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Margareta Lantow Ramya Sivakumar Leilani Zeumer Clive Wasserfall Ying-Yi Zheng Mark A Atkinson Laurence Morel 《Arthritis research & therapy》2013,15(2):R49
Introduction
An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF.Methods
Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus.Results
B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4+ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice.Conclusion
The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus. 相似文献63.
John F. Kalinich Ramya Ramakrishnan David E. McClain Narayani Ramakrishnan 《Free radical research》2013,47(4):349-358
4-Hydroxynonenal (HNE) is the major aldehydic product resulting from lipid peroxidation and has been implicated as involved in several pathological conditions. In our continuing studies on the role of membranes and lipid peroxidation in the induction of apoptosis, we investigated the effect of HNE on cultured human malignant immune system cells. Two cell lines were utilized; MOLT-4, a human T-cell leukemia cell line, and Reh, a human B-cell lymphoma cell line. A 10 min treatment with 0.01 mM HNE resulted in the apoptotic death, as determined by flow cytometric and morphological analyses, of both cell lines within 24 h. MOLT-4 cells exhibited the manifestations of impending apoptotic death much sooner than did Reh cells, indicating that MOLT-4 cells were more sensitive or not as efficient at detoxifying HNE than were Reh cells. These results suggest that peroxidative damage to cellular membranes resulting in the production of HNE may be a trigger for the induction of apoptosis in immune system cells. 相似文献
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Kwang‐Chul Kwon Ramya Nityanandam James S. New Henry Daniell 《Plant biotechnology journal》2013,11(1):77-86
Glucagon‐like peptide (GLP‐1) increases insulin secretion but is rapidly degraded (half‐life: 2 min in circulation). GLP‐1 analogue, exenatide (Byetta) has a longer half‐life (3.3–4 h) with potent insulinotropic effects but requires cold storage, daily abdominal injections with short shelf life. Because patients with diabetes take >60 000 injections in their life time, alternative delivery methods are highly desired. Exenatide is ideal for oral delivery because insulinotropism is glucose dependent, with reduced risk of hypoglycaemia even at higher doses. Therefore, exendin‐4 (EX4) was expressed as a cholera toxin B subunit (CTB)–fusion protein in tobacco chloroplasts to facilitate bioencapsulation within plant cells and transmucosal delivery in the gut via GM1 receptors present in the intestinal epithelium. The transgene integration was confirmed by PCR and Southern blot analysis. Expression level of CTB‐EX4 reached up to 14.3% of total leaf protein (TLP). Lyophilization of leaf material increased therapeutic protein concentration by 12‐ to 24‐fold, extended their shelf life up to 15 months when stored at room temperature and eliminated microbes present in fresh leaves. The pentameric structure, disulphide bonds and functionality of CTB‐EX4 were well preserved in lyophilized materials. Chloroplast‐derived CTB‐EX4 showed increased insulin secretion similar to the commercial EX4 in beta‐TC6, a mouse pancreatic cell line. Even when 5000‐fold excess dose of CTB‐EX4 was orally delivered, it stimulated insulin secretion similar to the intraperitoneal injection of commercial EX4 but did not cause hypoglycaemia in mice. Oral delivery of the bioencapsulated EX4 should eliminate injections, increase patient compliance/convenience and significantly lower their cost. 相似文献
