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1.
Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions
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Helen Ridley Carys A. Watts David J. Richardson Clive S. Butler 《Applied microbiology》2006,72(8):5173-5180
Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ~600 kDa. The individual subunit sizes are ~100 kDa (α), ~55 kDa (β), and ~36 kDa (γ), with a predicted overall subunit composition of α3β3γ3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be ~2 mM, with an observed Vmax of 500 nmol SeO42− min−1 mg−1 (kcat, ~5.0 s−1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase. 相似文献
2.
Studies of human NK cells and their role in tumor suppression have largely been restricted to in vitro experiments which lack the complexity of whole organisms, or mouse models which differ significantly from humans. In this study we showed that, in contrast to C57BL/6 Rag2−/−/γc
−/− and NOD/Scid mice, newborn BALB/c Rag2−/−/γc
−/− mice can support the development of human NK cells and CD56+ T cells after intrahepatic injection with hematopoietic stem cells. The human CD56+ cells in BALB/c Rag2−/−/γc
−/− mice were able to produce IFN-γ in response to human IL-15 and polyI:C. NK cells from reconstituted Rag2−/−/γc
−/− mice were also able to kill and inhibit the growth of K562 cells in vitro and were able to produce IFN-γ in response to stimulation with K562 cells. In vivo, reconstituted Rag2−/−/γc
−/− mice had higher survival rates after K562 challenge compared to non-reconstituted Rag2−/−/γc
−/− mice and were able to control tumor burden in various organs. Reconstituted Rag2−/−/γc
−/− mice represent a model in which functional human NK and CD56+ T cells can develop from stem cells and can thus be used to study human disease in a more clinically relevant environment. 相似文献
3.
Mingxuan Wang Jing Li Wenjie Cong Jianguo Zhang 《Current issues in molecular biology》2022,44(5):1828
Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min−1 mg−1 and 10.0 ± 0.5 nmol min−1 mg−1, respectively. The highest kcat/Km value of ω3 and ω6 desaturase was 0.41 µM−1 min−1 and 0.09 µM−1 min−1 when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina. 相似文献
4.
David C. Chiara Selwyn S. Jayakar Xiaojuan Zhou Xi Zhang Pavel Y. Savechenkov Karol S. Bruzik Keith W. Miller Jonathan B. Cohen 《The Journal of biological chemistry》2013,288(27):19343-19357
GABA type A receptors (GABAAR), the brain''s major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABAARs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the β+-α− subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R-[3H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human α1β3γ2 GABAARs. Protein microsequencing revealed that R-[3H]mTFD-MPAB did not photolabel the etomidate sites at the β+-α− subunit interfaces. Instead, it photolabeled sites at the α+-β− and γ+-β− subunit interfaces in the transmembrane domain. On the (+)-side, α1M3 was labeled at Ala-291 and Tyr-294 and γ2M3 at Ser-301, and on the (−)-side, β3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R-etomidate for the β+-α− interface relative to the α+-β−/γ+-β− interfaces was >100-fold, whereas that of R-mTFD-MPAB for its sites was >50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. We hypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics. 相似文献
5.
Sonia Kapur Mehran Amoui Chandrasekhar Kesavan Xiaoguang Wang Subburaman Mohan David J. Baylink K.-H. William Lau 《The Journal of biological chemistry》2010,285(48):37607-37618
This study investigated the role of leptin receptor (Lepr) signaling in determining the bone mechanosensitivity and also evaluated whether differences in the Lepr signaling may contribute to the differential osteogenic response of the C57BL/6J (B6) and C3H/HeJ (C3H) pair of mouse strains to mechanical stimuli. This study shows that a loading strain of ∼2,500 μϵ, which was insufficient to produce a bone formation response in B6 mice, significantly increased bone formation parameters in leptin-deficient ob−/ob− mice and that a loading strain of ∼3,000 μϵ also yielded greater osteogenic responses in Lepr-deficient db−/db− mice than in wild-type littermates. In vitro, a 30-min steady shear stress increased [3H]thymidine incorporation and Erk1/2 phosphorylation in ob−/ob− osteoblasts and db−/db− osteoblasts much greater than those in corresponding wild-type osteoblasts. The siRNA-mediated suppression of Lepr expression in B6 osteoblasts enhanced (but in osteoblasts of C3H (the mouse strain with poor bone mechanosensitivity) restored) their anabolic responses to shear stress. The Lepr signaling (leptin-induced Jak2/Stat3 phosphorylation) in C3H osteoblasts was higher than that in B6 osteoblasts. One of the three single nucleotide polymorphisms in the C3H Lepr coding region yielded an I359V substitution near the leptin binding region, suggesting that genetic variation of Lepr may contribute to a dysfunctional Lepr signaling in C3H osteoblasts. In conclusion, Lepr signaling is a negative modulator of bone mechanosensitivity. Genetic variations in Lepr, which result in a dysfunctional Lepr signaling in C3H mice, may contribute to the poor osteogenic response to loading in C3H mice. 相似文献
6.
