Role for mitogen-activated protein kinase p38 alpha in lung epithelial branching morphogenesis

In the early stages of lung development, the endoderm undergoes extensive and stereotypic branching morphogenesis. During this process, a simple epithelial bud develops into a complex tree-like system of tubes specialized for the transport and exchange of gas with blood. The endodermal cells in the distal tips of the developing lung express a special set of genes, have a higher proliferation rate than proximal part, undergo shape change and initiate branching morphogenesis. In this study, we found that of the four p38 genes, only p38α mRNA is localized specifically to the distal endoderm suggesting a role in the regulation of budding morphogenesis. Chemical inhibitors specific for the p38α and p38β isoforms suppress budding of embryonic mouse lung explants and isolated endoderm in vitro. Specific knockdown of p38α in cultured lung endoderm using shRNA also inhibited budding morphogenesis, consistent with the chemical inhibition of the p38 signaling pathway. Disruption of p38α did not affect proliferation or expression of the distal cell markers, Sox9 and Erm. However, the amount of E-cadherin protein increased significantly and ectopic expression of E-cadherin also impaired budding of endoderm in vitro. These results suggest that p38α modulates epithelial cell-cell interactions and possibly cell rearrangement during branching morphogenesis. This study provides the first evidence that p38α is involved in the morphogenesis of an epithelial organ.     

Vertical distribution and germination ability of Alexandrium spp. cysts (Dinophyceae) in the sediments collected from Kure Bay of the Seto Inland Sea, Japan

The vertical distribution of Alexandrium tamarense/ catenella (hereinafter Alexandrium spp.) cysts was investigated with special attention to living cysts filled with fresh protoplasm and empty cysts. In addition, based on the incubation experiments of Alexandrium spp. cysts, the germination ability of the cysts was examined. A sediment core 63 cm in length, collected from Kure Bay of the Seto Inland Sea, West Japan, in September 2000, was provided for an analysis on the vertical distribution of Alexandrium spp. cysts. Samples from every 1 cm interval depth from the top down to 13 cm depth of the same core were taken to examine the germination ability of the cysts. Results show that Alexandrium spp. cysts were continuously observed from 59 to 60 cm depth to the top. The cyst densities in the upper parts of the core (from 9 to 10 cm depth to the top) were much more abundant those that in the lower parts (below 10–11 cm depth). The relationship between living and empty cysts in each depth did not reveal a positive correlation with the sediment depth. Based on the sedimentation rate of the core sediment (approximately 1.6 cm/year), Alexandrium spp. cysts have been produced since 1962, and a remarkable increase of these cysts was observed from ca 1993. Such a rapid increase of Alexandrium spp. cysts has probably been as a result of dense blooms of A. tamarense occurring since 1992 in Hiroshima Bay, including Kure Bay. The germination of Alexandrium spp. cysts was observed in samples collected from the top to 12–13 cm depth of the core. It suggests that the Alexandrium spp. cysts can keep the germination ability for more than 8 years.     

Uptake of 3,4-methylenedioxymethamphetamine and its related compounds by a proton-coupled transport system in Caco-2 cells

3,4-Methylenedioxymethamphetamine (MDMA) is an illegal amphetamine-type stimulant (ATS) that is abused orally in the form of tablets for recreational purposes. The aim of this work is to investigate the absorption mechanism of MDMA and other related compounds that often occur together in ATS tablets, and to determine whether such tablet components interact with each other in intestinal absorption. The characteristics of MDMA uptake by the human intestinal epithelial Caco-2 cell line were investigated. The Michaelis constant and the maximal uptake velocity at pH 6.0 were 1.11 mM and 13.79 nmol/min/mg protein, respectively, and the transport was electroneutral. The initial uptake rate was regulated by both intra- and extracellular pH. MDMA permeation from the apical to the basolateral side was inferior to that in the reverse direction, and a decrease in apical pH enhanced MDMA permeation from the basolateral to the apical side. These facts indicate that this transport system may be an antiporter of H⁺. However, under physiological conditions, the proton gradient cannot drive the MDMA uptake because it is inwardly directed. Large concentration differences of MDMA itself drive this antiporter. Various compounds with similar amine moieties inhibited the uptake, but substrates of organic cation transporters (OCT1-3) and an H⁺-coupled efflux antiporter, MATE, were not recognized.     

Superoxide scavenging activity of pirfenidone-iron complex

Pirfenidone (PFD) is focused on a new anti-fibrotic drug, which can minimize lung fibrosis etc. We evaluated the superoxide () scavenging activities of PFD and the PFD-iron complex by electron spin resonance (ESR) spectroscopy, luminol-dependent chemiluminescence assay, and cytochrome c reduction assay. Firstly, we confirmed that the PFD-iron complex was formed by mixing iron chloride with threefold molar PFD, and the complex was stable in distillated water and ethanol. Secondary, the PFD-iron complex reduced the amount of produced by xanthine oxidase/hypoxanthine without inhibiting the enzyme activity. Thirdly, it also reduced the amount of released from phorbor ester-stimulated human neutrophils. PFD alone showed few such effects. These results suggest the possibility that the scavenging effect of the PFD-iron complex contributes to the anti-fibrotic action of PFD used for treating idiopathic pulmonary fibrosis.     

Applicability of new spin trap agent, 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide, in biological system

Electron spin resonance using spin-trapping is a useful technique for detecting direct reactive oxygen species, such as superoxide (). However, the widely used spin trap 2,2-dimethyl-3,4-dihydro-2H-pyrrole N-oxide (DMPO) has several fundamental limitations in terms of half-life and stability. Recently, the new spin trap 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO) was developed by us. We evaluated the biological applicability of DPhPMPO to analyze in both cell-free and cellular systems. DPhPMPO had a larger rate constant for and formed more stable spin adducts for than DMPO in the xanthine/xanthine oxidase (X/XO) system. In the phorbol myristate acetate-activated neutrophil system, the detection potential of DPhPMPO for was significantly higher than that of DMPO (k_DMPO = 13.95 M⁻¹ s⁻¹, k_DPhPMPO = 42.4 M⁻¹ s⁻¹). These results indicated that DPhPMPO is a potentially good candidate for trapping in a biological system.     

