A Conceptual Model to Describe the Decline of European Blackberry (Rubus anglocandicans), A Weed of National Significance in Australia.

Human activities have had an adverse impact on ecosystems on a global scale and have caused an unprecedented redispersal of organisms, with both plants and pathogens moving from their regions of origin to other parts of the world. Invasive plants are a potential threat to ecosystems globally, and their management costs tens of billions of dollars per annum. Rubus anglocandicans (European blackberry) is a serious invasive species in Australia. Herbicide and cultural control methods are generally inefficient or require multiple applications. Therefore, a biological control program using stem and leaf rust strains is the main option in Australia. However, biological control using rusts has been patchy, as host factors, climate, and weather can alter the impact of the rust at different locations. In 2007, Yeoh and Fontanini noticed that blackberry plants on the banks of the Donnelly and Warren rivers in the southwest of Western Australia were dying in areas that were being regularly monitored for the impact of rust as a biological control agent. The symptoms on blackberry became known as the disease "blackberry decline". Continuous and intensive investigations are required to discover the different biotic and abiotic components associated with specific declines in plant populations. The only agent so far introduced to Australia for the biological control of blackberry is the rust Phragmidium violaceum.

The effect of long term storage on tobacco smoke particulate matter in in vitro genotoxicity and cytotoxicity assays.

Particulate matter (PM) collected from mainstream tobacco smoke is a test article commonly used for in vitro genotoxicity and cytotoxicity testing of combustible tobacco products. However, little published data exists concerning the stability of PM. We completed a 2 year study to quantify the effect of PM storage at -80 °C, on the genotoxicity and cytotoxicity of PM generated from 3R4F and M4A reference cigarettes. The Ames test, Micronucleus assay (MNvit), Mouse Lymphoma assay (MLA) and the Neutral Red Uptake assay (NRU) were used. The majority of M4A and 3R4F PMs were genotoxic and cytotoxic at the timepoints tested. Some minor but statistically significant differences were observed for stored versus freshly prepared PM, but the magnitude of changes were within the variability observed for repeat testing.

Searching for peptide ligands with an epitope library.

Tens of millions of short peptides can be easily surveyed for tight binding to an antibody, receptor or other binding protein using an "epitope library." The library is a vast mixture of filamentous phage clones, each displaying one peptide sequence on the virion surface. The survey is accomplished by using the binding protein to affinity-purify phage that display tight-binding peptides and propagating the purified phage in Escherichia coli. The amino acid sequences of the peptides displayed on the phage are then determined by sequencing the corresponding coding region in the viral DNA's. Potential applications of the epitope library include investigation of the specificity of antibodies and discovery of mimetic drug candidates.

Discovering peptide ligands using epitope libraries.

Epitope libraries are large collections of peptides. Each peptide is displayed on the surface of a bacteriophage particle and is encoded by a randomly mutated region of the phage genome, thus associating each unique peptide with the DNA molecule encoding it. Antibodies and other binding proteins are used to select specifically for rare, phage-bearing peptide ligands; sequencing of the corresponding viral DNA will reveal their amino acid sequences. Relatively high-affinity peptides for a variety of peptide- and non-peptide-binding ligates have been affinity-isolated from epitope libraries. This technology has been used to map epitopes on proteins and to find peptide mimics for non-peptide-binding ligates. The current challenge lies in developing epitope library technology so that tight-binding peptide ligands can be detected for a wider variety of ligates, including those that recognize folded proteins. Should this be accomplished, many powerful applications can be envisioned in the areas of drug design and the development of diagnostic markers and vaccines.

The c-myc protein is constitutively expressed at elevated levels in colorectal carcinoma cell lines.

In this study, we have employed both indirect immunofluorescence and ELISA assays to compare the relative levels of c-myc protein in cell lines derived from normal human colon and colon adenocarcinomas. We show that the levels of protein found in the majority of carcinoma cell lines are consistent with the levels of mRNA expressed, and that both are significantly elevated with respect to the levels found in normal cells. Growing populations of fibroblastic and epithelial cell lines derived from normal colonic mucosa exhibit small numbers of steady-state transcripts and immunofluorescence signals which are weak and confined to the nucleus. The adenocarcinoma cell lines, however, express 5- to 10-fold elevated levels of c-myc mRNA and exhibit correspondingly intense immunofluorescence signals which appear to reside principally in the nucleus. Quantitation of c-myc protein levels in these tumor cell lines by ELISA assay indicates that they are 8- to 37-fold higher than the levels of protein in normal cells. Elevated expression of the c-myc gene at both the mRNA and protein levels occurs constitutively in the colorectal carcinoma cell lines during their growth in culture, in contrast to the transiently elevated levels of expression observed in normal cells which have been subjected to a mitogenic stimulus. The constitutively elevated expression of the c-myc protein in colorectal carcinoma cell lines is not typically accompanied by gross rearrangement or amplification of the gene.

