Phosphatidylinositol 3-kinase plays a vital role in regulation of rice seed vigor via altering NADPH oxidase activity

Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination.     

NADPH oxidase plays a central role in cone cell death in retinitis pigmentosa

Retinitis pigmentosa (RP) is a collection of diseases in which rod photoreceptors die from a variety of mutations. After rods die, the level of tissue oxygen in the outer retina becomes elevated and there is progressive oxidative damage to cones that ultimately triggers apoptosis. In this study, we investigated the hypothesis that NADPH oxidase (Nox) and/or xanthine oxidase serve as critical intermediaries between increased tissue oxygen and the generation of excessive reactive oxygen species that cause oxidative damage to cones. Apocynin, a blocker of Nox, but not allopurinol, a blocker of xanthine oxidase, markedly reduced the superoxide radicals visualized by hydroethidine in the outer retina in the retinal degeneration-1 ( rd1 ^+/+ ) model of RP. Compared to rd1 ^+/+ mice treated with vehicle, those treated with apocynin, but not those treated with allopurinol, had significantly less oxidative damage in the retina measured by ELISA for carbonyl adducts. Apocynin-treated, but not allopurinol-treated, rd1 ^+/+ mice had preservation of cone cell density, increased mRNA levels for m- and s-cone opsin, and increased mean photopic b-wave amplitude. In Q344ter mice, a model of dominant RP in which mutant rhodopsin is expressed, apocynin treatment preserved photopic electroretinogram b-wave amplitude compared to vehicle-treated controls. These data indicate that Nox, but not xanthine oxidase, plays a critical role in generation of the oxidative stress that leads to cone cell death in RP and inhibition of Nox provides a new treatment strategy.     

The role of oxidative stress and NADPH oxidase in cerebrovascular disease

The study of reactive oxygen species (ROS) and oxidative stress remains a very active area of biological research, particularly in relation to cellular signaling and the role of ROS in disease. In the cerebral circulation, oxidative stress occurs in diverse forms of disease and with aging. Within the vessel wall, ROS produce complex structural and functional changes that have broad implications for regulation of cerebral perfusion and permeability of the blood-brain barrier. These oxidative-stress-induced changes are thought to contribute to the progression of cerebrovascular disease. Here, we highlight recent findings in relation to oxidative stress in the cerebral vasculature, with an emphasis on the emerging role for NADPH oxidases as a source of ROS and the role of ROS in models of disease.     

Arg305 of Streptomyces L-glutamate oxidase plays a crucial role for substrate recognition

Recently, we have solved the crystal structure of L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward L-glutamate. By a docking simulation using L-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and Trp564 as candidates of the residues associating with recognition of L-glutamate. The activity of LGOX toward L-glutamate was significantly reduced by substitution of selected residues with Ala. However, the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various L-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the interaction of enzyme and side chain of substrate.     

The cell membrane plays a crucial role in survival of bacteria and archaea in extreme environments

The cytoplasmic membrane of bacteria and archaea determine to a large extent the composition of the cytoplasm. Since the ion and in particular the proton and/or the sodium ion electrochemical gradients across the membranes are crucial for the bioenergetic conditions of these microorganisms, strategies are needed to restrict the permeation of these ions across their cytoplasmic membrane. The proton and sodium permeabilities of all biological membranes increase with the temperature. Psychrophilic and mesophilic bacteria, and mesophilic, (hyper)thermophilic and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains low and constant (homeo-proton permeability). Thermophilic bacteria, however, have more difficulties to restrict the proton permeation across their membrane at high temperatures and these organisms have to rely on the less permeable sodium ions for maintaining a high sodium-motive force for driving their energy requiring membrane-bound processes. Transport of solutes across the bacterial and archaeal membrane is mainly catalyzed by primary ATP driven transport systems or by proton or sodium motive force driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary ATP-driven uptake systems for their carbon and energy sources. Several high-affinity ABC transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phytochelatin synthase OsPCS1 plays a crucial role in reducing arsenic levels in rice grains

Reduction of the level of arsenic (As) in rice grains is an important challenge for agriculture. A recent study reported that the OsABCC1 transporter prevents the accumulation of As in grains by sequestering As–phytochelatin complexes into vacuoles in the upper nodes. However, how phytochelatins are provided in response to As remains unclear. Here, we show that the phytochelatin synthase OsPCS1 plays a crucial role in reducing As levels in rice grains. Using a forward genetic approach, we isolated two rice mutants (has1 and has2) in which As levels were much higher in grains but significantly lower in node I compared with the wild type. Map‐based cloning identified the genes responsible as OsABCC1 in has1 and OsPCS1 in has2. The levels of As in grains and node I were similar between the two mutants, suggesting that OsABCC1 preferentially cooperates with OsPCS1 to sequester As, although rice has another phytochelatin synthase, OsPCS2. An in vitro phytochelatin synthesis assay indicated that OsPCS1 was more sensitive to activation by As than by cadmium, whereas OsPCS2 was more weakly activated by As than by cadmium. Transgenic plants highly expressing OsPCS1 showed significantly lower As levels in grains than did wild‐type plants. Our results provide new knowledge of the relative contribution of rice PCS paralogs to As sequestration and suggest a good candidate tool to reduce As levels in rice grains.     

