Polyspecific malaria antibodies present at the time of infection inhibit the development of immunity to malaria but antibodies specific for the malaria merozoite surface protein, MSP1, facilitate immunity

Serum taken from mice immune to malaria as a result of infection and drug cure, or from mice immunized with a recombinant form of the merozoite surface protein, MSP1, can provide passive protection of recipient mice against the lethal parasite, Plasmodium yoelii YM. However, recipients of MSP1-immune serum go on to develop long-term immunity, whereas recipients of serum from mice naturally immune to malaria rapidly lose their resistance to infection. We demonstrate that 'infection/cure' serum suppresses the development of both antibody and cell-mediated parasite-specific responses in recipients, whereas these develop in recipients of MSP1-specific antibodies. These data have profound implications for our understanding of the development of malaria immunity in babies who passively acquire antibodies from their mothers.

Testing the applicability of molecular genetic markers to population analyses of scleratinian corals

The abundance of coral reefs worldwide is in decline, and despite the ecological importance of reefs, only a limited number of DNA markers have been identified for scleractinian coral genetic studies. This paper addresses the search for new coral molecular markers and investigates the applicability of the cytochrome c oxidase subunit I (COI), the internal transcribed spacer region 1 (ITS1), and the pocilloporin gene to the question of intraspecific variation in the scleractinian coral Pocillopora verrucosa along the southeast African coastline. The COI fragment was 710 bp long and was identical for P. verrucosa (n = 10) and P. damicornis (n = 3). Only two different ITS1 sequences were found (differing by 13 bp insertion), but more importantly, 24% of the sequences were heterogenous indicating that different multiple copies of the sequence exist. Pocilloporin is an intronless gene that was absolutely conserved throughout all P. verrucosa populations (n = 50). Thus, the three DNA regions studied appear unsuitable for the population genetic analyses of P. verrucosa.

Development and characterization of polymorphic microsatellite markers in taro (Colocasia esculenta)

Microsatellite-containing sequences were isolated from enriched genomic libraries of taro (Colocasia esculenta (L.) Schott). The sequencing of 269 clones yielded 77 inserts containing repeat motifs. The majority of these (81.7%) were dinucleotide or trinucleotide repeats. The GT/CA repeat motif was the most common, accounting for 42% of all repeat types. From a total of 43 primer pairs designed, 41 produced markers within the expected size range. Sixteen (39%) were polymorphic when screened against a restricted set of taro genotypes from Southeast Asia and Oceania, with an average of 3.2 alleles detected on each locus. These markers represent a useful resource for taro germplasm management, genome mapping, and marker-assisted selection.

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx(2c), either alone (16%) or in combination with stx(1) (74%) or stx(2) (3%) PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.

A rapid PCR protocol for marker assisted detection of heterozygotes in segregating generations involving 1BL/1RS translocation and normal wheat lines

A rapid and reliable polymerase chain reaction (PCR)-based protocol was developed for detecting zygosity of the 1BL/1RS translocation in hexaploid wheat. The protocol involved a multiplex PCR with 2 pairs of oligonucleotide primers, rye-specific Ris-1 primers, and consensus 5S intergenic spacer (IGS) primers, and digestion of the PCR products with the restriction enzyme, MseI. A small piece of alkali-treated intact leaf tissue is used as a template for the PCR, thereby eliminating the necessity for DNA extraction. The test is simple, highly sensitive, and rapid compared with the other detection systems of 1BS1RS heterozygotes in hexaploid wheat. PCR results were confirmed with AFLP analyses. Diagnostic tests for 1BL/1RS translocation based on Sec-1-specific ELISA, screening for chromosome arm 1RS controlled rust resistance locus Yr9, and the PCR test differed in their ability to detect heterozygotes. The PCR test and rust test detected more heterozygotes than the ELISA test. The PCR test is being used to facilitate S1 family recurrent selection in the Germplasm Enhancement Program of the Australian Northern Wheat Improvement Program. A combination of the PCR zygosity test with other markers currently being implemented in the breeding program makes this test economical for 1BL/1RS characterisation of S1 families.

Donor-derived b2a2-specific T cells for immunotherapy of patients with chronic myeloid leukemia

The BCR-ABL fusion proteins, b2a2 and b3a2, are potential targets for a beneficial graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation for chronic myeloid leukemia (CML). This study demonstrates that CD4(+) T cells specific to the b2a2 peptide can be generated from a normal allogeneic stem cell transplant donor after stimulation with monocyte-derived dendritic cells (Mo-DC) using culture conditions applicable to clinical use. Stimulation of donor T-cell enriched mononuclear cells (MNC) with b2a2-pulsed Mo-DC produced approximately 3 x 10(9) b2a2-specific CD4(+) T cells. The CD4(+) T cells were HLA-DR7 restricted. These results confirm that the generation of donor derived b2a2-specific T cells for clinical use is feasible and warrants clinical testing after stem cell transplantation.

