共查询到20条相似文献,搜索用时 15 毫秒
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Microsomal Electron Transfer in Higher Plants: Cloning and
Heterologous Expression of NADH-Cytochrome
b5
Reductase from Arabidopsis 总被引:2,自引:0,他引:2 下载免费PDF全文
Masako Fukuchi-Mizutani Masaharu Mizutani Yoshikazu Tanaka Takaaki Kusumi Daisaku Ohta 《Plant physiology》1999,119(1):353-362
AtCBR, a cDNA encoding NADH-cytochrome (Cyt) b5 reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b5, were isolated from Arabidopsis. The primary structure deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b5 reductases of yeast and mammals. A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b5 reductase and was used to study its electron-transfer activity. The recombinant NADH-Cyt b5 reductase was functionally active and displayed strict specificity to NADH for the reduction of a recombinant Cyt b5 (AtB5-A), whereas no Cyt b5 reduction was observed when NADPH was used as the electron donor. Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cyt b5 with NADPH but not with NADH. To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b5 reductase and NADPH-Cyt P450 reductase can reduce Cyt b5 and have clear specificities in terms of the electron donor, NADH or NADPH, respectively. This substrate specificity of the two reductases is discussed in relation to the NADH- and NADPH-dependent activities of microsomal fatty acid desaturases. 相似文献
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Rheumatoid arthritis (RA) is a chronic, systematic autoimmune disease that mainly affects joints and bones. Although the precise etiology is still unknown, Th17 cell is being recognized as an important mediator in pathogenesis of RA. VSTM1-v2 is a novel cytokine which has recently been reported to promote the differentiation of Th17 cells. This study is performed to study whether VSTM1-v2 can be recognized as a biomarker of RA, and is correlated to IL-17 expression. We obtained peripheral blood mononuclear cells (PBMCs) from 40 patients with RA and 40 age- and sex- matched healthy controls by standard Ficoll-Paque Plus density centrifugation. The mRNA expression levels of VSTM1-v2 and IL-17A in PBMCs were detected by real time-PCR. Disease activity parameters of RA were measured by routine methods. Our results showed that VSTM1-v2 mRNA expression in PBMCs from RA patients was significantly increased in comparison of that in healthy individuals. The VSTM1-v2 mRNA expression level was positively correlated with IL-17A mRNA expression level, DAS28, CRP and ESR, but was not correlated to RF, Anti-CCP or ANA. VSTM1-v2 might be a biomarker of RA and a novel factor in the pathogenesis of RA. 相似文献
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Modification of Carbon Partitioning, Photosynthetic Capacity, and
O2 Sensitivity in Arabidopsis Plants with Low
ADP-Glucose Pyrophosphorylase Activity 下载免费PDF全文
Wild-type Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were evaluated by noninvasive in situ methods for their capacity for net CO2 assimilation, true rates of photosynthetic O2 evolution (determined from chlorophyll fluorescence measurements of photosystem II), partitioning of photosynthate into sucrose and starch, and plant growth. Compared with wild-type plants, the starch mutants showed reduced photosynthetic capacity, with the largest reduction occurring in mutant TL25 subjected to high light and increased