Search

Main conclusion: The Arabidopsis ppGpp-overproducing mutant indicates a larger biomass than wild type by modulated amino-acid metabolism under nitrogen-limiting conditions. The regulatory nucleotide, guanosine 3’, 5’-bis(pyrophosphate; ppGpp)—originally identified in Escherichia coli—controls gene expression and enzyme activities in the bacteria and plastids of plant cells. We recently reported that the ppGpp over-producing mutant of Arabidopsis thaliana had a larger shoot weight than wild type (WT), especially under nutrient-deficient conditions. However, the mechanisms behind the influence of ppGpp on plant growth and biomass remain elusive. To understand the impact of the ppGpp accumulation on plant growth, we characterized metabolic changes in the ppGpp-overproducing mutant upon transition from nitrogen-rich to nitrogen-limiting concentrations. We found that the fresh weight of the mutant was significantly larger than WT when the total nitrogen source (KNO3 and NH4NO3) concentration was below 0.9 mM. When the nitrogen content in the medium decreased, aromatic and branched-chain amino acids increased in WT due to accelerated protein degradation and/or attenuated protein synthesis. These amino-acid levels in the ppGpp over-accumulating mutant decreased upon nitrogen deficiency. The results suggest that the ppGpp-overaccumulation affects amino-acid and protein homeostasis and facilitates growth under nitrogen-limiting conditions. [ABSTRACT FROM AUTHOR]

Guanosine 3′,5′-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the evidence of the existence and function of the ppGpp-dependent stringent response in animals. Ito et al. succeed in detecting guanosine tetraphosphate (ppGpp) in measurable levels in metazoan, specifically in Drosophila. They further demonstrate that the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), is necessary, at least in certain conditions, to maintain low ppGpp levels, hence providing insights into the role of Mesh1 as a ppGpp hydrolase in vivo. [ABSTRACT FROM AUTHOR]

Precise control of the proton concentration gradient across thylakoid membranes (ΔpH) is essential for photosynthesis and its regulation because the gradient contributes to the generation of the proton motive force used for ATP synthesis and also for the fast and reversible induction of non-photochemical quenching (NPQ) to avoid photoinhibition and photodamage. However, the regulatory mechanism(s) controlling ΔpH in response to fluctuating light has not been fully elucidated. We previously described a new NPQ-regulatory chloroplastic protein, Fluctuating-Light-Acclimation Protein1 (FLAP1), which is important for plant growth and modulation of ΔpH under fluctuating light conditions. For this report, we further characterized FLAP1 activity by individually crossing an Arabidopsis flap1 mutant with npq4 and pgr5 plants; npq4 is defective in PsbS-dependent NPQ, and pgr5 is defective in induction of steady-state proton motive force (pmf) and energy-dependent quenching (qE). Both npq4 and npq4 flap1 exhibited similar NPQ kinetics and other photosynthetic parameters under constant or fluctuating actinic light. Conversely, pgr5 flap1 had recovered NPQ, photosystem II quantum yield and growth under fluctuating light, each of which was impaired in pgr5. Together with other data, we propose that FLAP1 activity controls proton homeostasis under steady-state photosynthesis to manipulate luminal acidification levels appropriately to balance photoprotection and photochemical processes. [ABSTRACT FROM AUTHOR]

The proton motive force (PMF) across the chloroplast thylakoid membrane that is generated by electron transport during photosynthesis is the driving force for ATP synthesis in plants. The PMF mainly arises from the oxidation of water in photosystem II and from electron transfer within the cytochrome b6f complex. There are two electron transfer pathways related to PMF formation: linear electron flow and cyclic electron flow. Proton gradient regulation 5 (PGR5) is a major component of the cyclic electron flow pathway, and the Arabidopsis pgr5 mutant shows a substantial reduction in the PMF. How the PGR5-dependent cyclic electron flow contributes to ATP synthesis has not, however, been fully delineated. In this study, we monitored in vivo ATP levels in Arabidopsis chloroplasts in real time using a genetically encoded bioluminescence-based ATP indicator, Nano-lantern(ATP1). The increase in ATP in the chloroplast stroma of pgr5 leaves upon illumination with actinic light was significantly slower than in wild type, and the decrease in ATP levels when this illumination stopped was significantly faster in pgr5 leaves than in wild type. These results indicated that PGR5-dependent cyclic electron flow around photosystem I helps to sustain the rate of ATP synthesis, which is important for growth under fluctuating light conditions. [ABSTRACT FROM AUTHOR]

