Isolation of arboviruses from mosquitoes (Diptera : Culicidae) collected from the Gulf Plains region of northwest Queensland, Australia

As part of investigations into Japanese encephalitis (JE) virus and related flaviviruses in northern Australia, 153,529 mosquitoes were collected and processed for virus isolation from the Gulf Plains region of northwest Queensland. Collections front within 30 km of each of the townships of Croydon, Normanton and Karumba yielded 3,087 (2.0%), 66,009 (43.0%), and 84,433 (55.0%) mosquitoes, respectively, from which 16 viruses were isolated. Four isolates of Murray Valley encephalitis (MVE), two of Kunjin (KUN), three of Ross River (1111), and one of Sindbis (SIN) viruses were obtained from Culex sitiens subgroup mosquitoes. Molecular identification of the mosquito species composition of these virus positive pools revealed that most isolates were from pools containing mainly Culex annulirostris Skuse and low numbers of Cidex palpalis (Taylor). Only three pools, one each of MVE, KUN, and RR, were from mosquitoes identified exclusively as Cx. annulirostris. Other viruses isolated include one Edge Hill Virus from Ochlerotatus normanensis (Taylor), an isolate of SIN from Anopheles meraukensis Venhuis, two isolates of RR from Anopheles amictus Edwards, and single isolates of RR from Anopheles bancroftii Giles and Aedes lineatopennis (Ludlow). The isolate of RR from Ae. lineatopennis was the first reported from this species. The public health implications of these isolations in the Gulf Plains region are discussed briefly.

Rapid isolation of total RNA and genomic DNA from Hakea actities

Tissues of the Australian native plant species Hakea actities (Proteaceae) contain numerous metabolites and structural compounds that hinder the isolation of nucleic acids. Separate RNA and genomic DNA extraction procedures were developed to isolate high quality nucleic acids from H. actities. Total RNA was extracted from leaves, roots and cluster roots of H. actities grown in low nutrient levels. Cluster root formation in H. actities only occurs when the plants are grown in low nutrient concentrations. However, under these conditions, nucleic acid extraction becomes increasingly difficult. The new procedures are faster than many of the published nucleic acid extraction protocols, and avoid the use of hazardous chemicals. The RNA extraction method was used successfully on another Australian species and a crop species, suggesting that the procedure is useful for molecular studies of a broad range of plants.

Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically, important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at similar to2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450,in, it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K-D = 0.7 mum), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.

Isolation of Gemmata-like and Isosphaera-like planctomycete bacteria from soil and freshwater

New cultured strains of the planctomycete division (order Planctomycetales) of the domain Bacteria related to species in the genera Gemmata and Isosphaera were isolated from soil, freshwater, and a laboratory ampicillin solution. Phylogenetic analysis of the 16S rRNA gene from eight representative isolates showed that all the isolates were members of the planctomycete division. Six isolates clustered with Gemmata obscuriglobus and related strains, while two isolates clustered with Isosphaera pallida. A double-membrane-bounded nucleoid was observed in Gemmata-related isolates but not in Isosphaera-related isolates, consistent with the ultrastructures of existing species of each genus. Two isolates from this study represent the first planctomycetes successfully cultivated from soil.

Parenthood and pay in Australia and the UK: evidence from workplace surveys

This article addresses the effect of parenthood on pay, examining potential reasons for-differences between Australia and the UK that are evident in spite of their similarly minimalist. male, breadwinner style approaches to work/family issues, Although cross-national differences reflect complex intersections of policy combinations, institutional frameworks, patterns of employment and gender contracts that cannot be assessed in a single analysis, the data used in this analysis uncover some of the factors that contribute to different outcomes. Motherhood penalties in the UK appear to be associated primarily with the comparatively low level of part-time earnings in that country, while higher premiums to fatherhood at least in part reflect a wider overall wage distribution. These findings reinforce the heed to interpret earnings effects of parenthood within the context of national patterns of employment and wage distribution; and highlight the breadth of strategies needed to deliver more equitable outcomes.

Isolation and characterisation of Indian Ocean ciguatoxin

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3 H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean. (C) 2002 Elsevier Science Ltd. All rights reserved.

Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: a platform for cancer immunotherapy

The fundamental role of dendritic cells (DC in initiating and directing the primary immune response is well established. Furthermore, it is now accepted that DC may be useful in new vaccination strategies for preventing certain malignant and infectious diseases. As blood DC (BDC physiology differs from that of the DC homologues generated in vitro from monocyte precursors, it is becoming more relevant to consider BDC for therapeutic interventions. Until recently, protocols for the isolation of BDC were laborious and inefficient; therefore, their use for investigative cancer immunotherapy is not widespread. In this study, we carefully documented BDC counts, yields and subsets during apheresis (Cobe Spectra), the initial and essential procedure in creating a BDC isolation platform for cancer immunotherapy. We established that an automated software package (Version 6,0 AutoPBPC) provides an operator-independent reliable source of motionuclear cells (MNC for BDC preparation. Further, we observed that BDC might be recovered in high yields, often greater than 100% relative to the number of circulating BDC predicted by blood volume. An average of 66 million (range, 17-179) BDC per 10-1 procedure were obtained, largely satisfying the needs for immunization. Higher yields were possible on total processed blood volumes of 151. BDC were not activated by the isolation procedure and, more importantly, both BDC subsets (CD11c(+)CD123(low) and CD11c(-)CD123(high)) were equally represented. Finally, we established that the apheresis product could be used for antibody-based BDC immunoselection and demonstrated that fully functional BDC can be obtained by this procedure. (C) 2002 Published by Elsevier Science B.V.

Social rules for managing attempted interpersonal domination in the workplace: Influence of status and gender.

The present study utilized a social rules approach to investigate the relative influence of gender and status on managers' self-evaluations of their effectiveness in handling a dominating subordinate. In the first study 84 White middle-class participants identified the prescriptive and proscriptive rules for socially appropriate responding to a stimulus situation involving a pushy subordinate. Four rule sets were identified for female and male managers and subordinates, respectively. Rule-sets shared a number of common rules and showed some variation according to gender roles. In the second study, 91 White middle-class participants rated the individual rules for importance and also rated their personal and managerial effectiveness when responding to the stimulus situation using gender- and status-consistent and gender-and status-inconsistent response strategies. Both men and women rated the female gender and status- consistent strategy as most effective, and rated the status-inconsistent strategy as less effective than a gender-inconsistent response. Results were interpreted as providing more support for a situational gender-related theory of workplace behavior, rather than a traditional gender role perspective.

Isolation and characterisation of a novel rabbit sulfotransferase isoform belonging to the SULT1A subfamily

Sulfotransferases (SULTs) catalyse the sulfonation of both endogenous and exogenous compounds including hormones, catecholamines. drugs and xenobiotics. While in most occasions, sulfonation is a detoxication pathway. in the case of certain drugs and carcinogens. it leads to metabolic activation. Since, the rabbit has been extensively used for both pharmacological and toxicological studies, the purpose of this study was to further characterise the sulfotransferase system of this animal. In the present study, a novel sulfotransferase isoform (GenBank Accession no. AF360872) was isolated from a rabbit liver cDNA lambdaZAP 11 library. The full-length sequence of the clone was 1138 bp long and contained a coding region of 888 bp encoding a cytosolic protein of 295 amino acids (deduced molecular weight 34,193 Da). The amino acid sequence of this novel SULT isoform showed >70% identity with members of the SULT1A subfamily of sulfotransferases from other species. Upon expression of the encoded rabbit sulfotransferase in Escherchia coli (E. coli), it was shown that the enzyme was capable of sulfonating both p-nitrophenot (K-m and V-max values of 0.15 muM and 897.5 nmol/min/mg protein. respectively) and dopamine (K-m and V-max values of 175.3 muM and 151.1 nmol/min/mg protein, respectively). Based on the sequence data obtained and substrate specificity, this new rabbit sulfotransferase was named rabSULT1A1. Immunoblotting was used to demonstrate that rabSULT1A1 protein is expressed in liver, duodenum, jejunum, ileum, colon and recturm. (C) 2002 Elsevier Science Ltd. All rights reserved.

Isolation and characterization of microsatellite loci in Bemisia tabaci

Bemisia tabaci (Hemiptera: Aleyrodidae) is a haplo-diploid species with a global distribution demonstrating strong geographical structure with eight recognizable genetic groups. Fifteen microsatellite loci (335 alleles, 6-44 alleles per locus) were derived from four of the eight groups and were then screened across 33 populations. These loci clearly differentiate the populations. The microsatellites amplified best in individuals from genetic groups representing the Mediterranean, Middle East, Asia (three groups) and Australasia/Oceania and amplified less well with populations from sub-Saharan Africa and the New World. This differential amplification pattern is a direct result of the relatedness to the microsatellite source material.