Functional role of a putative carbonic anhydrase II-binding domain in the electrogenic Na+ -HCO₃- cotransporter NBCe1 expressed in Xenopus oocytes

The electrogenic Na+ -HCO?? cotransporter NBCe1 plays essential roles in the regulation of systemic and/or local pH. Homozygous inactivating mutations in NBCe1 cause proximal renal tubular acidosis associated with ocular abnormalities. We recently showed that defective membrane expression of NBCe1, caused by several mutations such as Delta65bp (S982NfsX4), is also associated with familial migraine. The Delta65bp mutant is quite unique in that it lacks a putative carbonic anhydrase (CA) II-binding domain but still shows an apparently normal transport activity in Xenopus oocytes. In this addendum, we show that the co-expression of CAII together with the wild-type NBCe1 or the Delta65bp mutant does not enhance the NBCe1 activities in oocytes. Moreover, a carbonic anhydrase inhibitor acetazolamide fails to inhibit the wild-type or the Delta65bp activities co-expressed with CAII. These results indicate that a bicarbonate transport metabolon proposed for the interaction between CAII and NBCe1 does not work at least in Xenopus oocytes.     

Critical role of neuropeptides B/W receptor 1 signaling in social behavior and fear memory

Neuropeptide B/W receptor 1 (NPBWR1) is a G-protein coupled receptor, which was initially reported as an orphan receptor, and whose ligands were identified by this and other groups in 2002 and 2003. To examine the physiological roles of NPBWR1, we examined phenotype of Npbwr1 ^−/− mice. When presented with an intruder mouse, Npbwr1 ^−/− mice showed impulsive contact with the strange mice, produced more intense approaches toward them, and had longer contact and chasing time along with greater and sustained elevation of heart rate and blood pressure compared to wild type mice. Npbwr1 ^−/− mice also showed increased autonomic and neuroendocrine responses to physical stress, suggesting that impairment of NPBWR1 leads to stress vulnerability. We also observed that these mice show abnormality in the contextual fear conditioning test. These data suggest that NPBWR1 plays a critical role in limbic system function and stress responses. Histological and electrophysiological studies showed that NPBWR1 acts as an inhibitory regulator on a subpopulation of GABAergic neurons in the lateral division of the CeA and terminates stress responses. These findings suggest important roles of NPBWR1 in regulating amygdala function during physical and social stress.     

Disease-associated single amino acid mutation in the calf-1 domain of integrin α3 leads to defects in its processing and cell surface expression

Integrin α3β1, a receptor for laminins, is involved in the structural and functional organization of epithelial organs, including the lung, kidney, and skin. Recently, a missense mutation that causes substitution of Arg628 with Pro (R628P) in the calf-1 domain of human α3 was shown to be associated with disorders of the lung, kidney, and skin. Here, we found that the R628P mutation leads to aberrations in the posttranslational processing of α3. Specifically, α3 with the R628P mutation showed hardly any cleavage at the calf-2 domain, which usually occurs in the Golgi apparatus during the delivery of de novo-synthesized α3. The mutant α3 retained the ability to associate with integrin β1, but not with the tetraspanin CD151, and the bound β1 was a partially glycosylated immature form, the maturation of which also takes place in the Golgi apparatus. Furthermore, the cell surface expression of the mutant protein was markedly reduced. These results suggest that the R628P mutation leads to a deficit in the transport of α3β1 from the ER to the Golgi apparatus. When Arg628 was mutated to Gln or Glu, instead of Pro, the resulting mutants did not display aberrations in processing or CD151 binding, indicating that the presence of Pro, rather than the absence of Arg, at amino acid residue 628 of α3 is important for the abnormalities in the R628P mutant. In support of this notion, a homology modeling analysis of the calf-1 domain of α3 showed that replacement with Pro, but not with Gln or Glu, caused partial disruption of the β-sheet structures. Furthermore, the ER-associated degradation of the R628P mutant was not enhanced compared with that of the wild-type protein, suggesting that the deficits in the posttranslational processing and cell surface expression of the R628P mutant are independent of the ER-associated degradation, but arise from the defect in its export from the ER. We conclude that the calf-1 domain is required for the transport of α3 from the ER to the Golgi apparatus to maintain the integrity of epithelial tissues, and hence the impairment of the calf-1 domain by the R628P mutation leads to severe diseases of the kidneys, lungs, and skin.     

