ATR autophosphorylation as a molecular switch for checkpoint activation

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.     

A HIF-1 target, ATIA, protects cells from apoptosis by modulating the mitochondrial thioredoxin, TRX2

The regulation of apoptosis is critical for controlling tissue homeostasis and preventing tumor formation and growth. Reactive oxygen species (ROS) generation plays a key role in such regulation. Here, we describe a HIF-1 target, Vasn/ATIA (anti-TNFα-induced apoptosis), which protects cells against TNFα- and hypoxia-induced apoptosis. Through the generation of ATIA knockout mice, we show that ATIA protects cells from apoptosis through regulating the function of the mitochondrial antioxidant, thioredoxin-2, and ROS generation. ATIA is highly expressed in human glioblastoma, and ATIA knockdown in glioblastoma cells renders them sensitive to hypoxia-induced apoptosis. Therefore, ATIA is not only a HIF-1 target that regulates mitochondrial redox pathways but also a potentially diagnostic marker and therapeutic target in human glioblastoma.     

Uremic Retention Solute Indoxyl Sulfate Level Is Associated with Prolonged QTc Interval in Early CKD Patients

Total mortality and sudden cardiac death is highly prevalent in patients with chronic kidney disease (CKD). In CKD patients, the protein-bound uremic retention solute indoxyl sulfate (IS) is independently associated with cardiovascular disease. However, the underlying mechanisms of this association have yet to be elucidated. The relationship between IS and cardiac electrocardiographic parameters was investigated in a prospective observational study among early CKD patients. IS arrhythmogenic effect was evaluated by in vitro cardiomyocyte electrophysiological study and mathematical computer simulation. In a cohort of 100 early CKD patients, patients with corrected QT (QTc) prolongation had higher IS levels. Furthermore, serum IS level was independently associated with prolonged QTc interval. In vitro, the delay rectifier potassium current (IK) was found to be significantly decreased after the treatment of IS in a dose-dependent manner. The modulation of IS to the IK was through the regulation of the major potassium ion channel protein Kv 2.1 phosphorylation. In a computer simulation, the decrease of IK by IS could prolong the action potential duration (APD) and induce early afterdepolarization, which is known to be a trigger mechanism of lethal ventricular arrhythmias. In conclusion, serum IS level is independently associated with the prolonged QTc interval in early CKD patients. IS down-regulated I_K channel protein phosphorylation and the I_K current activity that in turn increased the cardiomyocyte APD and QTc interval in vitro and in the computer ORd model. These findings suggest that IS may play a role in the development of arrhythmogenesis in CKD patients.     

64Cu-ATSM Hypoxia Positron Emission Tomography for Detection of Conduit Ischemia in an Experimental Rat Esophagectomy Model

Background

We designed a hypoxia-imaging modality to detect ischemia of the gastric conduit after esophagectomy.

Materials and Methods

A rat esophagectomy model was created using 12-16-week-old, 300-350 g male Sprague-Dawley rats. In the operation group (n=6), partial gastric devascularization was performed by ligating the left gastric artery and the short gastric arteries and an esophagogastric anastomosis was performed. In the control group (n=6), the esophageal-gastric junction was incised and suturing was performed without gastric devascularization. Positron emission tomography (PET) images were taken using a microPET rodent model scanner, 24 h after the initial operation, after injection of 200 μCi 64Cu-diacetyl-bis (N4-methylsemicarbazone) (64Cu-ATSM) and pimonidazole 120 mg/kg. After microPET imaging, autoradiography and immunohistochemistry were performed.

Results

The PET image revealed 64Cu-ATSM uptake at the fundus in the operation group 3 h after 64Cu-ATSM injection. The maximum percentage of the injected dose per gram of tissue was higher in the operation group (0.047±0.015 vs. 0.026±0.006, p=0.021). The fundus/liver ratio was also higher in the operation group (0.541±0.126 vs. 0.278±0.049, p=0.002). Upon autoradiography, 64Cu-ATSM uptake was observed in the fundus in the operation group, and was well-correlated to that observed on the PET image. Upon immunohistochemistry, expression of hypoxia-inducible factor 1a and pimonidazole were significantly increased at the fundus and lesser curvature compared to the greater curvature in the operation group.

