The cloning and characterization of a second alpha-amylase of A-hydrophila JMP636

Aims: The aim of this study was to identify, clone and characterize the second amylase of Aeromonas hydrophila JMP636, AmyB, and to compare it to AmyA. Methods and Results: The amylase activity of A. hydrophila JMP636 is encoded by multiple genes. A second genetically distinct amylase gene, amyB, has been cloned and expressed from its own promoter in Escherichia coli. AmyB is a large alpha-amylase of 668 amino acids. Outside the conserved domains of alpha-amylases there is limited sequence relationship between the two alpha-amylases of A. hydrophila JMP636 AmyA and AmyB. Significant (80%) similarity exists between amyB and an alpha-amylase of A. hydrophila strain MCC-1. Differences in either the functional properties or activity under different environmental conditions as possible explanations for multiple copies of amylases in JMP636 is less likely after an examination of several physical properties, with each of the properties being very similar for both enzymes (optimal pH and temperature, heat instability). However the reaction end products and substrate specificity did vary enough to give a possible reason for the two enzymes being present. Both enzymes were confirmed to be alpha-type amylases. Conclusions: AmyB has been isolated, characterized and then compared to AmyA. Significance and Impact of Study: The amylase phenotype is rarely encoded by more than one enzyme within one strain, this study therefore allows the better understanding of the unusual amylase production by A. hydrophila.

Automatic construction of explicit R matrices for the one-parameter families of irreducible typical highest weight (0(m)vertical bar alpha(n)) representations of U-q[gl(m vertical bar n)]

We detail the automatic construction of R matrices corresponding to (the tensor products of) the (O-m\alpha(n)) families of highest-weight representations of the quantum superalgebras Uq[gl(m\n)]. These representations are irreducible, contain a free complex parameter a, and are 2(mn)-dimensional. Our R matrices are actually (sparse) rank 4 tensors, containing a total of 2(4mn) components, each of which is in general an algebraic expression in the two complex variables q and a. Although the constructions are straightforward, we describe them in full here, to fill a perceived gap in the literature. As the algorithms are generally impracticable for manual calculation, we have implemented the entire process in MATHEMATICA; illustrating our results with U-q [gl(3\1)]. (C) 2002 Published by Elsevier Science B.V.

High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever

Infection with any 1 of 4 dengue viruses produces a spectrum of clinical illness ranging from a mild undifferentiated febrile illness to dengue fever (DF) to dengue hemorrhagic fever (DHF), a potentially life-threatening disease. The morbidity and mortality of DHF can be reduced by early hospitalization and careful supportive care. To determine its usefulness as a predictor of DHF, plasma levels of the secreted dengue virus nonstructural protein NS1 (sNS1) were measured daily in 32 children with dengue-2 virus infections participating in a prospective, hospital-based study. Free sNS1 levels in plasma correlated with viremia levels and were higher in patients with DHF than in those with DF. An elevated free sNS1 level (greater than or equal to600 ng/mL) within 72 h of illness onset identified patients at risk for developing DHF.

Effects of molecular crowding by saccharides on alpha-chymotrypsin dimerization

Given the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of alpha-chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the ostuolyte-mediated stabilization of the a-chymotrypsin homodimer, we have used models based on binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer-free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar-mediated stabilization of the alpha-chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the a-chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars.

Sediment yield and impacts from river catchments to the Great Barrier Reef lagoon

Land use intensification is estimated to result in an overall increase in sediment delivery to the Great Barrier Reef lagoon by a factor of approximately four. Modelling suggests that, following land use intensification, croplands cause the greatest increase of sediment yield and sediment concentration, whereas erosion of grazing land is the main contemporary source of sediments, primarily owing to the large spatial extent of this land use. The spatial pattern of sediment yield to the coast after land use intensification is strongly correlated with the pattern under natural conditions, although the greatest increase is estimated to have occurred in the wet-dry catchments. Sediment transport and resuspension processes have led to the development of a strongly sediment-partitioned shelf, with modern mud-rich sediments almost exclusively restricted to the inner and inner-middle shelf, northward-facing embayments and in the lee of headlands. Elevated sediment concentrations increase the potential transport rates of nutrients and other pollutants. Whether increased sediment supply to the coastal zone has impacted on reefs remains a point of contention. More sediment load data need to be collected and analysed in order to make detailed estimates of catchment yields and establish the possible sediment impact on the Great Barrier Reef.