Characterization of antifungal activity of the GH-46 subclass III chitosanase from Bacillus circulans MH-K1

We characterized the antifungal activity of the Bacillus circulans subclass III MH-K1 chitosanase (MH-K1 chitosanase), which is one of the most intensively studied glycoside hydrolases (GHs) that belong to GH family 46. MH-K1 chitosanase inhibited the growth of zygomycetes fungi, Rhizopus and Mucor, even at 10 pmol (0.3 μg)/ml culture probably via its fungistatic effect. The amino acid substitution E37Q abolished the antifungal activity of MH-K1 chitosanase, but retained binding to chitotriose. The E37Q mutant was fused with green fluorescent protein (GFP) at its N-terminus and proved to act as a chitosan probe in combination with wheat-germ agglutinin (WGA), which is a chitin-specific binding lectin. The GFP-fused MH-K1 chitosanase mutant E37Q (GFP-E37Q) bound clearly to the hyphae of the Rhizopus and Mucor strains, indicating the presence of chitosan. In contrast, Cy5-labelled WGA (Cy5-WGA), but not GFP-E37Q, stained the hyphae of non-zygomycetes species, i.e. Fusarium oxysporum, Penicillium expansum, and Aspergillus awamori. When the mycelia of Rhizopus oryzae were treated with wild type MH-K1 chitosanase, they could not bind to GFP-E37Q but were stained instead by Cy5-WGA. We conclude that chitin is covered by chitosan in the cell walls of R. oryzae.     

Interleukin-1 and Tumor Necrosis Factor-α Trigger Restriction of Hepatitis B Virus Infection via a Cytidine Deaminase Activation-induced Cytidine Deaminase (AID)

Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.     

Transformation of Thylakoid Membranes during Differentiation from Vegetative Cell into Heterocyst Visualized by Microscopic Spectral Imaging