Sigma-1 receptor (σ1R) has been reported to be decreased in nigrostriatal motor system of Parkinson''s disease patients. Using heterozygous and homozygous σ1R knockout (σ1R+/− and σ1R−/−) mice, we investigated the influence of σ1R deficiency on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-impaired nigrostriatal motor system. The injection of MPTP for 5 weeks in wild-type mice (MPTP-WT mice), but not in σ1R+/− or σ1R−/− mice (MPTP-σ1R+/− or MPTP-σ1R−/− mice), caused motor deficits and ~40% death of dopaminergic neurons in substantia nigra pars compacta with an elevation of N-methyl-d-aspartate receptor (NMDAr) NR2B phosphorylation. The σ1R antagonist NE100 or the NR2B inhibitor Ro25-6981 could alleviate the motor deficits and the death of dopaminergic neurons in MPTP-WT mice. By contrast, MPTP-σ1R+/− mice treated with the σ1R agonist PRE084 or MPTP-σ1R−/− mice treated with the NMDAr agonist NMDA appeared to have similar motor deficits and loss of dopaminergic neurons as MPTP-WT mice. The pharmacological or genetic inactivation of σ1R suppressed the expression of dopamine transporter (DAT) in substantia nigra, which was corrected by NMDA. The activation of σ1R by PRE084 enhanced the DAT expression in WT mice or σ1R+/− mice. By contrast, the level of vesicular monoamine transporter 2 (VMAT2) in σ1R+/− mice or σ1R−/− mice had no difference from WT mice. Interestingly, MPTP-WT mice showed the reduction in the levels of DAT and VMAT2, but MPTP-σ1R−/− mice did not. The inactivation of σ1R by NE100 could prevent the reduction of VMAT2 in MPTP-WT mice. In addition, the activation of microglia cells in substantia nigra was equally enhanced in MPTP-WT mice and MPTP-σ1R−/− mice. The number of activated astrocytes in MPTP-σ1R−/− mice was less than that in MPTP-WT mice. The findings indicate that the σ1R deficiency through suppressing NMDAr function and DAT expression can reduce MPTP-induced death of dopaminergic neurons and parkinsonism.Parkinson''s disease (PD) is a neurodegenerative disorder characterized by motor symptoms, including bradykinesia and tremor, and a progressive loss of dopaminergic neurons in substantia nigra pars compacta (SNpc).1, 2 Sigma-1 receptor (σ1R), previously named the opioid receptor sigma-1, is found primarily in motoneurons localized in the brainstem and spinal cord.3 The σ1R is expressed in dopaminergic neurons and astrocytes.4 The σ1R agonist PRE084 has been reported to exert neurorestorative effects on 6-hydroxydopamine (6-OHDA)-induced parkinsonism.4 Using positron emission tomography, the σ1R-binding sites are found to be reduced in the brains of early-phase PD patients.5 However, the influence of σ1R deficiency on the pathogenesis of PD has not yet been reported.Dopamine toxicity is involved in the etiology of PD.6 The σ1R-binding sites on dopaminergic nerve terminals are involved in increasing dopamine release by enhancing N-methyl-d-aspartate receptors (NMDAr).7 The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolized to 1-methyl-4-phenylpyridinium in glial cells selectively impairs dopaminergic neurons in SNpc through disrupting respiratory enzymes and causing oxidative damage.8 The dopamine transporter (DAT), a high-affinity transmembrane protein, is responsible for dopamine reuptake from the synaptic cleft and the transportation of 1-methyl-4-phenylpyridinium into dopaminergic nerve terminals.9 The σ1R is co-expressed with DAT in dopaminergic neurons.4 Furthermore, the low density of DAT has been confirmed in the brains of PD patients.5The activation of σ1R enhances the Ca2+ influx across NMDAr through increasing the phosphorylation of NR2B or the trafficking NMDAr to the plasma membrane.10, 11 The NMDAr NR2B inhibitor can attenuate MPTP- or 6-OHDA-induced parkinsonian symptoms and neurodegeneration.12 The σ1R deficiency has been demonstrated to reduce Aβ-induced neuronal cell death through suppressing NR2B phosphorylation.13 The