CO2 partial pressure. The extent of stimulation of CO2 assimilation by increasing CO2 or by reducing O2 partial pressure was significantly less for the starch mutants than for wild-type plants. Under high light and moderate to high levels of CO2, the rates of CO2 assimilation and O2 evolution and the percentage inhibition of photosynthesis by low O2 were higher for the wild type than for the mutants. The relative rates of 14CO2 incorporation into starch under high light and high CO2 followed the patterns of photosynthetic capacity, with TL46 showing 31% to 40% of the starch-labeling rates of the wild type and TL25 showing less than 14% incorporation. Overall, there were significant correlations between the rates of starch synthesis and CO2 assimilation and between the rates of starch synthesis and cumulative leaf area. These results indicate that leaf starch plays an important role as a transient reserve, the synthesis of which can ameliorate any potential reduction in photosynthesis caused by feedback regulation. 相似文献
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硝酸盐对球形棕囊藻生长和硝酸还原酶活性的影响 总被引:9,自引:0,他引:9
以我国南海海域分离的赤潮原因种——球形棕囊藻(Phaeocystisglobosa)为材料,研究了不同硝酸盐浓度下藻细胞生长及硝酸还原酶活性的变化。当培养基中不含硝酸盐时,藻细胞内硝酸还原酶的活性保持在非常低的水平,藻细胞的生长受到限制,不能形成正常的生长曲线:当培养基中硝酸盐浓度为3.62μmol.L-1时,藻细胞的硝酸还原酶活性和比生长速率达到最大。在含有硝酸盐的培养基中,接种培养后第9天藻细胞硝酸还原酶活性达到最大值,并且在4种不同硝酸盐浓度下,藻细胞硝酸还原酶活性的差异性达到极显著水平(P<0.01)。在接种培养第16天藻细胞密度达到最大值,并且4种不同硝酸盐浓度培养的藻细胞密度之间的差异性也达到极显著水平(P<0.01)。实验结果表明,在培养基中添加不同浓度的硝酸盐,对球形棕囊藻细胞硝酸还原酶的活性和藻细胞的生长有极显著的影响,含有较高硝酸盐的富营养化海域有利于球形棕囊藻细胞的持续生长。 相似文献
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硝酸盐对球形棕囊藻生长和硝酸还原酶活性的影响 总被引:1,自引:0,他引:1
以我国南海海域分离的赤潮原因种——球形棕囊藻(Phaeocystis globosa)为材料, 研究了不同硝酸盐浓度下藻细胞生长及硝酸还原酶活性的变化。当培养基中不含硝酸盐时, 藻细胞内硝酸还原酶的活性保持在非常低的水平, 藻细胞的生长受到限制, 不能形成正常的生长曲线: 当培养基中硝酸盐浓度为3.62 mmol.L-1时, 藻细胞的硝酸还原酶活性和比生长速率达到最大。在含有硝酸盐的培养基中, 接种培养后第9天藻细胞硝酸还原酶活性达到最大值, 并且在4种不同硝酸盐浓度下, 藻细胞硝酸还原酶活性的差异性达到极显著水平(P<0.01)。在接种培养第16天藻细胞密度达到最大值, 并且4种不同硝酸盐浓度培养的藻细胞密度之间的差异性也达到极显著水平(P<0.01)。实验结果表明, 在培养基中添加不同浓度的硝酸盐, 对球形棕囊藻细胞硝酸还原酶的活性和藻细胞的生长有极显著的影响, 含有较高硝酸盐的富营养化海域有利于球形棕囊藻细胞的持续生长。 相似文献
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Yang Wang Dan Li Roza Nurieva Justin Yang Mehmet Sen Roberto Carre?o Sijie Lu Bradley W. McIntyre Jeffrey J. Molldrem Glen B. Legge Qing Ma 《The Journal of biological chemistry》2009,284(19):12645-12653
The activation of LFA-1 (lymphocyte function-associated antigen) is a
critical event for T cell co-stimulation. The mechanism of LFA-1 activation
involves both affinity and avidity regulation, but the role of each in T cell
activation remains unclear. We have identified antibodies that recognize and
block different affinity states of the mouse LFA-1 I-domain. Monoclonal
antibody 2D7 preferentially binds to the low affinity conformation, and this
specific binding is abolished when LFA-1 is locked in the high affinity
conformation. In contrast, M17/4 can bind both the locked high and low
affinity forms of LFA-1. Although both 2D7 and M17/4 are blocking antibodies,
2D7 is significantly less potent than M17/4 in blocking LFA-1-mediated
adhesion; thus, blocking high affinity LFA-1 is critical for preventing
LFA-1-mediated adhesion. Using these reagents, we investigated whether LFA-1
affinity regulation affects T cell activation. We found that blocking high
affinity LFA-1 prevents interleukin-2 production and T cell proliferation,
demonstrated by TCR cross-linking and antigen-specific stimulation.
