Activator Protein-1 and Epidermal Growth Factor Receptor Interplay : In Vivo & In Vitro Studies

Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.

Alamethicin and synthetic peptide fragments as uncouplers of mitochondrial oxidative phosphorylation. Effect of chain length and change

Alamethicin, its derivatives and some synthetic fragments have been shown to be uncouplers of oxidative phosphorylation in rat liver mitochondria. A minimum peptide chain length of 13 residues is necessary for this activity. Peptide esters are more efficient uncouplers than the corresponding peptide acids. Esterification of the Glu(18) γ-COOH group in alamethicin does not diminish uncoupling activity. The structural requirements for uncoupling activity parallel those determined for ionophoretic action in small, unilamellar liposomes. Aib, α-aminoisobutyric acid; Z, benzyloxycarbonyl; OMe, methyl ester; OBz, benzyl ester; Ac, acetyl; CTC, chlortetracycline.

Detection and differentiation of bovine herpesvirus 1 and 5 using a multiplex real-time polymerase chain reaction.

A multiplex real-time PCR was developed for the detection and differentiation of two closely related bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5). The multiplex real-time PCR combines a duplex real-time PCR that targets the DNA polymerase gene of BoHV-1 and BoHV-5 and a real-time PCR targeting mitochondrial DNA, as a house-keeping gene, described previously by Cawthraw et al. (2009). The assay correctly identified 22 BoHV-1 and six BoHV-5 isolates from the Biosecurity Sciences Laboratory virus collection. BoHV-1 and BoHV-5 were also correctly identified when incorporated in spiked semen and brain tissue samples. The detection limits of the duplex assay were 10 copies of BoHV-1 and 45 copies of BoHV-5. The multiplex real-time PCR had reaction efficiencies of 1.04 for BoHV-1 and 1.08 for BoHV-5. Standard curves relating Ct value to template copy number had correlation coefficients of 0.989 for BoHV-1 and 0.978 for BoHV-5. The assay specificity was demonstrated by testing bacterial and viral DNA from pathogens commonly isolated from bovine respiratory and reproductive tracts. The validated multiplex real-time PCR was used to detect and differentiate BoHV-1 and BoHV-5 in bovine clinical samples with known histories.

Preferred conformations and flexibility of aminoacyl side chain of penicillins

Possible conformations of penicillin G; d and l isomers of ampicillin; α-amino-α-methyl-benzyl penicillins and 3- pyridyl methyl penicillin have been studied by an energy minimization procedure using empirical potential functions. The preferred conformations of these antibiotics have been correlated with their biological activity. The conformational requirement of the antibiotic to be active against Gram-positive and Gram-negative (β-lactamase-negative) bacterial strains seems to be the same. The reduced activity of penicillin G against Gram-negative bacteria has been attributed to its lower ability to permeate the outer membrane. The flexibility of the sidechains of these antibiotics is also shown to be important for the desired biological activity.

A Huge Effect of Weak dc Electrical Fields on Grain Growth in Zirconia

We show that the application of a modest dc electrical field, about 4 V/cm, can significantly reduce grain growth in yttria-stabilized polycrystalline zirconia. These measurements were made by annealing samples, for 10 h at 1300°C, with and without an electrical field. The finding adds a new dimension to the role of applied electrical fields in sintering and superplasticity, phenomena that are critical to the net-shape processing of ceramics. Grain-growth retardation will considerably enhance the rates of sintering and superplasticity, leading to significant energy efficiencies in the processing of ceramics.

Self-assembly of three new coordination complexes: Formation of 2-D square grid, 1-D chain and tape structures