inflammation is a predominant aspect of PD, manifested by glial activation with the expression of pro-inflammatory mediators.14 Sustained neuro-inflammation can exacerbate the degeneration of dopaminergic neurons.15 The blockade of σ1R has been reported to inhibit methamphetamine-induced astrogliosis.16 Moreover, the 6-OHDA-induced spontaneous rotations or decline of dopaminergic fibers in σ1R knockout mice seem to be less than those in wild-type (WT) mice.4 Paquette et al. reported that the blockade of σ1R could attenuate abnormal involuntary movements induced by 6-OHDA.17In this study, we employed heterozygous and homozygous σ1R knockout (σ1R+/− and σ1R−/−) mice to investigate the influence of σ1R deficiency on MPTP-induced parkinsonism and death of dopaminergic neurons, and the underlying molecular mechanisms. Using the experimental PD models of MPTP-treated σ1R+/− mice and σ1R−/− mice, the present study provides in vivo evidence that the σ1R deficiency through suppressing NMDAr function and DAT expression can attenuate MPTP-induced dopaminergic neurodegeneration and parkinsonism. 相似文献
7.
Katsuya Tanabe Zhonghao Liu Satish Patel Bradley W Doble Lin Li Corentin Cras-Mneur Sara C Martinez Cris M Welling Morris F White Ernesto Bernal-Mizrachi James R Woodgett M. Alan Permutt 《PLoS biology》2008,6(2)
Despite treatment with agents that enhance β-cell function and insulin action, reduction in β-cell mass is relentless in patients with insulin resistance and type 2 diabetes mellitus. Insulin resistance is characterized by impaired signaling through the insulin/insulin receptor/insulin receptor substrate/PI-3K/Akt pathway, leading to elevation of negatively regulated substrates such as glycogen synthase kinase-3β (Gsk-3β). When elevated, this enzyme has antiproliferative and proapoptotic properties. In these studies, we designed experiments to determine the contribution of Gsk-3β to regulation of β-cell mass in two mouse models of insulin resistance. Mice lacking one allele of the insulin receptor (Ir+/−) exhibit insulin resistance and a doubling of β-cell mass. Crossing these mice with those having haploinsufficiency for Gsk-3β (Gsk-3β+/−) reduced insulin resistance by augmenting whole-body glucose disposal, and significantly reduced β-cell mass. In the second model, mice missing two alleles of the insulin receptor substrate 2 (Irs2−/−), like the Ir+/− mice, are insulin resistant, but develop profound β-cell loss, resulting in early diabetes. We found that islets from these mice had a 4-fold elevation of Gsk-3β activity associated with a marked reduction of β-cell proliferation and increased apoptosis. Irs2−/− mice crossed with Gsk-3β+/− mice preserved β-cell mass by reversing the negative effects on proliferation and apoptosis, preventing onset of diabetes. Previous studies had shown that islets of Irs2−/− mice had increased cyclin-dependent kinase inhibitor p27kip1 that was limiting for β-cell replication, and reduced Pdx1 levels associated with increased cell death. Preservation of β-cell mass in Gsk-3β+/−Irs2−/− mice was accompanied by suppressed p27kip1 levels and increased Pdx1 levels. To separate peripheral versus β-cell–specific effects of reduction of Gsk3β activity on preservation of β-cell mass, mice homozygous for a floxed Gsk-3β allele (Gsk-3F/F) were then crossed with rat insulin promoter-Cre (RIP-Cre) mice to produce β-cell–specific knockout of Gsk-3β (βGsk-3β−/−). Like Gsk-3β+/− mice, βGsk-3β−/− mice also prevented the diabetes of the Irs2−/− mice. The results of these studies now define a new, negatively regulated substrate of the insulin signaling pathway specifically within β-cells that when elevated, can impair replication and increase apoptosis, resulting in loss of β-cells and diabetes. These results thus form the rationale for developing agents to inhibit this enzyme in obese insulin-resistant individuals to preserve β-cells and prevent diabetes onset. 相似文献
8.
