A high throughput substrate binding assay reveals hexachlorophene as an inhibitor of the ER-resident HSP70 chaperone GRP78

Glucose-regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.     

Gradual Phenotypic Conversion Associated with Immortalization of Cultured Human Mammary Epithelial Cells

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20–30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-β (TGFβ); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFβ. A strong correlation between capacity to maintain growth in the presence of TGFβ and expression of telomerase activity was observed. We have used the term “conversion” to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFβ and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.     

Okadaic acid induces the release of Ca from intracellular stores in ECV304 endothelial cells

The effects of serine/threonine phosphatase inhibition on endothelial cell cytosolic free Ca²⁺ ([Ca²⁺]_c) were investigated using okadaic acid and Fura-2-loaded ECV304 endothelial cells. When added to confluent adherent cells, 500 nM okadaic acid induced a transient and oscillatory elevation of [Ca²⁺]_c both in the presence and absence of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, depletion of the intracellular Ca²⁺ stores with either ATP (1 μM) or thapsigargin (100 nM) prevented any further release of Ca²⁺ on the subsequent addition of okadaic acid. Likewise (in the absence of extracellular Ca²⁺), a prior release of Ca²⁺ induced by okadaic acid reduced the magnitude of the response to ATP (1 μM). Taken together these observations indicate that okadaic acid induces Ca²⁺ release from the agonist-sensitive pool. The okadaic acid-induced Ca²⁺ release was mimicked by another potent phosphatase inhibitor, calyculin A (10 nM), and also the less potent analogue of okadaic acid, 1-nor-okadone (500 nM). The response to okadaic acid was characterised by a series of asynchronous [Ca²⁺]_c oscillations, which at their peak resulted in 40–100% cells, at any one time, having an elevated [Ca²⁺]_c. The response appeared to propagate between adjacent cells and the elevation of [Ca²⁺]_c. appeared initially in the cell periphery. In adherent cells, the release of Ca²⁺ induced by okadaic acid was found to be dependent upon cell density, as the proportion of cells responding to okadaic acid increased as the cell density increased. The response to okadaic acid was not observed in ECV304 cell suspensions. The data suggest that a kinase activity stimulated either directly or indirectly by cell-cell interactions can lead to the release of Ca²⁺ from the agonist- and thapsigargin-sensitive intracellular stores.     

A comparison of the electromechanical properties of structurally diverse proteins by molecular dynamics simulation

Proteins are subjected to electric fields both within the cell and during routine biochemical analysis. We have used atomistic molecular dynamics simulations to study conformational changes within three structurally diverse proteins subjected to high electric fields. At electric fields in excess of .5?V/nm, major structural changes were observed in all three proteins due to charge redistribution within the biomolecule. However, the electromechanical resilience was found to be highly dependent on the protein secondary structure, with α-helices showing a particularly high susceptibility to deformation by the applied electric field.     

Cell Optical Density and Molecular Composition Revealed by Simultaneous Multimodal Label-Free Imaging

We show how Raman imaging can be combined with independent but simultaneous phase measurements of unlabeled cells, with the resulting data providing information on how the light is retarded and/or scattered by molecules in the cell. We then show, for the first time to our knowledge, how the chemistry of the cell highlighted in the Raman information is related to the cell quantitative phase information revealed in digital holographic microscopy by quantifying how the two sets of spatial information are correlated. The results show that such a multimodal implementation is highly useful for the convenience of having video rate imaging of the cell during the entire Raman measurement time, allowing us to observe how the cell changes during Raman acquisition. More importantly, it also shows that the two sets of label-free data, which result from different scattering mechanisms, are complementary and can be used to interpret the composition and dynamics of the cell, where each mode supplies label-free information not available from the other mode.     

Phenotypes and environment predict seedling survival for seven co‐occurring Great Basin plant taxa growing with invasive grass

Trait–environment correlations can arise from local adaptation and can identify genetically and environmentally appropriate seeds for restoration projects. However, anthropogenic changes can disrupt the relationships between traits and fitness. Finding the best seed sources for restoration may rely on describing plant traits adaptive in disturbed and invaded environments, recognizing that while traits may differ among species and functional groups, there may be similarities in the strategies that increase seedling establishment. Focusing on three grass genera, two shrub species, and two forb genera, we collected seeds of all taxa from 16 common sites in the sagebrush steppe of the western United States. We measured seed and seedling characteristics, including seed size, emergence timing, and root and shoot traits, and compiled a suite of environmental variables for each collection site. We described trait–environment associations and asked how traits or environment of origin were associated with seedling survival in invaded gardens. Sampling seven taxa from the same sites allowed us to ask how trait–environment–performance associations differ among taxa and whether natural selection favors similar traits across multiple taxa and functional groups. All taxa showed trait–environment associations consistent with local adaptation, and both environment of origin and phenotypes predicted survival in competitive restoration settings, with some commonalities among taxa. Notably, rapid emergence and larger seeds increased survival for multiple taxa. Environmental factors at collection sites, including lower slopes (especially for grasses), greater mean annual temperatures (especially for shrubs and forbs), and greater precipitation seasonality were frequently associated with increased survival. We noted one collection site with high seedling survival across all seven taxa, suggesting that conditions within some sites may result in selection for traits that increase establishment for multiple species. Thus, choosing native plant sources with the most adaptive traits, along with matching climates, will likely improve the restoration of invaded communities.     

A fast and accessible methodology for micro-patterning cells on standard culture substrates using Parafilm™ inserts

Micropatterning techniques provide direct control over the spatial organization of cells at the sub-mm scale. Regulation of these spatial parameters is important for controlling cell fate and cell function. While micropatterning has proved a powerful technique for understanding the impact of cell organization on cell behaviour, current methods for micropatterning cells require complex, specialized equipment that is not readily accessible in most biological and bioengineering laboratories. In addition, currently available methods require significant protocol optimization to ensure reliable and reproducible patterning. The inaccessibility of current methods has severely limited the widespread use of micropatterning as a tool in both biology and tissue engineering laboratories. Here we present a simple, cheap, and fast method to micropattern mammalian cells into stripes and circular patterns using Parafilm?, a common material found in most biology and bioengineering laboratories. Our method does not require any specialized equipment and does not require significant method optimization to ensure reproducible patterning. Although our method is limited to simple patterns, these geometries are sufficient for addressing a wide range of biological problems. Specifically, we demonstrate i) that using our Parafilm? insert method we can pattern and co-pattern ARPE-19 and MDCK epithelial cells into circular and stripe micropatterns in tissue culture polystyrene (TCPS) wells and on glass slides, ii) that we can contain cells in the desired patterns for more than one month and iii) that upon removal of the Parafilm? insert we can release the cells from the containment pattern and allow cell migration outward from the original pattern. We also demonstrate that we can exploit this confinement release feature to conduct an epithelial cell wound healing assay. This novel micropatterning method provides a reliable and accessible tool with the flexibility to address a wide range of biological and engineering problems that require control over the spatial and temporal organization of cells.     

Vagility and the Monsoon Rain Forest Archipelago of Northern Australia: Patterns of Genetic Diversity in Syzygium nervosum (Myrtaceae)1

Syzygium nervosum is a common monsoon rain forest tree. Its habitat in Australia consists of small rain forest patches that are scattered through a savanna matrix. It is a mast flowering canopy species that produces large quantities of fruits fed on by mobile frugivores such as birds and fruit bats. The genetic diversity of this species was investigated, especially in relation to rain forest patch size, geographic isolation, and geographic distribution. Syzygium nervosum was found to have high levels of genetic diversity within populations (H_e= 0.307). Diversity among populations, however, was relatively low (Fsr = 0.118), and was not spatially structured across its geographic range in Australia. This is thought to have been caused by relatively frequent gene flow among populations (N_m= 1.67), mediated primarily by mobile frugivores. Genetic diversity was not correlated with patch size or isolation. It is thought that seed dispersal by frugivores has acted to expand the effective population size of this species beyond the individual rain forest patch, and thus has prevented the substantial loss of genetic diversity that otherwise would have been observed. Thus this species is dependent upon these frugivores for the maintenance of its genetic diversity and hence its long-term viability. These results lend support to theories of post-Holocene expansion of rain forest by vagile species in northern Australia.     

A biphasic model for micro-indentation of a hydrogel-based contact lens

The stiffness and hydraulic permeability of soft contact lenses may influence its clinical performance, e.g., on-eye movement, fitting, and wettability, and may be related to the occurrence of complications; e.g., lesions. It is therefore important to determine these properties in the design of comfortable contact lenses. Micro-indentation provides a nondestructive means of measuring mechanical properties of soft, hydrated contact lenses. However, certain geometrical and material considerations must be taken into account when analyzing output force-displacement (F-D) data. Rather than solely having a solid response, mechanical behavior of hydrogel contact lenses can be described as the coupled interaction between fluid transport through pores and solid matrix deformation. In addition, indentation of thin membranes ( approximately 100 microm) requires special consideration of boundary conditions at lens surfaces and at the indenter contact region. In this study, a biphasic finite element model was developed to simulate the micro-indentation of a hydrogel contact lens. The model accounts for a curved, thin hydrogel membrane supported on an impermeable mold. A time-varying boundary condition was implemented to model the contact interface between the impermeable spherical indenter and the lens. Parametric studies varying the indentation velocities and hydraulic permeability show F-D curves have a sensitive region outside of which the force response reaches asymptotic limits governed by either the solid matrix (slow indentation velocity, large permeability) or the fluid transport (high indentation velocity, low permeability). Using these results, biphasic properties (Young's modulus and hydraulic permeability) were estimated by fitting model results to F-D curves obtained at multiple indentation velocities (1.2 and 20 microm/s). Fitting to micro-indentation tests of Etafilcon A resulted in an estimated permeability range of 1.0 x 10(-15) to 5.0 x 10(-15) m(4)N s and Young's modulus range of 130 to 170 kPa.