CRTC2 and Nedd4 ligase involvement in FSH and TGFß1 upregulation of connexin43 gap junction.

The major mission of the ovarian follicle is the timely production of the mature fertilizable oocyte, and this is achieved by gonadotropin-regulated, gap junction-mediated cell-cell communication between the oocyte and surrounding nurturing granulosa cells. We have demonstrated that FSH and transforming growth factor beta 1 (TGFß1) stimulate Gja1 gene-encoded connexin43 (Cx43) gap junction formation/function in rat ovarian granulosa cells is important for their induction of steroidogenesis; additionally, cAMP-protein kinase A (PKA)- and calcium-calcineurin-sensitive cAMP response element-binding (CREB) coactivator CRTC2 plays a crucial role during steroidogenesis. This study was to explore the potential molecular mechanism whereby FSH and TGFß1 regulate Cx43 synthesis and degradation, particularly the involvement of CRTC2 and ubiquitin ligase Nedd4. Primary culture of granulosa cells from ovarian antral follicles of gonadotropin-primed immature rats was used. At 48 h post-treatment, FSH plus TGFß1 increased Cx43 level and gap junction function in a PKA- and calcineurin-dependent manner, and TGFß1 acting through its type I receptor modulated FSH action. Chromatin-immunoprecipitation analysis reveals FSH induced an early-phase (45 min) and FSH+TGFß1 further elicited a late-phase (24 h) increase in CRTC2, CREB and CBP binding to the Gja1 promoter. Additionally, FSH+TGFß1 increased the half-life of hyper-phosphorylated Cx43 (Cx43-P2). Also, the proteasome inhibitor MG132 prevented the brefeldin A (blocker of protein transport through Golgi)-reduced Cx43-P2 level and membrane Cx43 gap junction plaque. This is associated with FSH+TGFß1-attenuated Cx43 interaction with Nedd4 and Cx43 ubiquitination. In all, this study uncovers that FSH and TGFß1 upregulation of Cx43 gap junctions in ovarian granulosa cells critically involves enhancing CRTC2/CREB/CBP-mediated Cx43 expression and attenuating ubiquitin ligase Nedd4-mediated proteosomal degradation of Cx43 protein.

Detection of organophosphate pesticides using a prototype liquid crystal monitor.

The precision and accuracy of a prototype wearable liquid crystal monitor (LCM) for the measurement of airborne organophosphate pesticide concentrations was explored in a series of laboratory experiments. LCM response to vapor-phase and aerosol diazinon was compared to concentrations obtained using a standard reference method (NIOSH 5600) at concentrations ranging from approximately 8 to 108 ppb (parts per billion) over durations of 2 to 80 hours. Temperature ( approximately 25, 30, and 35 degrees C) and relative humidity (15, 50, and 85%) were varied to estimate the effect of these factors on LCM performance. The LCM response to vapor phase pesticide exposure was linear for concentrations in the range of 8-20 ppb. At exposure concentrations above approximately 20 ppb, however, there was a decline in monitor response and measurement precision. Elevated temperatures improved diazinon vapor-only measurement precision, while increased relative humidity reduced LCM response at the extremes of tested temperatures. Compared to vapor-only exposures, the LCM was less sensitive to diazinon aerosol concentrations, but displayed reasonable precision over a relatively large range of exposures (29 to 1190 ppb-hr). Further efforts to characterize temperature and humidity effects and improve low-end sensitivity would likely provide a portable personal exposure monitor or environmental sensor for this widely used class of pesticides.

Microfluidic enzyme-linked immunosorbent assay technology.

In this chapter, we have presented an overview of microfluidic enzyme-linked immunosorbent assay (ELISA) by first introducing the principle of immunoassay, ELISA, and microfabricated devices, followed by a discussion of microfabrication technology and the characterization of microfluidic components. Significant advances in laboratory technology are contributing to the further understanding of microfluidic function, surface modification and immobilization, which lead to the development of improved biomolecule detection methods and prospective applications. For the future, the exploitation of more robust-manufacturing processes and integrated assay systems in an automatic fashion with much reduced assay time and reagent consumption will allow for the effective detection and quantification of biological agents that are of interest in medical diagnostics, food safety surveillance, and environmental monitoring.

Lindane, a gap junction blocker, suppresses FSH and transforming growth factor beta1-induced connexin43 gap junction formation and steroidogenesis in rat granulosa cells.

The present study was designed to explore the role of gap junctions in follicle-stimulating hormone (FSH) and transforming growth factor beta1 (TGF beta1)-stimulated steroidogenesis in ovarian granulosa cells of gonadotropin-primed immature rats. There were three specific aims. First, we investigated the effect of FSH and TGF beta1 as well as lindane (a general gap junction blocker) on the level of connexin43 (Cx43), the major gap junction constituent in granulosa cells, and on gap junction function. The second aim was to determine the effect of lindane on FSH and TGF beta1-stimulated progesterone production and the levels of two critical players, cytochrome P450 side-chain cleavage (P450scc) enzyme and steroidogenic acute regulatory (StAR) protein. The third aim was to further investigate the specific involvement of Cx43 gap junctions in FSH and TGF beta1-stimulated steroidogenesis using a Cx43 mimetic peptide blocker. Immunoblotting analysis showed that FSH plus TGF beta1 dramatically increased the levels of phosphorylated Cx43 without significantly influencing the level of nonphosphorylated Cx43, and this stimulatory effect was completely suppressed by lindane. Also, immunofluorescence analysis showed that Cx43 immuno-reactivity increased in the FSH plus TGF beta1-treated group and predominantly appeared in a punctate pattern at cell-cell contact sites, and lindane reduced such cell periphery immunostaining. Furthermore, TGF beta1 enhanced the FSH-induced gap junction intercellular communication and lindane completely suppressed this effect. In addition, lindane suppressed the FSH and TGF beta1-stimulated increases in progesterone production and the levels of P450scc enzyme and StAR protein. This study demonstrates a clear temporal association between the Cx43 protein level/gap junction communication and progesterone production in rat ovarian granulosa cells in response to FSH and TGF beta1 as well as lindane. Furthermore, a specific Cx43 gap junction blocker suppressed FSH plus TGF beta1-stimulated progesterone production. In conclusion, this study suggests that Cx43 gap junctions may play a critical role in FSH plus TGF beta1-stimulated progesterone production in rat ovarian granulosa cells.

Investigation into the applicability of the centrifugal microfluidics platform for the development of protein-ligand binding assays incorporating enhanced green fluorescent protein as a fluorescent reporter.

The incorporation of a protein-ligand binding assay into a centrifugal microfluidics platform is described. The platform itself is a disc-shaped polymer substrate, upon which a series of microfluidic channels and reservoirs have been machined. Centrifugal microfluidics platforms require no internal moving parts, and fluid propulsion is achieved solely through rotation of the disc. Fluid flow is controlled by passive valves, the opening of which is dependent on the angular frequency of the rotating platform, the channel dimensions, and the physical properties of the fluid. To evaluate the effectiveness of incorporating a protein-based assay onto the centrifugal microfluidics analytical platform, a class-selective, homogeneous assay for the detection of phenothiazine antidepressants was employed. This class of drugs is known to bind to calmodulin, a calcium binding protein. Specifically, a fusion protein between calmodulin and enhanced green fluorescent protein was utilized. Calmodulin undergoes a conformational change upon binding to phenothiazines that alters the fluorescence properties of the attached fluorescent protein, which can be correlated to the concentration of the drug present. Another important aspect of this work was to study the efficacy of the platform to perform reconstitution assays. To do this, the biological reagent was dried on the platform and rehydrated to carry out the assay. The ability to prealiquot reagents on the platform should enhance its versatility and portability. The integration of protein-based assays in this platform should be useful in the design of analytical systems for high-throughput screening of pharmaceuticals and clinical diagnostics.

Design of a compact disk-like microfluidic platform for enzyme-linked immunosorbent assay.

This paper presents an integrated microfluidic device on a compact disk (CD) that performs an enzyme-linked immunosorbent assay (ELISA) for rat IgG from a hybridoma cell culture. Centrifugal and capillary forces were used to control the flow sequence of different solutions involved in the ELISA process. The microfluidic device was fabricated on a plastic CD. Each step of the ELISA process was carried out automatically by controlling the rotation speed of the CD. The work on analysis of rat IgG from hybridoma culture showed that the microchip-based ELISA has the same detection range as the conventional method on the 96-well microtiter plate but has advantages such as less reagent consumption and shorter assay time over the conventional method.

A packaging technique for polymer microfluidic platforms.

A new technique, resin-gas injection, has been developed for bonding and surface modification of polymer microfluidic devices. This method can easily bond biochips with complex flow patterns. A cascade micromixer and a multichannel DNA sequencing chip were demonstrated experimentally. By adding surface modification agents, the interfacial free energy of the substrate with water can be controlled. Local modification of the channel surface can also be achieved through sequential resin-gas injection in conjunction with a masking technique. For application, this technique is used to form a layer of dry monolithic stationary hydrogel on the walls of a microchannel, serving as a sieving material for electrophoresis separation of DNA fragments. The reagent loading and the electrophoresis separation efficiency of this technique were compared experimentally with the conventional linear polymer solution method used in the microchannel-based DNA sequencing process. It is found that our method has the advantages of more user-friendly operation, easier and faster sample loading, but slightly less separation efficiency.