K+ is an indispensable cofactor for GrpE stimulation of ATPase activity of DnaK·DnaJ complex from Thermus thermophilus

AbstractK+ is an indispensable cofactor for ATPase activity of eukaryotic cytosolic Hsp70 chaperone systems which lack a GrpE homolog. In the case of the bacterial Hsp70 (DnaK) system, GrpE, a nucleotide exchange factor, stimulates ATPase activity but little is known about the effect of K+. Here, we have cloned a grpE gene from a thermophile, Thermus thermophilus, and purified a homodimeric GrpE protein. Using proteins of this bacterium, we found that the GrpE stimulation of ATPase activity of DnaK·DnaJ complex was absolutely dependent on the presence of K+

Identification of lysophospholipid receptors in human platelets: the relation of two agonists, lysophosphatidic acid and sphingosine 1-phosphate

AbstractLysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph-1-P) are known as structurally related bio-active lipids activating platelets through their respective receptors. Although the receptors for LPA and Sph-1-P have been recently identified in various cells, the identification and characterization of ones in platelets have been reported only preliminarily. In this report, we first investigated the distinct modes of LPA and Sph-1-P actions in platelet activation and found that LPA functioned as a much stronger agonist than Sph-1-P, and high concentrations of Sph-1-P specifically desensitized LPA-induced intracellular Ca2+ mobilization. In order to identify the responsible receptors underlying these observations, we analyzed the LPA and Sph-1-P receptors which might be expressed in human platelets, by RT-PCR. We found for the first time that Edg2, 4, 6 and 7 mRNA are expressed in human platelets

Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents.

Highly sensitive and low-cost DNA agarose gel detection systems were developed using non-mutagenic and loading dye-type DNA-staining reagents. The DNA detection system that used Midori Green Direct and Safelook Load-Green, both with an optimum excitation wavelength at ~490 nm, could detect DNA-fragments at the same sensitivity to that of the UV (312 nm)-transilluminator system combined with ethidium bromide, after it was excited by a combination of cyan LED light and a shortpass filter (510 nm). The cyan LED system can be also applied to SYBR Safe that is widely used as a non-toxic dye for post-DNA-staining. Another DNA-detection system excited by black light was also developed. Black light used in this system had a peak emission at 360 nm and caused less damage to DNA due to lower energy of UV rays with longer wavelength when compared to those of short UV rays. Moreover, hardware costs of the black light system were ~$100, less than 1/10 of the commercially available UV (365 nm) transilluminator (>$1,000). EZ-Vision and Safelook Load-White can be used as non-mutagenic and loading dye-type DNA-staining reagents in this system. The black light system had a greater detection sensitivity for DNA fragments stained by EZ-Vision and Safelook Load-White compared with the commercially available imaging system using UV (365 nm) transilluminator

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

Simple and low-cost recombinant enzyme-free seamless DNA cloning methods have recently become available. In vivo Escherichia coli cloning (iVEC) can directly transform a mixture of insert and vector DNA fragments into E. coli, which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation cloning extract (SLiCE) cloning uses the endogenous recombination activity of E. coli cellular extracts in vitro to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless cloning method as a useful tool. In this study, both seamless cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15–39 bp) end homology regions. However, colony formation was 30–60-fold higher with SLiCE cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. E. coli AQ3625 strains, which harbor a sbcA gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions in vivo. Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE cloning were still higher. In addition, the efficiency of seamless cloning methods depends on the intrinsic competency of E. coli cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE cloning permits the use of both homemade and commercially available competent cells because it can use general E. coli recA− strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid engineering

Outcomes and precautions of endoscopic submucosal dissection for undifferentiated-type early gastric cancer

Objectives: Since the development of techniques for endoscopic submucosal dissection (ESD), the indication range of endoscopic resection (ER) has been extended in early gastric cancer (EGC) treatment. For undifferentiated-type EGC, tumors with an intramucosal depth of invasion, no ulceration and a diameter of 20 mm or less were included in the expanded indications for ER in the Japanese Gastric Cancer Treatment Guidelines 2010. Nonetheless, because of difficulty in detecting lesions that meet the criteria for ER, the number of endoscopically resected cases of undifferentiated-type EGC is less than that of differentiated-type EGC. Methods: We retrospectively investigated the outcomes of ESD in 38 patients with 40 lesions of EGC in which the dominant pathological type was confirmed to be undifferentiated (signet ring cell carcinoma, poorly differentiated adenocarcinoma, mucinous adenocarcinoma) on histological examination of resected specimens. Results: Margin involvement and submucosal infiltration were common noncurative factors. Precise evaluation of the area and depth of lesions is a problem to be solved. Among a total of five patients with involved or uncertain horizontal margins, one of two patients who underwent additional surgery had residual cancer, and one of three patients who were observed had recurrence. Conclusions: Undifferentiated-type EGC with a positive horizontal margin may relapse after ESD. It is therefore essential to precisely evaluate the area of the lesion and to perform resection with an adequate safety margin to decrease the risk of recurrence