Ephrin-A5 induces rounding, blebbing and deadhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling

Eph receptor tyrosine kinases and ephrins regulate morphogenesis in the developing embryo where they effect adhesion and motility of interacting cells. Although scarcely expressed in adult tissues, Eph receptors and ephrins are overexpressed in a range of tumours. In malignant melanoma, increased Eph and ephrin expression levels correlate with metastatic progression. We have examined cellular and biochemical responses of EphA3-expressing melanoma cell lines and human epithelial kidney 293T cells to stimulation with polymeric ephrin-A5 in solution and with surfaces of defined ephrin-A5 densities. Within minutes, rapid reorganisation of the actin and myosin cytoskeleton occurs through activation of RhoA, leading to the retraction of cellular protrusions, membrane blebbing and detachment, but not apoptosis. These responses are inhibited by monomeric ephrin-A5, showing that receptor clustering is required for this EphA3 response. Furthermore, the adapter CrkII, which associates with tyrosine-phosphorylated EphA3 in vitro, is recruited in vivo to ephrin-A5-stimulated EphA3. Expression of an SH3-domain mutated CrkII ablates cell rounding, blebbing and detachment. Our results suggest that recruitment of CrkII and activation of Rho signalling are responsible for EphA3-mediated cell rounding, blebbing and de-adhesion, and that ephrin-A5-mediated receptor clustering and EphA3 tyrosine kinase activity are essential for this response.

Movement through slits: cellular migration via the Slit family

First isolated in the fly and now characterised in vertebrates, the Slit proteins have emerged as pivotal components controlling the guidance of axonal growth cones and the directional migration of neuronal precursors. As well as extensive expression during development of the central nervous system (CNS), the Slit proteins exhibit a striking array of expression sites in non-neuronal tissues, including the urogenital system, limb primordia and developing eye. Zebrafish Slit has been shown to mediate mesodermal migration during gastrulation, while Drosophila slit guides the migration of mesodermal cells during myogenesis. This suggests that the actions of these secreted molecules are not simply confined to the sphere of CNS development, but rather act in a more general fashion during development and throughout the lifetime of an organism. This review focuses on the non-neuronal activities of Slit proteins, highlighting a common role for the Slit family in cellular migration.

Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cells

Background: Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor Cell proliferation and, following clonal expansion of these cells. promotion of differentiation along the osteogenic lineage. Objectives: We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. Method: The cell populations were assessed for mineralization potential after long-term culture in media containing beta-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogcnic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin. osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. Results: As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP. osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. Conclusion: The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers.

Melanocytic nevi, solar keratoses, and divergent pathways to cutaneous melanoma

Background: Some melanomas form on sun-exposed body sites, whereas others do not. We previously proposed that melanomas at different body sites arise through different pathways that have different associations with melanocytic nevi and solar keratoses. We tested this hypothesis in a case-case comparative study of melanoma patients in Queensland, Australia. Methods: We randomly selected patients from among three prespecified groups reported to the population-based Queensland Cancer Registry: those with superficial spreading or nodular melanomas of the trunk (n = 154, the reference group), those with such melanomas of the head and neck (n = 77, the main comparison group), and those with lentigo maligna melanoma (LMM) (n = 75, the chronic sun-exposed group). Each participant completed a questionnaire, and a research nurse counted melanocytic nevi and solar keratoses. We calculated exposure odds ratios (ORs) and 95% confidence intervals (CIs) to quantify the association between factors of interest and each melanoma group. Results: Patients with head and neck melanomas, compared with patients with melanomas of the trunk, were statistically significantly less likely to have more than 60 nevi (OR = 0.34, 95% CI = 0.15 to 0.79) but were statistically significantly more likely to have more than 20 solar keratoses (OR = 3.61, 95% CI = 1.42 to 9.17) and also tended to have a past history of excised solar skin lesions (OR = 1.87, 95% CI = 0.89 to 3.92). Patients with LMM were also less likely than patients with truncal melanomas to have more than 60 nevi (OR = 0.32, 95% CI = 0.14 to 0.75) and tended toward more solar keratoses (OR = 2.14, 95% CI = 0.88 to 5.16). Conclusions: Prevalences of nevi and solar keratoses differ markedly between patients with head and neck melanomas or LMM and patients with melanomas of the trunk. Cutaneous melanomas may arise through two pathways, one associated with melanocyte proliferation and the other with chronic exposure to sunlight.

Tyrosinase-Cre mice for tissue-specific gene ablation in neural crest and neuroepithelial-derived tissues

This study describes the derivation of two new lines of transgenic mice that express Cre recombinase under the control of tyrosinase transcriptional elements. To determine the suitability of the Tyrosinase-Cre transgene for tissue-specific gene ablation studies, a fate map of Cre expression domains was determined using the Z/AP reporter strain. It was shown that Cre-expressing cells contribute to a wide array of neural crest and neuroepithelial-derived lineages. The melanocytes of the harderian gland and eye choroid, sympathetic cephalic ganglia, leptomeninges of the telencephalon, as well as cranial nerves (V), (VII), and (IX) are derived either fully or partly from Cre-expressing cephalic crest. The cells contributing to the cranial nerves were the first to exhibit Cre expression at E10.5 as they were migrating into the branchial arches. The melanocytes, chromaffin cells of the adrenal medulla, and dorsal root ganglia are derived from trunk neural crest that either express Cre or were derived from Cre-expressing precursors. An array of brain tissue including the basal forebrain, hippocampus, olfactory bulb, and the granule cell layer of the lateral cerebellum, as well as the retinal pigmented epithelium and glia of the optic nerve originate from Cre-expressing neuroepithelial cells. (C) 2003 Wiley-Liss, Inc.

