Gene expression changes associated with cytotoxicity identified using cDNA arrays

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes. Electronic Publication     

Advances in Tools to Determine the Glycan-Binding Specificities of Lectins and Antibodies

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Highlights

• •Lectins and glycan-binding antibodies are valuable as probe of glycans.
• •Advanced bioinformatics tools enable the mining of glycan-array data.
• •New insights into protein-glycan interactions have value in biological research.

     

Models for contact-mediated pattern formation: cells that form parallel arrays

Kinetic continuum models are derived for cells that crawl over a 2D substrate, undergo random reorientation, and turn in response to contact with a neighbor. The integro-partial differential equations account for changes in the distribution of orientations in the population. It is found that behavior depends on parameters such as total mass, random motility, adherence, and sloughing rates, as well as on broad aspects of the contact response. Linear stability analysis, and numerical, and cellular automata simulations reveal that as parameters are varied, a bifurcation leads to loss of stability of a uniform (isotropic) steady state, in favor of an (anisotropic) patterned state in which cells are aligned in parallel arrays.     

A novel tubular array associated with protein bodies in the rough endoplasmic reticulum ofopaque-2 maize

Summary The seed storage proteins of maize (Zea mays L.) are synthesized during endosperm development on membrane-bound polyribosomes. Protein body formation in normal genotypes occurs via a sequential deposition of the various types of zeins, and leads to the formation of spherical structures with a diameter of about l m. In the endosperm mutantopaque-2 the level of one zein class is reduced; these kernels exhibit an opaque phenotype instead of the vitreous phenotype displayed in normal genotypes, presumably due to the decrease in total zein protein at the time of desiccation. Previous microscopic examination ofopaque-2 protein bodies at 22 DAP (days after pollination) showed that the protein bodies were morphologically similar to those of normal genotypes. However, the endosperm ofopaque-2 maize at 14 DAP contains tubular arrays within the rough endoplasmic reticulum. These tubular arrays are tightly associated with the developing protein bodies. Long strands of tubules, sometimes 10 m in length, are observed in the endosperm, and partially formed protein bodies often seem to be forming directly from these tubular arrays. No immunostaining is associated with this tubular material when any of the anti-zein antibodies are used.Abbreviations BSA bovine serum albumin - DAP days after pollination - IgG immunoglobulin G Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Astrocytes alter their polarity in organotypic slice cultures of rat visual cortex

The ultrastructure of astrocytes in an organotypic slice culture of the rat visual cortex was investigated using ultrathin sections and freeze-fracture replicas. After a culture period of 9–15 days, a glial scaffold formed that separated the bulk of the slice neuropil from the medium and the underlying plasma clot. However, the glial cells and processes did not build a dense barrier but allowed the outgrowth of neurites. A basal lamina covering the medium-oriented surface of the astrocytes was not found. In freeze-fracture replicas, orthogonal arrays of particles (OAP) were characteristic components of astrocytic membranes. The OAP density in membranes bordering the medium was 35±13 OAP/m², corresponding to 2.5% of this membrane area; the OAP density in membranes within the slice neuropil was 22±12 OAP/², corresponding to 1.4% of this membrane area. Although the difference was significant, it was greatly reduced when comparing OAP densities in endfoot and non-endfoot membranes in vivo. Another mode of polarity was recognized in astrocytes of the organotypic slice culture. In membranes of astrocytes bordering upon the medium, the density of non-OAP intramembranous particles (IMP) was clearly higher (1130±136 IMP/ m²) than in membranes of astrocytes in the center of the slice (700±172 IMP/m²). This pronounced IMP-related polarity was observed neither in vivo nor in cultured astrocytes. The present study suggests, together with data from the literature, that the distribution of astrocytic OAP across the cell surface is influenced by the existence of a basal lamina and neuronal activity, and that astrocytes possess a more remarkable plasticity of membrane structure than previously suspected.     

Self-assembly and two-dimensional patterning of cell arrays by electrophoretic deposition.

Using Saccharomyces cerevisiae as a demonstration system, we present a method to form two-dimensional, patternable cellular arrays. The method does not require surface chemical templating of the substratum to produce arrays or patterns. By virtue of their colloidal characteristics, S. cerevisiae cells may be induced to form dense, quasi-ordered two-dimensional clusters adjacent to an electrode surface by electrophoretic deposition (EPD). Using ac EPD, dense two-dimensional cell clusters may be formed in minutes from extremely dilute cell suspensions. The arrays may be induced to form geometric patterns by focusing the electric field during deposition. These monolayer arrays are reversible, dissipating by diffusion on removal of the electric field, and are not in adhesive contact with the electrode surface. Brief application of a modest dc current density adheres the arrays tightly to the surface.     

Competitive strategies between Anastrepha fraterculus and Anastrepha obliqua (Diptera: Tephritidae) regulating the use of host fruits

Interspecific competition between individuals of different species can result in reductions in their fecundity, growth or survival, reflecting differential exploitation of resources that become intensified due to spatial co-occurrence, ecological similarity and increased population densities. As two species cannot occupy the same niche, coexistence is only possible if the available resources are used in non-overlapping manners such as niche partitioning or the use of refuges. Among agricultural insect pests, such as fruit flies of the family Tephritidae, competitive interactions can result in competitive displacement, host changes, or the expansion or restriction of the numbers of hosts utilized that can have negative consequences for human agricultural activities. We evaluated the competitive interactions between two fruit fly species of the genus Anastrepha, Anastrepha obliqua (Macquart, 1835) and Anastrepha fraterculus (Wiedmann, 1830), on their respective preferred hosts (mangoes and guava). Experiments of larval competition and competition for ovipositioning sites by adult females were performed to compare the parameters of larval development time, numbers of pupae and emerged adults and numbers of ovipositions in the presence or absence of interspecific competition. We observed that the interactions between those species were asymmetrical and hierarchical, and our results suggest a competitive displacement of A. fraterculus by A. obliqua when those two species are present on the same fruit, whether mangoes or guavas.     

Soft tissue structures resisting anterior instability in a computational glenohumeral joint model

The glenohumeral joint is the most dislocated joint in the body due to the lack of bony constraints and the dependence on soft tissue for stability. The roles that various structures provide to joint function are important for understanding injury treatment and orthopaedic device design purposes. The goal of this study was to develop a computational model of the glenohumeral joint whereby joint behaviour was dictated by articular contact, ligamentous constraints, muscle loading and external perturbations. The bone structure of the computational model consisted of assembled computer tomographic images of the scapula, humerus and clavicle. The soft tissue elements were composed of forces and tension-only springs that represented muscles and ligaments. Validation of this model was achieved by comparing computational predictions to the results of a cadaveric experiment in which the relative contribution of muscles and ligaments to anterior joint stability was examined. The computational model predicted an anterior subluxation force that was similar to the cadaveric experimental results in humeral external rotation. The individual structure results showed the subscapularis to be critical to stabilisation in both neutral and external rotations, the biceps stabilised the joint in neutral but not in external rotation, and the inferior glenohumeral ligament resisted anterior displacement only in external rotation. The model's predictions were similar to the conclusions of the cadaveric experiment and the literature. Knowledge gained from this type of model could assist in further understanding the contribution of soft tissue stabilisers to joint function, pre-operative planning or the design of orthopaedic implants.     

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.