Spectroscopic studies of perturbed T1 Cu sites in the multicopper oxidases Saccharomyces cerevisiae Fet3p and Rhus vernicifera laccase: allosteric coupling between the T1 and trinuclear Cu sites

The multicopper oxidases catalyze the 4e- reduction of O2 to H2O coupled to the 1e- oxidation of 4 equiv of substrate. This activity requires four Cu atoms, including T1, T2, and coupled binuclear T3 sites. The T2 and T3 sites form a trinuclear cluster (TNC) where O2 is reduced. The T1 is coupled to the TNC through a T1-Cys-His-T3 electron transfer (ET) pathway. In this study the two T3 Cu coordinating His residues which lie in this pathway in Fet3 have been mutated, H483Q, H483C, H485Q, and H485C, to study how perturbation at the TNC impacts the T1 Cu site. Spectroscopic methods, in particular resonance Raman (rR), show that the change from His to Gln to Cys increases the covalency of the T1 Cu-S Cys bond and decreases its redox potential. This study of T1-TNC interactions is then extended to Rhus vernicifera laccase where a number of well-defined species including the catalytically relevant native intermediate (NI) can be trapped for spectroscopic study. The T1 Cu-S covalency and potential do not change in these species relative to resting oxidized enzyme, but interestingly the differences in the structure of the TNC in these species do lead to changes in the T1 Cu rR spectrum. This helps to confirm that vibrations in the cysteine side chain of the T1 Cu site and the protein backbone couple to the Cu-S vibration. These changes in the side chain and backbone provide a possible mechanism for regulating intramolecular T1 to TNC ET in NI and partially reduced enzyme forms for efficient turnover.     

Assessing the performance of volunteers in monitoring streams

1. Citizens are concerned about the quality of water resources and many participate in monitoring activities, though doubts remain about the quality of the data volunteers collect. We trained volunteers to collect benthic macroinvertebrates using professional protocols. Of the seven stream sites sampled by volunteer crews, six sites were also sampled by professional crews.
2. In the laboratory, volunteers used morphological features to identify as many different taxa as possible within the major insect orders; their identification was approximately to family. Volunteers calculated five metrics: total taxon richness, richness of three key groups (Ephemeroptera, Plecoptera and Trichoptera), and percentage dominance of the three most abundant taxa. All metrics were strongly correlated with (a) the percentage of urbanized area in the catchment and (b) the metrics derived from a more complete taxonomic identification by a professional scientist. Taxon richness metrics declined with urban development, while percent dominance increased.
3. An overall summary multimetric index was used to compare the field and laboratory procedures of volunteers and professionals. Using an ANOVA model, we detected no significant difference between field samples collected by volunteers and professionals. The variance of index values associated with differences between crews was zero. The ability of the index to detect significant differences among sites (statistical power) improved by only 13% for assessments based on professional laboratory identification instead of volunteer laboratory identification.
4. Citizen volunteers, when properly trained, can collect reliable data and make stream assessments that are comparable to those made by professionals. Data collected by volunteers can supplement information used by government agencies to manage and protect rivers and streams.     

Interaction of drought and high temperature on photosynthesis and grain-filling of wheat

Drought and high temperature often occur simultaneously, but their effects on crops are usually investigated individually. Our objective was to compare effects of drought, high temperature, and their interactions on photosynthesis and grain-growth of wheat (Triticum aestivum L.). Plants (cv. Len) were grown uniformly in well-watered soil at 25/20 ± 2 °C day/night until anthesis, when they were subjected to regimes of no drought (soil at field capacity) and drought (plant water potential of –.0 to –2.4 MPa) at 15/10, 25/20, and 35/30 °C in controlled environments until physiological maturity. Drought decreased photosynthesis, stomatal conductance, viable leaf area, shoot and grain mass, and weight and soluble sugar content of kernels but increased plant water-use efficiency. High temperature hastened the decline in photosynthesis and leaf area, decreased shoot and grain mass as well as weight and sugar content of kernels, and reduced water-use efficiency. Interactions between the two stresses were pronounced, and consequences of drought on all physiological parameters were more severe at high temperature than low temperature. The synergistic interactions indicated that productivity of wheat is reduced considerably more by the combined stresses than by either stress alone, and that much of the effect is on photosynthetic processes.     

Spatial and temporal distributions of submarine groundwater discharge rates obtained from various types of seepage meters at a site in the Northeastern Gulf of Mexico

Direct measurements of submarine groundwaterdischarge (SGD) were taken by three different(continuous heat, heat pulse, and ultrasonic)types of automated seepage meters as well asstandard Lee-type manually operated meters. SGD flux comparisons and the spatial andtemporal variations in groundwater flow wereanalyzed. Seepage rates measured by thedifferent meters agree relatively well witheach other (more than 80% agreement). Comparisons of flux rates as a function ofdistance offshore using exponentialapproximations show that more than fivemeasurement locations (200 m offshore) areneeded for a precise integrated estimation ofSGD offshore within an accuracy of ±10%. Thedominant period of seepage variations isestimated to be about 12 hours, which closelymatches the semidiurnal tides in this area. Our analysis also shows that short durationmeasurement periods may cause significantunderestimates or overestimates of the dailyaveraged groundwater flow rates (±25%–±60% difference when the measurement durationis less than 12 hours). Thus, continuousmeasurements of SGD using automated seepagemeters with high time resolution should enableus to evaluate temporal and spatial variationsof dissolved material transports viagroundwater pathways. Such inputs may affectbiogeochemical phenomena in the coastal zone.     

Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus

Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which far exceeds that of other herpesviruses. Few of these proteins have been characterized. We have investigated the gene product(s) of reading frame 10, which is present in both the internal and terminal repeat regions of HCMV strain AD169 and only once in clinical isolates. The putative protein product is a 171-amino-acid glycoprotein with a theoretical mass of 20.5 kDa. We characterized the protein encoded by this reading frame in the laboratory strain AD169 and a recent isolate, TB40E. The results from both strains were comparable. Northern blot analyses showed that the gene was transcribed with early/late kinetics. Two proteins of 22 and 23.5-kDa were detected in virus-infected cells and in cells transiently expressing recombinant TRL10. Both forms contained only high-mannose-linked carbohydrate modifications. In addition, virus-infected cells expressed small amounts of the protein modified with complex N-linked sugars. Image analysis localized transiently expressed TRL10 to the endoplasmic reticulum. Immunoblot analyses as well as immunoelectron microscopy of purified virions demonstrated that TRL10 represents a structural component of the virus particle. Immunoblot analysis in the absence of reducing agents indicated that TRL10, like the other HCMV envelope glycoproteins, is present in a disulfide-linked complex. Sequence analysis of the TRL10 coding region in nine low-passage clinical isolates revealed strain-specific variation. In summary, the protein product of the TRL10 open reading frame represents a novel structural glycoprotein of HCMV and was termed gpTRL10.     

Phylogeny of multidrug transporters.

We currently recognize five large ubiquitous superfamilies and one small eukaryotic-specific family in which cellular multidrug efflux pumps occur. One, the ABC superfamily, includes members that use ATP hydrolysis to drive drug efflux, but the MFS, RND, MATE and DMT superfamilies include members that are secondary carriers, functioning by drug:H(+)or drug:Na(+)antiport mechanisms. The small MET family seems to be restricted to endosomal membranes of eukaryotes, and only a single such system has been functionally characterized. In this review article, these families of drug transporters are discussed and evaluated from phylogenetic standpoints.     

Characterization of a biologically active arabinogalactan from the leaves of Plantago major L.

PMIa is a Type II arabinogalactan with anti-complementary activity isolated from the leaves of Plantago major L. It has a molecular weight of 77000–80000 Da and consists of arabinose (38%), galactose (49%), rhamnose (6%), galacturonic acid (7%) and 1.5% protein with hydroxyproline, alanine and serine as the main amino acids. Characterization of PMIa by methylation and GC-MS, methanolysis and GC, Smith degradation, weak acid hydrolysis, ¹³C-NMR, ¹H-NMR, two-dimensional heteronuclear NMR and DEPT show that it consists of 1,3-linked galactan chains with 1,6-linked galactan side chains attached to position 6. The side chains are further branched in position 3 with 1,3-linked galactose residues which have 1,6-linked galactose attached to position 6; these 1,3- and 1,6-linked galactose chains altogether probably form a network. Terminal and 1,5-linked arabinose in furanose form are attached to the galactan mainly through position 3 of the 1,6-linked galactose side chains.     

Semi-continuous monitoring of the capacity of sea water for supporting growth of phytoplankton

Overlapping series of dialysis cultures of three marine planktonic algae were grown in running sea water for several months and responded systematically to the variations in the nutrient level of the water. Under the right conditions the algal cultures showed three growth phases, namely, exponential, linear, and asymptotic as predicted for the dialysis system used. The transition point between exponential and linear growth was well correlated with the concentration of the limiting nutrient in the sea water. Furthermore, linear line correlation between growth rate in the linear phase and the nitrate content of the sea water was obtained for the three of the species involved. It was also found that the final cell density of the asymptotic growth phase was determined by the nitrate content of the sea water. The yield coefficients for conversion of nitrate to cells calculated for linear growth agreed well with the protein content of the various algae.For several cultures of one of the algal species the “maintenance requirement” for nitrate was estimated on the basis of the linear correlation obtained between final cell yield and the nitrate content of the sea water.     

Phenotypic Profiling of Scedosporium aurantiacum,an Opportunistic Pathogen Colonizing Human Lungs

Genotyping studies of Australian Scedosporium isolates have revealed the strong prevalence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibrosis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using a Galleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (saccharose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two different temperature conditions tested (28 and 37°C). Phenotype microarray data were integrated with the whole-genome sequence data of S. aurantiacum to reconstruct a pathway map for the metabolism of selected substrates to further elucidate differences between the strains.     

Purification and characterization of a growth inhibitory hepatic peptide. A preliminary note

A pentapeptide isolated from normal mouse liver seems to inhibit DNA synthesis (3H-thymidine incorporation into liver DNA and labeling indices) and the mitotic rate (G2-M cell flux) in regenerating mouse liver. The inhibitor is somewhat similar to the growth inhibitory pentapeptides previously reported for granulocytes and epidermis. It is active at very low dose levels, showing a bell-shaped dose-response curve.