Photoreceptor proteins have been used to study how protein conformational changes are induced by alterations in their environments and how their signals are transmitted to downstream factors to dictate physiological responses. These proteins are attractive models because their signal transduction aspects and structural changes can be precisely regulated in vivo and in vitro based on light intensity. Among the known photoreceptors, members of the blue light-using flavin (BLUF) protein family have been well characterized with regard to how they control various light-dependent physiological responses in several microorganisms. Herein, we summarize our current understanding of their photoactivation and signal-transduction mechanisms. For signal transduction, we review recent studies concerning how the BLUF protein, PixD, transmits a light-induced signal to its downstream factor, PixE, to modulate phototaxis of the cyanobacterium Synechocystis sp. PCC6803. [ABSTRACT FROM AUTHOR]

The regulatory nucleotides, guanosine 5′-triphosphate 3′-diphosphate (pppGpp) and guanosine 5′-diphosphate 3′-diphosphate (ppGpp), were originally identified in Escherichia coli, and control a large set of gene expression and enzyme activities. The (p)ppGpp-dependent control of cell activities is referred to as the stringent response. A growing number of (p)ppGpp synthase/hydrolase homologs have been identified in plants, which are localized in plastids in Arabidopsis thaliana. We recently reported that the Arabidopsis mutant overproducing ppGpp in plastids showed dwarf chloroplasts, and transcript levels in the mutant plastids were significantly suppressed. Furthermore, the mutant showed more robust growth than the wild type (WT), especially under nutrient-deficient conditions, although the mechanisms are unclear. To better understand the impact of the ppGpp accumulation on plant responses to nutrient deficiency, photosynthetic activities and metabolic changes in the ppGpp-overproducing mutant were characterized here. Upon transition to the nitrogen-deficient conditions, the mutant showed reduction of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) contents, and effective and maximum quantum yield of photosystem II compared with WT. The mutant also showed more obvious changes in key metabolite levels including some amino acid contents than WT; similar metabolic change is known to be critical for plants to maintain carbon-nitrogen balance in their cells. These results suggest that artificially overproducing ppGpp modulates the organelle functions that play an important role in controlling photosynthetic performance and metabolite balance during nitrogen starvation. [ABSTRACT FROM AUTHOR]

Main conclusion: Respective contributions of PGR5- and NDH-dependent cyclic electron flows around photosystem I for generating the proton gradient across the thylakoid membrane are ~30 and ~5%. The proton concentration gradient across the thylakoid membrane (ΔpH) produced by photosynthetic electron transport is the driving force of ATP synthesis and non-photochemical quenching. Two types of electron transfer contribute to ΔpH formation: linear electron flow (LEF) and cyclic electron flow (CEF, divided into PGR5- and NDH-dependent pathways). However, the respective contributions of LEF and CEF to ΔpH formation are largely unknown. We employed fluorescence quenching analysis with the pH indicator 9-aminoacridine to directly monitor ΔpH formation in isolated chloroplasts of Arabidopsis mutants lacking PGR5- and/or NDH-dependent CEF. The results indicate that ΔpH formation is mostly due to LEF, with the contributions of PGR5- and NDH-dependent CEF estimated as only ~30 and ~5%, respectively. [ABSTRACT FROM AUTHOR]

The nucleotide (p)ppGpp is a second messenger that controls the stringent response in bacteria. The stringent response modifies expression of a large number of genes and metabolic processes and allows bacteria to survive under fluctuating environmental conditions. Recent genome sequencing analyses have revealed that genes responsible for the stringent response are also found in plants. These include (p)ppGpp synthases and hydrolases, RelA/SpoT homologs (RSHs), and the pppGpp-specific phosphatase GppA/Ppx. However, phylogenetic relationship between enzymes involved in bacterial and plant stringent responses is as yet generally unclear. Here, we investigated the origin and evolution of genes involved in the stringent response in plants. Phylogenetic analysis and primary structures of RSH homologs from different plant phyla (including Embryophyta, Charophyta, Chlorophyta, Rhodophyta and Glaucophyta) indicate that RSH gene families were introduced into plant cells by at least two independent lateral gene transfers from the bacterial Deinococcus-Thermus phylum and an unidentified bacterial phylum; alternatively, they were introduced into a proto-plant cell by a lateral gene transfer from the endosymbiotic cyanobacterium followed by gene loss of an ancestral RSH gene in the cyanobacterial linage. Phylogenetic analysis of gppA/ ppx families indicated that plant gppA/ ppx homologs form an individual cluster in the phylogenetic tree, and show a sister relationship with some bacterial gppA/ ppx homologs. Although RSHs contain a plastidial transit peptide at the N terminus, GppA/Ppx homologs do not, suggesting that plant GppA/Ppx homologs function in the cytosol. These results reveal that a proto-plant cell obtained genes for the stringent response by lateral gene transfer events from different bacterial phyla and have utilized them to control metabolism in plastids and the cytosol. [ABSTRACT FROM AUTHOR]

