EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.     

In vivo correlation between c-Fos expression and corticotroph stimulation by adrenocorticotrophic hormone secretagogues in rat anterior pituitary gland

In the anterior pituitary gland, c-Fos expression is evoked by various stimuli. However, whether c-Fos expression is directly related to the stimulation of anterior pituitary cells by hypothalamic secretagogues is unclear. To confirm whether the reception of hormone-releasing stimuli evokes c-Fos expression in anterior pituitary cells, we have examined c-Fos expression of anterior pituitary glands in rats administered with synthetic corticotrophin-releasing hormone (CRH) intravenously or subjected to restraint stress. Single intravenous administration of CRH increases the number of c-Fos-expressing cells, and this number does not change even if the dose is increased. Double-immunostaining has revealed that most of the c-Fos-expressing cells contain adrenocorticotrophic hormone (ACTH); corticotrophs that do not express c-Fos in response to CRH have also been found. However, restraint stress evokes c-Fos expression in most of the corticotrophs and in a partial population of lactotrophs. These results suggest that c-Fos expression increases in corticotrophs stimulated by ACTH secretagogues, including CRH. Furthermore, we have found restricted numbers of corticotrophs expressing c-Fos in response to CRH. Although the mechanism underlying the different responses to CRH is not apparent, c-Fos is probably a useful immunohistochemical marker for corticotrophs stimulated by ACTH secretagogues. This work was supported by the Jichi Medical University young investigator award.     

Microdissected region-specific gene expression analysis with methacarn-fixed, paraffin-embedded tissues by real-time RT-PCR.

We have previously shown methacarn to be a versatile fixative for analysis of proteins, DNA, and RNA in paraffin-embedded tissues (PETs). In this study we analyzed its suitability for quantitative mRNA expression analysis of microdissected PET specimens using a real-time RT-PCR technique. Fidelity of expression in the methacarn-fixed PET sections, with reference to dose-dependent induction of cytochrome P450 2B1 in the phenobarbital-treated rat liver, was high in comparison with the unfixed frozen tissue case, even after hematoxylin staining. RNA yield from methacarn-fixed PET sections was equivalent to that in unfixed cryosections and was also not significantly affected by hematoxylin staining. Correlations between the expression levels of target genes and input amounts of extracted RNA in the range of 1-1000 pg were very high (correlation coefficients >0.98), the regression curves being similar to those with unfixed cryosections. Although cell numbers should be optimized for each target gene/tissue, >/=200 cells were necessary for accurate measurement in 10-microm-thick rat liver sections judging from the variation of measured value in small microdissected areas. These results indicate high performance with methacarn, close to that of unfixed tissues, regarding quantitative expression analysis of mRNAs in microdissected PET-specimens.     

Repetitive prenatal glucocorticoid therapy reduces oxidative stress in the lungs of preterm lambs

Repetitive coursesof maternal prenatal glucocorticoids are often used in high-riskpregnancies with threatening preterm labor to induce lung maturation,but the effects on the cellular oxidant-antioxidant balance in thefetal lung have not been evaluated. We investigated the effect ofrepetitive treatment with glucocorticoids, beginning early ingestation, on oxidative stress in the preterm ovine lung. Pregnant eweswere randomized to receive one, two, three, or four doses of 0.5 mg/kgbetamethasone or saline placebo at 7-day intervals on 104, 111, 118, and 124 days gestation (n = 11 foreach group). All lambs were delivered preterm at 125 days gestation,and lung tissue was assayed for antioxidant enzymes, lipidhydroperoxides, and carbonyl proteins. Lung manganese superoxidedismutase, catalase, and glutathione peroxidase activity increasedafter 1 dose of betamethasone given at 104 days gestation, whereascopper-zinc superoxide dismutase activity increased after 2 doses givenat 104 and 111 days gestation. The activity of all four antioxidant enzymes further increased with additional doses and was maximal afterfour doses of betamethasone. Lung lipid hydroperoxide levels andcarbonyl protein content decreased stepwise after each dose ofbetamethasone and were lowest after four doses. Repetitive prenatalglucocorticoid therapy increases antioxidant enzyme activity andreduces oxidative stress in the lungs of preterm lambs, and theseeffects begin early in gestation and persist for 2-3wk.

