Structure of bradavidin-C-terminal residues act as intrinsic ligands

Bradavidin is a homotetrameric biotin-binding protein from Bradyrhizobium japonicum, a nitrogen fixing and root nodule-forming symbiotic bacterium of the soybean. Wild-type (wt) bradavidin has 138 amino acid residues, whereas the C-terminally truncated core-bradavidin has only 118 residues. We have solved the X-ray structure of wt bradavidin and found that the C-terminal amino acids of each subunit were uniquely bound to the biotin-binding pocket of an adjacent subunit. The biotin-binding pocket occupying peptide (SEKLSNTK) was named "Brad-tag" and it serves as an intrinsic stabilizing ligand in wt bradavidin. The binding of Brad-tag to core-bradavidin was analysed by isothermal titration calorimetry and a binding affinity of ～25 μM was measured. In order to study the potential of Brad-tag, a green fluorescent protein tagged with Brad-tag was prepared and successfully concentrated from a bacterial cell lysate using core-bradavidin-functionalized Sepharose resin.     

Alanine scanning mutagenesis of a type 1 insulin-like growth factor receptor ligand binding site

The high resolution crystal structure of an N-terminal fragment of the IGF-I receptor, has been reported. While this fragment is itself devoid of ligand binding activity, mutational analysis has indicated that its N terminus (L1, amino acids 1-150) and the C terminus of its cysteine-rich domain (amino acids 190-300) contain ligand binding determinants. Mutational analysis also suggests that amino acids 692-702 from the C terminus of the alpha subunit are critical for ligand binding. A fusion protein, formed from these fragments, binds IGF-I with an affinity similar to that of the whole extracellular domain, suggesting that these are the minimal structural elements of the IGF-I binding site. To further characterize the binding site, we have performed structure directed and alanine-scanning mutagenesis of L1, the cysteine-rich domain and amino acids 692-702. Alanine mutants of residues in these regions were transiently expressed as secreted recombinant receptors and their affinity was determined. In L1 alanine mutants of Asp(8), Asn(11), Tyr(28), His(30), Leu(33), Leu(56), Phe(58), Arg(59), and Trp(79) produced a 2- to 10-fold decrease in affinity and alanine mutation of Phe(90) resulted in a 23-fold decrease in affinity. In the cysteine-rich domain, mutation of Arg(240), Phe(241), Glu(242), and Phe(251) produced a 2- to 10-fold decrease in affinity. In the region between amino acids 692 and 702, alanine mutation of Phe(701) produced a receptor devoid of binding activity and alanine mutations of Phe(693), Glu(693), Asn(694), Leu(696), His(697), Asn(698), and Ile(700) exhibited decreases in affinity ranging from 10- to 30-fold. With the exception of Trp(79), the disruptive mutants in L1 form a discrete epitope on the surface of the receptor. Those in the cysteine-rich domain essential for intact affinity also form a discrete epitope together with Trp(79).     

Biochemical characterization and structural analysis of a new cold-active and salt-tolerant esterase from the marine bacterium <Emphasis Type="Italic">Thalassospira</Emphasis> sp.

A gene encoding an esterase, ThaEst2349, was identified in the marine psychrophilic bacterium Thalassospira sp. GB04J01. The gene was cloned and overexpressed in E. coli as a His-tagged fusion protein. The recombinant enzyme showed optimal activity at 45 °C and the thermal stability displayed a retention of 75 % relative activity at 40 °C after 2 h. The optimal pH was 8.5 but the enzyme kept more than 75 % of its maximal activity between pH 8.0 and 9.5. ThaEst2349 also showed remarkable tolerance towards high concentrations of salt and it was active against short-chain p-nitrophenyl esters, displaying optimal activity with the acetate. The enzyme was tested for tolerance of organic solvents and the results are suggesting that it could function as an interesting candidate for biotechnological applications. The crystal structure of ThaEst2349 was determined to 1.69 Å revealing an asymmetric unit containing two chains, which also is the biological unit. The structure has a characteristic cap domain and a catalytic triad comprising Ser158, His285 and Asp255. To explain the cold-active nature of the enzyme, we compared it against thermophilic counterparts. Our hypothesis is that a high methionine content, less hydrogen bonds and less ion pairs render the enzyme more flexible at low temperatures.     