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Under physiological conditions, circulating platelets are discoid in shape.1 On these platelets, the fibrinogen receptor (integrin αIIbβ3) is in a low-affinity state, unable to bind soluble fibrinogen (Fg). Activation by agonists such as ADP and thrombin leads to a change in the conformation of the integrin αIIbβ3 through a process known as inside-out signaling. This enables the integrin to bind soluble Fg, which initiates a cascade of events referred to as outside-in signaling.2 Outside-in signaling control processes, such as platelet spreading and clot retraction, by regulating small G-proteins such as RhoA, Rac and cdc42.Key words: platelets, integrin αIIbβ3, Galpha13, RhoA, clot retraction, thrombin, fibrinogenThe majority of the physiological platelet agonists (except collagen) induce inside-out signaling by binding to specific G-protein-coupled receptors (GPCRs). A G-protein plays a crucial role in translating the signal from GPCR to downstream effector molecules, ultimately leading to affinity modulation of integrin αIIbβ3. Platelets express nine Gα subunits; namely Gq, Gi1, Gi2, Gi3, Gz, G12, G13, Gs and G16. Previous studies have shown that a small G-protein, RhoA, is activated by the G12/13 family and plays a crucial role in calcium-independent platelet shape change.3 However, RhoA is also activated by αIIbβ3 and inhibits platelet spreading to trigger clot retraction.4 Recently, in a series of elegant experiments, Gong et al. have described the dynamic regulation of RhoA through a signaling crosstalk between Gα13 and αIIbβ3.5By generating mice in which the platelets were depleted of Gα13 using siRNA technology, Gong et al. investigated the role of Gα13-mediated signaling on platelet spreading on immobilized Fg.5 The confocal images very clearly showed that, in the absence of Gα13, platelets spread poorly on Fg, which was rescued by pretreatment with the Rho-kinase inhibitor Y27632, confirming previous findings that RhoA activated downstream of integrin αIIbβ3 inhibits platelet spreading. Interestingly, Gα13-depleted platelets failed to activate c-Src but accelerated RhoA activation. From these observations, the authors infer that Gα13 is important for integrin-mediated c-Src activation and RhoA inhibition, leading to increased cell spreading.5Since Gα13 regulates integrin-mediated cell spreading and c-Src activation, Gong et al. examined the interaction of Gα13 with αIIbβ3 using co-immunoprecipitation and GST pull-down assays.5 They found that the GTP-bound form of Gα13 shows enhanced interaction with the integrin β3 subunit. This interaction is required for the activation of c-Src and the inhibition of RhoA. However, they found that the inhibition of RhoA is transient. RhoA activation is suppressed for the first 15 min of platelet spreading, after which RhoA is activated. This initial suppression is rescued by blocking Gα13 and β3 cytoplasmic domain (β3-CD) interaction. Furthermore, they observed that RhoA activation parallels clot retraction.5 These findings indicate that Gα13 is a key regulator of platelet spreading and clot retraction phenomena.According to Gong et al., thrombin-induced inside-out signaling through GPCR leads to GTP loading of Gα13 (Fig. 1A). This GTP-bound Gα13 interacts with integrin β3-CD of ligand-bound integrin, thus facilitating c-Src activation, which leads to platelet spreading. Blockade of the interaction between Gα13 and β3-CD or cleavage of β3-CD by calpain results in clot retraction (Fig. 1B).Open in a separate windowFigure 1Schematic representation of the dynamic regulation of RhoA by Gα13 during platelet activation. (A) Activation of platelets by thrombin receptors coupled to Gα13 leads to the activation of RhoA, leading to platelet shape change. (B) The change in the conformation of integrin to a high-affinity form results in fibrinogen binding to αIIbβ3. Active Gα13 binds to the cytoplasmic domain of β3 leading to the activation of c-Src, resulting in platelet spreading. The rise in intracellular calcium activates calpain, which cleaves the β3 cytoplasmic domain, releasing c-Src, which, resulting in the activation of RhoA, leads to cell retraction. *Denotes GTP-bound active form of G-proteins.Perhaps the most significant and novel finding of the study is the identification of integrin αIIbβ3 as an effector of Gα13. The study also convincingly shows that Gα13 bound to integrin regulates RhoA via c-Src. Furthermore, achieving 80% knockdown of Gα13 in an in vivo setting using siRNA represents a technological advancement. Since Gα13 binds to integrin β3-CD in a 1:1 stoichiometry, it appears that only a small population of integrin is regulated by Gα13, as there are far less Gα13 molecules in a single platelet than the number of αIIbβ3 molecules. This will require further investigation. Gong et al. also finds that an appreciable amount of Gα13 is associated with β3 in resting platelets, which requires some explanation.5 It is also not clear if Gα13 remains bound to β3-CD or dissociates from the integrin during clot retraction.Overall, this is a paradigm-shifting study that establishes the importance of the dynamic regulation of RhoA by Gα13 in order to achieve efficient platelet spreading and clot retraction. 相似文献
