the chitooligosaccharides was also plotted in a polyacrylamide gel-like format to aid visual interpretation. Moreover, the logD value of the NMR signal resonances of a chitin-binding protein (hevein) changed as a function of a given titrated ligand, (GlcNAc)(6). Evidence for a 2:1 hevein:(GlcNAc)(6) complex is detected by DOSY at high hevein:(GlcNAc)(6) ratios. This data is consistent with published analytical ultracentrifugation and isothermal titration calorimetry data. A 1:1 complex is preferred at higher ligand concentrations. DOSY can complement size exclusion chromatography in carbohydrate research with the advantage that oligosaccharides are more readily detected by NMR.     

Relationship of size at return with environmental variation,hatchery production,and productivity of wild pink salmon in Prince William Sound,Alaska: does size matter?

Pink salmon (Oncorhynchus gorbuscha) returning to Prince William Sound (PWS), Alaska, have increased to historically high levels of abundance in recent years, but average body size at return has declined. We examined how body size at return of PWS pink salmon was related to 10 biophysical factors, including the scale of hatchery production. We also examined the effect of body size at return on productivity of wild pink salmon in PWS. For the 1975–1999 brood years, we found that an index of total abundance of pink salmon in the Gulf of Alaska and sea surface temperature during the year of return best explained the variation in pink salmon body size over time. Body size at return was significantly correlated with productivity of wild pink salmon. We used stepwise-regression to fit a generalized linear version of the Ricker spawner-recruit model to determine if body size would explain significant variation in wild-stock productivity in context with other environmental variation, including hatchery production. The results indicate that variability in wild-stock productivity is primarily driven by density-independent factors in the marine environment, but that body size of wild spawners also significantly affects productivity of wild PWS pink salmon. We conclude that the success of large-scale enhancement increasing the total run in PWS may have contributed to the decline in body size because of density-dependent growth in the Gulf of Alaska. We used a simulation model to estimate the impact of hatchery-induced changes in adult body size on wild-stock production in PWS. We estimated an annual wild-stock yield loss of 1.03 million pink salmon, less than 5% of the annual hatchery return of 24.2 million adult pink salmon for brood years 1990–1999.     

Effects of live coral,epilithic algal communities and substrate type on algal recruitment

The recruitment of algae is a critical process during algal colonization and invasions, including coral-to-algal phase shifts. Although algae are widely assumed to colonize and kill corals, there is very little known about the recruitment dynamics of coral reef algae. This study tested the ability of two dominant macroalgae (Fucales including Sargassum spp. and Lobophora variegata) to settle and grow on healthy coral tissue. The study also explored the effects of interactions with prior occupants, and of abiotic substrate properties (texture, and ceramic and carbonate material). The results indicate that healthy corals were able to prevent attachment or survival of recruits of these macroalgae. This is a significant point, since it suggests that the replacement of corals by algae may often require prior stress or death in the coral tissue. Pre-conditioning of plates at different sites had some effects, but these were relatively minor, whereas there was considerable variation within sites. Some of this variation appeared to be related to the amount of turf algae or crustose coralline algae on the plates. Recruitment was generally, but not always, higher on plates with rougher texture. Overall, this preliminary exploration indicates considerable potential for variability in outcomes of algal colonization, with implications for the dynamics of algal invasions. In particular, the results do not support suggestions that planktonic algal propagules can directly settle on and colonize healthy coral tissue.Communicated by Topic Editor D. Barnes     

Metal-humic complexes and plant micronutrient uptake: a study based on different plant species cultivated in diverse soil types

There are several studies in the literature dealing with the effect of metal-humic complexes on plant metal uptake, but none of them correlate the physicochemical properties of the complexes with agronomic results. Our study covers both aspects under various experimental conditions. A humic extract (SHE) obtained from a sapric peat was selected for preparing the metal–humic complexes used in plant experiments. Fe–, Zn– and Cu–humic complexes with a reaction stoichiometry of 2:0.25 (humic:metal, w/w) were chosen after studying their stability and solubility with respect to pH (6–9) and the humic:metal reaction stoichiometry. Wheat and alfalfa plants were greenhouse cultured in pots containing one of three model soils: an acid, sandy soil and two alkaline, calcareous soils. Treatments were: control (no additions), SHE (53 mg kg^–1 of SHE), and metal (Cu, Zn and Fe)–SHE complexes (2.5 and 5 mg kg^–1 of metal rate and a SHE concentration to make 53 mg kg ^–1). Cu- and Zn–humic complexes significantly (p0.05) increased the plant uptake and the DTPA-extractable soil fraction of complexed micronutrients in most plant–soil systems. However, these effects were associated with significant increases (p0.05) of shoot and root dry weight only in alfalfa plants. In wheat, significant increases of root and shoot dry matter were only observed in the Cu–humic treated plants growing in the acid soil, where Cu deficiency was more intense. The Fe–humic complex did not increase Fe plant assimilation in any plant–soil system, but SHE increased Fe-uptake and/or DTPA-extractable soil Fe in the wheat–calcareous soil systems. These results, taken together with those obtained from the study of the pH- and SHE:metal ratio-dependent SHE complex solubility and stability, highlight the importance of the humic:Fe complex stoichiometry on iron bioavailability as a result of its influence on complex solubility.     

Baby-transfer and other interactions between its mother and grandmother in a captive social group of lowland gorillas

This report describes the responses of an experienced gorilla mother to inappropriate maternal behavior displayed by her young adult daughter toward a newborn baby and repeated acts of baby-transfer between these two females in a captive social group of lowland gorillas (Gorilla g. gorilla). The quality of infant care by the young adult daughter clearly improved during the first 4 days after birth, and this improvement was at least partly based on her mothers encouragement. Thus, the mothers activities can be considered scaffolding for her daughter with regard to maternal infant care.     

Mitochondrial Respiratory Chain Complex Patterns from Acanthamoeba castellanii and Lycopersicon esculentum: Comparative Analysis by BN-PAGE and Evidence of Protein–Protein Interaction Between Alternative Oxidase and Complex III

We have previously shown that a kinetic interplay exists between the cytochrome pathway and the alternative oxidase in mitochondria from amoeba Acanthamoeba castellanii . Native interaction analyses using blue native gel electrophoresis coupled to denaturating electrophoresis and immunodetection have indicated associations between alternative oxidase and oxidative phosphorylation complexes in both amoeba and tomato mitochondria. These associations are dependent on the expression level of alternative oxidase according to the physiological state in both organisms. Alternative oxidase associates broadly with large complexes of the respiratory chain when it is expressed in large amount, i.e., in ripe tomato and exponentially growing amoeba. On the contrary, alternative oxidase interacts specifically with complex III even if expression of the oxidase is low, i.e., in green tomato and stationary phase amoeba. This specific interaction represents a higher level of regulation driven by protein-protein interactions leading to a direct kinetic interplay between the cytochrome pathway and alternative oxidase in both plant and amoeba mitochondria.