Chloride intracellular channel 4 (CLIC4) expression profile in the mouse medial prefrontal cortex and its regulation by ethanol.

BACKGROUND: Chloride intracellular channel 4 (CLIC4) is a multifunctional metamorphic protein for which a growing body of evidence supports a major role in the brain's molecular and behavioral responses to ethanol (EtOH). Although key to understanding the functional biology underlying this role, little is known about the cellular and subcellular expression patterns of CLIC4 in brain and how they are affected by EtOH. METHODS: We used qRT-PCR to assess Clic4 mRNA expression in the medial prefrontal cortex (mPFC) of C57BL/6J mice in the absence and presence of acute EtOH exposure. Two complementary immunohistochemical techniques were employed to assess the subcellular localization of the CLIC4 protein and its pattern of expression across brain cell types in the mPFC in the absence and presence of acute EtOH. RESULTS: Through immunohistochemical and stereological techniques, we show that CLIC4 protein is robustly expressed by oligodendrocytes (most abundant), microglia, and astrocytes, with minimal expression in neurons. Following acute EtOH exposure, we observed a rapid increase in Clic4 mRNA expression in female but not male mice and an overall increase in the number of oligodendrocytes and astrocytes expressing the CLIC4 protein. CONCLUSIONS: These findings suggest that Clic4 functions as an early response gene for acute EtOH in brain, which likely underlies its ability to modulate EtOH behavior. Our results also suggest that the role of CLIC4 in the brain's response to EtOH is mediated through oligodendrocytes.

Inhibiting pollen reduced nicotinamide adenine dinucleotide phosphate oxidase-induced signal by intrapulmonary administration of antioxidants blocks allergic airway inflammation.

BACKGROUND: Ragweed extract (RWE) contains NADPH oxidases that induce oxidative stress in the airways independent of adaptive immunity (signal 1) and augment antigen (signal 2)-induced allergic airway inflammation. OBJECTIVE: To test whether inhibiting signal 1 by administering antioxidants inhibits allergic airway inflammation in mice. METHODS: The ability of ascorbic acid (AA), N-acetyl cystenine (NAC), and tocopherol to scavenge pollen NADPH oxidase-generated reactive oxygen species (ROS) was measured. These antioxidants were administered locally to inhibit signal 1 in the airways of RWE-sensitized mice. Recruitment of inflammatory cells, mucin production, calcium-activated chloride channel 3, IL-4, and IL-13 mRNA expression was quantified in the lungs. RESULTS: Antioxidants inhibited ROS generation by pollen NADPH oxidases and intracellular ROS generation in cultured epithelial cells. AA in combination with NAC or Tocopherol decreased RWE-induced ROS levels in cultured bronchial epithelial cells. Coadministration of antioxidants with RWE challenge inhibited 4-hydroxynonenal adduct formation, upregulation of Clca3 and IL-4 in lungs, mucin production, recruitment of eosinophils, and total inflammatory cells into the airways. Administration of antioxidants with a second RWE challenge also inhibited airway inflammation. However, administration of AA+NAC 4 or 24 hours after RWE challenge failed to inhibit allergic inflammation. CONCLUSION: Signal 1 plays a proinflammatory role during repeated exposure to pollen extract. We propose that inhibiting signal 1 by increasing antioxidant potential in the airways may be a novel therapeutic strategy to attenuate pollen-induced allergic airway inflammation. CLINICAL IMPLICATIONS: Administration of antioxidants in the airways may constitute a novel therapeutic strategy to prevent pollen induced allergic airway inflammation.

Cloning and localisation of an avermectin receptor-related subunit from Haemonchus contortus.

Ivermectin is believed to exert its anthelminthic effects by binding to glutamate-gated chloride channels (Glu-Cl) and several cDNAs encoding subunits of Glu-Cl have been cloned from Caenorhabditis elegans. We report the cloning of cDNAs encoding a putative Glu-Cl subunit (HG4) from the parasite Haemonchus contortus. The HG4 cDNAs were isolated using RT-PCR and the sequence of the predicted polypeptide has 82% amino-acid identity with the C. elegans Glu-Cl beta subunit. Individual HG4 cDNAs showed up to 4% sequence variation at the nucleotide level, but the vast majority of these polymorphisms were translationally silent. A synthetic peptide corresponding to sequence near the N-terminus of the mature polypeptide was used to raise an antiserum that recognised the N-terminal domain of HG4 expressed in E. coli. Affinity purified antibodies reacted with motor neuron commissures in immuno-localisation studies: these commissures were limited to the anterior portion of the worms, from a region level with the nerve ring to just anterior of the vulva. Some possible nerve cord staining was also observed, but no expression of HG4 on pharyngeal muscle could be detected.

Electron probe X-ray microanalysis of rabbit ciliary epithelium.

Rabbit iris-ciliary bodies were preincubated in control and experimental Ringer's solutions before quick freezing, cryosectioning, dehydration and electron probe X-ray microanalysis. After preincubation in a baseline bicarbonate-free Cl- Ringer's solution, the ciliary epithelial intracellular Na+, K+ and Cl- concentrations were estimated to be 15 +/- 3, 162 +/- 14 and 46 +/- 5 mmol kg-1 intracellular water, respectively. The water and elemental Na, K, Cl and P contents were similar in the non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells. As expected, inhibition of the Na,K-exchange pump by preincubation with ouabain markedly increased the intracellular Na content, and markedly reduced the intracellular K content, verifying the validity of the experimental analysis. The Cl- channels of the NPE cells likely play a critical role in determining the rate of aqueous humor formation. Therefore, we have examined the effects of altering Cl- transport on the intracellular composition in this initial microprobe study of the ciliary epithelium. As expected, exposure to bicarbonate increased the intracellular Cl and water contents. Replacement of external Cl- by NO3- was twice as effective as replacement by gluconate in leaching Cl- out of the intracellular compartment. An unexpected finding was that NO3- replacement of internal Cl- substantially increased the intracellular Na and decreased the intracellular K content, possibly by stabilizing the Na,K-pump in the E1P form and inhibiting enzyme activity.

Production and characterisation of monoclonal and polyclonal antibodies to different regions of the cystic fibrosis transmembrane conductance regulator (CFTR): detection of immunologically related proteins.

We have raised mouse monoclonal antibodies to eight synthetic peptides corresponding to different regions of the human cystic fibrosis transmembrane conductance regulator (CFTR) and rabbit polyclonal antisera to beta-galactosidase fusion proteins which encompass three different regions of CFTR. Immunoblot, immunoprecipitation, immunofluorescence and immunocytochemical experiments demonstrate that, in addition to recognising CFTR, these antibodies recognise one or more immunologically related proteins with a similar molecular mass, calcium responsiveness and tissue distribution to CFTR.