Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.

Folding, calcium binding, and structural characterization of a concatemer of the first and second ligand-binding modules of the low-density lipoprotein receptor

The ligand-binding domain of the low-density lipoprotein (LDL) receptor is comprised of seven tandemly repeated ligand-binding modules, each being approximately 40 amino acids long and containing six conserved cysteine residues. We have expressed and characterized a concatemer of the first two modules (LB1 and LB2) of the human LDL receptor. Oxidative folding of the recombinant concatemer (rLB(1-2)), in the presence of calcium ions, gave a single dominant isomer with six disulfide bonds. Peptic cleavage of the short Linker region that connects the last cysteine residue of LB1 and the first cysteine residue of LB2 yielded two discrete fragments, thus excluding the presence of intermodule disulfide bonds. The N-terminal module, LB1, reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDL receptor. From this, we concluded that the first module was correctly folded, with the same set of disulfide bonds as LB1 of the LDL receptor. The disulfide bond connections of LB2 were identified from mass spectral analysis of fragments formed by digestion of the C-terminal peptic fragment with elastase. These data showed that the disulfide bonds of LB2 connected Cys(I) and Cys(III), Cys(II) and Cys(V), and Cys(IV) and Cys(VI). This pattern is identical to that found for recombinant LB1 and LB2. The concatemer has two high-affinity calcium-binding sites, one per module. An analysis of the secondary chemical shifts of C alpha protons shows that the conformations of LB1 and LB2 in the concatemer are very similar to those of the individual modules, with no evidence for strong interactions between the two modules.

Control of nitrate recirculation flow in predenitrification systems

The control of the nitrate recirculation flow in a predenitrification system is addressed. An elementary mass balance analysis on the utilisation efficiency of the influent biodegradable COD (bCOD) for nitrate removal indicates that the control problem can be broken down into two parts: maintaining the anoxic zone anoxic (i.e. nitrate is present throughout the anoxic zone) and maximising the usage of influent soluble bCOD for denitrification. Simulation studies using the Simulation Benchmark developed in the European COST program show that both objectives can be achieved by maintaining the nitrate concentration at the outlet of the anoxic zone at around 2 mgN/L. This setpoint appears to be robust towards variations in the influent characteristics and sludge kinetics.

Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41 -, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.

Ar-40/-Ar-39 geochronology at the Instituto de Geociencias, USP: instrumentation, analytical procedures, and calibration

Laser heating Ar-40/Ar-39 geochronology provides high analytical precision and accuracy, mum-scale spatial resolution. and statistically significant data sets for the study of geological and planetary processes, A newly commissioned Ar-40/Ar-39 laboratory at CPGeo/USP, Sao Paulo, Brazil, equips the Brazilian scientific community with a new powerful tool applicable to the study of geological and cosmochemical processes. Detailed information about laboratory layout, environmental conditions, and instrumentation provides the necessary parameters for the evaluation of the CPGeo/USp Ar-40/Ar-39 suitability to a diverse range of applications. Details about analytical procedures, including mineral separation, irradiation at the IPEN/CNEN reactor at USP, and mass spectrometric analysis enable potential researchers to design the necessary sampling and sample preparation program suitable to the objectives of their study. Finally, the results of calibration tests using Ca and K salts and glasses, international mineral standards, and in-house mineral standards show that the accuracy and precision obtained at the Ar-40/Ar-39 laboratory at CPGeo/USP are comparable to results obtained in the most respected laboratories internationally. The extensive calibration and standardization procedures under-taken ensure that the results of analytical studies carried out in our laboratories will gain immediate international credibility, enabling Brazilian students and scientists to conduct forefront research in earth and planetary sciences.

Observations of foehn onset in the Southern Alps, New Zealand

Local scale windfield and air mass characteristics during the onset of two foehn wind events in an alpine hydro-catchment are presented. Grounding of the topographically modified foehn was found to be dependent on daytime surface heating and topographic channelling of flow. The foehn front was observed to advance down-valley until the valley widened significantly. The foehn wind appeared to decouple from the surface downstream of the accelerated flow associated with the valley constriction. and to be lifted above local thermally generated circulations including a lake breeze. Towards evening. the foehn front retreated up valley in response to reduced surface heating and the intrusion into the study area of a deep and cool air mass associated with a regional scale mountain-plain circulation. Differences in the local windfield observed during both case study events reflect the importance of different thermal and dynamic forcings on airflow in complex terrain. These are the result of variation in surface energy exchanges, channelling and blocking of airflow. Observations presented here have both theoretical and applied implications with regard to forecasting foehn onset, wind hazard management, recreational activities and air quality management in alpine settings.

Rabbit CYP4B1 engineered for high-level expression in Escherichia coli: ligand stabilization and processing of the N-terminus and heme prosthetic group

Modifications at the N-terminus of the rabbit CYP4B1 gene resulted in expression levels in Escherichia coli of up to 660 nmol/L. Solubilization of the enzyme from bacterial membranes led to substantial conversion to cytochrome P420 unless alpha-naphthoflavone was added as a stabilizing ligand. Mass spectrometry analysis and Edman sequencing of purified enzyme preparations revealed differential N-terminal post-translational processing of the various constructs expressed. Notably, bacterial expression of CYP4B1 produced a holoenzyme with >98.5% of its heme prosthetic group covalently linked to the protein backbone. The near fully covalently linked hernoproteins exhibited similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies shed new light on the consequences of covalent heme processing in CYP4B1 and provide a facile system for future mechanistic and structural studies with the enzyme. (C) 2003 Elsevier Science (USA). All rights reserved.