Glycerol‐3‐phosphate metabolism plays a role in stress response in the red alga Pyropia haitanensis

Glycerol‐3‐phosphate (G3P) has been suggested as a novel regulator of plant defense signaling, however, its role in algal resistance remains largely unknown. The glycerol kinase (also designated as NHO1) and NAD‐dependent G3P dehydrogenase (GPDH) are two key enzymes involved in the G3P biosynthesis. In our study, we cloned the full‐length cDNA of NHO1 (NHO1_Ph) and GPDH (GPDH_Ph) from the red alga Pyropia haitanensis (denoted as NHO1_Ph and GPDH_Ph) and examined their expression level under flagellin peptide 22 (flg22) stimulation or heat stress. We also measured the level of G3P and floridoside (a downstream product of G3P in P. haitanensis) under flg22 stimulation or heat stress. Both NHO1_Ph and GPDH_Ph shared high sequence identity and structural conservation with their orthologs from different species, especially from red algae. Phylogenetic analysis showed that NHO1s and GPDHs from red algae were closely related to those from animals. Under flg22 stimulation or heat stress, the expression levels of NHO1_Ph and GPDH_Ph were up‐regulated, G3P levels increased, and the contents of floridoside decreased. But the floridoside level increased in the recovery period after heat stress. Taken together, we found that G3P metabolism was associated with the flg22‐induced defense response and heat stress response in P. haitanensis, indicating the general conservation of defense response in angiosperms and algae. Furthermore, floridoside might also participate in the stress resistance of P. haitanensis.     

NADH‐dependent glutamate synthase plays a crucial role in assimilating ammonium in the Arabidopsis root

Plant roots under nitrogen deficient conditions with access to both ammonium and nitrate ions, will take up ammonium first. This preference for ammonium rather than nitrate emphasizes the importance of ammonium assimilation machinery in roots. Glutamine synthetase (GS) and glutamate synthase (GOGAT) catalyze the conversion of ammonium and 2‐oxoglutarate to glutamine and glutamate. Higher plants have two GOGAT species, ferredoxin‐dependent glutamate synthase (Fd‐GOGAT) and nicotinamide adenine dinucleotide (NADH)‐GOGAT. While Fd‐GOGAT participates in the assimilation of ammonium, which is derived from photorespiration in leaves, NADH‐GOGAT is highly expressed in roots and its importance needs to be elucidated. While ammonium as a minor nitrogen form in most soils is directly taken up, nitrate as the major nitrogen source needs to be converted to ammonium prior to uptake. The aim of this study was to investigate and quantify the contribution of NADH‐GOGAT to the ammonium assimilation in Arabidopsis (Arabidopsis thaliana Columbia) roots. Quantitative real‐time polymerase chain reaction (PCR) and protein gel blot analysis showed an accumulation of NADH‐GOGAT in response to ammonium supplied to the roots. In addition the localization of NADH‐GOGAT and Fd‐GOGAT did not fully overlap. Promoter–β‐glucuronidase (GUS) fusion analysis and immunohistochemistry showed that NADH‐GOGAT was highly accumulated in non‐green tissue like vascular bundles, shoot apical meristem, pollen, stigma and roots. Reverse genetic approaches suggested a reduction in glutamate production and biomass accumulation in NADH‐GOGAT transfer DNA (T‐DNA) insertion lines under normal CO₂ condition. The data emphasize the importance of NADH‐GOGAT in the ammonium assimilation in Arabidopsis roots.     

GLUT1‐mediated glucose uptake plays a crucial role during Plasmodium hepatic infection

Intracellular pathogens have evolved mechanisms to ensure their survival and development inside their host cells. Here, we show that glucose is a pivotal modulator of hepatic infection by the rodent malaria parasite Plasmodium berghei and that glucose uptake via the GLUT1 transporter is specifically enhanced in P. berghei‐infected cells. We further show that ATP levels of cells containing developing parasites are decreased, which is known to enhance membrane GLUT1 activity. In addition, GLUT1 molecules are translocated to the membrane of the hepatic cell, increasing glucose uptake at later stages of infection. Chemical inhibition of GLUT1 activity leads to a decrease in glucose uptake and the consequent impairment of hepatic infection, both in vitro and in vivo. Our results reveal that changes in GLUT1 conformation and cellular localization seem to be part of an adaptive host response to maintain adequate cellular nutrition and energy levels, ensuring host cell survival and supporting P. berghei hepatic development.     

