The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress

Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear localization regions in the N-terminal region of RECQL4 and a nucleolar localization signal at amino acids 376-386. RECQL4 localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and gamma irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 accumulated in nucleoli. Using a T7 phage display screen, we determined that RECQL4 interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and signaling pathways. The RECQL4 nucleolar localization was inhibited by pretreatment with a PARP-1 inhibitor. The C-terminal portion of RECQL4 was found to be an in vitro substrate for PARP-1. These results demonstrate changes in the intracellular localization of RECQL4 in response to oxidative stress and identify an interaction between RECQL4 and PARP-1.     

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to approximately 200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation.     

WFS1 protein modulates the free Ca(2+) concentration in the endoplasmic reticulum

The WFS1 gene, encoding an endoplasmic reticulum (ER) membrane glycoprotein, is mutated in Wolfram syndrome characterized by diabetes mellitus and optic atrophy. Herein, Ca(2+) dynamics were examined in WFS1-knockdown and -overexpressing HEK293 cells. Studies using ER-targeted Ca(2+)-sensitive photoprotein aequorin demonstrated WFS1 protein to positively modulate ER Ca(2+) levels by increasing the rate of Ca(2+) uptake. Furthermore, Ca(2+) imaging with Fura-2 showed the magnitude of the store-operated Ca(2+) entry to parallel WFS1 expression levels. These data indicate that WFS1 protein participates in the regulation of cellular Ca(2+) homeostasis, at least partly, by modulating the filling state of the ER Ca(2+) store.     

Role of numb in dendritic spine development with a Cdc42 GEF intersectin and EphB2

Numb has been implicated in cortical neurogenesis during nervous system development, as a result of its asymmetric partitioning and antagonizing Notch signaling. Recent studies have revealed that Numb functions in clathrin-dependent endocytosis by binding to the AP-2 complex. Numb is also expressed in postmitotic neurons and plays a role in axonal growth. However, the functions of Numb in later stages of neuronal development remain unknown. Here, we report that Numb specifically localizes to dendritic spines in cultured hippocampal neurons and is implicated in dendritic spine morphogenesis, partially through the direct interaction with intersectin, a Cdc42 guanine nucleotide exchange factor (GEF). Intersectin functions as a multidomain adaptor for proteins involved in endocytosis and cytoskeletal regulation. Numb enhanced the GEF activity of intersectin toward Cdc42 in vivo. Expression of Numb or intersectin caused the elongation of spine neck, whereas knockdown of Numb and Numb-like decreased the protrusion density and its length. Furthermore, Numb formed a complex with EphB2 receptor-type tyrosine kinase and NMDA-type glutamate receptors. Knockdown of Numb suppressed the ephrin-B1-induced spine development and maturation. These results highlight a role of Numb for dendritic spine development and synaptic functions with intersectin and EphB2.     

Acute effect of static stretching on power output during concentric dynamic constant external resistance leg extension

The purpose of the present study was to clarify the effect of static stretching on muscular performance during concentric isotonic (dynamic constant external resistance [DCER]) muscle actions under various loads. Concentric DCER leg extension power outputs were assessed in 12 healthy male subjects after 2 types of pretreatment. The pretreatments included (a) static stretching treatment performing 6 types of static stretching on leg extensors (4 sets of 30 seconds each with 20-second rest periods; total duration 20 minutes) and (b) nonstretching treatment by resting for 20 minutes in a sitting position. Loads during assessment of the power output were set to 5, 30, and 60% of the maximum voluntary contractile (MVC) torque with isometric leg extension in each subject. The peak power output following the static stretching treatment was significantly (p < 0.05) lower than that following the nonstretching treatment under each load (5% MVC, 418.0 +/- 82.2 W vs. 466.2 +/- 89.5 W; 30% MVC, 506.4 +/- 82.8 W vs. 536.4 +/- 97.0 W; 60% MVC, 478.6 +/- 77.5 W vs. 523.8 +/- 97.8 W). The present study demonstrated that relatively extensive static stretching significantly reduces power output with concentric DCER muscle actions under various loads. Common power activities are carried out by DCER muscle actions under various loads. Therefore, the result of the present study suggests that relatively extensive static stretching decreases power performance.     

Conferring cadmium resistance to mature tobacco plants through metal-adsorbing particles of tomato mosaic virus vector

Tomato mosaic virus vectors were designed that produced, by a translational readthrough, a fusion protein consisting of coat protein and metal-binding peptide, as a result of which particles were expected to present the metal-binding peptides on their surface. When inoculated in plants, they were expected to replicate and form a metal-adsorbing artificial sink in the cytoplasm, so as to reduce metal toxicity. Vectors were constructed harbouring sequences encoding various lengths of polyhistidine as a metal-binding peptide. One of the vectors, TLRT6His, which contains a 6 x histidine sequence, moved systemically in tobacco plants, and its particles were shown to retain cadmium ions by an in vitro assay. When a toxic amount of cadmium was applied, the toxic effect was much reduced in TLRT6His-inoculated tobacco plants, probably as a result of cadmium adsorption by TLRT6His particles in the cytosol. This shows the possible use of an artificial sink for metal tolerance and the advantage of employing a plant viral vector for phytoremediation.