Movements of satellite-monitored humpback whales on their feeding ground along the Antarctic Peninsula

Humpback whales were instrumented with satellite transmitters off the western Antarctic Peninsula in January of 2004–2006 to examine their movement patterns and habitat use. Whales were tracked from 4 to 80 days (mean = 36.5 days). Distance and travel rate estimates for nine individuals ranged from 223 to 4,356 km and from 17 to 75 km/day, respectively. Considerable individual variation was observed in direction, speed and range of movements. The overall pattern was characterized by short- and long-distance movements between presumed foraging areas with relatively short residency times. Travel rates were lower at these sites, characterized by erratic movements, than during traveling between them. Area usage for six individuals based on the 95% fixed kernel home range with least squares cross-validation ranged from 2,771 to 172,356 km². The management boundary between the feeding grounds associated with Breeding Stocks G and A needs revision, as current available data suggest it should be located to the east of 50°W. This study is the first to present detailed information on the movements of humpback whales in the Southern Ocean.     

Outbreaking forest insect drives phase synchrony among sympatric folivores: Exploring potential mechanisms

We explore a common feature of insect population dynamics, interspecific synchrony, which refers to synchrony in population dynamics among sympatric populations of different species. Such synchrony can arise via several possible mechanisms, including shared environmental effects and shared trophic interactions, but distinguishing the relative importance among different mechanisms can be challenging. We analyze interannual time series of population densities of the larch budmoth, Zeiraphera griseana (Lepidoptera: Tortricidae), along with six sympatric larch-feeding folivores from a site in the European Alps 1952–1979. These species include five lepidopterans, Exapate duratella, Ptycholomoides aeriferana, Spilonota laricana, Epirrita autumnata and Teleiodes saltuum, and one hymenopteran sawfly Pristiphora laricis. We document that the highly regular oscillatory behavior (period 9–10 years) of Z. griseana populations is similarly evident in the dynamics of most of the sympatric folivores. We also find that all of the sympatric species are phase synchronized with Z. griseana populations with half of the sympatric species exhibiting nonlagged phase synchrony and three of the species exhibiting 2–5 year lags behind Z. griseana populations. We adapt a previously developed tritrophic model of Z. griseana dynamics to explore possible mechanisms responsible for observed phase synchronization. Results suggest that either shared stochastic influences (e.g., weather) or shared parasitoid impacts are likely causes of nonlagged phase synchronization. The model further indicates that observed patterns of lagged phase synchronization are most likely caused by either shared delayed induced host plant defenses or direct density-dependent effects shared with Z. griseana.     

Possible evolution of alliarinoside biosynthesis from the glucosinolate pathway in Alliaria petiolata

Nitrile formation in plants involves the activity of cytochrome P450s. Hydroxynitrile glucosides are widespread among plants but generally do not occur in glucosinolate producing species. Alliaria petiolata (garlic mustard, Brassicaceae) is the only species known to produce glucosinolates as well as a γ-hydroxynitrile glucoside. Furthermore, A. petiolata has been described to release diffusible cyanide, which indicates the presence of unidentified cyanogenic glucoside(s). Our research on A. petiolata addresses the molecular evolution of P450s. By integrating current knowledge about glucosinolate and hydroxynitrile glucoside biosynthesis in other species and new visions on recurrent evolution of hydroxynitrile glucoside biosynthesis, we propose a pathway for biosynthesis of the γ-hydroxynitrile glucoside, alliarinoside. Homomethionine and the corresponding oxime are suggested as shared intermediates in the biosynthesis of alliarinoside and 2-propenyl glucosinolate. The first committed step in the alliarinoside pathway is envisioned to be catalysed by a P450, which has been recruited to metabolize the oxime. Furthermore, alliarinoside biosynthesis is suggested to involve enzyme activities common to secondary modification of glucosinolates. Thus, we argue that biosynthesis of alliarinoside may be the first known case of a hydroxynitrile glucoside pathway having evolved from the glucosinolate pathway. An intriguing question is whether the proposed hydroxynitrile intermediate may also be converted to novel homomethionine-derived cyanogenic glucoside(s), which could release cyanide. Elucidation of the pathway for biosynthesis of alliarinoside and other putative hydroxynitrile glucosides in A. petiolata is envisioned to offer significant new knowledge on the emerging picture of P450 functional dynamics as a basis for recurrent evolution of pathways for bioactive natural product biosynthesis.     