Recently, a bacterial second messenger, guanosine 5′-diphosphate 3′-diphosphate (ppGpp), has been detected in chloroplasts. However, because ppGpp concentration in plants is much lower than that in bacteria, detailed analysis of ppGpp in plants has not been performed. A highly sensitive quantification method is required for further characterization of ppGpp function in chloroplasts. Here, we report a new method that allows for the highly sensitive and selective high-throughput quantification of ppGpp by ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI). This method requires only ~100 mg of plant tissue for ppGpp quantification. We used this method to measure ppGpp levels in Arabidopsis thaliana under different light conditions. A. thaliana accumulated ppGpp during dark periods. This method will be helpful to further characterize the stringent response in higher plants. [ABSTRACT FROM AUTHOR]

Non-alcoholic steatohepatitis (NASH) is the progressive phenotype of non-alcoholic fatty liver disease associated with the metabolic syndrome. The existence of autoimmune features in NASH has been reported, but its significance remains unclear. We herein report the autoantibody profile of 54 patients with histologically proven NASH and further determined the development of autoimmunity in three different murine NASH models (monosodium glutamate, CDAA (choline-deficient l-amino acid-defined), and TSOD (Tsumura Suzuki, Obese Diabetes)) at 48 weeks of age. Forty-eight percent (26/54) of NASH cases were positive for antinuclear (ANA) or antimitochondrial antibody and manifested histological signs of overlap with autoimmune hepatitis and primary biliary cirrhosis, respectively. These patients were significantly older (60 ± 10 versus 50 ± 16 years), more frequently women (81 % versus 43 %), and with more severe portal inflammatory infiltrate compared with patients without autoimmunity. In one third of mice, regardless of the model, we observed a marked lymphoid infiltrate with non-suppurative cholangitis, and several cases were ANA-positive, but none AMA-positive. Our data suggest that autoimmunity may share some pathogenetic traits with the chronic inflammation of NASH, possibly related to advanced age. [ABSTRACT FROM AUTHOR]

Jasmonates are phytohormones derived from oxygenated fatty acids that regulate a broad range of plant defense and developmental processes. In Arabidopsis, hypocotyl elongation under various light conditions was suppressed by exogenously supplied methyl jasmonate (MeJA). Moreover, this suppression by MeJA was particularly effective under red light condition. Mutant analyses suggested that SCF-mediated proteolysis was involved in this function. However, MeJA action still remained in the coi1 mutant, and (+)-7-iso-JA-L-Ile, a well-known active form of jasmonate, had a weaker effect than MeJA under the red light condition, suggesting that unknown signaling pathway are present in MeJA-mediated inhibition of hypocotyl elongation. EMS mutant screening identified two MeJA-insensitive hypocotyl elongation mutants, jasmonate resistance long hypocotyl 1 ( jal1) and jal36, which had mutations in the phytochrome B ( PHYB) gene. These analyses suggested that inhibition of hypocotyl elongation by jasmonates is enhanced under red light in phyB dependent manner. [ABSTRACT FROM AUTHOR]

To assist physicians in recognizing the potentially fatal onset of symptoms in cases of fulminant bacterial infection, we analyzed 11 autopsy cases of such infection (four caused by Streptococcus pneumoniae, four by S. pyogenes, one by S. dysgalactiae subsp. equisimilis, one by Staphylococcus aureus, and one by Vibrio vulnificus). Clinicohistopathologic features were evaluated. All patients experienced sudden onset of hypotension and multiple organ failure, leading to unexpected death. Blood culture confirmed bacteremia. The main chief complaints were gastrointestinal symptoms (45%) and limb pain (36%). All had an underlying chronic illness (82%), e.g., a hematologic disorder (36.3%) or liver cirrhosis (27.2%). Necrotizing fasciitis occurred in only 55% of cases, with none involving pneumococcal infection. Laboratory tests typically showed C-reactive protein elevation but without leukocytosis, indicating a high-level inflammatory state. In ten cases, death was attributed to circulatory collapse due to sepsis; severe pulmonary congestion and hemorrhage were present in these cases. The onset of fulminant bacterial infection depends on both virulence of the bacterium and status of the host defense system. [ABSTRACT FROM AUTHOR]