Identification of research to improve the efficiency of breeding strategies for white clover in Australia - a review

A major challenge faced by today's white clover breeder is how to manage resources within a breeding program. It is essential to utilise these resources with sufficient flexibility to build on past progress from conventional breeding strategies, but also take advantage of emerging opportunities from molecular breeding tools such as molecular markers and transformation. It is timely to review white clover breeding strategies. This background can then be used as a foundation for considering how to continue conventional plant improvement activities and complement them with molecular breeding opportunities. In this review, conventional white clover breeding strategies relevant to the Australian dryland target population environments are considered. Attention is given to: (i) availability of genetic variation, (ii) characterisation of germplasm collections, (iii) quantitative models for estimation of heritability, (iv) the role of multi-environment trials to accommodate genotype-by-environment interactions, (v) interdisciplinary research to understand adaptation to dryland environments, (vi) breeding and selection strategies, and (vii) cultivar structure. Current achievements in biotechnology with specific reference to white clover breeding in Australia are considered, and computer modelling of breeding programs is discussed as a useful integrative tool for the joint evaluation of conventional and molecular breeding strategies and optimisation of resource use in breeding programs. Four areas are identified as future research priorities: (i) capturing the potential genetic diversity among introduced accessions and ecotypes that are adapted to key constraints such as summer moisture stress and the use of molecular markers to assess the genetic diversity, (ii) understanding the underlying physiological/morphological root and shoot mechanisms involved in water use efficiency of white clover, with the objective of identifying appropriate selection criteria, (iii) estimation of quantitative genetic parameters of important morphological/physiological attributes to enable prediction of response to selection in target environments, and (iv) modelling white clover breeding strategies to evaluate the opportunities for integration of molecular breeding strategies with conventional breeding programs.

Characterization of a distinct plasma membrane macrodomain in differentiated adipocytes

Caveolae are small invaginations of the cell surface that are abundant in mature adipocytes. A recent study (Kanzaki, M., and Pessin, J. E. (2002) J. Biol Chem 277, 25867-25869) described novel caveolin- and actin-containing structures associated with the adipocyte cell surface that contain specific signaling proteins. We have characterized these structures, here termed caves, using light and electron microscopy and observe that they represent surface-connected wide invaginations of the basal plasma membrane that are sometimes many micrometers in diameter. Rather than simply a caveolar domain, these structures contain all elements of the plasma membrane including clathrin-coated pits, lipid raft markers, and non-raft markers. GLUT4 is recruited to caves in response to insulin stimulation. Caves can occupy a significant proportion of the plasma membrane area and are surrounded by cortical actin. Caveolae density in caves is similar to that on the bulk plasma membrane, but because these structures protrude much deeper into the plane of focus of the light microscope molecules such as caveolin and other plasma membrane proteins appear more concentrated in caves. We conclude that the adipocyte surface membrane contains numerous wide invaginations that do not represent novel caveolar structures but rather large surface caves.

Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities

Increasingly, cystic fibrosis (CF) is regarded as an inflammatory disorder where the response of the lung to Pseudomonas aeruginosa is exaggerated as a consequence of processes mediated by the product of the CF gene, CFTR. Of importance to any gene-replacement strategy for treatment of CF is the identification of the cell type(s) within the lung milieu that need to be corrected and an indication whether this is sufficient to restore a normal inflammatory response and bacterial clearance. We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages. Following chronic pulmonary infection with P. aeruginosa, CF mice with epithelial-expressed but not macrophage-specific CFTR showed an improvement in pathogen clearance and inflammatory markers compared with control CF animals. Additionally, these data indicate the general role for epithelial cell-mediated events in the response of the lung to bacterial pathogens and the importance of CFTR in mediating these processes.

Characterization of highly conserved microsatellite loci in Araucaria cunninghamii and related species

Ten microsatellite loci are described in Araucaria cunninghamii, the first reported in the Araucariaceae. Eight were tested in sections Eutacta and Bunya, which diverged more than 200 MYA, and to the sister genus Agathis. Specific amplification products within the expected size range were obtained for six to eight loci in section Eutacta (depending on species), five loci in section Bunya and three. loci in Agathis. Two of the loci (CRCAc1 and CRCAc2, both GA repeats) produced specific amplification products in all taxa, with orthology confirmed by sequence analysis. The repeats were perfect in all taxa. The flanking sequences were extremely conserved, with sequence divergence of 0% to 2.0% within Araucaria species and 2.9% to 7.5% between Araucaria and Agathis. These microsatellites represent some of the most conserved microsatellite loci reported in plants. This may be due to a low evolutionary rate in Araucariaceae genome or the loci may be closely associated with highly conserved, unreported genes.