     

New records and distributions of two North American branchiobdellidan species (Annelida: Clitellata) from introduced signal crayfish, <Emphasis Type="Italic">Pacifastacus leniusculus,</Emphasis> in Japan

This is the first report of two North American branchiobdellidans, Sathodrilus attenuatus Holt, 1981, and Xironogiton victoriensis Gelder and Hall, 1990, on the signal crayfish, Pacifastacus leniusculus (Dana, 1852) introduced into Japan from the Columbia River system, northwestern North America. Signal crayfish from 12 localities in eastern and northern Hokkaido, Japan, were examined and each supported S. attenuatus. In addition, an individual of this species was found on preserved material from Ishikawa Prefecture, central Honshu. All of these branchiobdellidans reported in Hokkaido most probably came from the original population of signal crayfish introduced into Lake Mashu, Hokkaido, Japan, in 1930. It is suggested that the use of non-pathogenic branchiobdellidans, when present, provides an easy method for tracing the spread of crayfishes around Japan and could also be applied in other countries and continents. Specimens of X. victoriensis were only found on crayfish in a stream at Akashina in Nagano Prefecture, central Honshu, Japan. Although the signal crayfish appears to be displacing the endemic Japanese crayfish, C. japonicus, no native branchiobdellidans were found on any of the introduced signal crayfish examined.     

Proteolytic activity of HtpX, a membrane-bound and stress-controlled protease from Escherichia coli

Escherichia coli HtpX is a putative membrane-bound zinc metalloprotease that has been suggested to participate in the proteolytic quality control of membrane proteins in conjunction with FtsH, a membrane-bound and ATP-dependent protease. Here, we biochemically characterized HtpX and confirmed its proteolytic activities against membrane and soluble proteins. HtpX underwent self-degradation upon cell disruption or membrane solubilization. Consequently, we purified HtpX under denaturing conditions and then refolded it in the presence of a zinc chelator. When supplemented with Zn2+, the purified enzyme exhibited self-cleavage activity. In the presence of zinc, it also degraded casein and cleaved a solubilized membrane protein, SecY. We verified its ability to cleave SecY in vivo by overproducing both HtpX and SecY. These results showed that HtpX is a zinc-dependent endoprotease member of the membrane-localized proteolytic system in E. coli.     

Minor but key chlorophylls in Photosystem II

A ‘metal-free’ chlorophyll (Chl) a, pheophytin (Phe) a, functions as the primary electron acceptor in PS II. On the basis of Phe a/PS II = 2, Phe a content is postulated as an index for estimation of the stoichiometry of pigments and photosystems. We found Phe a in a Chl d-dominant cyanobacterium Acaryochloris marina, whereas Phe d was absent. The minimum Chl a:Phe a ratio was 2:2, indicating that the primary electron donor is Chl a, accessory is Chl d, and the primary electron acceptor is Phe a in PS II of A. marina. Chl d was artificially formed by the treatment of Chl a with papain in aqueous organic solvents. Further, we will raise a key question on the mechanisms of water oxidation in PS II.     

Early stage of establishment of persistent Sendai virus infection: unstable dynamic phase and then selection of viruses which are tightly cell associated, temperature sensitive, and capable of establishing persistent infection

We obtained 157 cloned cell lines persistently infected with Sendai virus; these cell lines were generated independently of each other. Infectious viruses could be isolated from 123 of these cloned cell lines by inoculation of culture fluids or infected cells into embryonated eggs. The majority of the viruses carried by cells persistently infected with viruses showed high cytotoxicity and did not have the ability to establish persistent infection. The association of carried virus with cells became stronger and virus isolation correspondingly became more difficult as cells persistently infected with virus were subcultured. Viruses derived from virus-infected cells eventually acquired the ability to establish persistent infection, although the ways in which the viruses acquired this ability varied. The viruses also acquired temperature sensitivity as persistently infected cells were subcultured. First, the hemagglutinin-neuraminidase and M proteins acquired temperature sensitivity, and then the polymerase(s) did so. The M proteins were localized in the nuclei of cells infected with cloned viruses that had the ability to establish persistent infection. Cells infected with viruses capable of establishing persistent infection showed no or slight staining by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. Specific amino acid substitutions accumulated in the M protein and the L protein as virus-infected cells were subcultured. This study shows that there is an unstable dynamic phase at an early stage of the establishment of persistent Sendai virus infection (steady state), and then viruses capable of establishing persistent infection are selected.