First detection of piscine reovirus (PRV) in marine fish species

Heart and skeletal muscle inflammation (HSMI) is a disease that affects farmed Atlantic salmon Salmo salar L. several months after the fish have been transferred to seawater. Recently, a new virus called piscine reovirus (PRV) was identified in Atlantic salmon from an outbreak of HSMI and in experimentally challenged fish. PRV is associated with the development of HSMI, and has until now only been detected in Atlantic salmon. This study investigates whether the virus is also present in wild fish populations that may serve as vectors for the virus. The virus was found in few of the analyzed samples so there is probably a more complex relationship that involves several carriers and virus -reservoirs.     

The eclipse period of Escherichia coli

The minimal time between successive initiations on the same origin (the eclipse) in Escherichia coli was determined to be approximately 25-30 min. An inverse relationship was found between the length of the eclipse and the amount of Dam methyltransferase in the cell, indicating that the eclipse corresponds to the period of origin hemimethylation. The SeqA protein was absolutely required for the eclipse, and DnaA titration studies suggested that the SeqA protein prevented the binding of multiple DnaA molecules on oriC (initial complex formation). No correlation between the amount of SeqA and eclipse length was revealed, but increased SeqA levels affected chromosome partitioning and/or cell division. This was corroborated further by an aberrant nucleoid distribution in SeqA-deficient cells. We suggest that the SeqA protein's role in maintaining the eclipse is tied to a function in chromosome organization.     

Temporal changes in species diversity and composition in abandoned fields in a <Emphasis Type="Italic">trans</Emphasis>-Himalayan landscape,Nepal

Secondary succession is an increasing phenomenon due to global changes in agriculture policies and practices. The empirical findings are biased towards the temperate zone. Abandonment of agriculture fields is less frequent in the subtropical and tropical zones where agriculture areas are, in general, expanding. But there are exceptions; a rapid rate of abandonment of agricultural fields have taken place in the arid trans-Himalayan region, due to today’s globalization of economy. We analysed agriculture fields that were abandoned between 1950 and 2003 in a large u-valley in central Nepal (3400 m a.s.l.). The potential forest vegetation is dominated by Pinus wallichina and shrubs of junipers and cotoneaster species. We tested the intermediate richness hypothesis in relation to vegetation cover, soil development and whether old-field succession is convergent or divergent with species data from 242 1 m² plots in 5 age-classes. The main species compositional turnover expressed by Detrended Correspondence Analyses (DCA) correlated, as expected, with time after abandonment. Fields that were abandoned a long time ago are closer to forest at the periphery of the agricultural landscape. Moisture of the soil significantly increased with age of abandonment, but total vegetation cover and pH were negatively related to age. Beta diversity expressed in DCA SD-units showed an increasing trend with age of abandonment, supporting the divergence pattern in old-field succession. The reason why the succession is not converging may be due to browsing by domestic animals that prevent a closed canopy of pines and juniper to develop. There was a significant hump-shaped pattern in species richness along the temporal gradient, which agrees with the intermediate species-richness hypothesis. There was a rapid increase in species richness in plots close to the villages that were used for haymaking which increased the seed input significantly.