Loss of caveolin-1 in bronchiolization in lung fibrosis.

Bronchiolization is a key process in fibrosing lung in which the proliferative status of bronchiolar epithelium changes, leading to abnormal epithelial morphology. Within the context that caveolin-1 acts to suppress epithelial proliferation, we postulated that stimulating epithelial injury would lead to caveolin-1 downregulation and encourage proliferation. The present study evaluates the expression of caveolin-1, especially in bronchiolization, in C57BL/6J mice with bleomycin-induced lung fibrosis and in various types of re-epithelialization in human interstitial pneumonias (IPs). Immunohistochemically, levels of caveolin-1 decreased in the bronchiolar epithelium of mice treated with bleomycin. Levels of caveolin-1 mRNA in the whole lung were decreased at 7 and 14 days. Caveolin-1 mRNA was also decreased in laser-capture microdissection- retrieved bronchiolar epithelial cells at 7 days. Among patients with 12 IPs, including four usual IPs (UIPs) and eight nonspecific IPs (NSIPs), whole lung caveolin-1 was significantly decreased compared with 12 controls at both mRNA and protein levels. By scoring immunointensity, caveolin-1 was significantly reduced in bronchiolization and squamous metaplasia as well as in bronchiolar epithelium in 23 IPs (12 UIPs and 11 NSIPs) compared with bronchiolar epithelium from seven controls. These data suggested that loss of caveolin-1 is associated with abnormal re-epithelialization in lung fibrosis.     

Co-expression of two distinct polysialic acids, α2,8- and α2,9-linked polymers of N-acetylneuraminic acid, in distinct glycoproteins and glycolipids in sea urchin sperm

Naturally occurring polysialic acid (polySia) structures have a large diversity, primarily arising from the diversity in the sialic acid components as well as in the intersialyl linkages. In 2004, we demonstrated the presence of a new type of polySia, 8-O-sulfated N-acetylneuraminic acid (Neu5Ac) capped α2,9-linked polyNeu5Ac, on the O-glycans of a major 40-80 kDa sialoglycoprotein, flagellasialin, in sea urchin sperm. In this study, we demonstrated that another type of polySia, the α2,8-linked polyNeu5Ac, exclusively occurs on O-glycans of a 190 kDa glycoprotein (190 kDa-gp), whereas the α2,9-linked polyNeu5Ac is exclusively present on flagellasialin. The 190 kDa-gp is localized in both flagellum and head of sperm. We also demonstrated that polysialogangliosides containing the α2,8-linked polyNeu5Ac are present in sperm head. Thus, this study shows two novel features of the occurrence of polySia in nature, the co-localization of polySia with different intersialyl linkages, the α2,8- and α2,9-linkages, in a single cell and the occurrence of α2,8-linked polyNeu5Ac in glycolipids. Anti-α2,8-linked polyNeu5Ac antibody had no effect on fertilization, which contrasted with the previous results that anti-α2,9-linked polyNeu5Ac antibody inhibited sperm motility and fertilization. Based on these properties, distinct functions of α2,8- and α2,9-polySia structures are implicated in fertilization.     

Relative biological effectiveness of fission neutrons for induction of micronucleus formation in mouse reticulocytes in vivo

Following whole-body irradiation of ICR mice with various doses of fission neutrons or X-rays, the frequency of micronuclei (MNs) in peripheral blood reticulocytes was measured at 12 h intervals beginning immediately after irradiation and ending at 72 h after irradiation. The resulting time-course curve of MN frequency had a clear peak 36 h after irradiation, irrespective of the type of radiation applied and the dose used. The MN frequency, averaged as the unweighted mean over the experimental time course, showed a linear increase with increasing dose of either fission neutrons or X-rays. The linear response to X-rays supports reported conclusion that induction of MN formation in reticulocytes is a dose-rate independent phenomenon. The relative biological effectiveness (RBE) of fission neutrons to X-rays for MN induction was estimated to be 1.9 +/- 0.3. This value is considerably lower than the RBE value of 4.6 +/- 0.5 reported for the same fission neutrons for induction of lymphocyte apoptosis in the thymus of ICR mice that represents dose-rate independent, one-track event. Based on these results, we propose that MNs increased in reticulocytes after irradiation mostly represent acentric fragments caused by single chromosome breaks, and that some confounding factor is operating in erythroblasts for the formation of aberrations from non-rejoining DNA double-strand breaks more severely after high-LET radiation than after low-LET radiation.     

Susceptibility of male and female medaka (Oryzias latipes) fish to spontaneous and X-ray induced micronucleus formation in gill cells

The frequency of micronucleated cells (MNCs) was measured in acridine-orange (AO) stained RNA-rich gill cells from male and female medaka (Oryzias latipes) fish of known body weight. Spontaneous MNC frequencies were not significantly correlated with body weight, despite the fact that the heaviest of the 30 fish used outweighed the lightest by a factor of 3. Average MNC frequencies were identical in males and females at 0.8 per thousand. An X-ray dose of 4 Gy increased the frequency of MNCs over the spontaneous level in all 30 of the fish used, reaching a level of 7.2 per thousand on average when assayed 24 h after exposure. In X-ray treated fish, MNC frequency and body weight were not significantly correlated, nor was there any difference between the sexes. These and other results support our primary conclusion that AO-staining is suitable for the medaka micronucleus assay in gill cells, and indicate that male and female medaka fish are similarly and size-independently susceptible to both spontaneous and X-ray induced micronucleus formation in gill cells.     

