Vertical Migration and Mortality of Marine Benthos in Dredged Material: A Synthesis

This account describes the comparative response of four species of benthic invertebrates to burial in terms of vertical migration and mortality, and provides a synthesis of studies and recommendations upon which to assess future impacts. The species featured were the bivalve Mercenaries mercenaria, the amphipod crustacean Parahaustorius longimerus, and the polychaetes Scoloplos fragilis and Nereis succinea. There was evidence of synergistic effects on burrowing activity and mortality with changes in time of burial, sediment depth, sediment type and temperature. In sediment with silt-clay, N. succinea was the most resistant species followed by M. mercenaria, S. fragilis and P. longimerus. In sediment without silt-clay the order of percent mortality was reversed. Studies of surface water chemistry and sediment geochemistry showed that dissolved oxygen decreased significantly and ammonia and sulfide increased significantly between the surface and below 2.0 cm within a 15-day period. Based on these results and physiological tolerances from the literature it was concluded that M. mercenaria and N. succinea would be relatively resistant to chemical effects of spoil disposal, whereas S. fragilis and P. longimerus would be less resistant to such effects. Vertical migration of benthic invertebrates through dredge disposal can be a viable mechanism of recolonization under certain conditions. Some effects of burial of benthos can be predicted based on morphology, behavior and physiology. These biological features were discussed with examples dealing with molluscs, crustaceans, and polychaetes. Finally, recommendations were made concerning the type of studies to provide additional data to aid management agencies in decision making about future dredging and disposal practices.     

Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes

This study describes the effect of ethanol and the presence of poly(ethylene) glycol (PEG) lipids on the interaction of nucleotide-based polyelectrolytes with cationic liposomes. It is shown that preformed large unilamellar vesicles (LUVs) containing a cationic lipid and a PEG coating can be induced to entrap polynucleotides such as antisense oligonucleotides and plasmid DNA in the presence of ethanol. The interaction of the cationic liposomes with the polynucleotides leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. The degree of lamellarity as well as the size and polydispersity of the liposomes formed increases with increasing polynucleotide-to-lipid ratio. A direct correlation between the entrapment efficiency and the membrane-destabilizing effect of ethanol was observed. Although the morphology of the liposomes is still preserved at the ethanol concentrations used for entrapment (25-40%, v/v), entrapped low-molecular-weight solutes leak rapidly. In addition, lipids can flip-flop across the membrane and exchange rapidly between liposomes. Furthermore, there are indications that the interaction of the polynucleotides with the cationic liposomes in ethanol leads to formation of polynucleotide-cationic lipid domains, which act as adhesion points between liposomes. It is suggested that the spreading of this contact area leads to expulsion of PEG-ceramide and triggers processes that result in the formation of multilamellar systems with internalized polynucleotides. The high entrapment efficiencies achieved at high polyelectrolyte-to-lipid ratios and the small size and neutral character of these novel liposomal systems are of utility for liposomal delivery of macromolecular drugs.     

A high-throughput screen for MscL channel activity and mutational phenotyping

A novel fluorescence-based screen for bacterial mechanosensitive ion-channel activity has been developed. This assay is capable of clearly distinguishing the previously observed gain of function and loss of function phenotypes for the Escherichia coli mechanosensitive channel of large conductance (Ec-MscL). The method modifies Molecular Probes' Live/Dead BacLight bacterial viability assay to monitor MscL channel activity as a function of bacterial survival from osmotic downshock.     

Cloning,expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The gene encoding CYP102A2, a novel P450 monooxygenase from Bacillus subtilis, was cloned and expressed in Escherichia coli. The recombinant enzyme formed was purified by immobilised metal chelate affinity chromatography (IMAC) and characterised. CYP102A2 is a 119-kDa self-sufficient monooxygenase, consisting of an FMN/FAD-containing reductase domain and a heme domain. The deduced amino acid sequence of CYP102A2 exhibits a high level of identity with the amino acid sequences of CYP102A1 from B. megaterium (59%) and CYP102A3 from B. subtilis (60%). In reduced, CO-bound form, the enzyme shows a typical Soret band at 449 nm. It catalyses the oxidation of even- and odd-chain saturated and unsaturated fatty acids. In all reactions investigated, the products were the respective -3, -2 and -1 hydroxylated fatty acids. Activity was highest towards oleic acid (K_M=17.36±1.4 M, k_cat=2,244±72 min^–1) and linoleic acid (K_M=12.25±1.8 M, k_cat=1,950±84 min^–1). Comparison of a CYP102A2 homology model with the CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in the catalytic properties of these two enzymes.     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.     

Neutral and non-neutral macroecology

Because of the multiscalar nature of processes underlying biodiversity dynamics, macroecology has emerged as a discipline that seeks to build an understanding of this complexity by examining statistical patterns in large assemblages of species in geographic space and ecological time. Models that assume individual organisms within trophically defined assemblages are ecologically equivalent can produce many patterns identified by macroecology. Neutral models predict two important dynamical patterns that can be tested in real assemblages. First, they predict that species diversity will decline within an assemblage over time. The rate of this decay in species diversity can be predicted from estimates of migration rates from a “metacommunity” or species pool. Second, neutral models predict a divergence of species composition among local communities over time. The rate and degree of divergence among communities also depend on the migration rate. The few studies that have been done to date imply that the rate of migration in real species assemblages is much lower than that required to explain the degree of community similarity maintained in space and time. There are at least two alternative ways to extend neutral models to incorporate more biological realism. First, competitive asymmetries among species may be introduced to allow for the possibility that individuals of some species may have an advantage in replacing individuals that die. Second, environmental heterogeneity can be introduced by assuming sites available to individuals differ in quality to individuals of different species. The neutral model, because of its conceptual simplicity and rigor, should be considered as a null model for baseline comparison to actual patterns of distribution, abundance, species composition, and beta diversity.

