Electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM)

A microbial array chip with collagen gel spots entrapping living Escherichia coli (E. coli) DH5alpha was applied for the screening of recombinant protein solubilities. The alpha-fragment of beta-galactosidase (betaGal) was fused to the target protein, namely, maltose-binding protein (MBP), to monitor the solubility of MBP. Scanning electrochemical microscopy (SECM) was used to detect the release of p-aminophenol from E. coli cells catalyzed by intracellular betaGal. Comparison of the SECM-based method with the Western blotting-based method indicated that the current response obtained using SECM increased with an increase in the betaGal activity and therefore, with the soluble fraction of MBP in the host cells.     

A real-time method of imaging glucose uptake in single, living mammalian cells

This protocol details a method for monitoring glucose uptake into single, living mammalian cells using a fluorescent D-glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), as a tracer. The specifically designed chamber and superfusion system for evaluating 2-NBDG uptake into cells in real time can be combined with other fluorescent methods such as Ca2+ imaging and the subsequent immunofluorescent classification of cells exhibiting divergent 2-NBDG uptake. The whole protocol, including immunocytochemistry, can be completed within 2 d (except for cell culture). The procedure for 2-NBDG synthesis is also presented.     

Work behaviors of artificial muscle based on cation driven polypyrrole

A soft actuator mimicking natural muscles (artificial muscle) has been developed using a flexible conducting polymer of polypyrrole films, which were driven by electrical stimulus in a saline solution. The work characteristics were studied under various load stresses and found to behave like natural muscles. The artificial muscles shrunk and stiffened by the positive electrical stimulus by 2-3% at the maximum force of 5 MPa, and relaxed by application of negative voltages. At larger load stresses, the artificial muscle shrunk slowly as natural muscles do. The driving current also lasted longer at larger loads, indicating that the muscle sensed the magnitude of the load stress. During contraction of the muscle, the conversion efficiency from the electrical input and mechanical output energies was estimated to be around 0.06%. The maximum volumetric work was approximately estimated to be 100 kJ m(-3). These figures are unexpectedly small compared with those of natural muscles.     

Sex Change in Protogynous Fish Red-Belted Anthias Pseudanthias rubrizonatus (Serranidae) in Kagoshima Bay,Japan

Journal of Ichthyology - The Serranidae are well known for protogynous sex change. The red-belted anthias Pseudanthias rubrizonatus inhabits Kagoshima Bay. We aimed to estimate the body size and...     

Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.     

Chromosomal effects of newly identified water pollutants PBTA-1 and PBTA-2 and their possible mother compounds (azo dyes) and intermediates (non-ClPBTAs) in two Chinese hamster cell lines

We performed the in vitro micronucleus (MN) test on 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)-ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), which are newly identified water pollutants from the Nishitakase river in Kyoto, Japan, and on their possible mother compounds (AZO DYE) and intermediates (non-ClPBTAs). We tested these compounds in the absence and presence of S9 mix in two Chinese hamster cell lines CHL and V79-MZ and scored MN, polynuclear and karyorrhectic (PN), and mitotic (M) cells. PBTA-2 in the absence of S9 mix induced the strongest responses in both cell lines. It was also a strong inducer of binucleate cells in PN cells in both cell lines, which suggested that it induced polyploidy. PBTA-1 showed clear positive results only in the absence of S9 mix and only in V79-MZ cells, inducing aneuploidy. In CHL cells AZO DYE-1 significantly induced MN cells in the presence of S9 mix, and AZO DYE-2 induced MN and PN cells, including binucleate cells and cells with a multilobed nucleus, in the absence of S9 mix. In V79-MZ cells, AZO DYE-1 and -2 induced primarily M cells in the presence of S9 mix. 9% of the M cells treated with 50 microg/ml AZO DYE-1 showed endoreduplication. AZO DYE-2 at 200 microg/ml condensed the chromatin in 100% of the cells. The non-ClPBTAs were a bit more cytotoxic than the other compounds and induced a slight increase in MN cells in both cell lines. Some of the chemicals tested induced a characteristic karyomorphology that might reflect abnormal cell division. Abnormalities of cell division could be detected in PN and M cells as well as in MN cells. Structure-activity relationships have also been discussed.     

In vivo gene transfer of an extracellular domain of platelet-derived growth factor beta receptor by the HVJ-liposome method ameliorates bleomycin-induced pulmonary fibrosis

A number of investigators have reported augmented expression of PDGF in lungs with idiopathic pulmonary fibrosis (IPF) or with other types of pulmonary fibrosis. To accomplish such a regulation of PDGF activity, we constructed an expression plasmid of the extracellular domain of PDGF receptor beta chain (XR), which lacks intracellular tyrosine kinase domain and transmembrane portions, and estimated the therapeutic effects of XR gene transfer through the trachea on bleomycin-induced lung fibrosis of C57BL/6 mice using the hemagglutinating virus of Japan(HVJ)-liposome method. The XR gene transfer ameliorated the increases in the wet weight and hydroxyproline content and the histopathologic changes of the lung induced by bleomycin. These findings suggest that PDGF plays a crucial role in the pathogenesis of pulmonary fibrosis, and that XR gene transfer using the HVJ-liposome method may limit the progression of pulmonary fibrosis.     

Molecular evidence of the existence of two sibling species within the echinothurioid echinoid Asthenosoma ijimai from Japanese waters

The echinoid, Asthenosoma ijimai belonging to the order Echinothurioida from Japanese waters shows the geographical variation in morphological and ecological characters. The echinothurioid from Ryukyu Islands in southern Japan is cleary different from that of Sagami Bay and Suruga Bay in the middle part of Japan at non-molecular level.Their phylogenetic and taxonomic relationships were studied at the molecular level by allozyme analysis. The results demonstrated that the two echinothurioids from Ryukyu Islands and Sagami Bay do not share gene pools with each other, and they were fixed for different alleles at five genetic loci (Mdh, G6pd, Po, Alk-3 and Est-7) in a total of 23 enzyme genetic loci scored. This indicates no gene flow between the two echinothurioids, and is a molecular evidence for that they are reproductively isolated and genetically distinct species. The Nei's genetic distance (D=0.181) between the two were significantly higher than those between conspecific local populations, and comparable to those between closely related species in many other animals containing echinoderms. The present molecular data are well consistent with the non-molecular evidence from morphology, developmental biology and ecology. Putting these data together, we propose that the two echinothurioids should be classified as two sibling species of the genus Asthenosoma and would like to give the following scientific names: the echinothurioid species from Sagami Bay is Asthenosoma ijimai and that from Ryukyu Islands is A. ijimai R.