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

Investigations into the PCDD contamination of topsoil, river sediments and kaolinite clay in Queensland, Australia

Recent findings of elevated PCDDs from an unknown source in the coastal marine environment of Queensland, Australia has instigated further investigations into the distribution of, and environments associated with the PCDD contamination. This study presents data for OCDD concentrations in the coastal, mountainous and inland environment of Queensland. Additionally, full 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and dibenzofuran (PCDD/F) profiles from different land-use types and environments in the coastal region were analysed. Distinct east-west gradients were detected in topsoil collected from various bushland regions with elevated OCDD concentrations confined to the coastal region. However, PCDD/F results from topsoil and river sediments collected in the Queensland coastal region suggest that elevated OCDD concentrations cannot be attributed to any of the environments, land-use or industry types investigated. PCDD/F congener profiles from select samples were remarkably similar to those previously described in marine sediments collected along the entire Queensland coastline. In addition, kaolinite clay samples from Queensland exhibited elevated OCDD concentrations, and PCDD/F profiles in these samples were similar to those detected in kaolinite clays elsewhere. Natural formation processes have been hypothesised as the source of elevated PCDDs in Queensland and other locations, where similar PCDD/F profiles and the general lack of anthropogenic sources are evident. This study presents additional data supporting this hypothesis and provides further information that may assist in the identification of the processes involved in the natural formation of PCDDs. (C) 2002 Elsevier Science Ltd. All rights reserved.

Catalytically active Dengue virus NS3 protease forms aggregates that are separable by size exclusion chromatography

An active form of the Dengue virus protease NS3 (CF40.Gly.NS3pro) was expressed in Escherichia coli. This construct consists of a critical 40 amino acid cofactor domain from NS2B fused to the N-terminal 184 amino acid protease domain of NS3 via a flexible, covalent linker (Gly(4)SerGly(4)). The recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the N-terminus for ease of purification using metal affinity chromatography. However, the presence of lower molecular weight impurities after affinity chromatography indicated the need for additional purification steps. The consistent appearance of these impurities suggested that they may be the products of proteolysis and/or auto-proteolysis. The latter possibility was subsequently excluded by the observation of the same impurities in a purified, catalytically inactive form of the recombinant protease (CF40.Gly.NS3pro.SA). Further analysis indicated that these impurities may represent premature translation termination products. Regardless of their origin, they were shown to form various sized aggregates with full-length CF40.Gly.NS3pro that can be separated by size exclusion chromatography, yielding fractions of active protease of sufficient purity for crystallisation trials. The ultimate goal of these studies is to obtain a crystal structure of a catalytically active form of the Dengue virus NS3 protease for structure-based drug design. (C) 2002 Elsevier Science (USA). All rights reserved.

Peroxisome proliferator-activated receptor alpha in the human breast cancer cell lines MCF-7 and MDA-MB-231

Peroxisome proliferator-activated receptor (PPAR) alpha is a ligand-activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARalpha mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARalpha mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARalpha is expressed and dynamically regulated in human breast cancer MCF-7 and MDA-MB-231 cells. Having established the presence of PPARalpha in both cell types, we then examined the consequence of PPARa activation, by its ligands Wy-14,643 and clofibrate, on proliferation. With real-time reverse transcriptase-polymerase chain reaction, we showed that PPARalpha mRNA was dynamically regulated in MDA-MB-231 cells and that PPARalpha activation significantly increased proliferation of the cell line. In contrast, PPARalpha expression in MCF-7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA-MB-231 cells. However, PPARalpha ligand activation still significantly increased the proliferation of MCF-7 cells. The promotion of proliferation in breast cancer cell lines following PPARalpha activation was in stark contrast to the effects of PPARgamma-activating ligands that decrease proliferation in human breast cancer cells. our results established the presence of PPARalpha in human breast cancer cell lines and showed for the first time that activation of PPARalpha in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis. (C) 2002 Wiley-Liss, Inc.

New graptolite faunas from the Llandovery, lower Silurian of the Graveyard Creek Subprovince, Broken River region, Queensland, Australia

A continuous biostratigraphic sequence has been established, possibly from as low as the sedgwickii Biozone but certainly throughout the Telychian Stage (?sedgwickii, turriculatus, ?crispus, griestoniensis and crenulata biozones). Thirty-three taxa have been recorded, including the new genus Wandograptus wandovalensis sp. nov., the new species Pseudostreptograptus queenslandensis and the new subspecies Dictyonema paululum australis and Stimulograptus sedwickii loydelli. A number of species have been recorded from Australia for the first time: Dictyonema cf. geniculatum Bulman, Petalolithus kurcki (Rickards), Pristiograptus initialis (Kirste), Monoclimacis directa Zalasciewicz, Monograptus aff. distans (Portlock), Monograptus sartorius Törnquist, and Torquigraptus pragensis (Bouček). Nine forms have been left under open nomenclature. The type section for the Poley Cow Formation yields a griestoniensis biozone fauna close to the top, and this level can be correlated with a griestoniensis Biozone fauna in the Broken River crossing section, again just below the top of the formation. Further north, griestoniensis Biozone faunas have also been recognized, beyond Diggers Creek, and in the Quinton Formation at Top Hut, enabling a reliable time plane throughout the region. At Top Hut, the highest graptolitic strata yield a crenulata Biozone fauna; and stratigraphically lower sequences, referable to the ?sedgwickii, turriculatus and ?crispus biozones, have been established on the Broken River section. It is likely that a full Telychian (Upper Llandovery) sequence occurs in the Poley Cow and Quinton formations enabling accurate correlation with other parts of the world.