Some filamentous cyanobacteria carry out oxygenic photosynthesis in vegetative cells and nitrogen fixation in specialized cells known as heterocysts. Thylakoid membranes in vegetative cells contain photosystem I (PSI) and PSII, while those in heterocysts contain predominantly PSI. Therefore, the thylakoid membranes change drastically when differentiating from a vegetative cell into a heterocyst. The dynamics of these changes have not been sufficiently characterized in situ. Here, we used time-lapse fluorescence microspectroscopy to analyze cells of Anabaena variabilis under nitrogen deprivation at approximately 295 K. PSII degraded simultaneously with allophycocyanin, which forms the core of the light-harvesting phycobilisome. The other phycobilisome subunits that absorbed shorter wavelengths persisted for a few tens of hours in the heterocysts. The whole-thylakoid average concentration of PSI was similar in heterocysts and nearby vegetative cells. PSI was best quantified by selective excitation at a physiological temperature (approximately 295 K) under 785-nm continuous-wave laser irradiation, and detection of higher energy shifted fluorescence around 730 nm. Polar distribution of thylakoid membranes in the heterocyst was confirmed by PSI-rich fluorescence imaging. The findings and methodology used in this work increased our understanding of how photosynthetic molecular machinery is transformed to adapt to different nutrient environments and provided details of the energetic requirements for diazotrophic growth.The most essential pigment-protein complexes for oxygenic photosynthesis are PSI and PSII, which are embedded in the thylakoid membranes of chloroplasts and cyanobacteria. Cooperation between PSI and PSII achieves light-driven noncyclic electron transport from the oxidative splitting of water to the reduction of ferredoxin and is accompanied by the generation of a proton gradient for ATP synthesis. Phycobilisomes (PBS), another pigment-protein complex, are attached to the stromal side of the thylakoid membrane in cyanobacteria and red algae; they work as light-harvesting antennae to transfer electronic excitation energy mainly to PSII and, in some cases, to PSI (Gantt 1994). The integration of these pigment-protein complexes changes in response to light conditions, nutrient status, and developmental stage (Fujita et al., 1994; Grossman et al., 1994; Wolk et al., 1994).Some cyanobacteria, including Anabaena variabilis, are able to grow diazotrophically using the nitrogen-fixing enzyme nitrogenase. Because nitrogenase is sensitive to oxygen, oxygenic photosynthesis is not readily compatible with diazotrophic growth. When this filamentous cyanobacterium is grown under fixed nitrogen-deficient conditions, approximately 1 in 10 to 20 vegetative cells differentiates into a heterocyst, in which oxygenic photosynthesis is suppressed and nitrogenase becomes operative (Haselkorn, 1978; Wolk et al., 1994). The other vegetative cells continue oxygenic photosynthesis. The differentiation of heterocysts from chains of vegetative cells has been studied extensively (Golden and Yoon, 2003; Toyoshima et al., 2010). The abundances of PSII and PBS decrease during the transition. PSI appears to persist in the heterocyst to produce ATP by cyclic electron transport, because nitrogen fixation demands a large amount of ATP (Wolk et al., 1994). However, the mechanisms by which PBS and PSII are degraded during heterocyst differentiation remain unclear, and whether the amount of PSI per cell changes is unknown.The PBS of A. variabilis contain three types of phycobiliproteins, pigment-protein complexes with distinct absorption and fluorescence spectra. The core PBS contains allophycocyanin (APC), which absorbs around 654 nm (Ying and Xie, 1998); the core is most closely connected to PSII. More peripherally in the PBS, the so-called rod contains phycoerythrocyanin (PEC) and phycocyanin (PC), which absorb maximally around 575 and 604 to 620 nm, respectively (Switalski and Sauer, 1984; Zhang et al., 1998). Photon energy is absorbed by PEC, then transferred downhill through PC and APC and finally to PSII. The structure of PBS is probably optimized not only for efficient energy transfer to PSII and/or PSI but also for transformation and/or degradation under various nutrient conditions. However, the order in which these subunits degrade during heterocyst differentiation remains unknown. One strategy to address this question is to isolate heterocysts at several stages during differentiation and quantify their proteomes via mass spectrometry. However, such isolation procedures work well only when there is a good understanding of the properties of cells at different stages. Ideally, noninvasive methods should be used to understand changes in the integrity of PSII and PBS in intact cells in filaments.In principle, time-lapse microscopic observations can clarify the process of differentiation from a vegetative cell into a mature heterocyst. Spectral microscopy is an ideal tool to analyze physiological state and/or amounts of pigment-protein complexes under various conditions. Acquiring microscopic fluorescence spectra of individual cells is a natural extension of laser scanning confocal fluorescence microscopy, which has been applied to several types of cyanobacterial cells, including heterocysts (Peterson et al., 1981; Ying et al., 2002; Wolf and Schüssler, 2005; Kumazaki et al., 2007; Vermaas et al., 2008; Sukenik et al., 2009; Bordowitz and Montgomery, 2010; Collins et al., 2012, Sugiura and Itoh, 2012). Microscopic fluorescence spectra reflect the concentration of pigment-protein complexes and the energy transfer dynamics between photosynthetic pigments. However, to date, there have been no thorough time-lapse investigations of the fluorescence spectra of heterocysts and vegetative cells during the differentiation process.In this study, we investigated the dynamic changes in thylakoid membranes of A. variabilis during heterocyst differentiation. Our unique microscopic system can acquire fluorescence spectra from an entire linearly illuminated region with about 2-nm wavelength resolution in a single exposure (Kumazaki et al., 2007). Heterocyst formation was induced by transferring vegetative cell filaments from fixed-nitrogen-sufficient incubation medium to nitrogen-deprived medium. We conducted long-term observations (60–96 h) on identical filaments. Another unique feature of our setup is that it uses a near-infrared (NIR) excitation laser source. Our previous microspectroscopic study of chloroplasts of a higher plant, maize (Zea mays), and a green alga (Parachlorella kessleri) showed that continuous wave (CW) laser light emitting at 785 to 820 nm excited PSI with high selectivity under the one-photon excitation (OPE) mode. This enabled us to observe highly PSI-rich fluorescence spectra and images with signals around 710 to 740 nm, even at approximately 295 K (Hasegawa et al., 2010, 2011). We used this technique to quantify PSI in individual heterocysts compared with its parental and contiguous vegetative cells. Pigment fluorescence under OPE qualitatively differed from that under two-photon excitation (TPE) using a pulsed NIR laser (typically achieved with picosecond or femtosecond pulses), because TPE using 800 to 830 nm resulted in spectra with contributions from PBS, PSII, and PSI, as typically observed by visible light excitation (Kumazaki et al., 2007; Hasegawa et al., 2010, 2011). The advantages of our microscopic system are the high wavelength resolution and coverage of the entire fluorescence spectrum, the availability of fluorescence spectra at several differentiation stages, and the multiple excitation modes with different selectivities for pigment-protein complexes. Together, these analyses allowed us to characterize spectral decomposition and to understand the time dependence of different pigment-protein complexes, even at a physiological temperature. Microscopic absorption spectra were also obtained from single cells. These data were tentatively used to estimate the absolute concentrations of PSI and PSII in heterocysts and vegetative cells.     

