Spatial variation of phosphorus fractions in bottom sediments and the potential contributions to eutrophication in shallow lakes

Spatial variation of phosphorus fractions in bottom sediment, pore water and overlying water in three shallow eutrophic lakes, Nishiura, Kitaura and Sotonasakaura, Japan, and the contributions of the fractional P to mobilization of phosphorus from sediment were examined in this study. The vertical distributions of dissolved inorganic phosphorus (DIP) concentrations in overlying and pore water differed with lake and sampling site. In particular, DIP was high in pore water in the surface layer of the sediment for the middle to downlake areas of Lake Kitaura. DIP release flux calculated from a gradient of the concentrations at the sediment–water interface was high compared with other sites. The distribution of fractional P content in sediments was highly variable. The citrate–dithionite–bicarbonate–non-reactive phosphorus (CDB–NRP) fraction, in particular, differed greatly among the three lakes. According to correlation in the ratios between CDB–NRP and loss on ignition, sediments of these lakes were classified in three clusters. The CDB–NRP fraction was suggested to play a role in DIP release from sediment. The possibility of nitrate concentration playing a role in the control of DIP release was considered.     

PARP1 gene knock-out increases resistance to retinal degeneration without affecting retinal function

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP.     

Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12

We investigated the effects of IL-18 on the development of CD8+ effector T cells in DBA/2 anti-BDF1 whole spleen cell MLC and compared the results with those of IL-12. Addition of IL-18 to the MLC resulted in a twofold increase in CD8/CD4 ratios compared with the control cultures when cells were expanded in IL-2-containing medium following MLC. Purified CD8+ T cells recovered from the IL-18-stimulated MLC produced 20- to 30-fold more IFN-gamma after secondary stimulation with C57BL/6 spleen cells or anti-CD3 mAb, and exhibited strong allospecific CTL activity. Neither IL-18 nor IL-18-supplemented culture supernatants from DBA/2 anti-BDF1 MLC induced type I CD8+ effector T cells when purified CD8+ T cells were used as responder cells in primary MLC. Furthermore, CD4+ T cell depletion from the responder cells abrogated the IL-18-induced increase in secondary IFN-gamma production by CD8+ T cells, suggesting that IL-18-induced type I effector CD8+ T cell development was CD4+ T cell dependent. In marked contrast, adding IL-12 to primary MLC decreased CD8/CD4 ratios by 50% and suppressed secondary IFN-gamma production and CTL activity by CD8+ T cells regardless of concentration, whereas Th1 development was promoted by IL-12. Moreover, both IL-12 and IL-18 efficiently induced type I CD8+ effector T cells in C57BL/6 anti-BDF1 MLC. These findings show that IL-18 plays an important role in the generation of type I CD8+ effector T cells, and further suggest that functional maturation of CD8+ T cells is differentially regulated by IL-18 and IL-12.     

Altered hydrogen bonding of Arg82 during the proton pump cycle of bacteriorhodopsin: a low-temperature polarized FTIR spectroscopic study

Light-driven proton transport in bacteriorhodopsin (BR) is achieved by dynamic rearrangement of the hydrogen-bonding network inside the membrane protein. Arg82 is located between the Schiff base region and proton release group, and has a major influence on the pK(a) values of these groups. It is believed that Arg82 changes its hydrogen-bonding acceptors during the pump cycle of BR, stages of which are correlated with proton movement along the transport pathway. In this study, we compare low-temperature polarized FTIR spectra of [eta(1,2)-(15)N]arginine-labeled BR in the 2750-2000 cm(-1) region with those of unlabeled BR for the K, L, M, and N intermediates. In the K-minus-BR difference spectra, (15)N-shifted modes were found at 2292 (-)/2266 (+) cm(-1) and at 2579 (-)/2567 (+) cm(-1). The former corresponds to strong hydrogen bonding, while the latter corresponds to very weak hydrogen bonding. Both N-D stretches probably originate from Arg82, the former oriented toward water 406 and the latter toward the extracellular side, and both hydrogen bonds are somewhat strengthened upon retinal photoisomerization. This perturbation of arginine hydrogen bonding is entirely relaxed in the L intermediate where no (15)N-isotope shifts are observed in the difference spectrum. In the M intermediate, the frequency is not significantly altered from that in BR. However, the polarized FTIR spectra strongly suggest that the dipolar orientation of the strongly hydrogen bonded N-D group of Arg82 is changed from perpendicular to parallel to the membrane plane. Such a change is presumably related to the motion of the Arg82 side chain from the Schiff base region to the extracellular proton release group. Additional bands corresponding to weak hydrogen bonding were observed in both the M-minus-BR and N-minus-BR spectra. Changes in hydrogen-bonding structures involving Arg82 are discussed on the basis of these FTIR observations.     