Random-peptide libraries and antigen-fragment libraries for epitope mapping and the development of vaccines and diagnostics.

Random peptide libraries and antigen-fragment libraries (also known as gene-fragment libraries) have been used to identify epitopes on protein antigens. These technologies promise to make significant contributions to diagnostic and vaccine development. Researchers in a number of labs have shown that phage selected from libraries with protective antibodies, raised against whole antigen, can be used as immunogens to stimulate antibody responses that bind native antigen and provide protection in vivo. Others have used the sera of patients with idiopathic diseases to screen libraries, and by this approach have identified candidate antigens involved in immune disease. These may prove useful for diagnosis and, possibly, in determining disease etiology.

Homodimeric peptides displayed by the major coat protein of filamentous phage.

Peptide libraries displayed by filamentous bacteriophage have proven a powerful tool for the discovery of novel peptide agonists, antagonists and epitope mimics. Most phage-displayed peptides are fused to the N terminus of either the minor coat protein, pIII, or the major coat protein, pVIII. We report here that peptides containing cysteine residues, displayed as N-terminal fusions to pVIII, can form disulfide-bridged homodimers on the phage coat. Phage clones were randomly selected from libraries containing one or two fixed Cys residues, and surveyed for the presence of peptide-pVIII homodimers by SDS-PAGE analysis that involved pretreatment of the phage with reducing or thiol-modifying agents. For all phage whose recombinant peptide contained a single Cys residue, a significant fraction of the peptide-pVIII molecules were displayed as dimers on the phage coat. The dimeric form was in greater abundance than the monomer in almost all cases in which both forms could be reliably observed. Occasionally, peptides containing two Cys residues also formed dimers. These results indicate that, for a given pVIII-displayed peptide bearing a single Cys residue, a significant fraction of the peptide (>40 %) will dimerize regardless of its sequence; however, sequence constraints probably determine whether all of the peptide will dimerize. Similarly, only occasionally do peptides bearing two Cys residues form intermolecular disulfide bridges instead of intramolecular ones; this indicates that sequence constraints may also determine dimerization versus cyclization. Sucrose-gradient analysis of membranes from cells expressing pVIII fused to a peptide containing a single Cys residue showed that dimeric pVIII is present in the cell prior to its assembly onto phage. A model of the peptide-pVIII homodimer is discussed in light of existing models of the structure and assembly of the phage coat. The unique secondary structures created by the covalent association of peptides on the phage surface suggest a role for homo- and heterodimeric peptide libraries as novel sources of bioactive peptides.

The role of structure in antibody cross-reactivity between peptides and folded proteins.

Peptides have the potential for targeting vaccines against pre-specified epitopes on folded proteins. When polyclonal antibodies against native proteins are used to screen peptide libraries, most of the peptides isolated align to linear epitopes on the proteins. The mechanism of cross-reactivity is unclear; both structural mimicry by the peptide and induced fit of the epitope may occur. The most effective peptide mimics of protein epitopes are likely to be those that best mimic both the chemistry and the structure of epitopes. Our goal in this work has been to establish a strategy for characterizing epitopes on a folded protein that are candidates for structural mimicry by peptides. We investigated the chemical and structural bases of peptide-protein cross-reactivity using phage-displayed peptide libraries in combination with computational structural analysis. Polyclonal antibodies against the well-characterized antigens, hen eggwhite lysozyme and worm myohemerythrin, were used to screen a panel of phage-displayed peptide libraries. Most of the selected peptide sequences aligned to linear epitopes on the corresponding protein; the critical binding sequence of each epitope was revealed from these alignments. The structures of the critical sequences as they occur in other non-homologous proteins were analyzed using the Sequery and Superpositional Structural Assignment computer programs. These allowed us to evaluate the extent of conformational preference inherent in each sequence independent of its protein context, and thus to predict the peptides most likely to have structural preferences that match their protein epitopes. Evidence for sequences having a clear structural bias emerged for several epitopes, and synthetic peptides representing three of these epitopes bound antibody with sub-micromolar affinities. The strong preference for a type II beta-turn predicted for one peptide was confirmed by NMR and circular dichroism analyses. Our strategy for identifying conformationally biased epitope sequences provides a new approach to the design of epitope-targeted, peptide-based vaccines.

Probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage.

The structural requirements for peptide binding to an antibody may be elucidated by probing it with a variety of peptides having different constraints. To this end, we have constructed and screened a panel of peptide libraries displayed by filamentous bacteriophage. The peptides in most of the libraries have the potential for constraint by fixed Cys residues, which have been placed at different sites within a randomized amino acid sequence of varying length. When taken together, the binding data obtained from screening the panel with a given antibody allow one to determine the types of constraints that promote binding, as well as the residues that are critical for binding. We describe the construction of 11, pVIII-displayed, peptide libraries, whose sizes range from 150 million to 10 billion clones. The libraries were screened with a number of polyclonal and monoclonal antibodies against peptides, proteins and carbohydrates. Cross-reactivity with peptides was always found for antibodies produced against peptides, linear epitopes on folded proteins and, surprisingly, carbohydrates, whereas antibodies against discontinuous epitopes on proteins were found less frequently. The implications of these results are discussed in terms of the structural basis for cross-reactivity with peptides.