Pumice rafting and faunal dispersion during 2001-2002 in the Southwest Pacific: record of a dacitic submarine explosive eruption from Tonga

A new influx of sea-rafted pumice reached the eastern coast of Australia in October 2002, approximately 1 year after a felsic, shallow-marine explosive eruption at a previously unknown volcano (0403-091) along the Tofua volcanic arc (Tonga). The eruption produced floating pumice rafts that first became stranded in Fiji in November 2001, approximately I month after the eruption. Strandings of sea-rafted pumice along shorelines have been the only record of products from this submarine explosive eruption at the remote, submerged volcano. Computed drift trajectories of the sea-rafted pumice using numerical models of southwest Pacific surface wind fields and ocean currents indicate two cyclonic systems disturbed the drift of pumice to eastern Australia, as well as the importance of the combined wave and direct wind effect on pumice trajectory. Pumice became stranded along at least two-thirds (>2000 km) of the coastline of eastern Australia, being deposited on beaches during a sustained period of fresh onshore winds. Typical amounts of pumice initially stranded on beaches were 500-4000 individual clasts per in, and a minimum volume estimate of pumice that arrived to eastern Australia is 1.25 x 10(5) m(3). Pumice was beached below maximum tidal/storm surge levels and was quickly reworked back into the ocean, such that the concentration of beached pumice rapidly dissipated within weeks of the initial stranding, and little record of this stranding event now exists. Most stranded pumice clasts ranged in size from 2 to 5 cm in diameter; the largest measured clasts were 10 cm in Australia and 20 cm in Fiji. The pumice has a low phenocryst content (3500 km) and period of pumice floatation (greater than or equal to1 year), confirm the importance of sea-rafted pumice as a long-distance dispersal mechanism for marine organisms including marine pests and harmful invasive species. Billions of individual rafting pumice clasts can be generated in a single small-volume eruption, such as observed here, and the geological implications for the transport of sessile taxa over large distances are significant. An avenue for future research is to examine whether speciation events and volcanicity are linked; the periodic development of globalism for some taxa (e.g., corals, gastropods, bryozoa) may correlate in time and/or space with voluminous silicic igneous events capable of producing >10(6) km(3) of silicic pumice-rich pyroclastic material and emplaced into ocean basins. (C) 2004 Elsevier B.V. All rights reserved.

Aeration for plant root respiration in a tidal marsh

This paper investigates the tidal effects on aeration conditions for plant root respiration in a tidal marsh. We extend the work of Ursino et al. ( 2004) by using a two-phase model for air and water flows in the marsh. Simulations have been conducted to examine directly the link between the airflow dynamics and the aeration condition in the marsh soil. The results show that the effects of entrapped air on water movement in the vadose zone are significant in certain circumstances. Single-phase models based on Richards' equation, which neglect such effects, may not be adequate for quantifying the aeration condition in tidal marsh. The optimal aeration condition, represented by the maximum of the integral magnitude of tidally advected air mass ( TAAM) flux, is found to occur near the tidal creek for the four soil textures simulated. This may explain the observation that some salt marsh plant species grow better near tidal creeks than in the inner marsh areas. Our analyses, based on the two-phase model and predicted TAAM flux magnitude, provide further insight into the positive feedback'' mechanism proposed by Ursino et al. ( 2004). That is, pioneer plants may grow successfully near the creek where the root aeration condition is optimal. The roots of the pioneer plants can soften and loosen the rhizosphere soil, which increases the evapotranspiration rate, the soil porosity, and absolute permeability and weakens the capillary effects. These, in turn, improve further the root aeration conditions and may lead to colonization by plants less resistant to anaerobic conditions.

Experimental Analysis of Froude Number Effect on Air Entrainment in the Hydraulic Jump

A hydraulic jump is characterized by strong energy dissipation and mixing, large-scale turbulence, air entrainment, waves and spray. Despite recent pertinent studies, the interaction between air bubbles diffusion and momentum transfer is not completely understood. The objective of this paper is to present experimental results from new measurements performed in rectangular horizontal flume with partially-developed inflow conditions. The vertical distributions of void fraction and air bubbles count rate were recorded for inflow Froude number Fr1 in the range from 5.2 to 14.3. Rapid detrainment process was observed near the jump toe, whereas the structure of the air diffusion layer was clearly observed over longer distances. These new data were compared with previous data generally collected at lower Froude numbers. The comparison demonstrated that, at a fixed distance from the jump toe, the maximum void fraction Cmax increases with the increasing Fr1. The vertical locations of the maximum void fraction and bubble count rate were consistent with previous studies. Finally, an empirical correlation between the upper boundary of the air diffusion layer and the distance from the impingement point was provided.

Adsorption of supercritical fluids in non-porous and porous carbons: analysis of adsorbed phase volume and density

In this paper, we revisit the surface mass excess in adsorption studies and investigate the role of the volume of the adsorbed phase and its density in the analysis of supercritical gas adsorption in non-porous as well as microporous solids. For many supercritical fluids tested (krypton, argon, nitrogen, methane) on many different carbonaceous solids, it is found that the volume of the adsorbed phase is confined mostly to a geometrical volume having a thickness of up to a few molecular diameters. At high pressure the adsorbed phase density is also found to be very close to but never equal or greater than the liquid phase density. (C) 2003 Elsevier Science Ltd. All rights reserved.