Residual chemoresponsiveness to acids in the superior laryngeal nerve in "taste-blind" (P2X2/P2X3 double-KO) mice

Mice lacking both the P2X2 and the P2X3 purinergic receptors (P2X-dblKO) exhibit loss of responses to all taste qualities in the taste nerves innervating the tongue. Similarly, these mice exhibit a near total loss of taste-related behaviors in brief access tests except for a near-normal avoidance of acidic stimuli. This persistent avoidance of acids despite the loss of gustatory neural responses to sour was postulated to be due to continued responsiveness of the superior laryngeal (SL) nerve. However, chemoresponses of the larynx are attributable both to taste buds and to free nerve endings. In order to test whether the SL nerve of P2X-dblKO mice remains responsive to acids but not to other tastants, we recorded responses from the SL nerve in wild-type (WT) and P2X-dblKO mice. WT mice showed substantial SL responses to monosodium glutamate, sucrose, urea, and denatonium-all of which were essentially absent in P2X-dblKO animals. In contrast, the SL nerve of P2X-dblKO mice exhibited near-normal responses to citric acid (50 mM) although responsiveness of both the chorda tympani and the glossopharyngeal nerves to this stimulus were absent or greatly reduced. These results are consistent with the hypothesis that the residual avoidance of acidic solutions by P2X-dblKO mice may be attributable to the direct chemosensitivity of nerve fibers innervating the laryngeal epithelium and not to taste.     

Translation termination factor eRF3 is targeted for caspase-mediated proteolytic cleavage and degradation during DNA damage-induced apoptosis

Polypeptide chain release factor eRF3 plays pivotal roles in translation termination and post-termination events including ribosome recycling and mRNA decay. It is not clear, however, if eRF3 is targeted for the regulation of gene expression. Here we show that DNA-damaging agents (UV and etoposide) induce the immediate cleavage and degradation of eRF3 in a caspase-dependent manner. The effect is selective since the binding partners of eRF3, eRF1 and PABP, and an unrelated control, GAPDH, were not affected. Point mutations of aspartate residues within overlapping DXXD motifs near the amino terminus of eRF3 prevented the appearance of the UV-induced cleavage product, identifying D32 as the major cleavage site. The cleavage and degradation occurred in a similar time-dependent manner to those of eIF4G, a previously established caspase-3 target involved in the inhibition of translation during apoptosis. siRNA-mediated knockdown of eRF3 led to inhibition of cellular protein synthesis, supporting the idea that the decrease in the amount of eRF3 caused by the caspase-mediated degradation contributes to the inhibition of translation during apoptosis. This is the first report showing that eRF3 could serve as a target in the regulation of gene expression.     

Expression patterns of hedgehog, wingless, and decapentaplegic during gut formation of Gryllus bimaculatus (cricket).

We observed expression patterns of hedgehog (hh), wingless (wg), and decapentaplegic (dpp) during gut development of Gryllus bimaculatus (the cricket), a typical hemimetabolous insect, and compared with those observed in Drosophila, a typical holometabolous insect. Gryllus hh(Gbhh) and Gbwg are expressed in both foregut and hindgut, while Gbdpp is expressed only in the hindgut: at the boundaries between the small and large intestine, and between the large intestine and rectum. Although the expression patterns of Gbhh and Gbwg are essentially comparable to those observed in Drosophila, the expression pattern of Gbdpp differs from those of the Drosophila dpp.     

Theoretical calculation of triazolam hydroxylation and endogenous steroid inhibition in the active site of CYP3A4

CYP3A4 has unusual kinetic characteristics because it has a large active site. CYP3A4 produced more 4-hydroxytriazolam than alpha-hydroxytriazolam at concentrations of more than 60 muM triazolam, and different steroids had different inhibitory effects on the system. To clarify these interesting observations, the interactions between substrate and substrate/steroid were investigated by theoretical calculations. When two triazolam molecules were docked into the active site, the distance between the O-atom and the 4-hydroxylated site was less than the distance to the alpha-hydroxylated site because of interaction between the two triazolam molecules. Estradiol inhibited both alpha- and 4-hydroxytriazolam formation by 50%. Dehydroepiandrosterone (DHEA) inhibited alpha-hydroxylation more than 4-hydroxytriazolam formation, whereas aldosterone had no effect. When one triazolam molecule and one steroid molecule were simultaneously docked, estradiol increased the distance between the O-atom and the two hydroxylated sites, DHEA only increased the distance between the O-atom and alpha-hydroxylated site, and aldosterone did not change the distances. The relevant angles of Fe-O-C in the hydroxylated site of triazolam also widened, together with increased distance. These findings indicate that formation of a substrate and substrate/effector complex in the active site may be a factor for determining the enzyme kinetic parameters of CYP3A4.     

Human stem cells for modeling neurological disorders: accelerating the drug discovery pipeline

The availability of human stem cells heralds a new era for modeling normal and pathologic tissues and developing therapeutics. For example, the in vitro recapitulation of normal and aberrant neurogenesis holds significant promise as a tool for de novo modeling of neurodevelopmental and neurodegenerative diseases. Translational applications include deciphering brain development, function, pathologies, traditional medications, and drug discovery for novel pharmacotherapeutics. For the latter, human stem cell-based assays represent a physiologically relevant and high-throughput means to assess toxicity and other undesirable effects early in the drug development pipeline, avoiding late-stage attrition whilst expediting proof-of-concept of genuine drug candidates. Here we consider the potential of human embryonic, adult, and induced pluripotent stem cells for studying neurological disorders and preclinical drug development.