Variation of bulk properties of anaerobic granules with wastewater type

Development of a granular sludge with high strength, high biological activity and a narrow settling distribution is necessary for optimal operation of high-rate upflow anaerobic treatment systems. Several studies have compared granules produced from different wastewaters but these have largely been from laboratory-fed reactors or compared granules from full-scale reactors fed similar wastewater types. Though two authors have commented on the inferiority of granules produced by a protein-based feed, the properties of these granules have not been characterised. In this paper, granules from full-scale reactors treating fruit and vegetable cannery effluent, two brewery effluents and a pig abattoir (slaughterhouse) were compared in terms of basic composition, size distribution, density, settling velocity, shear strength, and EPS content. The results supported previous qualitative observations by other researchers that indicate granule properties depend more on wastewater type rather than reactor design or operating conditions such as pre-acidification level. The cannery-fed granules bad excellent shear strength, settling distribution and density. Granules from the two brewery-fed reactors had statistically the same bulk properties, which were still acceptable for upflow applications. The protein-grown granule had poor strength and settling velocity. (C) 2001 Elsevier Science Ltd. All rights reserved.

Chapter 16: Prophylactic Human Papillomavirus Vaccines

Candidate prophylactic vaccines based on papillomavirus L1 virus-like particles (VLPs) are currently in human clinical trials. The main long-term goal of the vaccine is to reduce the incidence of cervical cancer and its precursors. In animal papillomavirus models, systemic immunization with L1 VLPs can induce high titers of neutralizing antibodies that confer protection against high-dose experimental papillomavirus challenge. In humans, systemic vaccination with L1 VLPs has been well tolerated and induced high serum antibody titers (at least 40 times higher than titers seen following natural infection). A recent proof of principle HPV16 L1 VLP efficacy trial has shown excellent protection against persistent HPV16 infection and associated cytological abnormalities. Large scale efficacy trials of L1 VLPs from HPV16 and 18 (the HPV types found most frequently in cervical cancer), with or without HPV6 and 11 (the HPV types responsible for most genital warts), are planned. If the results of these large trials support the encouraging results of the early trials, they should lead to a commercial prophylactic HPV vaccine. Implementation issues may include how to make the vaccine available in the developing world, where the majority of cervical cancer cases occur, the appropriate age of vaccination, and the role of male vaccination. Because a VLP vaccine is likely to provide type-specific protection, increasing the number of cancer-associated HPV types in the vaccine is a likely approach to broadening the protection to additional types. There will probably also be efforts to develop alternative vaccine formulations better suited to implementation in developing countries as well as attempts to develop vaccines with a therapeutic activity against established HPV infection because a combined prophylactic/therapeutic vaccine may be expected to have an even greater impact than a purely prophylactic vaccine on HPV induced disease.

Hyperinsulinism and overgrowth without obesity

We report a 5 year old girl with postnatal overgrowth (height velocity >97th centile), hyperinsulinaemia, and increased insulin-like growth factor 1 for age, without evidence of bioactive or immunoreactive growth hormone excess or pituitary abnormality. Although her overgrowth may be a result of hyperinsulinism, her serum contains a factor (neither insulin nor IGF-1) which is able to stimulate the proliferation of lymphocyte precursors, and this could also account for the overgrowth. Over the course of two years observation she has developed acanthosis nigricans and diabetes mellitus.

Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3

The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

A comprehensive insight into floc characteristics and their impact on compressibility and settleability of activated sludge

This paper presents a comprehensive study of sludge floc characteristics and their impact on compressibility and settleability of activated sludge in full scale wastewater treatment processes. The sludge flocs were characterised by morphological (floc size distribution, fractal dimension, filament index), physical (flocculating ability, viscosity, hydrophobicity and surface charge) and chemical (polymeric constituents and metal content) parameters. Compressibility and settleability were defined in terms of the sludge volume index (SVI) and zone settling velocity (ZSV). The floc morphological and physical properties had important influence on the sludge compressibility and settleability. Sludges containing large flocs and high quantities of filaments, corresponding to lower values of fractal dimension (D-f), demonstrated poor compressibility and settleability. Sludge flocs with high flocculating ability had lower SVI and higher ZSV, whereas high values of hydrophobicity, negative surface charge and viscosity of the sludge flocs correlated to high SVI and low ZSV. The quantity of the polymeric compounds protein. humic substances and carbohydrate in the sludge and the extracted extracellular polymeric substances (EPS) had significant positive correlations with SVI. The ZSV was quantitatively independent of the polymeric constituents. High concentrations of the extracted EPS were related to poor compressibility and settleability. The cationic ions Ca, Mg, Al and Fe in the sludge improved significantly the sludge compressibility and settleability. (C) 2003 Elsevier Science B.V. All rights reserved.

