Phorbol ester is a strong inducer for both cell cornification and squamous-cell marker SPRR1 gene expression in conducting airway epithelial cells. However, the signaling pathways involved in the regulation of both events have not been completely elucidated. The current study focuses on the common and divergent pathways involved in the induction of these two activities by phorbol-13-myristate-12-acetate (PMA). Using a protein kinase (PK) C inhibitor, bisindolylmaleimide I, PMA-induced cell cornification and SPRR1 gene expression were abolished. Further, a PKC activator, indolactam V, induced cell cornification in the absence of PMA treatment. These results suggest a PKC-dependent signaling pathway for both gene induction and enhanced cell cornification by PMA. However, a mitogen-activated protein kinase-specific inhibitor, PD98059, could only block the gene induction event but failed to prevent cell cornification induced by PMA. These results suggest that diverse signaling pathways after PKC activation by PMA are involved in the regulation of these two events.
Infectivity is a fundamental property of viral pathogens such as human rhinoviruses (HRVs). This chapter describes two methods for measuring the infectivity of HRV-A and -B serotypes: end point dilution (TCID50) assay and plaque assay. End point dilution assay is a quantal, not quantitative, assay that determines the dilution of the sample at which 50 % of the aliquots have infectious virus. It can be used for all the HRV-A and -B serotypes and related clinical isolates that grow in cell culture and induce cytopathic effect (CPE), degenerative changes in cells that are visible under a microscope. Plaque assay is a quantitative assay that determines the number of infectious units of a virus in a sample. After an infectious unit of virus infects one cell, the infected cell produces progeny viruses that then infect and kill a circle of adjacent cells. This circle of dead cells detaches from the dish and thus leaves a clear hole in a cell monolayer. Plaque assay works only for HeLa-adapted HRV-A and -B serotypes that can make visible plaques on the cell monolayer. Currently the end point dilution assay and plaque assay have not been developed for the newly discovered HRV-C.
β-Defensin is one of the major host defense shields produced by various tissues and organs against microbial infection. To date, four human β-defensins (DEFBs) gene products that share a consensus six-cysteine motif have been discovered. The hidden Markov model (HMM) profile was constructed from the common features of those known β-defensin peptides to search for additional novel DEFB genes. A genome-wide search of the profile against ORFeome-based peptide databases (e.g., Ensembl project) led to the identification of six new DEFB members that also shared the conserved six-cysteine motif. Phylogenetic analysis supported a close relationship of these six new members with existing DEFB genes. Polymerase Chain Reaction studies of human tissue cDNA panels confirmed the expression of all six novel DEFB genes in various tissues. Two of them, DEFB106 and DEFB109, were expressed in the lung. A pilot study with cRNA probes for in situ hybridization and a synthetic propeptide for the functional characterization demonstrated the tissue-/ cell-specific expression and the strong antimicrobial activity of DEFB106. These results support the utility of ORFeome-based HMM search in gene discovery for members of a specific gene family. The novel DEFB genes identified in this study may significantly contribute to overall antimicrobial host defenses.
PMID: 21531775;PMCID: PMC3129903;Abstract:
Exposure to environmental pollutants has been linked to various airway diseases and disease exacerbations. Almost all chronic airway diseases uch as chronic obstructive pulmonary disease and asthma are aused by complicated interactions between gene and environment. ne of the major hallmarks of those diseases is airway mucus verproduction (MO). Excessive mucus causes airway obstruction and significantly increases morbidity and mortality. Metals are major components of environmental particulate matters (PM). Among them, vanadium has been suggested to play an important role in PM-induced mucin production. Vanadium pentoxide (V2O5) is the most common commercial source of vanadium, and it has been associated with occupational chronic bronchitis and asthma, both of which are MO diseases. However, the underlying mechanism is not entirely clear. In this study, we used both in vitro and in vivo models to demonstrate the robust inductions of mucin production by V2O5. Furthermore, the follow-up mechanistic study revealed a novel v-raf-1 murine leukemia viral oncogene homolog 1-IKK-NF-κB pathway that mediated V2O5- induced mucin production. Most interestingly, the reactive oxygen species and the classical mucin-inducing epidermal growth factor receptor (EGFR)-MAPK pathway appeared not to be involved in this process. Thus the V2O5-induced mucin production may represent a novel EGFR-MAPK-independent and environmental toxicant-associated MO model. Complete elucidation of the signaling pathway in this model will not only facilitate the development of the treatment for V2O5-associated occupational diseases but also advance our understanding on the EGFR-independent mucin production in other chronic airway diseases. © 2011 the American Physiological Society.
Using a BLAST-searching approach, we identified a mouse expressed sequence tag (EST) clone (AA038672) showing great similarity to the 3′ end of the human MUC5B gene. The clone was named "3pmmuc5b-1" after complete nucleotide sequencing (Genbank Accession, AF369933). A subsequent search of the mouse genome database with the 3pmmuc5b-1 sequence identified two overlapping genomic clones (AC020817 and AC020794) that contained the sequence of both 3pmmuc5b-1 and the mouse Muc5ac gene. Like their human homologs, the genomic order of the mouse Muc genes is 5′-Muc5ac-Muc5b-3′. These results suggest that the newly identified EST clone, 3pmmuc5b-1, is part of the 3′ portion of the mouse Muc5b gene. In situ hybridization demonstrated that this putative mouse Muc5b message was expressed in a restricted manner in the sublingual gland region of the tongue and the submucosal gland region of the mouse trachea in a normal animal. However, the gene expression was greatly enhanced in airway surface epithelium and the submucosal gland region in ovalbumin-induced asthmatic mice. These results were consistent with previous studies of human airway tissues. We therefore conclude that this newly cloned mouse Muc5b gene could be used as a marker for studying aberrant mucin gene expression in mouse models of various airway diseases.