Structural changes in the O-decay accelerated mutants of pharaonis phoborhodopsin

pharaonis phoborhodopsin ( ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronomonas pharaonis. The X-ray crystallographic structure of ppR is very similar to those of the ion-pumping rhodopsins, bacteriorhodopsin (BR) and halorhodopsin (hR). However, the decay processes of the photocycle intermediates such as M and O are much slower than those of BR and hR, which is advantageous for the sensor function of ppR. Iwamoto et al. previously found that, in a quadruple mutant (P182S/P183E/V194T/T204C; denoted as SETC) of ppR, the decay of the O intermediate was accelerated by approximately 100 times ( t 1/2 approximately 6.6 ms vs 690 ms for the wild type of ppR), being almost equal to that of BR (Iwamoto, M., et al. (2005) Biophys. J. 88, 1215-1223). The mutated residues are located on the extracellular surface (Pro182, Pro183, and Val194) and near the Schiff base (Thr204). The present Fourier-transform infrared (FTIR) spectroscopy of SETC revealed that protein structural changes in the K and M states were similar to those of the wild type. In contrast, the ppR O minus ppR infrared difference spectra of SETC are clearly different from those of the wild type in amide-I (1680-1640 cm (-1)) and S-H stretching (2580-2520 cm (-1)) vibrations. The 1673 (+) and 1656 (-) cm (-1) bands newly appear for SETC in the frequency region typical for the amide-I vibration of the alpha II- and alpha I-helices, respectively. The intensities of the 1673 (+) cm (-1) band of various mutants were well correlated with their O-decay half-times. Since the alpha II-helix possesses a considerably distorted structure, the result implies that distortion of the helix is required for fast O-decay. In addition, the characteristic changes in the S-H stretching vibration of Cys204 were different between SETC and T204C, suggesting that structural change near the Schiff base was induced by mutations of the extracellular surface. We conclude that the lifetime of the O intermediate in ppR is regulated by the distorted alpha-helix and strengthened hydrogen bond of Cys204.     

Association of pharaonis phoborhodopsin with its cognate transducer decreases the photo-dependent reactivity by water-soluble reagents of azide and hydroxylamine

pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. In halobacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm. In the present work, the truncated transducer, t-Htr, was used which interacts with ppR [Sudo et al. (2001) Photochem. Photobiol. 74, 489-494]. Two water-soluble reagents, hydroxylamine and azide, reacted both with the transducer-free ppR and with the complex ppR/t-Htr (the complex between ppR and its truncated transducer). In the dark, the bleaching rates caused by hydroxylamine were not significantly changed between transducer-free ppR and ppR/t-Htr, or that of the free ppR was a little slower. Illumination accelerated the bleach rates, which is consistent with our previous conclusion that the reaction occurs selectively at the M-intermediate, but the rate of the complex was about 7.4-fold slower than that of the transducer-free ppR. Azide accelerated the M-decay, and its reaction rate of ppR/t-Htr was about 4.6-fold slower than free ppR. These findings suggest that the transducer binding decreases the water accessibility around the chromophore at the M-intermediate. Its implication is discussed.     

Internal water molecules of pharaonis phoborhodopsin studied by low-temperature infrared spectroscopy.

In the Schiff base region of bacteriorhodopsin (BR), a light-driven proton-pump protein, three internal water molecules are involved in a pentagonal cluster structure. These water molecules constitute a hydrogen-bonding network consisting of two positively charged groups, the Schiff base and Arg82, and two negatively charged groups, Asp85 and Asp212. Previous infrared spectroscopy of BR revealed stretching vibrations of such water molecules under strong hydrogen-bonding conditions using spectral differences in D2O and D2(18O) [Kandori and Shichida (2000) J. Am. Chem. Soc. 122, 11745-11746]. The present study extends the infrared analysis to another archaeal rhodopsin, pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin-II, psR-II), involved in the negative phototaxis of Natronobacterium pharaonis. Despite functional differences between ppR and BR, similar spectral features of water bands were observed before and after photoisomerization of the retinal chromophore at 77 K. This implies that the structure and the structural changes of internal water molecules are similar between ppR and BR. Higher stretching frequencies of the bridged water in ppR suggest that the water-containing pentagonal cluster structure is considerably distorted in ppR. These observations are consistent with the crystallographic structures of ppR and BR. The water structure and structural changes upon photoisomerization of ppR are discussed here on the basis of their infrared spectra.     

Modern dinoflagellate cysts in hypertrophic coastal waters of Tokyo Bay, Japan

A survey of dinoflagellate resting cysts in surface sedimentsamples was carried out in Tokyo Bay, Japan, to document theirhorizontal distribution. At least 21 different cyst types werefound. Dominant cyst types allowed the recognition of assemblageswhich form three different dinoflagellate cyst communities:the innermost part of the Bay, the central area and the moutharea. In all stations in Tokyo Bay, heterotrophic dinoflagellatecysts always occupied more than half of the cyst populations.Cysts of Polykrikos schwartzii/kofoidii are the most abundantheterotrophic species. These assemblages may reflect highlynutrient-enriched (hypertrophic) and turbulent water conditions.Among the cyst types found were probable ellipsoidal cysts ofAlexandrium tamarense. This is the first record of toxic Alexandriumspecies cysts in Tokyo Bay sediments.