The influence of key chemical constituents in activated sludge on surface and flocculating properties

This paper examines the influence of the chemical constituents of activated sludge and extracted extracellular polymeric substances (EPS) on the surface properties, hydrophobicity, surface charge (SC) and flocculating ability (FA) of activated sludge floes. Activated sludge samples from 7 different full-scale wastewater treatment plants were examined. Protein and humic substances were found to be the dominant polymeric compounds in the activated sludges and the extracted EPS, and they significantly affected the FA and surface properties, hydrophobicity and SC, of the sludge floes. The polymeric compounds proteins, humic substances and carbohydrates in the sludge floes and the extracted EPS contributed to the negative SC, but correlated negatively to the hydrophobicity of sludge floes. The quantity of protein and carbohydrate within the sludge and the extracted EPS was correlated positively to the FA of the sludge floes, while increased amounts of humic substances resulted in lower FA. In contrast, increased amounts of total extracted EPS had a negative correlation to FA. The results reveal that the quality and quantity of the polymeric compounds within the sludge floes is more informative, with respect to understanding the mechanisms involved in flocculation, than if only the extracted EPS are considered. This is an important finding as it indicates that extracting EPS may be insufficient to characterise the EPS. This is due to the low extraction efficiency and difficulties involved in the separation of EPS from other organic compounds. Correlations were observed between the surface properties and FA of the sludge floes., This confirms that the surface properties of the, sludge flocs play an important role in the bioflocculation process but that also other interactions like polymer entanglement are important. (C) 2002 Elsevier Science Ltd. All rights reserved.

Assessing forest fire as a potential PCDD/F source in Queensland, Australia

Forest fires are suggested as a potential and significant source of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs), even though no studies to date provide sufficient evidence to confirm forest fires as a source of PCDD/Fs. Recent investigations in Gueensland, Australia have identified a widespread contamination of PCDDs (in particular OND) in soils and sediments in the coastal region from an unknown source of PCDD/Fs. Queensland is predominately rural; it has few known anthropogenic sources of PCDD/Fs, whereas forest fires are a frequent occurrence. This study was conducted to assess forest fires as a potential source of the unknown PCDD/F contamination in Queensland. A combustion experiment was designed to assess the overall mass of PCDD/Fs before and after a simulated forest fire. The results from this study did not identify an increase in Sigma-PCDD/Fs or OCDD after the combustion process. However, specific non-2,3,7,8 substituted lower chlorinated PCDD/Fs were elevated after the combustion process, suggesting formation from a precursor. The results from this study indicate that forest fires are unlikely to be the source of the unknown PCDD contamination in Gueensland, rather they are a key mechanism for the redistribution of PCDD/Fs from existing sources and precursors.

Thermal and hydrolytic degradation of electrospun fish gelatin membranes

The thermal and hydrolytic degradation of electrospun gelatin membranes cross-linked with glutaraldehyde in vapor phase has been studied. In vitro degradation of gelatin membranes was evaluated in phosphate buffer saline solution at 37 ºC. After 15 days under these conditions, a weight loss of 68 % was observed, attributed to solvation and depolymerization of the main polymeric chains. Thermal degradation kinetics of the gelatin raw material and as-spun electrospun membranes showed that the electrospinning processing conditions do not influence polymer degradation. However, for cross-linked samples a decrease in the activation energy was observed, associated with the effect of glutaraldehyde cross-linking reaction in the inter- and intra-molecular hydrogen bonds of the protein. It is also shown that the electrospinning process does not affect the formation of the helical structure of gelatin chains.

Molecular dynamics computer simulation of scratch resistance testing of polymers: visualization

Experimental scratch resistance testing provides two numbers: the penetration depth Rp and the healing depth Rh. In molecular dynamics computer simulations, we create a material consisting of N statistical chain segments by polymerization; a reinforcing phase can be included. Then we simulate the movement of an indenter and response of the segments during X time steps. Each segment at each time step has three Cartesian coordinates of position and three of momentum. We describe methods of visualization of results based on a record of 6NX coordinates. We obtain a continuous dependence on time t of positions of each of the segments on the path of the indenter. Scratch resistance at a given location can be connected to spatial structures of individual polymeric chains.

Simulating scratch behavior of polymers with mesoscopic molecular dynamics

Part replacement and repair is needed in structures with moving parts because of scratchability and wear. In spite of some accumulation of experimental evidence, scratch resistance is still not well understood. We have applied molecular dynamics to study scratch resistance of amorphous polymeric materials through computer simulations. As a first approach, a coarse grain model was created for high density polyethylene at the mesoscale. We have also extended the traditional approach and used real units rather than reduced units (to our knowledge, for the first time), which enable an improved quantification of simulation results. The obtained results include analysis of penetration depth, residual depth and recovery percentage related to indenter force and size. Our results show there is a clear effect from these parameters on the tribological properties. We also discuss a "crooked smile" effect on the scratched surface and the reasons for its appearance.