A ferrocene functionalised macrocyclic receptor for cations and anions

The isolation and characterisation of a new macrocyclic hexaamine trans-6,13-bis(ferrocenylmethylamino)-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane (L-2) bearing two ferrocenyl groups appended to its exocyclic amines is reported. The crystal structures of L-2 and its dihydrochloride salt L-2. 2HCl . 2H(2)O have been determined. In the latter case cation-anion hydrogen bonding is observed in the solid state. Substrate binding by the electroactive L-2 in MeCN-CH2Cl2 solution has been examined by cyclic voltammetry and reveals the receptor electrochemically to recognise benzoate and chloride anions. The macrocyclic N-donors may also bind transition metal cations such as Cu-II and Zn-II.

Human pigmentation genes: identification, structure and consequences of polymorphic variation

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones a-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation. (C) 2001 Elsevier Science B.V. All rights reserved.

A practical method for identifying intermetallic phase particles in aluminium alloys by electron probe microanalysis

Aluminium alloys that contain Si, Mg, Fe, Mn and/or Cu usually contain one or more types of intermetallic phases that are not readily distinguishable in the microstructure by conventional microscopy methods. It has thus been a challenge to develop a method that will unambiguously identify them. A practical approach has been developed that is based on an inherent linear relationship revealed for the overall distribution of any two elements in a precipitate/matrix geometry and the first-order approximation of electron probe microanalysis (EPMA) results. Application of this approach to a direct chill cast 6082 alloy is demonstrated, and its major limitations are discussed.

Immune responses to ISCOM (R) formulations in animal and primate models

ISCOMs(R) are typically 40 nm cage-like structures comprising antigen, saponin, cholesterol and phospholipid. ISCOMs(R) have been shown to induce antibody responses and activate T helper cells and cyrolytic T lymphocytes in a number of animal species, including non-human primates. Recent clinical studies have demonstrated that ISCOMs(R) are also able to induce antibody and cellular immune responses in humans. This review describes the current understanding of the ability of ISCOMs(R) to induce immune responses and the mechanisms underlying this property. Recent progress in the characterisation and manufacture of ISCOMs(R) will also be discussed. (C) 2001 Elsevier Science Ltd. All rights reserved.

Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability

Attempts to classify colorectal cancer into subtypes based upon molecular characterisation are overshadowed by the classical stepwise model in which the adenoma-carcinoma sequence serves as the morphological counterpart. Clarity is achieved when cancers showing DNA microsatellite instability (MSI) are distinguished as sporadic MSI-low (MSI-L), sporadic MSI-high (MSI-H) and hereditary non-polyposis colorectal cancer (HNPCC). Divergence of the 'methylator' pathway into MSI-L and MSI-H is at least partly determined by the respective silencing of MGMT and hMLH1. Multiple differences can be demonstrated between sporadic and familial (HNPCC) MSI-H colorectal cancer with respect to early mechanisms, evolution, molecular characterisation, demographics and morphology. By acknowledging the existence of multiple pathways, rapid advances in the fields of basic and translational research will occur and this will lead to improved strategies for the prevention, early detection and treatment of colorectal cancer. (C) 2002 Elsevier Science Ltd. All rights reserved.

A portable load cell for in-situ ore impact breakage testing

This paper discusses the design and characterisation of a short, and hence portable impact load cell for in-situ quantification of ore breakage properties under impact loading conditions. Much literature has been published in the past two decades about impact load cells for ore breakage testing. It has been conclusively shown that such machines yield significant quantitative energy-fragmentation information about industrial ores. However, documented load cells are all laboratory systems that are not adapted for in-situ testing due to their dimensions and operating requirements. The authors report on a new portable impact load cell designed specifically for in-situ testing. The load cell is 1.5 m in height and weighs 30 kg. Its physical and operating characteristics are detailed in the paper. This includes physical dimensions, calibration and signal deconvolution. Emphasis is placed on the deconvolution issue, which is significant for such a short load cell. Finally, it is conclusively shown that the short load cell is quantitatively as accurate as its larger laboratory analogues. (C) 2062 Elsevier Science B.V. All rights reserved.

On the importance function in splitting simulation

The splitting method is a simulation technique for the estimation of very small probabilities. In this technique, the sample paths are split into multiple copies, at various stages in the simulation. Of vital importance to the efficiency of the method is the Importance Function (IF). This function governs the placement of the thresholds or surfaces at which the paths are split. We derive a characterisation of the optimal IF and show that for multi-dimensional models the natural choice for the IF is usually not optimal. We also show how nearly optimal splitting surfaces can be derived or simulated using reverse time analysis. Our numerical experiments illustrate that by using the optimal IF, one can obtain a significant improvement in simulation efficiency.

The anammox case - A new experimental manifesto for microbiological eco-physiology

The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater. It is also a new microbial physiology that was previously believed to be impossible. The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach. That approach is the focus of this paper. The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology. It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems. The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation. We argue that this was impossible using molecular ecology or conventional 'cultivation based techniques' alone. We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers.