Phosphorylation of small subunit plays a crucial role in the regulation of RuBPCase in moss and spinach

RuBPCase has been purified to electrophoretic homogeneity from moss and spinach. On denaturing SDS-polyacrylamide gels the purified enzyme revealed two discrete bands, thereby indicating the presence of large and small subunits. The phosphoprotein nature of RuBPCase was proved by in vivo labelling of enzyme with [³²P]orthophosphate. Autoradiographic analysis of ³²P-labelled RuBPCase on SDS-PAG demonstrated that phosphorylation was restricted to the small subunit. Dephosphorylation of purified RuBPCase with alkaline phosphatase resulted in a dramatic decline (70% decrease) in the biological activity of the enzyme. Fractionation of the dephosphorylated enzyme on denaturing gels revealed only the presence of large subunits of RuBPCase. Thus it became evident that dephosphorylation of RuBPCase brings about the dissociation of small subunits from the catalytic large subunits (octamer). The dephosphorylated small subunits were isolated as dimers. These results clearly indicate that phosphorylation of small subunits is mandatory for the reconstitution of holoenzyme and hence crucial for the activation of RuBPCase.     

The role of PI3Ks in the regulation of the neutrophil NADPH oxidase

The NADPH oxidase complex of neutrophils and macrophages is an important weapon used by these cells to kill microbial pathogens. The regulation of this enzyme complex is necessarily complicated by the diverse receptor types that are needed to trigger its activation and also the tight control that is required to deliver this activation at the appropriate time and place. As such, several signalling pathways have been established to regulate the NADPH oxidase downstream of cell surface receptors. Central amongst these are PI3K- (phosphoinositide 3-kinase)-dependent pathways, blockade of which severely limits activation of the oxidase to several soluble and particulate stimuli. The precise roles of the phosphoinositide products of PI3K activity in regulating NADPH oxidase assembly and activation are still unclear, but there is emerging evidence that they play a key role via regulation of guanine nucleotide exchange on Rac, a key component in the oxidase complex. There is also very strong evidence that the PI3K products PtdIns(3,4)P2 and PtdIns3P can bind directly to the PX (Phox homology) domains of the core oxidase components p47phox and p40phox respectively. However, the significance of these interactions in terms of membrane localization or allosteric consequences for the oxidase complex remains to be established.     

MTBP plays a crucial role in mitotic progression and chromosome segregation

Murine double minute 2 (MDM2) binding protein (MTBP) has been implicated in tumor cell proliferation, but the underlying mechanisms remain unclear. The results of MTBP expression analysis during cell cycle progression demonstrated that MTBP protein was rapidly degraded during mitosis. Immunofluorescence studies revealed that a portion of MTBP was localized at the kinetochores during prometaphase. MTBP overexpression delayed mitotic progression from nuclear envelope breakdown (NEB) to anaphase onset and induced abnormal chromosome segregation such as lagging chromosomes, chromosome bridges, and multipolar chromosome segregation. Conversely, MTBP downmodulation caused an abbreviated metaphase and insufficient mitotic arrest, resulting in abnormal chromosome segregation, aneuploidy, decreased cell proliferation, senescence, and cell death, similar to that of Mad2 (mitotic arrest-deficient 2) downmodulation. Furthermore, MTBP downmodulation inhibited the accumulation of Mad1 and Mad2, but not BubR1 (budding uninhibited by benzimidazoles related 1), on the kinetochores, whereas MTBP overexpression inhibited the release of Mad2 from the metaphase kinetochores. These results may imply that MTBP has an important role in recruiting and/or retaining the Mad1/Mad2 complex at the kinetochores during prometaphase, but its degradation is required for silencing the mitotic checkpoint. Together, this study indicates that MTBP has a crucial role in proper mitotic progression and faithful chromosome segregation, providing new insights into regulation of the mitotic checkpoint.     

Epidermal growth factor plays a crucial role in mitogenic regulation of human brain tumor stem cells

A cancer stem cell population in malignant brain tumors takes an essential part in brain tumor initiation, growth, and recurrence. Growth factors, such as epidermal growth factor, fibroblast growth factor-2, vascular endothelial growth factor, platelet-derived growth factor, and hepatocyte growth factor, are shown to support the proliferation of neural stem cells and also may play key roles in gliomagenesis. However, the responsible growth factor(s), which controls maintenance of brain tumor stem cells, is not yet uncovered. We have established three cancer stem cell lines from human gliomas. These cells were immunoreactive with the neuronal progenitor markers, nestin and CD133, and established tumors that closely resembled the features of original tumor upon transplantation into mouse brain. Three cell lines retained their self-renewal ability and proliferation only in the presence of epidermal growth factor (>2.5 ng/ml). In sharp contrast, other growth factors, including fibroblast growth factor-2, failed to support maintenance of these cells. The tyrosine kinase inhibitors of epidermal growth factor signaling (AG1478 and gefitinib) suppressed the proliferation and self-renewal of these cells. Gefitinib inhibited phosphorylation of epidermal growth factor receptor as well as Akt kinase and extracellular signal-regulated kinase 1/2. Flow cytometric analysis revealed that epidermal growth factor concentration-dependently increased the population of CD133-positive cells. Gefitinib significantly reduced CD133-positive fractions and also induced their apoptosis. These results indicate that maintenance of human brain tumor stem cells absolutely requires epidermal growth factor and that tyrosine kinase inhibitors of epidermal growth factor signaling potentially inhibit proliferation and induce apoptosis of these cells.