Three distinct coordination complexes, viz., [Co(imi)(2)(tmb)(2)] (1) [where imi = imidazole], {[Ni(tmb)(2)(H2O)(3)]center dot 2H(2)O}(n) (2) and [Cu-2(mu-tmb)(4)(CH3OH)(2)] (3), have been synthesized hydrothermally by the reactions of metal acetates,2,4,6-trimethylbenzoic acid (Htmb) and with or without appropriate amine. The Ni analogue of 1 and the Co analogue of 2 have also been synthesized. X-ray single-crystal diffraction suggests that complex 1 represents discrete mononuclear species and complex 2 represents a 1D chain coordination polymer in which the Ni(H) ions are connected by the bridging water molecules. Complex 3 represents a neutral dinuclear complex. In 1, the central metal ions are associated by the carboxylate moiety and imidazole ligands, whereas the central metal atom is coordinated to the carboxylate moiety and the respective solvent molecules in 2 and 3. In 3, the four 2,4,6-trimethylbenzoate moieties act as a bridge connecting two copper (11) ions and the 0 atoms of methanol coord geometry, with the methanol molecule at the apical position. In all the three structures the central metal atom sits on a crystallographic inversion centre. In all the cases, the coordination entities are further organized via hydrogen bonding interactions to generate multifarious supramolecular networks. Complexes 1, 2 and 3 have also been characterized by spectroscopic (UV/Vis and IR) and thermal analysis (TGA). In addition, the complexes were found to exhibit antimicrobial activity. The magnetic susceptibility measurements, measured from 8 to 300 K, revealed antiferromagnetic interactions between the Co(II) ions in compound 1 and the Ni(II) ions in la, respectively.

Hierarchical Bayesian modelling of plant pest invasions with human-mediated dispersal

Hierarchical Bayesian models can assimilate surveillance and ecological information to estimate both invasion extent and model parameters for invading plant pests spread by people. A reliability analysis framework that can accommodate multiple dispersal modes is developed to estimate human-mediated dispersal parameters for an invasive species. Uncertainty in the observation process is modelled by accounting for local natural spread and population growth within spatial units. Broad scale incursion dynamics are based on a mechanistic gravity model with a Weibull distribution modification to incorporate a local pest build-up phase. The model uses Markov chain Monte Carlo simulations to infer the probability of colonisation times for discrete spatial units and to estimate connectivity parameters between these units. The hierarchical Bayesian model with observational and ecological components is applied to a surveillance dataset for a spiralling whitefly (Aleurodicus dispersus) invasion in Queensland, Australia. The model structure provides a useful application that draws on surveillance data and ecological knowledge that can be used to manage the risk of pest movement.

Influence of soil organic amendments on suppression of the burrowing nematode, Radopholus similis, on the growth of bananas.

Radopholus similis is a major constraint to banana production in Australia and growers have relied on nematicides to manage production losses. The use of organic amendments is one method that may reduce the need for nematicides, but there is limited knowledge of the influence of organic amendments on endo-migratory nematodes, such as R. similis. Nine different amendments, namely, mill mud, mill ash, biosolids, municipal waste compost, banana residue, grass hay, legume hay, molasses and calcium silicate were applied to the three major soil types of the wet tropics region used for banana production. The nutrient content of the amendments was also determined. Banana plants were inoculated with R. similis and grown in the soil-amendment mix for 12-weeks in a glasshouse experiment. Assessments of plant growth, plant-parasitic nematodes and soil nematode community characteristics were made at the termination of the experiment. Significant suppression of plant-parasitic nematodes occurred in soils amended with legume hay, grass hay, banana residue and mill mud relative to untreated soil. These amendments were found to have the highest N and C content. The application of banana residue and mill mud significantly increased shoot dry weight at the termination of the experiment relative to untreated soil. Furthermore, the applications of banana residue, grass hay, mill mud and municipal waste compost increased the potential for suppression of plant-parasitic nematodes through antagonistic activity. The application of amendments that are high in C and N appeared to be able to induce suppression of plant-parasitic nematodes in bananas, by developing a more favourable environment for antagonistic organisms.

A novel breeding programme for improved growth in barramundi Lates calcarifer (Bloch) using foundation stock from progeny-tested parents

Rapid genetic gains for growth in barramundi ( Lates calcarifer) appear achievable by starting a breeding programme using foundation stock from progeny tested broodstock. The potential gains of this novel breeding design were investigated using biologically feasible scenarios tested with computer simulation models. The design involves the production of a large number of full-sib families using artificial mating which are compared in common growout conditions. The estimated breeding values of their paternal parents are calculated using a binomial probit analysis to assess their suitability as foundation broodstock. The programme can theoretically yield faster rates of genetic gain compared to other breeding programmes for aquaculture species. Assuming a heritability of 0.25 for growth, foundation broodstock evaluated in two years had breeding values for faster growth ranging from 21% to 51% depending on the genetic diversity of stock under evaluation. As a comparison it will take between nine and twenty-two years to identify broodstock with similar breeding values in a contemporary barramundi breeding programme.