The recognition of a noncanonical RNA base pair by a zinc finger protein.

BACKGROUND: The zinc finger (ZF) is the most abundant nucleic-acid-interacting protein motif. Although the interaction of ZFs with DNA is reasonably well understood, little is known about the RNA-binding mechanism. We investigated RNA binding to ZFs using the Zif268-DNA complex as a model system. Zif268 contains three DNA-binding ZFs; each independently binds a 3 base pair (bp) subsite within a 9 bp recognition sequence. RESULTS: We constructed a library of phage-displayed ZFs by randomizing the alpha helix of the Zif268 central finger. Successful selection of an RNA binder required a noncanonical base pair in the middle of the RNA triplet. Binding of the Zif268 variant to an RNA duplex containing a G.A mismatch (rG.A) is specific for RNA and is dependent on the conformation of the mismatched middle base pair. Modeling and NMR analyses revealed that the rG.A pair adopts a head-to-head configuration that counterbalances the effect of S-puckered riboses in the backbone. We propose that the structure of the rG.A duplex is similar to the DNA in the original Zif268-DNA complex. CONCLUSIONS: It is possible to change the specificity of a ZF from DNA to RNA. The ZF motif can use similar mechanisms in binding both types of nucleic acids. Our strategy allowed us to rationalize the interactions that are possible between a ZF and its RNA substrate. This same strategy can be used to assess the binding specificity of ZFs or other protein motifs for noncanconical RNA base pairs, and should permit the design of proteins that bind specific RNA structures.

Random peptide libraries.

Over the past year, great strides have been made in the design of peptide libraries, and new approaches have been developed for identifying peptide ligands. The libraries comprise large collections of peptides, ranging from 1 million to 1 billion different sequences, which can be screened using monoclonal and polyclonal antibodies, receptors, enzymes or other target molecules. The power of this technology stems from the chemical diversity of the amino acids coupled with the large number of sequences in a library. As such, peptide libraries may be useful for finding ligands that can serve as leads for pharmaceutical development and other purposes.

Phage-displayed peptide libraries.

Over the past year, significant advances have been achieved through the use of phage-displayed peptide libraries. A wide variety of bioactive molecules, including antibodies, receptors and enzymes, have selected high-affinity and/or highly-specific peptide ligands from a number of different types of peptide library. The demonstrated therapeutic potential of some of these peptides, as well as new insights into protein structure and function that peptide ligands have provided, highlight the progress made within this rapidly-expanding field.

The developing occlusion of children and young people in general practice: when to watch and when to refer.

This paper discusses the assessment of the developing occlusions of children and adolescents in the general practice setting; that is, reviewing the potential of interceptive orthodontics. In particular we will illustrate the management of these individuals with case examples. We have also provided a handy pull-out guide with this issue of the Journal which can be used in the GDP's surgery for quick reference.

S-carboxymethylcysteine in the fluidification of sputum and treatment of chronic airway obstruction.

The clinical results and changes in sputum found in both a short-term inpatient trial and a subsequent long-term outpatient investigation (three-month double-blind controlled study) of 82 patients with chronic bronchitis treated with a new mucolytic agent, S-carboxymethylcysteine (Mucodyne), are reported. Fluidification of sputum with reduction in certain measurements of the viscosity of morning sputum aliquots, associated with improvement in the ability to cough up bronchial secretions, significant increase in sputum volume output, and improvement in ventilation (as estimated by the forced expiratory volume in one second), were observed in both trials as dose-related responses, with an increase in the ease of expectoration and a reduction in cough frequency and dyspnea. Therapy with S-carboxymethylcysteine was well tolerated, and there were no serious adverse effects, either immediate or delayed. We suggest that the effect of the drug in fluidifying sputum may be due to a mucoregulatory mechanism which reverses the sputum macromolecular disturbances seen in chronic bronchitis.

International medicine and air pollution.

Environmental pollution worldwide has been increasing rapidly as a result of massive increases in industrial production, especially since 1950. Although the United States has begun to address the problem, many nations have not been able to, such as those formerly part of the Communist bloc and many emerging nations. The International Society of Doctors for the Environment was established to promote the information flow about environmental impacts on health. The National Association of Physicians for the Environment is a member of that organization.

Upper lip blanching and diplopia associated with local anaesthesia of the inferior alveolar nerve.

A case of transient left lateral rectus nerve palsy, following an inferior alveolar nerve block to enable the surgical removal of a permanent mandibular left third molar tooth, is reported. The anatomy related to this case is considered together with suggestions for management of such patients.