Neuroendocrine neoplasms in hepatobiliary organs are very rare, but several cases of mixed adenoneuroendocrine carcinoma (MANEC) have been reported. In this study, we characterized the neuroendocrine component of biliary MANEC. A total of 274 cases of biliary cancer including 17 intrahepatic cholangiocarcinomas (CCs), 15 hepatic hilar CCs without preceding hepatobiliary disease, 55 hepatic hilar CCs with hepatolithiasis, 49 gallbladder cancers, 53 extrahepatic CCs, and 85 hepatocellular carcinomas were examined for a neuroendocrine component using immunohistochemistry with neuroendocrine markers (chromogranin A and synaptophysin). In the MANEC cases, in addition to a close histological examination, the proliferative activity and the expression of somatostatin receptor 2A were also evaluated. In addition to an ordinary adenocarcinoma, a neuroendocrine component occupying more than 30% of the entire tumor was also found in 4% (2/55 cases) of hepatic hilar cholangiocarcinomas with hepatolithiasis, 10% (5/49 cases) of gallbladder cancers, and 4% (2/53 cases) of extrahepatic cholangiocarcinomas, but not in the intrahepatic cholangiocarcinomas, hilar cholangiocarcinomas without preceding hepatobiliary disease, and hepatocellular carcinomas. Two cases were positive for somatostatin receptor 2A. The adenocarcinoma components were predominately located at the surface of the tumors, and the majority of stromal and vascular invasion and lymph node metastasis involved neuroendocrine components, showing the features of neuroendocrine tumor G2 or neuroendocrine carcinomas (NECs). NEC components showed higher proliferative activity on Ki67 immunostaining, compared to the adenocarcinoma components. Biliary MANECs are found in hepatic hilar cholangiocarcinomas with hepatolithiasis, gallbladder cancers, and extrahepatic cholangiocarcinomas and show a characteristic histology. Since the neuroendocrine component in biliary MANEC defines the prognosis, it is important to identify it and consider the indications for adjunctive therapy with somatostatin analogues. [ABSTRACT FROM AUTHOR]

photo-induced radical pair of FADH and Y8 and in BLUF protein SyPixD was studied by pulsed electron paramagnetic resonance (EPR) spectroscopy. Blue light illumination at 150 K for 30 min followed by cooling to 50 K during illumination induced the stable radical pair. The EPR signal has been characterized by a Pake doublet signal with complete S = 1 spin state. The radical pair was utilized as a probe to analyze the oligomer of SyPixD. The relative arrangement of PixD proteins in the complex was investigated by pulsed electron-electron double resonance (PELDOR) with the orientation selection. Based on the decameric structure in the crystal, the possible structure for the PELDOR results was discussed. [ABSTRACT FROM AUTHOR]

This study aimed to assess the protein level of inhibitor of growth gene 5 ( ING5) in head and neck squamous cell carcinoma (HNSCC) and to explore its roles in tumorigenesis and cancer progression. ING5 expression was assessed in 172 cases of HNSCC by immunohistochemistry using tissue microarray, and in 3 oral SCC cell lines by immunohistochemistry and Western blot. Expression of ING5 was compared with clinicopathological variables, TUNEL assay staining, and the expression of several tumorigenic markers. In addition, double immunofluorescence labeling was performed in order to analyze the colocalization of ING5 with p300 and p21. ING5 expression was primarily observed in the nuclei, but was also occasionally found in the cytoplasm of both SCC cell lines and tissue samples of HNSCC. Nuclear expression of ING5 in HNSCC was significantly lower than that of non-cancerous epithelium, and was positively correlated with a well-differentiated status. In contrast, cytoplasmic expression of ING5 was significantly increased in HNSCC, and was inversely correlated with a well-differentiated status and nuclear ING5 expression. In addition, nuclear expression of ING5 was positively correlated with p21 and p300 expression, and with the apoptotic index. In contrast, cytoplasmic expression of ING5 was negatively correlated with the expression of p300, p21, and PCNA. Although no statistical association was found between the expression of nuclear ING5 and mutant p53 in HNSCC, patients with high expression of nuclear ING5 tended to have converse prognoses when grouped according to mutant p53 expression. Our results suggest that a decrease in nuclear ING5 localization and cytoplasmic translocation are involved in tumorigenesis and tumor differentiation in HNSCC. Nuclear ING5 may modulate the transactivation of target genes, and may promote apoptosis and cell cycle arrest by interacting with the p300 and p21 proteins. ING5 may function as a tumor suppressor gene or oncogene tightly linked with p53 status, and may play an important role in the prognosis of HNSCC patients. Therefore, we propose that ING5 represents a novel potential molecular therapeutic target for HNSCC. [ABSTRACT FROM AUTHOR]