Nilay Dey Mala Sinha Shivali Gupta Mariela Natacha Gonzalez Rong Fang Janice J. Endsley Bruce A. Luxon Nisha Jain Garg 《PloS one》2014,9(11)
In this study, we have utilized wild-type (WT), ASC−/−, and NLRP3−/− macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1β production in mφs in comparison to LPS-treated controls. When WT and ASC−/− macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3−/− macrophages, despite an inability to elicit IL-1β activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3−/− macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1β-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1β/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages. 相似文献
9.
Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG+/+ and UNG−/− cells. We report that despite identical growth rates and DSB repair capacities, UNG−/− cells accumulated significantly greater uracil and DSBs compared with UNG+/+ cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG+/+ cells. Furthermore, DSBs in UNG+/+ and UNG−/− cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG−/− cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG+/+ and UNG−/− cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG−/− but not UNG+/+ cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG−/− cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death. 相似文献
10.
Methylglyoxal, an endogenous aldehyde, crosslinks DNA polymerase and the substrate DNA 总被引:7,自引:0,他引:7
Methylglyoxal, a known endogenous and environmental mutagen, is a reactive α-ketoaldehyde that can modify both DNA and proteins. To investigate the possibility that methylglyoxal induces a crosslink between DNA and DNA polymerase, we treated a ‘primed template’ DNA and the exonuclease-deficient Klenow fragment (KFexo–) of DNA polymerase I with methylglyoxal in vitro. When the reaction mixtures were analyzed by SDS–PAGE, we found that methylglyoxal induced a DNA–KFexo– crosslink. The specific binding complex of KFexo– and ‘primed template’ DNA was necessary for formation of the DNA–KFexo– crosslink. Methylglyoxal reacted with guanine residues in the single-stranded portion of the template DNA. When 2′-deoxyguanosine was incubated with Nα-acetyllysine or N-acetylcysteine in the presence of methylglyoxal, a crosslinked product was formed. No other amino acid derivatives tested could generate a crosslinked product. These results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methylglyoxal-induced DNA–DNA polymerase crosslink. 相似文献
11.
Rafik Menasria Nicolas Boivin Manon Lebel Jocelyne Piret Jean Gosselin Guy Boivin 《Journal of virology》2013,87(13):7301-7308
Toll-like receptors (TLRs) and RNA helicases (RLHs) are important cell sensors involved in the immunological control of viral infections through production of type I interferon (IFN). The impact of a deficiency in the TRIF and IPS-1 adaptor proteins, respectively, implicated in TLR3 and RLH signaling pathways, was investigated during herpes simplex virus 1 (HSV-1) encephalitis. TRIF−/−, IPS-1−/−, and C57BL/6 wild-type (WT) mice were infected intranasally with 7.5 × 105 PFU of HSV-1. Mice were monitored for neurological signs and survival over 20 days. Groups of mice were sacrificed on days 3, 5, 7, 9, and 11 postinfection for determination of brain viral replication by quantitative PCR (qPCR), plaque assay, and immunohistochemistry and for alpha/beta interferon (IFN-α/β) levels and phosphorylation of interferon regulatory factors 3 and 7 (IRF-3 and -7) in brain homogenates by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. TRIF−/− and IPS-1−/− mice had higher mortality rates than WT mice (P = 0.02 and P = 0.09, respectively). Viral antigens were more disseminated throughout the brain, correlating with a significant increase in brain viral load for TRIF−/− (days 5 to 9) and IPS-1−/− (days 7 and 9) mice compared to results for the WT. IFN-β production was reduced in brain homogenates of TRIF−/− and IPS-1−/− mice on day 5 compared to results for the WT, whereas IFN-α levels were increased on day 7 in TRIF−/− mice. Phosphorylation levels of IRF-3 and IRF-7 were decreased in TRIF−/− and IPS-1−/− mice, respectively. These data suggest that both the TRIF and IPS-1 signaling pathways are important for the control of HSV replication in the brain and survival through IFN-β production. 相似文献
12.
Winifred O. Idigo Svetlana Reilly Mei Hua Zhang Yin Hua Zhang Raja Jayaram Ricardo Carnicer Mark J. Crabtree Jean-Luc Balligand Barbara Casadei 《The Journal of biological chemistry》2012,287(52):43665-43673
Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β3-adrenergic receptor (β3-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β3-AR signaling and found that the β3-AR-mediated reduction in cell shortening and [Ca2+]i transient amplitude was abolished both in eNOS−/− and nNOS−/− left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-l-thiocitrulline. LV superoxide (O2˙̄) production was increased in nNOS−/− mice and reduced by l-Nω-nitroarginine methyl ester (l-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS−/− myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O2˙̄, only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca2+]i transient response to β3-AR stimulation in nNOS−/− mice. In summary, our data show that increased O2˙̄ production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β3-AR stimulation in nNOS−/− myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS−/− mice result from disruption of eNOS signaling. 相似文献
13.
