The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by ...RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.
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► ATR is phosphorylated at Thr 1989 after DNA damage ► ATR autophosphorylates Thr 1989 in trans independently of TopBP1 ► TopBP1 engages phosphorylated ATR via BRCT domains 7 and 8 ► Binding of TopBP1 to ATR facilitates ATR stimulation and substrate recognition
Chk1 is a kinase crucial for genomic integrity and an effector of ATR (ATM and Rad3-related) in DNA damage response. Here, we show that Chk1 regulates the DNA damage-induced ubiquitination of ...proliferating cell nuclear antigen (PCNA), which facilitates the continuous replication of damaged DNA. Surprisingly, this Chk1 function requires the DNA replication protein Claspin but not ATR. Claspin, which is stabilized by Chk1, regulates the binding of the ubiquitin ligase Rad18 to chromatin. Timeless, a Claspin-associating protein, is also required for efficient PCNA ubiquitination. Thus, Chk1 and the Claspin-Timeless module of replication forks not only participate in ATR signaling, but also protect stressed forks independently of ATR.
Kidney function and blood pressure homeostasis are regulated by purinergic signaling mechanisms. These autocrine/paracrine signaling pathways are initiated by the release of cellular ATP, which ...influences kidney hemodynamics and steady-state renin secretion from juxtaglomerular cells. However, the mechanism responsible for ATP release that supports tonic inputs to juxtaglomerular cells and regulates renin secretion remains unclear. Pannexin 1 (Panx1) channels localize to both afferent arterioles and juxtaglomerular cells and provide a transmembrane conduit for ATP release and ion permeability in the kidney and the vasculature. We hypothesized that Panx1 channels in renin-expressing cells regulate renin secretion in vivo. Using a renin cell-specific Panx1 knockout model, we found that male Panx1 deficient mice exhibiting a heightened activation of the renin-angiotensin-aldosterone system have markedly increased plasma renin and aldosterone concentrations, and elevated mean arterial pressure with altered peripheral hemodynamics. Following ovariectomy, female mice mirrored the male phenotype. Furthermore, constitutive Panx1 channel activity was observed in As4.1 renin-secreting cells, whereby Panx1 knockdown reduced extracellular ATP accumulation, lowered basal intracellular calcium concentrations and recapitulated a hyper-secretory renin phenotype. Moreover, in response to stress stimuli that lower blood pressure, Panx1-deficient mice exhibited aberrant “renin recruitment” as evidenced by reactivation of renin expression in pre-glomerular arteriolar smooth muscle cells. Thus, renin-cell Panx1 channels suppress renin secretion and influence adaptive renin responses when blood pressure homeostasis is threatened.
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Cell mechanical properties that depend on cytoskeleton architecture are critical to the mechanotransduction process, and have great potential for cancer diagnosis and therapy. In this study, the ...morphological and mechanical properties of typical osteosarcoma microenvironment cells, including mesenchymal stem cells (MSC), normal human osteoblast cells (NHOst) and osteosarcoma cells (MG-63), were compared using atomic force microscopy (AFM). The MG-63 cells were smaller and thicker than the MSC and NHOst cells. The membrane roughness of MG-63 cells was higher than that of MSC and NHOst cells. The MG-63 cells had lower stiffness than their normal counterparts due to their reduced organization of the cytoskeleton structure. The cell stiffness influenced the mechanotransduction. The MG-63 cells had a lower percentage of nuclear YAP/TAZ compared with the MSC and NHOst cells. The F-actin assembly was disrupted by the cytochalasin D (cyto D) treatment used to investigate its influence on mechanotransduction. Disruption of the cytoskeleton leaded to a decrease of the cell stiffness, and reduced the nuclear YAP/TAZ percentage, indicating its inhibition in the cell mechanotransduction process. This study would shed light on the development of a novel cancer diagnosis strategy and would contribute to reveal the relationship between the cytoskeleton structure and the cell mechanical properties.
RATIONALE:Resistance arteries and conduit arteries rely on different relative contributions of endothelial-derived hyperpolarization versus nitric oxide to achieve dilatory heterocellular signaling. ...Anatomically, resistance arteries use myoendothelial junctions (MEJs), endothelial cell projections that make contact with smooth muscle cells. Conduit arteries have very few to no MEJs.
OBJECTIVE:Determine if the presence of MEJs in conduit arteries can alter heterocellular signaling.
METHODS AND RESULTS:We previously demonstrated that PAI-1 (plasminogen activator inhibitor-1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI-1 directly to conduit arteries (carotid arteries) to determine if this could induce formation of MEJs. We found a significant increase in endothelial cell projections resembling MEJs that correlated with increased biocytin dye transfer from endothelial cells to smooth muscle cells. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI-1-treated carotids underwent a switch from a conduit to resistance artery vasodilatory profile via diminished nitric oxide signaling and increased endothelial-derived hyperpolarization signaling in response to the endothelium-dependent agonists acetylcholine and NS309. After PAI-1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice with PAI-1, but lacking alpha globin (Hba1), demonstrated that L-nitro-arginine methyl ester, an inhibitor of nitric oxide signaling, was able to prevent arterial relaxation.
CONCLUSIONS:The presence or absence of MEJs is an important determinant for influencing heterocellular communication in the arterial wall. In particular, alpha globin expression, induced within newly formed endothelial cell projections, may influence the balance between endothelial-derived hyperpolarization and nitric oxide–mediated vasodilation.