Membrane translocation mechanism of the antimicrobial peptide buforin 2

The antimicrobial peptide magainin 2 isolated from the skin of the African clawed frog Xenopus laevis crosses lipid bilayers by transiently forming a peptide-lipid supramolecular complex pore inducing membrane permeabilization and flip-flop of membrane lipids [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In contrast, the antimicrobial peptide buforin 2 discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans efficiently crosses lipid bilayers without inducing severe membrane permeabilization or lipid flip-flop, and the Pro(11) residue plays a key role in this unique property [Kobayashi, S, Takeshima, K., Park, C. B., Kim, S. C., and Matsuzaki, K. (2000) Biochemistry 39, 8648-8654]. To elucidate the translocation mechanism, the secondary structure and the orientation of the peptide in lipid bilayers as well as the effects of the peptide concentration, the lipid composition, and the cis-trans isomerization of the Pro peptide bond on translocation efficiency were investigated. The translocation efficiencies of F10W-buforin 2 (BF2), P11A-BF2, and F5W-magainin 2 (MG2) across egg yolk L-alpha-phosphatidyl-DL-glycerol (EYPG)/egg yolk L-alpha-phosphatidylcholine (1/1) bilayers were dependent supralinearly on the peptide concentration, suggesting that the translocation mechanisms of these peptides are similar. The incorporation of the negative curvature-inducing lipid egg yolk L-alpha -phosphatidylethanolamine completely suppressed the translocation of BF2, indicating the induction of the positive curvature by BF2 on the membrane is related to the translocation process, similarly to MG2. In pure EYPG, where the repulsion between polycationic BF2 molecules is reduced, membrane permeabilization and coupling lipid flip-flop were clearly observed. Structural studies by use of Fourier transform infrared-polarized attenuated total reflection spectroscopy indicated that BF2 assumed distorted helical structures in EYPG/EYPC bilayers. A BF2 analogue with an alpha-methylproline, which fixed the peptide bond to the trans configuration, translocated similarly to the parent peptide, suggesting the cis-trans isomerization of the Pro peptide bond is not involved in the translocation process. These results indicate that BF2 crosses lipid bilayers via a mechanism similar to that of MG2. The presence of Pro(11) distorts the helix, concentrating basic amino acid residues in a limited amphipathic region, thus destabilizing the pore by enhanced electrostatic repulsion, enabling efficient translocation.     

The diversity of vacA and cagA genes of Helicobacter pylori in East Asia

It has been reported that Helicobacter pylori infection with the type I strain, which expresses the VacA and CagA antigens, is associated with duodenal ulcer. We examined the diversity of vacA and cagA genes in 143 isolates obtained from patients with duodenal ulcer or chronic gastritis in East Asia (two different areas of Japan, Fukui and Okinawa, and also in Hangzhou, China) by polymerase chain reaction (PCR) and sequence analysis. Diversities of cagA and vacA genes were detected in East Asia. The prevalence of cagA-positive H. pylori was significantly different between Fukui and Okinawa (P=0.0032). The prevalence of Western type CagA was significantly higher in Okinawa than in Fukui (P<0.0001). However, there was no significant association between the genotype of cagA and clinical outcome. In Japan, the predominant vacA genotype was s1c/m1b. In contrast, in Hangzhou, the predominant vacA genotype was s1c/m2, and they were all East Asian CagA-positive. These findings suggest that a distinct distribution of the vacA and cagA genotypes is present in East Asia, regardless of clinical outcome.     

Novel evidence suggesting Clostridium difficile is present in human gut microbiota more frequently than previously suspected

Prevalence rate of Clostridium difficile in healthy human adults is believed to be very low. Our RT-PCR system using glass powder, which can eliminate PCR inhibitors, detected C. difficile toxin B mRNA in 16 of 30 fecal samples (53.3%) from healthy human adults. In contrast, we failed to detect toxin B in the same fecal samples by PCR using DNA templates extracted with phenol-chloroform. Our results suggest that PCR inhibitors in feces carried through phenol-chloroform extraction procedure might suppress the sensitivity of PCR and that C. difficile is actually present in human gut microbiota more frequently than previously suspected.