The stringent response is one of the most important regulatory systems in bacteria for adaptation to environmental stresses such as amino acid starvation. This response is mediated by an unusual nucleotide, guanosine 3′,5′-bis(pyrophosphate) (ppGpp), the levels of which are controlled by two enzymes, RelA and SpoT. ppGpp dramatically influences a broad range of physiological activities in bacterial cells, including transcription and translation as well as the enzymatic activities of several metabolic pathways. Recently, a growing number of RelA/SpoT homologues, designated RSH, have been identified in plants. Arabidopsis has four RSHs ( AtRSH1, AtRSH2, AtRSH3 and AtCRSH). In order to reveal the specific roles of the individual Arabidopsis RSHs, we have analyzed the ppGpp synthase activities of the four RSHs and characterized the expression pattern of the RSH genes. Results from the present and previous studies indicate that all four RSH proteins are targeted into plastids. Complementation analysis of E. coli mutants revealed that AtRSH2, AtRSH3 and AtCRSH, but not AtRSH1, have ppGpp synthetase activity. Promoter analysis using reporter gene fusions indicated that the four Arabidopsis RSH genes are expressed in green tissues and flowers, suggesting the involvement of RSH functions in chloroplast development and reproduction. We also observed that all RSH transcripts exhibit a diurnal rhythm, and that induction of AtRSH1 and AtRSH2 transcripts are responsive to several environmental stresses. These results suggest that expression of the four RSH genes is coordinately regulated and that the ppGpp-dependent plastid stringent response has certain roles in the physiology of higher plants. [ABSTRACT FROM AUTHOR]

The photosynthetic bacterium Rhodobacter sphaeroides ( R. sphaeroides) f. sp. denitrificans IL106 accumulates trehalose as the major organic osmoprotectant in response to a salt stress. An analysis of the R. sphaeroides 2.4.1 genome sequence revealed the presence of five different genes encoding enzymes belonging to three putative trehalose biosynthesis pathways (OtsA-OtsB, TreY-TreZ, and TreS). The function of the different pathways of trehalose was studied by characterizing strains defective in individual trehalose biosynthetic routes. A phenotypic comparison revealed that trehalose synthesis in R. sphaeroides f. sp. denitrificans IL106 is mediated mainly by the OtsA-OtsB pathway and, to some extent, by the TreY-TreZ pathway. Strains with the simultaneous inactivation of these two pathways were completely unable to synthesize trehalose. On the other hand, treS mutants showed an increase in the trehalose level. These results suggest that treS plays a role in trehalose degradation. In addition, treS was found to be important in reducing trehalose after osmotic stress was removed. In this report, we show that the strains that accumulate the most trehalose adapt to salt stress earlier. This is the first report of an organism using multiple pathways to synthesize trehalose solely for use as a compatible solute against salt stress. [ABSTRACT FROM AUTHOR]

We succeeded to create the genetically modified purple photosynthetic bacterium capable of synthesizing chlorophyll a. The results indicate that not only chlorophyll synthase, but also an enzyme for galactolipid synthesis and reaction center proteins are required for accumulating chlorophyll a. [ABSTRACT FROM AUTHOR]

Two nodules of hepatic adenomatous hyperplasia (AH) resembling focal nodular hyperplasia were found in two patients with cirrhosis or chronic active hepatitis. Imaging techniques suggested that the nodules were hepatocellular carcinoma. Pathological examination showed that the nodules (approximately 1.0 cm in diameter) were clearly demarcated from the surrounding liver tissue, and contained foci of scar-like fibrosis in the centre of the nodules. Microscopically, they contained portal tracts and fulfilled the criteria of AH. A large number of arteries were present in the central scarlike fibrosis as well as in the parenchyma of the nodules. There were foci of mildly atypical hepatocytes in one nodule but no cellular atypia in the other. Morphometric analysis showed that the cumulative luminal area of arteries per unit area was much greater in the nodules than in the extranodular liver tissues, while the cumulative luminal area of portal veins per unit area was much less in the nodules than in the extranodular liver tissues. Although the pathogenesis is unclear, these nodules might have developed through localized vascular changes associated with chronic liver disease, may have arisen from pre-existing arterial malformation, or may represent the early stages of angiogenesis in hepatocarcinogenesis. [ABSTRACT FROM AUTHOR]