Measuring Airway Surface Liquid Depth in Ex Vivo Mouse Airways by X-Ray Imaging for the Assessment of Cystic Fibrosis Airway Therapies

In the airways of those with cystic fibrosis (CF), the leading pathophysiological hypothesis is that an ion channel defect results in a relative decrease in airway surface liquid (ASL) volume, producing thick and sticky mucus that facilitates the establishment and progression of early fatal lung disease. This hypothesis predicts that any successful CF airway treatment for this fundamental channel defect should increase the ASL volume, but up until now there has been no method of measuring this volume that would be compatible with in vivo monitoring. In order to accurately monitor the volume of the ASL, we have developed a new x-ray phase contrast imaging method that utilizes a highly attenuating reference grid. In this study we used this imaging method to examine the effect of a current clinical CF treatment, aerosolized hypertonic saline, on ASL depth in ex vivo normal mouse tracheas, as the first step towards non-invasive in vivo ASL imaging. The ex vivo tracheas were treated with hypertonic saline, isotonic saline or no treatment using a nebuliser integrated within a small animal ventilator circuit. Those tracheas exposed to hypertonic saline showed a transient increase in the ASL depth, which continued for nine minutes post-treatment, before returning to baseline by twelve minutes. These findings are consistent with existing measurements on epithelial cell cultures, and therefore suggest promise for the future development of in vivo testing of treatments. Our grid-based imaging technique measures the ASL depth with micron resolution, and can directly observe the effect of treatments expected to increase ASL depth, prior to any changes in overall lung health. The ability to non-invasively observe micron changes in the airway surface, particularly if achieved in an in vivo setting, may have potential in pre-clinical research designed to bring new treatments for CF and other airway diseases to clinical trials.     

Biological Activities of Triterpenoids and Phenolic Compounds from Myrica cerifera Bark

Seven triterpenoids, 1  –  7 , two diarylheptanoids, 8 and 9 , four phenolic compounds, 10  –  13 , and three other compounds, 14  –  16 , were isolated from the hexane and MeOH extracts of the bark of Myrica cerifera L. (Myricaceae). Among these compounds, betulin ( 1 ), ursolic acid ( 3 ), and myricanol ( 8 ) exhibited cytotoxic activities against HL60 (leukemia), A549 (lung), and SK‐BR‐3 (breast) human cancer cell lines (IC₅₀ 3.1 – 24.2 μm ). Compound 8 induced apoptotic cell death in HL60 cells (IC₅₀ 5.3 μm ) upon evaluation of the apoptosis‐inducing activity by flow cytometric analysis and by Hoechst 33342 staining method. Western blot analysis on HL60 cells revealed that 8 activated caspases‐3, ‐8, and ‐9 suggesting that 8 induced apoptosis via both mitochondrial and death receptor pathways in HL60. Upon evaluation of the melanogenesis‐inhibitory activity in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH), erythrodiol ( 7 ), 4‐hydroxy‐2‐methoxyphenyl β‐d ‐glucopyranoside ( 13 ), and butyl quinate ( 15 ) exhibited inhibitory effects (65.4 – 86.0% melanin content) with no, or almost no, toxicity to the cells (85.9 – 107.4% cell viability) at 100 μm concentration. In addition, 8 , myricanone ( 9 ), myricitrin ( 10 ), protocatechuic acid ( 11 ), and gallic acid ( 12 ) revealed potent DPPH radical‐scavenging activities (IC₅₀ 6.9 – 20.5 μm ).     

Two isoforms of chicken melanopsins show blue light sensitivity

Melanopsin is a vertebrate non-visual opsin and functions as a circadian photoreceptor in mammalian retinas. Here we found the expression of two kinds of melanopsin genes in the chicken pineal gland and identified the presence of five isoforms derived from these two genes. Reconstitution of the recombinant proteins with 11-cis-retinal revealed that at least two of these melanopsin protein isoforms can function as blue-sensitive photopigments with absorption maxima at 476-484nm. These values are consistent with maximal sensitivities of action spectra determined from the physiological and behavioral studies on mammalian melanopsins. The melanopsin isoforms found in this study may function as pineal circadian photoreceptors.     

Daily growth increments in otoliths of wild-caught honmoroko Gnathopogon caerulescens

Otolith growth increments in wild-caught alizarin complex one (ALC)-marked honmoroko Gnathopogon caerulescens were examined to verify the veracity of the age determination method in cyprinids. ALC-marked G. caerulescens recaptured from their natural environment had lapilli increment counts outside the ALC ring mark that had formed on a daily basis during the juvenile stage. This apparently being the first direct evidence of daily periodicity of otolith increment formation in wild-caught cyprinids.     

Hepatic neuroendocrine carcinoma in a Japanese macaque (Macaca fuscata)

Primary neuroendocrine neoplasm of the liver is extremely rare in both humans and non‐human primates. The present report describes the clinical and pathological findings of an aged Japanese macaque (Macaca fuscata) with hepatic neuroendocrine carcinoma. To our knowledge, this is the first report of hepatic neuroendocrine neoplasm in macaques.     

DNA Microarray for Rapid Detection of Mitochondrial DNA Haplotypes of Chum Salmon

For use in genetic stock identification, we developed an oligonucleotide (DNA) microarray hybridization method for rapid and accurate detection of nucleotide sequence variations in 20 previously identified variable nucleotide sites in about 500 bp within the 5 half of the control region of mitochondrial DNA of chum salmon (Oncorhynchus keta). The method includes immobilization of synthesized oligonucleotides containing respective polymorphic sites on a glass slide precoated with polycarbodiimide resin, a 2-hour hybridization with DNA microarray of biotinylated polymerase chain reaction fragments spanning the 5 variable portion followed by short washing, and visualization of hybridization signals by conventional ABC method and scanner-assisted computation of signal intensity on a computer. The entire process of hybridization and detection was completed within 4 hours. The resulting DNA microarray could detect all of the single nucleotide mutations and therefore could be used to identity the sequence variations defining 30 mtDNA haplotypes of chum salmon as revealed previously by nucleotide sequence analysis.     

Cutting edge: profile of chemokine receptor expression on human plasma cells accounts for their efficient recruitment to target tissues

We systematically examined the repertoire of chemokine receptors expressed by human plasma cells. Fresh bone marrow plasma cells and myeloma cells consistently expressed CXCR4, CXCR6, CCR10, and CCR3. Accordingly, plasma cells responded to their respective ligands in chemotaxis and very late Ag-4-dependent cell adhesion to fibronectin. Immobilized CXC chemokine ligand (CXCL)16, a novel transmembrane-type chemokine and CXCR6 ligand, also directly induced adhesion of plasma cells without requiring G(alpha i) signaling or divalent cations. Furthermore, we revealed consistent expression of CXCL12 (CXCR4 ligand), CXCL16 (CXCR6 ligand), and CC chemokine ligand 28 (CCR10 and CCR3 ligand) in tissues enriched with plasma cells including bone marrow, and constitutive expression of CXCL12, CXCL16, and CC chemokine ligand 28 by cultured human bone marrow stromal cells. Collectively, plasma cells are likely to be recruited to bone marrow and other target tissues via CXCR4, CXCR6, CCR10, and CCR3. CXCR6 may also contribute to tissue localization of plasma cells through its direct binding to membrane-anchored CXCL16.     

Stable high-producer cell clone expressing virus-like particles of the Japanese encephalitis virus e protein for a second-generation subunit vaccine

We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 micro g per 10(4) cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 x 10(6) PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.