Nicole Wydeven Ekaterina Posokhova Zhilian Xia Kirill A. Martemyanov Kevin Wickman 《The Journal of biological chemistry》2014,289(4):2440-2449
Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K+ channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4−/−, Rgs6−/−, and Rgs4−/−:Rgs6−/− mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6−/− mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6−/− and Gβ5−/− mice, suggest that the partial rescue of phenotypes in Rgs4−/−:Rgs6−/− mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice. 相似文献
14.
The influence of chloride and other univalent inorganic anions on the visible-absorption spectrum of aspartate aminotransferase
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1. The influence of Cl−, Br−, NO3− and F− ions on the visible-absorption spectrum of deionized aspartate aminotransferase was investigated. 2. Except for F−, these anions caused an increase of the extinction at 430mμ with a concomitant decrease of that at 362mμ. 3. The affinity constants for Cl− and NO3− ions were calculated by a procedure based on the assumption that the anion stabilizes the protonated form of the enzyme chromophore (λmax. 430mμ). 4. The true pK of the chromophore of the enzyme was found to be 5·25. 相似文献
15.
Sergio Roa Ziqiang Li Jonathan U. Peled Chunfang Zhao Winfried Edelmann Matthew D. Scharff 《PloS one》2010,5(6)
Mismatch repair of AID-generated dU:G mispairs is critical for class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The generation of a previously unavailable Msh2−/−Msh6−/− mouse has for the first time allowed us to examine the impact of the complete loss of MutSα on lymphomagenesis, CSR and SHM. The onset of T cell lymphomas and the survival of Msh2−/−Msh6−/− and Msh2−/−Msh6−/−Msh3−/− mice are indistinguishable from Msh2−/− mice, suggesting that MSH2 plays the critical role in protecting T cells from malignant transformation, presumably because it is essential for the formation of stable MutSα heterodimers that maintain genomic stability. The similar defects on switching in Msh2−/−, Msh2−/−Msh6−/− and Msh2−/−Msh6−/−Msh3−/− mice confirm that MutSα but not MutSβ plays an important role in CSR. Analysis of SHM in Msh2−/−Msh6−/− mice not only confirmed the error-prone role of MutSα in the generation of strand biased mutations at A:T bases, but also revealed an error-free role of MutSα when repairing some of the dU:G mispairs generated by AID on both DNA strands. We propose a model for the role of MutSα at the immunoglobulin locus where the local balance of error-free and error-prone repair has an impact in the spectrum of mutations introduced during Phase 2 of SHM. 相似文献
16.
Srikanth Kotapati Leena Maddukuri Susith Wickramaratne Uthpala Seneviratne Melissa Goggin Matthew G. Pence Peter Villalta F. Peter Guengerich Lawrence Marnett Natalia Tretyakova 《The Journal of biological chemistry》2012,287(46):38800-38811
The 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N6-γ-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N6-propano group on 1,N6-γ-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N6-γ-HMHP-dA lesions in the presence of human DNA polymerases (hpols) β, η, κ, and ι and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol η, κ, and Dpo4 were able to copy the modified template. In contrast, hpol ι inserted a single base opposite 1,N6-γ-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol β. Single nucleotide incorporation experiments indicated that although hpol η, κ, and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N6-γ-HMHP-dA and detected large amounts of −1 and −2 deletion products. Taken together, these results indicate that hpol η and κ enzymes bypass 1,N6-γ-HMHP-dA lesions in an error-prone fashion, potentially contributing to A→T and A→C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane. 相似文献
17.