Investigation of electrical conductivity as a function of dopant-ion radius in the systems Zr0.75Ce0.08M0.17O1.92 (M=Nd, Sm, Gd, Dy, Ho, Y, Er, Yb, Sc)

Electrical conductivity versus dopant ionic radius studies in zirconia- and ceria-based, solid oxide fuel cell (SOFC) electrolyte systems have shown that oxygen-ion conductivity is highest when the host and dopant ions are similar in size [J. Am. Ceram. Soc. 48 (1965) 286; Solid State Ionics 37 (1989) 67; Solid State Ionics 5 (1981) 547]. Under these conditions, it is thought that the conduction paths within the crystal lattice become less distorted [Solid State Ionics 8 (1983) 201]. In this study, binary ZrO2-M2O3 unit cells were expanded, via the partial substitution of Ce+4 for Zr+4 into the lattice, in an attempt to identify new, ternary, zirconia/ceria-based electrolyte systems with enhanced electrical conductivity. The compositions Zr0.75Ce0.08M0.17O1.92 (M = Nd, Sm, Gd, Dy, Ho, Y, Yb, Sc) were prepared using traditional solid state techniques. Bulk phase characterisation and precise lattice parameter measurements were performed with X-ray diffraction techniques. Four-probe DC conductivity measurements between 400 and 900 degreesC showed that the dopant-ion radius influenced electrical conductivity. The conductivity versus dopant-ion radius trends previously observed in zirconia-based, binary systems are clearly apparent in the ternary systems investigated in this study. The addition of ceria was found to have a negative influence on the electrical conductivity over the temperature range 400-900 degreesC. It is suggested that distortion of the oxygen-ion conduction path by the presence of the larger M+3 and Ce+4 species (relative to Zr+4) is the reason for the decreasing electrical conductivity as a function of increasing dopant size and ceria addition, respectively. (C) 2002 Elsevier Science B.V. All rights reserved.

Inducible system in human hepatoma cell lines for hepatitis C virus production

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetra cycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 In to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect naive Huh7 and stable Huh7-human CD81 cells. (C) 2002 Elsevier Science (USA).

Prediction of coal ash fusion temperatures with the F*A*C*T thermodynamic computer package

This paper outlines research on the processes taking place within the coal mineral matter at high temperatures and development of the relationship between ash fusion temperatures (AFT) and phase equilibria of the coal ash slags. A new thermodynamic database for the Al-Ca-Fe-O-Si system developed by the author was used in conjunction with the thermodynamic computer package F*A*C*T for these purposes. In addition, high temperature experimental studies were undertaken that involved heat treatment and quenching of the ash cones followed by the analyses using different techniques. The study provided new information on the processes taking place during AFT test and demonstrated the validity of the AFTs predictions with F*A*C*T. Examples of practical applications of the AFT prediction method are given in the paper. The results of this study are important not only for the AFT predictions, but also in general for the application of phase equilibrium science to the characterisation of the coal mineral matter interactions at high temperature. (C) 2002 Elsevier Science Ltd. All rights reserved.

Discovery and structures of the cyclotides: novel macrocyclic peptides from plants

Circular disulfide-rich polypeptides were unknown a decade ago but over recent years a large family of such molecules has been discovered, which we now refer to as the cyclotides. They are typically about 30 amino acids in size, contain an N- to C-cyclised backbone and incorporate three disulfide bonds arranged in a cystine knot motif. In this motif, an embedded ring in the structure formed by two disulfide bonds and their connecting backbone segments is penetrated by the third disulfide bond. The combination of this knotted and strongly braced structure with a circular backbone renders the cyclotides impervious to enzymatic breakdown and makes them exceptionally stable. This article describes the discovery of the cyclotides in plants from the Rubiaceae and Violaceae families, their chemical synthesis, folding, structural characterisation, and biosynthetic origin. The cyclotides have a diverse range of biological applications, ranging from uterotonic action, to anti-HIV and neurotensin antagonism. Certain plants from which they are derived have a history of uses in native medicine, with activity being observed after oral ingestion of a tea made from the plants. This suggests the possibility that the cyclotides may be orally bioavailable. They therefore have a range of potential applications as a stable peptide framework.

Molecular confirmation of an adenovirus in brushtail possums (Trichosurus vulpecula)

Partial genome characterisation of a non-cultivable marsupial adenovirus is described. Adenovirus-like particles were found by electron microscopy (EM) in the intestinal contents of brushtail possums (Trichosurus vulpecula) in New Zealand. Using degenerate PCR primers complementary to the most conserved genome regions of adenoviruses, the complete nucleotide sequence of the penton base gene, and partial nucleotide sequences of the DNA polymerase, hexon, and pVII genes were obtained. Phylogenetic analysis of the penton base gene strongly suggested that the brushtail possum adenovirus (candidate PoAdV-1) belongs to the recently proposed genus Atadenovirus. Sequence analysis of the PCR products amplified from the intestinal contents of brushtail possums originating from different geographical regions of New Zealand identified a single genotype. This is the first report of molecular confirmation of an adenovirus in a marsupial.

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.

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.