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Kandaswamy Ramya Kuppuswamy Ashok Ayyappa Saurabh Ghosh Viswanathan Mohan Venkatesan Radha 《Gene》2013
Objective
To investigate the genetic association of eight variants of the adiponectin gene with type 2 diabetes mellitus (T2DM), obesity and serum adiponectin level in the south Indian population.Methods
The study comprised of 1100 normal glucose tolerant (NGT) and 1100 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. The variants were screened by polymerase chain reaction-restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotype frequencies.Results
Of the 8 variants, four SNPs namely, + 276 G/T (rs1501299), − 4522 C/T (rs822393), − 11365 C/G (rs266729), and + 712 G/A (rs3774261) were significantly associated with T2DM in our study population. The −3971 A/G (rs822396) and − 11391 G/A (rs17300539) SNPs' association with T2DM diabetes was mediated through obesity (where the association with type 2 diabetes was lost after adjusting for BMI). There was an independent association of + 276 G/T (rs1501299) and − 3971 A/G (rs822396) SNPs with generalized obesity and + 349 A/G (rs2241767) with central obesity. Four SNPs, −3971 A/G (rs822396), + 276 G/T (rs1501299), − 4522 C/T (rs822393) and Y111H T/C (rs17366743) were significantly associated with hypoadiponectinemia. The haplotypes GCCATGAAT and AGCGTGGGT conferred lower risk of T2DM in this south Indian population.Conclusion
The adiponectin gene variants and haplotype contribute to the genetic risk towards the development of type 2 diabetes, obesity and hypoadiponectinemia in the south Indian population. 相似文献67.
Anodization and sol-gel treatments of titanium (Ti) were evaluated as biofilm control measures on surfaces exposed to seawater exposed to ultraviolet light. Anodized and sol-gel treated specimens were characterized using Raman spectroscopy to confirm the presence of TiO2. The single anatase phase was observed at the anodized surfaces whereas the anatase/rutile mixed phase was detected on the sol-gel coated surfaces. After exposure of the specimens to seawater, biofilms were characterized by total viable counts, and epifluorescence and Raman microscopy. These techniques confirmed the reduction in biofilm formation on both the anodized and sol-gel coated Ti specimens compared to the untreated specimens. Biofilm control by anodization was found to be more effective than by sol-gel treatment of the specimens. The higher particle size and the inhomogeneity at the sol-gel coated surfaces produced less effective biofilm control. 相似文献
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Sirinakis G Clapier CR Gao Y Viswanathan R Cairns BR Zhang Y 《The EMBO journal》2011,30(12):2364-2372
ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to reposition and reconfigure nucleosomes. Despite their diverse functions, all remodellers share highly conserved ATPase domains, many shown to translocate DNA. Understanding remodelling requires biophysical knowledge of the DNA translocation process: how the ATPase moves DNA and generates force, and how translocation and force generation are coupled on nucleosomes. Here, we characterize the real-time activity of a minimal RSC translocase 'motor' on bare DNA, using high-resolution optical tweezers and a 'tethered' translocase system. We observe on dsDNA a processivity of ~35 bp, a speed of ~25 bp/s, and a step size of 2.0 (±0.4, s.e.m.) bp. Surprisingly, the motor is capable of moving against high force, up to 30 pN, making it one of the most force-resistant motors known. We also provide evidence for DNA 'buckling' at initiation. These observations reveal the ATPase as a powerful DNA translocating motor capable of disrupting DNA-histone interactions by mechanical force. 相似文献