Variation of the Virus-Related Elements within Syntenic Genomes of the Hyperthermophilic Archaeon Aeropyrum

The increasing number of genome sequences of archaea and bacteria show their adaptation to different environmental conditions at the genomic level. Aeropyrum spp. are aerobic and hyperthermophilic archaea. Aeropyrum camini was isolated from a deep-sea hydrothermal vent, and Aeropyrum pernix was isolated from a coastal solfataric vent. To investigate the adaptation strategy in each habitat, we compared the genomes of the two species. Shared genome features were a small genome size, a high GC content, and a large portion of orthologous genes (86 to 88%). The genomes also showed high synteny. These shared features may have been derived from the small number of mobile genetic elements and the lack of a RecBCD system, a recombinational enzyme complex. In addition, the specialized physiology (aerobic and hyperthermophilic) of Aeropyrum spp. may also contribute to the entire-genome similarity. Despite having stable genomes, interference of synteny occurred with two proviruses, A. pernix spindle-shaped virus 1 (APSV1) and A. pernix ovoid virus 1 (APOV1), and clustered regularly interspaced short palindromic repeat (CRISPR) elements. Spacer sequences derived from the A. camini CRISPR showed significant matches with protospacers of the two proviruses infecting A. pernix, indicating that A. camini interacted with viruses closely related to APSV1 and APOV1. Furthermore, a significant fraction of the nonorthologous genes (41 to 45%) were proviral genes or ORFans probably originating from viruses. Although the genomes of A. camini and A. pernix were conserved, we observed nonsynteny that was attributed primarily to virus-related elements. Our findings indicated that the genomic diversification of Aeropyrum spp. is substantially caused by viruses.     

Replication‐coupled passive DNA demethylation for the erasure of genome imprints in mice

Genome‐wide DNA demethylation, including the erasure of genome imprints, in primordial germ cells (PGCs) is a critical first step to creating a totipotent epigenome in the germ line. We show here that, contrary to the prevailing model emphasizing active DNA demethylation, imprint erasure in mouse PGCs occurs in a manner largely consistent with replication‐coupled passive DNA demethylation: PGCs erase imprints during their rapid cycling with little de novo or maintenance DNA methylation potential and no apparent major chromatin alterations. Our findings necessitate the re‐evaluation of and provide novel insights into the mechanism of genome‐wide DNA demethylation in PGCs.