Zusammenfassung

Wegen der multiskalaren Natur der Prozesse, die der Biodiversitätsdynamik zugrunde liegen, entstand die Makroökologie als eine Disziplin, die anstrebt ein Verständnis dieser Komplexität zu schaffen, indem sie statistische Muster in großen Vergesellschaftungen von Arten im geografischen Raum und ökologischer Zeit untersucht. Modelle, die davon ausgehen, dass individuelle Organismen innerhalb trophisch definierter Vergesellschaftungen ökologisch äquivalent sind, können viele Muster erzeugen, die durch die Makroökologie indentifiziert werden. Neutrale Modelle sagen zwei wichtige dynamische Muster vorher, die in realen Vergesellschaftungen getestet werden können. Als Erstes sagen sie vorher, dass die Artendiversität in einer Vergesellschaftung mit der Zeit abnehmen wird. Die Rate der Abnahme der Artendiversität kann über Schätzungen der Migrationsraten aus einer Metagemeinschaft bzw. einem Artenpool vorhergesagt werden. Als Zweites sagen neutrale Modelle eine Divergenz der Artenzusammensetzung zwischen den lokalen Gemeinschaften mit der Zeit vorher. Die Rate und der Grad der Divergenz zwischen den Gemeinschaften hängt ebenfalls von der Migrationsrate ab. Die wenigen Untersuchungen, die bis heute gemacht wurden, implizieren, dass die Rate der Migration in realen Artenvergesellschaftungen viel geringer als erforderlich sind, um den Grad der Gemeinschaftsähnlichkeit zu erklären, der in Raum und Zeit aufrecht erhalten wird. Es gibt mindestens zwei alternative Weisen neutrale Modelle zu erweitern, um mehr biologische Realität mit einzubeziehen. Als Erstes können Asymmetrien der Konkurrenz unter Arten einbezogen werden, um die Möglichkeit zu zulassen, dass Individuen einiger Arten einen Vorteil bei der Ersetzung von sterbenden Individuen haben. Als Zweites kann die Umweltheterogenität mit einbezogen werden, indem angenommen wird, dass sich die verfügbaren Standorte in ihrer Qualität für Individuen verschiedener Arten unterscheiden. Wegen seiner konzeptuellen Einfachheit und Starrheit sollte das neutrale Modell als Null-Modell für grundlegende Vergleiche von Verbreitung, Abundanz, Artenzusammensetzung und Betadiversität angesehen werden.     

The nuclear export receptor Xpo1p forms distinct complexes with NES transport substrates and the yeast Ran binding protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin beta-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.     

Evaluating the roles of thrombin and calcium in the activation of coagulation factor XIII using H/D exchange and MALDI-TOF MS

Factor XIII catalyzes the formation of isopeptide bonds between noncovalently associated fibrin monomers in the final stages of the blood coagulation cascade. This results in a rigid, covalently linked network that is much more resistant to proteolytic degradation. Calcium ion is critical to this process, and its continued presence after activation aids in maintenance of Factor XIII activity. Hydrogen/deuterium exchange experiments were conducted on recombinant Factor XIII a(2) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The method revealed changes in the structure of Factor XIII a(2) localized to different areas of the protein that were related to the manner in which the enzyme was activated and the calcium environment in which it was maintained. A possible substrate recognition region in the catalytic core (220-230) shows an increase in deuteration upon activation. The degree of deuteration varies depending on the calcium environment in which the active enzyme is maintained. A portion of the beta-sandwich domain (98-104) exhibits a decrease in deuteration upon activation by exposure to calcium alone. A third change occurs in the beta-barrel 1 domain of the protein, a portion of which (526-546) shows a decrease in deuteration upon activation by calcium exposure, but almost none at all when the enzyme is activated by thrombin. The pattern of observed changes reveals individual contributions of calcium and thrombin to activating the enzyme toward substrate binding and exposure of the active site.     

Structural features associated with the binding of glutamine-containing peptides to Factor XIII

Activated Factor XIII a2 catalyzes the formation of intermolecular gamma-glutamyl- epsilon -lysyl cross-links in the fibrin network. Solution NMR studies were carried out to characterize, the structural features associated with the binding of glutamine-containing peptides to Factor XIII. A coupled uv/vis kinetic assay demonstrated that K9 peptide (1-10), alpha2-antiplasmin (1-15), and alpha2-antiplasmin (1-15 Q4N) all function as glutamine-containing substrates for activated Factor XIII a2. 2D TOCSY spectra of the peptides exhibit upfield chemical shifts for the glutamine protons in the presence of Factor XIII. These results indicate that the reactive peptide glutamines are encountering a distinctive environment within the Factor XIII active site. 1D proton line-broadening and 2D transferred-NOESY studies reveal that the glutamines and residues located C-terminally come in direct contact with the enzyme and adopt an extended conformation. Substrates with sequences similar to alpha2-antiplasmin (1-15) are proposed to bind both at the catalytic site and at a neighboring apolar region.     

NMR solution structure and dynamics of the peptidyl-prolyl cis-trans isomerase domain of the trigger factor from Mycoplasma genitalium compared to FK506-binding protein

We have solved the solution structure of the peptidyl-prolyl cis-trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy. Our results lead to a well-defined structure with a backbone rmsd of 0.23 A. As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold. Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar. Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the beta sheet. Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor. Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central beta sheet. Our data indicate, however, that this strand may be partially structured as "nascent" beta strand. This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases.