Imaging synaptic inhibition in transgenic mice expressing the chloride indicator, Clomeleon

We describe here a molecular genetic approach for imaging synaptic inhibition. The thy-1 promoter was used to express high levels of Clomeleon, a ratiometric fluorescent indicator for chloride ions, in discrete populations of neurons in the brains of transgenic mice. Clomeleon was functional after chronic expression and provided non-invasive readouts of intracellular chloride concentration ([Cl(-)](i)) in brain slices, allowing us to quantify age-dependent declines in resting [Cl(-)](i) during neuronal development. Activation of hippocampal interneurons caused [Cl(-)](i) to rise transiently in individual postsynaptic pyramidal neurons. [Cl(-)](i) increased in direct proportion to the amount of inhibitory transmission, with peak changes as large as 4 mM. Integrating responses over populations of pyramidal neurons allowed sensitive detection of synaptic inhibition. Thus, Clomeleon imaging permits non-invasive, spatiotemporally resolved recordings of [Cl(-)](i) in a large variety of neurons, opening up new opportunities for imaging synaptic inhibition and other forms of chloride signaling.     

Human body fluid proteome analysis

The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future.     

Interleukin-17 acts independently of TNF-alpha under arthritic conditions

The proinflammatory T cell cytokine IL-17 is a potent inducer of other cytokines such as IL-1 and TNF-alpha. The contribution of TNF in IL-17-induced joint inflammation is unclear. In this work we demonstrate using TNF-alpha-deficient mice that TNF-alpha is required in IL-17-induced joint pathology under naive conditions in vivo. However, overexpression of IL-17 aggravated K/BxN serum transfer arthritis to a similar degree in TNF-alpha-deficient mice and their wild-type counterparts, indicating that the TNF dependency of IL-17-induced pathology is lost under arthritic conditions. Also, during the course of the streptococcal cell wall-induced arthritis model, IL-17 was able to enhance inflammation and cartilage damage in the absence of TNF. Additional blocking of IL-1 during IL-17-enhanced streptococcal cell wall-induced arthritis did not reduce joint pathology in TNF-deficient mice, indicating that IL-1 is not responsible for this loss of TNF dependency. These data provide further understanding of the cytokine interplay during inflammation and demonstrate that, despite a strong TNF dependency under naive conditions, IL-17 acts independently of TNF under arthritic conditions.     

Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket

P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.     

Effects of hydrogen peroxide in a keratinocyte-fibroblast co-culture model of wound healing

Recently, there has been renewed interest in the role of reactive oxygen species (ROS), especially H(2)O(2), in wound healing. We previously showed that H(2)O(2) stimulates healing in a keratinocyte scratch wound model. In this paper, we used a more complex and physiologically relevant model that involves co-culturing primary keratinocytes and fibroblasts. We found that the two main cell types within the skin have different sensitivities to H(2)O(2) and to the widely used "antioxidant"N-acetyl-l-cysteine (NAC). Keratinocytes were very resistant to the toxicity of H(2)O(2) (250 and 500 μM) or NAC (5 mM). However, the viability of fibroblasts was decreased by both compounds. Using the co-culture model, we also found that H(2)O(2) increases re-epithelialization while NAC retards it. Our data further illustrate the possible role of ROS in wound healing and the co-culture model should be useful for screening agents that may influence the wound healing process.     

Catalytic promiscuity in Pseudomonas aeruginosa arylsulfatase as an example of chemistry-driven protein evolution

In recent years, it has become increasingly clear that many enzymes are catalytically "promiscuous". This can provide a springboard for protein evolution, allowing enzymes to acquire novel functionality without compromising their native activities. We present here a detailed study of Pseudomonas aeruginosa arylsulfatase (PAS), which catalyzes the hydrolysis of a number of chemically distinct substrates, with proficiencies comparable to that towards its native reaction. We demonstrate that the main driving force for the promiscuity is the ability to exploit the electrostatic preorganization of the active site for the native substrate, providing an example of chemistry-driven protein evolution.     

Perfusion characteristics of the human hepatic microcirculation based on three-dimensional reconstructions and computational fluid dynamic analysis

The perfusion of the liver microcirculation is often analyzed in terms of idealized functional units (hexagonal liver lobules) based on a porous medium approach. More elaborate research is essential to assess the validity of this approach and to provide a more adequate and quantitative characterization of the liver microcirculation. To this end, we modeled the perfusion of the liver microcirculation using an image-based three-dimensional (3D) reconstruction of human liver sinusoids and computational fluid dynamics techniques. After vascular corrosion casting, a microvascular sample (±0.134 mm(3)) representing three liver lobules, was dissected from a human liver vascular replica and scanned using a high resolution (2.6 μm) micro-CT scanner. Following image processing, a cube (0.15?×?0.15?×?0.15 mm(3)) representing a sample of intertwined and interconnected sinusoids, was isolated from the 3D reconstructed dataset to define the fluid domain. Three models were studied to simulate flow along three orthogonal directions (i.e., parallel to the central vein and in the radial and circumferential directions of the lobule). Inflow and outflow guidances were added to facilitate solution convergence, and good quality volume meshes were obtained using approximately 9?×?10(6) tetrahedral cells. Subsequently, three computational fluid dynamics models were generated and solved assuming Newtonian liquid properties (viscosity 3.5 mPa s). Post-processing allowed to visualize and quantify the microvascular flow characteristics, to calculate the permeability tensor and corresponding principal permeability axes, as well as the 3D porosity. The computational fluid dynamics simulations provided data on pressure differences, preferential flow pathways and wall shear stresses. Notably, the pressure difference resulting from the flow simulation parallel to the central vein (0-100 Pa) was clearly smaller than the difference from the radial (0-170 Pa) and circumferential (0-180 Pa) flow directions. This resulted in a higher permeability along the central vein direction (k(d,33)?=?3.64?×?10(-14) m(2)) in comparison with the radial (k(d,11)?=?1.56?×?10(-14) m(2)) and circumferential (k(d,22)?=?1.75?×?10(-14) m(2)) permeabilities which were approximately equal. The mean 3D porosity was 14.3. Our data indicate that the human hepatic microcirculation is characterized by a higher permeability along the central vein direction, and an about two times lower permeability along the radial and circumferential directions of a lobule. Since the permeability coefficients depend on the flow direction, (porous medium) liver microcirculation models should take into account sinusoidal anisotropy.     

Spontaneous autoimmune gastritis and hypochlorhydria are manifest in the ileitis-prone SAMP1/YitFcs mice

SAMP1/YitFcs mice serve as a model of Crohn's disease, and we have used them to assess gastritis. Gastritis was compared in SAMP1/YitFcs, AKR, and C57BL/6 mice by histology, immunohistochemistry, and flow cytometry. Gastric acid secretion was measured in ligated stomachs, while anti-parietal cell antibodies were assayed by immunofluorescence and enzyme-linked immunosorbent spot assay. SAMP1/YitFcs mice display a corpus-dominant, chronic gastritis with multifocal aggregates of mononuclear cells consisting of T and B lymphocytes. Relatively few aggregates were observed elsewhere in the stomach. The infiltrates in the oxyntic mucosa were associated with the loss of parietal cell mass. AKR mice, the founder strain of the SAMP1/YitFcs, also have gastritis, although they do not develop ileitis. Genetic studies using SAMP1/YitFcs-C57BL/6 congenic mice showed that the genetic regions regulating ileitis had comparable effects on gastritis. The majority of the cells in the aggregates expressed the T cell marker CD3 or the B cell marker B220. Adoptive transfer of SAMP1/YitFcs CD4(+) T helper cells, with or without B cells, into immunodeficient recipients induced a pangastritis and duodenitis. SAMP1/YitFcs and AKR mice manifest hypochlorhydria and anti-parietal cell antibodies. These data suggest that common genetic factors controlling gastroenteric disease in SAMP1/YitFcs mice regulate distinct pathogenic mechanisms causing inflammation in separate sites within the digestive tract.