Verticillium dahliae, a soil-borne pathogen, causes Verticillium wilt, one of the most serious diseases in cotton, deleteriously influencing crop's production and quality. Verticillium wilt has ...become a major obstacle in cotton production since Helicoverpa armigera, the cotton bollworm, became effectively controlled in recent years. The wilt is becoming a key subject of research in cotton-resistance genetics, breeding and plant pathology. This paper reviews the recent research progress on genetic methods of resistance, the status and existing problems, traditional breeding, the main resistance mechanism, molecular markers and genetic engineering of resistance genes. It is hoped that new breeding methods and new varieties resistant to Verticillium wilt will be developed in the very near future.
The DNA damage and replication checkpoints are signaling mechanisms that regulate and coordinate
cellular responses to genotoxic conditions. The activation of checkpoints not only attenuates cell
...cycle progression, but also facilitates DNA repair and recovery of faulty replication forks, thereby
preventing DNA lesions from being converted to inheritable mutations. It has become increasingly
clear that the activation and signaling of the checkpoint are intimately linked to the cellular processes
directly involved in chromosomal metabolism, such as DNA replication and DNA repair. Thus, the checkpoint
pathway is not just a surveillance system that monitors genomic integrity and regulates cell
proliferation, but also an integral part of the processes that work directly on chromosomes to maintain
genomic stability. In this article, we discuss the current models of DNA damage and replication checkpoints,
and highlight recent advances in the field.
Resistance arteries use different dilatory heterocellular signaling mechanisms than conduit arteries (endothelial derived hyperpolarization versus nitric oxide). Anatomically, resistance arteries use ...myoendothelial junctions (MEJs), endothelial cell (EC) extensions that make contact with smooth muscle cells (SMCs), which are not present in conduit arteries. The objective of this study was to determine if the presence of MEJs in conduit arteries can alter heterocellular signaling. We previously demonstrated that plasminogen activator inhibitor‐1 (PAI‐1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI‐1 directly to conduit arteries (carotid arteries, CAs) in live mice to determine if this could induce formation of MEJs. We found a significant increase in EC projections that resemble MEJs, correlating with increased biocytin dye transfer from ECs to SMCs and dilation to NS309. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI‐1‐treated CAs underwent a switch from conduit to resistance artery vasodilatory profile via increased endothelial‐derived hyperpolarization (EDH) signaling and diminished nitric oxide (NO) signaling. Following PAI‐1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice lacking alpha globin (Hba1−/−) demonstrated that L‐NAME, an inhibitor of NO signaling, was able to prevent vasodilation. Thus, the presence or absence of MEJs is an important determinant for protein expression to influence heterocellular communication. In particular, our data suggests that alpha globin may allow for EDH to predominate in resistance arteries.
Support or Funding Information
This work was supported by HL088554 (BEI) and the National Natural Science Foundation of China 81672945 (XHS).
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
To investigate the effectiveness and safety of tripterygium glycosides (TG) tablet for the treatment of Lupus nephritis (LN).
Several databases were systematically searched including PubMed, Embase, ...Cochrane, Wiley, China National Knowledge Infrastructure Database, SinoMed and Wanfang Library till June 20, 2020. Revman5.3 was utilized to analyze the data according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement.
In total, 8 randomized controlled trials involving 583 participants were identified. Meta-analyses showed that, compared with glucocorticoids (GC) alone, the combination with TG tablet provided a statistically significant improvement in total remission (TR) ( = 1.27, 95% : 1.08-1.50, = 0.004), complete remission (CR) ( = 1.61, 95% : 1.05-2.47, = 0.03) and C3 levels ( = 0.27, 95% : 0.14-0.39, < 0.000 1), C4 levels ( = 0.12, 95% : 0.07-0.17, < 0.000 01). No significant differences were seen in TR, CR, proteinuria, serum creatinine, C3 and C4 (TR: = 1.00, 95% : 0.87-1.16, = 0.95; CR: = 1.10, 95% : 0.78-1.56, = 0.58; proteinuria levels: = -0.06, 95% : -0.13 to 0.01, = 0.10; serum creatinine levels: = -0.01, 95%: -7.36 to 7.35, = 1.00; C3 levels: = 0.01, 95%: -0.06 to 0.07, = 0.84; C4 levels: = -0.01, 95%: -0.03 to 0.01, = 0.49) between azathioprine (AZA) / leflomit (LEF) + GC and TG tablet + GC. Adverse events (hepatic dysfunction, nausea, vomitting) showed no statistical differences between the TG tablet + GC group and the GC group. There were more new onset of irregular menstruation in the TG tablet + GC group than those in the AZA + GC ( = 3.57, 95% : 1.40-9.11, = 0.008) /LEF+ GC ( = 6.69, 95% : 2.42-18.46, = 0.000 2) group, but leucopenia lower than those in AZA + GC group ( = 0.38, 95% : 0.17-0.85, = 0.02) and alopecia ( = 0.14, 95% : 0.03-0.77, = 0.02) and rash ( = 0.09, 95% : 0.01-0.69, = 0.02) lower than those in LEF + GC group.
This review indicates that TG tablet maybe effective in LN treatment. Nevertheless, adverse events cannot be ignored. Large sample, multi-center, high-quality clinical studies are needed to verify the exact effects and safety of TG tablet in treatment of LN.
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