Molecular assays for detection of human metapneumovirus

The recent description of the respiratory pathogen human metapneumovirus (hMPV) has highlighted a deficiency in current diagnostic techniques for viral agents associated with acute lower respiratory tract infections. We describe two novel approaches to the detection of viral RNA by use of reverse transcriptase PCR (RT-PCR). The PCR products were identified after capture onto a solid-phase medium by hybridization with a sequence-specific, biotinylated oligonucleotide probe. The assay was applied to the screening of 329 nasopharyngeal aspirates sampled from patients suffering from respiratory tract disease. These samples were negative for other common microbial causes of respiratory tract disease. We were able to detect hMPV sequences in 32 (9.7%) samples collected from Australian patients during 2001. To further reduce result turnaround times we designed a fluorogenic TaqMan oligoprobe and combined it with the existing primers for use on the LightCycler platform. The real-time RT-PCR proved to be highly reproducible and detected hMPV in an additional 6 out of 62 samples (9.6%) tested during the comparison of the two diagnostic approaches. We found the real-time RT-PCR to be the test of choice for future investigation of samples for hMPV due to its speed, reproducibility, specificity, and sensitivity.

Mechanism of mitosis-specific activation of MEK1

Activation of cyclin B-Cdc2 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G(2)/M progression. The MAPK cascade has well established roles in entry and exit from mitosis in Xenopus, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. Here we report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G(2)/M. The focus of this pathway is the dramatic activation of an endomembrane-associated MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-Cdc2-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr(286). These results demonstrate that cyclin B-Cdc2 activity regulates signaling through the MAPK pathway in mitosis.

Immunogenicity of recombinant HBsAg/HCV particles in mice pre-immunised with hepatitis B virus-specific vaccine

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice. A high percentage of the human population is positive for anti-HBsAg antibodies (anti-HBs), either through vaccination or natural infection. We, therefore, determined if pre-existing anti-HBs could influence immunisation with modified VLPs. Mice were immunised with a commercial HBV vaccine, monitored to ensure an anti-HBs response, then immunised with HBsAg/HVR1-VLPs. The resulting anti-HVR1 antibody titre was similar in mice with or without pre-existing anti-HBs. This suggests that HBsAg/HVR1-VLPs induce a primary immune response to HVR1 in anti-HBs positive mice and, hence, they may be used successfully in individuals already immunised with the HBV vaccine. (C) 2003 Elsevier Science Ltd. All rights reserved.

eMelanoBase: An online locus-specific variant database for familial melanoma

A proportion of melanoma,prone individuals in both familial and non,familial contexts has been shown to carry inactivating mutations in either CDKN2A or, rarely, CDK4. CDKN2A is a complex locus that encodes two unrelated proteins from alternately spliced transcripts that are read in different frames. The alpha transcript (exons 1a, 2, and 3) produces the p16INK4A cyclin-dependent kinase inhibitor, while the beta transcript (exons 1beta and 2) is translated as p14ARF, a stabilizing factor of p53 levels through binding to MDM2. Mutations in exon 2 can impair both polypeptides and insertions and deletions in exons 1alpha, 1beta, and 2, which can theoretically generate p16INK4A,p14ARF fusion proteins. No online database currently takes into account all the consequences of these genotypes, a situation compounded by some problematic previous annotations of CDKN2A related sequences and descriptions of their mutations. As an initiative of the international Melanoma Genetics Consortium, we have therefore established a database of germline variants observed in all loci implicated in familial melanoma susceptibility. Such a comprehensive, publicly accessible database is an essential foundation for research on melanoma susceptibility and its clinical application. Our database serves two types of data as defined by HUGO. The core dataset includes the nucleotide variants on the genomic and transcript levels, amino acid variants, and citation. The ancillary dataset includes keyword description of events at the transcription and translation levels and epidemiological data. The application that handles users' queries was designed in the model,view. controller architecture and was implemented in Java. The object-relational database schema was deduced using functional dependency analysis. We hereby present our first functional prototype of eMelanoBase. The service is accessible via the URL www.wmi.usyd.e, du.au:8080/melanoma.html.