Conclusion

Hypoxia PET imaging with 64Cu-ATSM can detect ischemia in a rat esophagectomy model. Further clinical studies are needed to verify whether hypoxia imaging may be useful in humans.     

Floral Spray Transformation Can Efficiently Generate Arabidopsis

In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.     

Enhanced biological effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant, on HL60 cells

Granulocyte colony-stimulating factor (G-CSF) is a cytokine secreted by stromal cells and plays a role in the differentiation of bone marrow stem cells and proliferation of neutrophils. Therefore, G-CSF is widely used to reduce the risk of serious infection in immunocompromised patients; however, its use in such patients is limited because of its non-persistent biological activity. We created an N-linked glycosylated form of this cytokine, hG-CSF (Phe140Asn), to assess its biological activity in the promyelocyte cell line HL60. Enhanced biological effects were identified by analyzing the JAK2/STAT3/survivin pathway in HL60 cells. In addition, mutant hG-CSF (Phe140Asn) was observed to have enhanced chemoattractant effects and improved differentiation efficiency in HL60 cells. These results suggest that the addition of N-linked glycosylation was successful in improving the biological activity of hG-CSF. Furthermore, the mutated product appears to be a feasible therapy for patients with neutropenia.     

Delineation of critical amino acids in activation function 1 of progesterone receptor for recruitment of transcription coregulators

The activation functions AF1 and AF2 of nuclear receptors mediate the recruitment of coregulators in gene regulation. AF1 is mapped to the highly variable and intrinsically unstructured N terminal domain and AF2 lies in the conserved ligand binding domain. The unstructured nature of AF1 offers structural plasticity and hence functional versatility in gene regulation. However, little is known about the key functional residues of AF1 that mediates its interaction with coregulators. This study focuses on the progesterone receptor (PR) and reports the identification of K464, K481 and R492 (KKR) as the key functional residues of PR AF1. The KKR are monomethylated and function cooperatively. The combined mutations of KKR to QQQ render PR isoform B (PRB) hyperactive, whereas KKR to FFF mutations abolishes as much as 80% of PR activity. Furthermore, the hyperactive QQQ mutation rescues the loss of PR activity due to E911A mutation in AF2. The study also finds that the magnitudes of the mutational effect differ in different cell types as a result of differential effects on the functional interaction with coregulators. Furthermore, KKR provides the interface for AF1 to physically interact with p300 and SRC-1, and with AF2 at E911. Intriguingly, the inactive FFF mutant interacts strikingly stronger with both SRC-1 and AF2 than wt PRB. We propose a tripartite model to describe the dynamic interactions between AF1, AF2 and SRC-1 with KKR of AF1 and E911 of AF2 as the interface. An overly stable interaction would hamper the dynamics of disassembly of the receptor complex.     

Soil surface disturbance alters cyanobacterial biocrusts and soil properties in dry grassland and shrubland ecosystems

Plant and Soil - Biological soil crusts (biocrusts) dominate soil surfaces in drylands, providing services that include soil stabilization and carbon uptake. In this study, we investigated the...     

Crosslinking of uteroglobin by transglutaminase

Uteroglobin, a progesterone induced, pregnancy related protein, can be incorporated into higher molecular weight proteins by human placental Factor XIIIa. This process is time dependent, requires CaCl2 and can be inhibited by the addition of polylysine, dansylcadavarine or histamine. Crosslinking of uteroglobin into higher molecular weight proteins can also be brought about by guinea pig liver transglutaminase. Such a process may be involved in the modification of epididymal spermatozoa to suppress their antigenicity.     

Enzymatic synthesis of tea theaflavin derivatives and their anti-inflammatory and cytotoxic activities

Derivatives based on a benzotropolone skeleton (9-26) have been prepared by the enzymatic coupling (horseradish peroxidase/H2O2) of selected pairs of compounds (1-8), one with a vic-trihydroxyphenyl moiety, and the other with an ortho-dihydroxyphenyl structure. Some of these compounds have been found to inhibit TPA-induced mice ear edema, nitric oxide (NO) synthesis, and arachidonic acid release by LPS-stimulated RAW 264.7 cells. Their cytotoxic activities against KYSE 150 and 510 human esophageal squamous cell carcinoma and HT 29 human colon cancer cells were also evaluated.