Furthermore, there is a differential requirement of high affinity LFA-1 in the
activation of CD4+ and CD8+ T cells. Although
CD4+ T cell activation depends on both high and low affinity LFA-1,
only high affinity LFA-1 provides co-stimulation for CD8+ T cell
activation. Together, our data demonstrated that the I-domain of LFA-1 changes
to the high affinity state in primary T cells, and high affinity LFA-1 is
critical for facilitating T cell activation. This implicates LFA-1 activation
as a novel regulatory mechanism for the modulation of T cell activation and
proliferation.LFA-1 (lymphocyte function-associated antigen), an integrin family member,
is important in regulating leukocyte adhesion and T cell activation
(1,
2). LFA-1 consists of the
αL (CD11a) and β2 (CD18) heterodimer. The
ligands for LFA-1, including intercellular adhesion molecule
ICAM3-1, ICAM-2, and
ICAM-3, are expressed on antigen-presenting cells (APCs), endothelial cells,
and lymphocytes (1). Mice that
are deficient in LFA-1 have defects in leukocyte adhesion, lymphocyte
proliferation, and tumor rejection
(3–5).
Blocking LFA-1 with antibodies can prevent inflammation, autoimmunity, organ
graft rejection, and graft versus host disease in human and murine
models
(6–10).LFA-1 is constitutively expressed on the surface of leukocytes in an
inactive state. Activation of LFA-1 is mediated by inside-out signals from the
cytoplasm (1,
11). Subsequently, activated
LFA-1 binds to the ligands and transduces outside-in signals back into the
cytoplasm that result in cell adhesion and activation
(12,
13). The activation of LFA-1
is a critical event in the formation of the immunological synapse, which is
important for T cell activation
(2,
14,
15). The active state of LFA-1
is regulated by chemokines and the T cell receptor (TCR) through Rap1
signaling (16). LFA-1 ligation
lowers the activation threshold and affects polarization in CD4+ T
cells (17). Moreover,
productive LFA-1 engagement facilitates efficient activation of cytotoxic T
lymphocytes and initiates a distinct signal essential for the effector
function
(18–20).
Thus, LFA-1 activation is essential for the optimal activation of T cells.The mechanism of LFA-1 activation involves both affinity (conformational
changes within the molecule) and avidity (receptor clustering) regulation
(21–23).
The I-domain of the LFA-1 αL subunit is the primary
ligand-binding site and has been proposed to change conformation, leading to
an increased affinity for ligands
(24–26).
The structural basis of the conformational changes in the I-domain of LFA-1
has been extensively characterized
(27). Previously, we have
demonstrated that the conformation of the LFA-1 I-domain changes from the low
affinity to the high affinity state upon activation. By introducing disulfide
bonds into the I-domain, LFA-1 can be locked in either the closed or open
conformation, which represents the “low affinity” or “high
affinity” state, respectively
(28,
29). In addition, we
identified antibodies that are sensitive to the affinity changes in the
I-domain of human LFA-1 and showed that the activation-dependent epitopes are
exposed upon activation (30).
This study supports the presence of the high affinity conformation upon LFA-1
activation in cell lines. It has been demonstrated recently that therapeutic
antagonists, such as statins, inhibit LFA-1 activation and immune responses by
locking LFA-1 in the low affinity state
(31–34).