Mile-a-minute (Mikania micrantha): its distribution, growth and physical and socio-economic impacts in Papua New Guinea

Mikania micrantha, Kunth. H.B.K (Asteraceae) or mile-a-minute is a weed of Neotropical origin in 17 Pacific Island countries. It is becoming increasingly regarded as an invasive weed in Papua New Guinea and is now the focus of an Australian Government-funded biological control program. As part of the program, growth rates, distribution and physical and socia-economic impacts were studied to obtain baseline data and to assist with the field release of biological control agents. Through public awareness campaigns and dedicated surveys, mikania has been reported in most lowland provinces. It is particularly widespread in East New Britain and West New Britain Province. In field trials, mikania grew more than 1 metre per month in open sunny areas but slightly slower when growing under cocoa. The weed invades a wide range of land types, impacting on plantations and food gardens, smothering pawpaw, young cocoa, banana, taro, young oil palms and ornamental plants. In socia-economic surveys, mikania was found to have severe impacts on crop production and income generated through reduced yields and high weeding costs. These studies suggest that there would be substantial benefits to the community if biological control of mikania is successful.

Genetic alterations in microsatellite-unstable colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in Finland. Of all CRC tumors, 15% display microsatellite-instability (MSI) caused by defective cellular mismatch repair. Cells displaying MSI accumulate a high number of mutations genome-wide, especially in short repeat areas, microsatellites. When targeting genes essential for cell growth or death, MSI can promote tumorigenesis. In non-coding areas, microsatellite mutations are generally considered as passenger events. Since the discovery of MSI and its linkage to cancer, more that 200 genes have been investigated for a role in MSI tumorigenesis. Although various criteria have been suggested for MSI target gene identification, the challenge has been to distinguish driver mutations from passenger mutations. This study aimed to clarify these key issues in the research field of MSI cancer. Prior to this, background mutation rate in MSI cancer has not been studied in a large-scale. We investigated the background mutation rate in MSI CRC by analyzing the spectrum of microsatellite mutations in non-coding areas. First, semenogelin I was studied for a possible role in MSI carcinogenesis. The intronic T9 repeat of semenogelin I was frequently mutated but no evidence for selection during tumorigenesis was obtained. Second, a sequencing approach was utilized to evaluate the general background mutation rate in MSI CRC. Both intronic and intergenic repeats harbored extremely high mutation rates of ≤ 87% and intergenic repeats were more unstable than the intronic repeats. As mutation rates of presumably neutral microsatellites can be high in MSI CRC in the absence of apparent selection pressure, high mutation frequency alone is not sufficient evidence for identification of driver MSI target genes. Next, an unbiased approach was designed to identify the mutatome of MSI CRC. By combining expression array data and a database search we identified novel genes possibly related to MSI CRC carcinogenesis. One of the genes was studied further. In the functional analysis this gene was observed to cause an abnormal cancer-prone cellular phenotype, possibly through altered responses to DNA damage. In our recent study, smooth muscle myosin heavy chain 11 (MYH11) was identified as a novel MSI CRC gene. Additionally, MYH11 has a well established role in acute myeloid leukemia (AML) through an oncogenic fusion protein CBFB-MYH11. We investigated further the role of MYH11 in AML by sequencing. Three novel missense variants of MYH11 were identified. None of the variants were present in the population-based control material. One of the identified variants, V71A, lies in the N-terminal SH3-like domain of MYH11 of unknown function. The other two variants, K1059E and R1792Q are located in the coil-coiled myosin rod essential for the regulation and filament formation of MYH11. The variant K1059E lies in the close proximity of the K1044N that has been functionally assessed in our earlier work of CRC and has been reported to cause total loss of MYH11 protein regulation. As the functional significance of the three novel variants examined in this work remains unknown, future studies should clarify the further role of MYH11 in AML leukaemogenesis and in other malignancies.