Estimating Bacterioplankton Production by Measuring [3H]thymidine Incorporation in a Eutrophic Swedish Lake 总被引:3,自引:0,他引:3
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Bacterioplankton abundance, [3H]thymidine incorporation, 14CO2 uptake in the dark, and fractionated primary production were measured on several occasions between June and August 1982 in eutrophic Lake Norrviken, Sweden. Bacterioplankton abundance and carbon biomass ranged from 0.5 × 109 to 2.4 × 109 cells liter−1 and 7 to 47 μg of C liter−1, respectively. The average bacterial cell volume was 0.185 μm3. [3H]thymidine incorporation into cold-trichloroacetic acid-insoluble material ranged from 12 × 10−12 to 200 × 10−12 mol liter−1 h−1. Bacterial carbon production rates were estimated to be 0.2 to 7.1 μg of C liter−1 h−1. Bacterial production estimates from [3H]thymidine incorporation and 14CO2 uptake in the dark agreed when activity was high but diverged when activity was low and when blue-green algae (cyanobacteria) dominated the phytoplankton. Size fractionation indicated negligible uptake of [3H]thymidine in the >3-μm fraction during a chrysophycean bloom in early June. We found that >50% of the 3H activity was in the >3-μm fraction in late August; this phenomenon was most likely due to Microcystis spp., their associated bacteria, or both. Over 60% of the 14CO2 uptake in the dark was attributed to algae on each sampling occasion. Algal exudate was an important carbon source for planktonic bacteria. Bacterial production was roughly 50% of primary production. 相似文献
18.
The δ13C value of a tropical marine grass Thalassia testudinum is −9.04‰. This value is similar to the δ13C value of terrestrial tropical grasses. The δ13C values of the organic acid fraction, the amino acid fraction, the sugar fraction, malic acid, and glucose are: −11.2‰, −13.1‰, −10.1‰, −11.1‰, and −11.5‰, respectively. The δ13C values of malic acid and glucose of Thalassia are similar to the δ13C values of these intermediates in sorghum leaves and attest to the presence of the photosynthetic C4-dicarboxylic acid pathway in this marine grass. The inorganic HCO3− for the growth of the grass fluctuates between −6.7 to −2.7‰ during the day. If CO2 fixation in Thalassia is catalyzed by phosphoenolpyruvate carboxylase (which would result in a −3‰ fractionation between HCO3− and malic acid), the predicted δ13C value for Thalassia would be −9.7 to −5.7‰. This range is close to the observed range of −12.6 to −7.8‰ for Thalassia and agree with the operation of the C4-dicarboxylic acid pathway in this plant. The early products of the fixation of HCO3− in the leaf sections are malic acid and aspartic acid which are similar to the early products of CO2 fixation in C4 terrestrial plants. 相似文献
19.
Gammaherpesvirus infection is associated with an increased incidence of lymphoproliferative disease in immunocompromised hosts. Murine gammaherpesvirus 68 (γHV68) infection of BALB β2-microglobulin-deficient (BALB β2m−/−) mice provides an animal model for analysis of the mechanisms responsible for the induction of a lymphoproliferative disease, atypical lymphoid hyperplasia (ALH), that is pathologically similar to posttransplant lymphoproliferative disease associated with Epstein-Barr virus infection. Here we report that the γHV68 v-cyclin and v-bcl-2 genes are required for the efficient induction of γHV68-associated ALH in BALB β2m−/− mice, while the v-GPCR gene is dispensable for ALH induction. In contrast to these findings, deletion of the viral M1 gene enhanced ALH. Thus, γHV68 genes can either inhibit or enhance the induction of lymphoproliferative disease in immunocompromised mice. 相似文献
20.
Distinct involvement of the Jun-N-terminal kinase and NF-kappaB pathways in the repression of the human COL1A2 gene by TNF-alpha
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We used a gene knockout approach to elucidate the specific roles played by the Jun-N-terminal kinase (JNK) and NF-κB pathways downstream of TNF-α in the context of α(2) type I collagen gene (COL1A2) expression. In JNK1−/−-JNK2−/− (JNK−/−) fibroblasts, TNF-α inhibited basal COL1A2 expression but had no effect on TGF-β-driven gene transactivation unless jnk1 was introduced ectopically. Conversely, in NF-κB essential modulator−/− (NEMO−/−) fibroblasts, lack of NF-κB activation did not influence the antagonism exerted by TNF-α against TGF-β but prevented repression of basal COL1A2 gene expression. Similar regulatory mechanisms take place in dermal fibroblasts, as evidenced using transfected dominant-negative forms of MKK4 and IKK-α, critical kinases upstream of the JNK and NF-κB pathways, respectively. These results represent the first demonstration of an alternate usage of distinct signaling pathways by TNF-α to inhibit the expression of a given gene, COL1A2, depending on its activation state. 相似文献