Furthermore, high affinity LFA-1 has been shown to be important for mediating
the adhesion of human T cells
(35,
36). Thus, the affinity
regulation is a critical step in LFA-1 activation.LFA-1 is a molecule of great importance in the immune system, and its
activation state influences the outcome of T cell activation. Our previous
data using the activating LFA-1 I-domain-specific antibody MEM83 indicate that
avidity and affinity of the integrin can be coupled during activation
(37). However, whether
affinity or avidity regulation of LFA-1 contributes to T cell activation
remains controversial (23,
38,
39). Despite the recent
progress suggesting that conformational changes represent a key step in the
activation of LFA-1, there are considerable gaps to be filled. When LFA-1 is
activated, the subsequent outside-in signaling contributes to T cell
activation via immunological synapse and LFA-1-dependent signaling. It is
critical to determine whether high affinity LFA-1 participates in the
outside-in signaling and affects the cellular activation of T cells.
Nevertheless, the rapid and dynamic process of LFA-1 activation has hampered
further understanding of the role of high affinity LFA-1 in primary T cell
activation. The affinity of LFA-1 for ICAM-1 increases up to 10,000-fold
within seconds and involves multiple reversible steps
(23). In addition, the
activation of LFA-1 regulates both adhesion and activation of T cells, two
separate yet closely associated cellular functions. When LFA-1 is
constitutively expressed in the active state in mice, immune responses are
broadly impaired rather than hyperactivated, suggesting the complexity of
affinity regulation (40).
Therefore, it is difficult to dissect the mechanisms by which high affinity
LFA-1 regulates stepwise activation of T cells in the whole animal system.In the present study, we identified antibodies recognizing and blocking
different affinity states of mouse LFA-1. These reagents allowed us to
determine the role of affinity regulation in T cell activation. We found that
blocking high affinity LFA-1 inhibited IL-2 production and proliferation in T
cells. Furthermore, there is a differential requirement of high affinity LFA-1
in antigen-specific activation of CD4+ and CD8+ T cells.
The activation of CD4+ T cells depends on both high and low
affinity LFA-1. For CD8+ T cell activation, only high affinity
LFA-1 provides co-stimulation. Thus, affinity regulation of LFA-1 is critical
for the activation and proliferation of naive T cells. 相似文献
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Differential Expression and Internal Feedback Regulation
of
1-Aminocyclopropane-1-Carboxylate Synthase,
1-Aminocyclopropane-1-Carboxylate Oxidase, and
Ethylene Receptor
Genes in Tomato Fruit during Development and Ripening 总被引:21,自引:1,他引:21 下载免费PDF全文
Akira Nakatsuka Shiho Murachi Hironori Okunishi Shinjiro Shiomi Ryohei Nakano Yasutaka Kubo Akitsugu Inaba 《Plant physiology》1998,118(4):1295-1305
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Nitrate Reductase and Nitrite Reductase Activity in Dark-Grown Radish Seedlings: Relation to the Supply of NADPH 总被引:1,自引:0,他引:1
Functioning of nitrate reductase and nitrite reductase was measured in intact cotyledons from radish seedlings (Raphanus sativus L.) grown in the dark in a nitrate medium. Reduction of nitrate to nitrate did proceed during the whole period of 45 h, whereas the reduction of nitrite in the intact cotyledons dropped abruptly between 20 and 23 h after exposing the roots to nitrate. The activity of the enzymes glucose-6-P dehydrogenase (G6PDH) and 6-P-gluconate dehydrogenase (6PGDH), measured in cotyledon extracts, showed a sharp decline simultaneously with the drop in nitrite reductase activity of the intact cotyledons. It was concluded that the amount of NADPH generated by the enzymes G6PDH and 6PGDH is not sufficient to allow continuous functioning of nitrite reductase after 20 h in cotyledons of seedlings grown in the dark. Therefore, the results from our experiments point to the functioning of nitrite reductase as the rate limiting step in the reduction pathway of nitrate in the dark. 相似文献
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An N-terminally modified form of the Arabidopsis NADPH–cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2′,5′-ADP–agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 μmol min−1 mg−1 and the Km for cytochrome c reduction was 15 ± 2 μM. The purified NADPH–cytochrome P450 reductase was able to support function of CYP79B2. 相似文献
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