A possible role of glutathione and glutathione disulfide in tracheary element differentiation in the cultured mesophyll cells of Zinnia elegans.

We investigated the relationship between the cellular redox state of GSH or GSSG and tracheary element (TE) differentiation using a Zinnia experimental system, in which isolated mesophyll cells transdifferentiate to TEs. TE differentiation was suppressed by the application of L-buthionine sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at the early stage of cell culture. Application of GSSG at the early culture stage promoted the differentiation, but that of GSH or GSSG at an advanced period of culture suppressed the differentiation. Application of GSH and GSSG nullified the TE differentiation-suppressing effect of BSO. The results suggest that changes in the redox states of GSH and GSSG have a role in TE differentiation.     

Effects of lipophorin and 20-hydroxyecdysone on in vitro development of the larval endoparasitoid Venturia canescens (Hymenoptera: Ichneumonidae)

We previously reported that development of the embryos of the larval endoparasitoid Venturia canescens proceeded in the insect cell culture medium, MGM-450, and was promoted by the addition of a pupal extract from their host Galleria mellonella. The developmental promotion also was obtained by supplementing an equal amount of chicken egg yolk instead of the pupal extract to the medium. In this case, approximately 30% of the embryos developed into the second instar, but the value increased to more than 90% by treatment with 20-hydroxyecdysone. The medium supplemented with a G. mellonella pupal extract obtained by using Carlson's solution displayed growth-promoting ability, and in the extract, apolipophorin I was electrophoretically detected in large amounts. Both lipophorin purified from G. mellonella pupae and low density lipoprotein from chicken egg yolk acted as a growth-promoting substance for parasitoid development, although fetal bovine serum and 20-hydroxyecdysone were required as supplements to the medium for the expression of the ability. This indicated that lipophorin or lipophorin-transported lipids could act as a substance closely related to the growth-promoting factor(s) putatively involved in the host extract.     

Cell-surface hydrophobicity and scum formation of Rhodococcus rhodochrous strains with different colonial morphologies

Rhodococcus rhodochrous has been reported to be one of the micro-organisms responsible for the formation of scum which is thick and viscous biological foam in activated sludge plants. The hydrophobicity of mycolic acids present on the cell surface and the long-branched shape of the hyphae have been thought to contribute to the scum formation. Cell surface hydrophobicity and scum formation of four R. rhodochrous strains with different colony morphologies were determined, and the results showed that the two rough strains had strong cell surface hydrophobicity and produced scum, whereas the weakly hydrophobic smooth strain and the hydrophilic mucoidal strain did not. All four strains displayed long, branched hyphae, and their electrophoretic mobilities were similar, between pH 4 and 9. These data suggest that changes in the cell surface hydrophobicity of the R. rhodochrous result in changes in the culture characteristics and the formation of scum.     

PKU-beta/TLK1 regulates myosin II activities, and is required for accurate equaled chromosome segregation

Tousled-like kinase 1 (or protein kinase ubiquitous, PKU-beta/TLK1) is a serine/threonine protein kinase that is implicated in chromatin remodeling, DNA replication and mitosis. RNAi-mediated PKU-beta/TLK1-depleted human cells showed aneuploidy, and immunofluorescence analysis of these cells revealed the unequal segregation of daughter chromosomes. Immunoblots indicated a substantial reduction in the phosphorylation level of Ser19/Thr18 on the myosin II regulatory light chain (MRLC) in PKU-beta/TLK1-depleted cells, with no change in total MRLC protein. To confirm the relationship between mitotic aberration and MRLC dysfunction, we expressed wild type MRLC or DD-MRLC (mimics diphosphorylation; substitution of both Thr18 and Ser19 with aspartate) in PKU-beta/TLK1-depleted cells. DD-MRLC expression dramatically reduced the unequal segregation of chromosomes. Our data suggest that human PKU-beta/TLK1 plays an important role in chromosome integrity via the regulation of myosin II dynamics by phosphorylating MRLC during mitosis.     

Culture of insect cells contracting spontaneously; research moving toward an environmentally robust hybrid robotic system

Here we propose an environmentally robust hybrid (biotic-abiotic) robotic system that uses insect heart cells. Our group has already presented a hybrid actuator using rat heart muscle cells, but it is difficult to keep rat heart muscle cells contracting spontaneously without maintaining the culture conditions carefully. Insect cells, by contrast, are robust over a range of culture conditions (temperature, osmotic pressure and pH) compared to mammalian cells. Therefore, a hybrid robotic system using not mammalian cells but insect cells can be driven without precise environmental control. As a first step toward the realization of this robotic system, the larvae of two lepidopteran species, Bombyx mori (BM) and Thysanoplusia intermixta (TI) were excised and the culture conditions of their dorsal vessel (insect heart) cells were examined. As a result, spontaneously contracting TI cells derived from the dorsal vessel were obtained. The contraction of TI cells started on the 7th day and continued for more than 18 days. Spontaneously contracting BM cells were not obtained in this study. These experimental results suggest the possibility of constructing an environmentally robust hybrid robotic system with living cells in the near future.     

Cell sorting analysis of cell cycle-dependent X-ray sensitivity in end joining-deficient human cells

Non-homologous end joining (NHEJ) plays a major role in the repair of ionizing radiation-induced DNA double-strand breaks (DSBs), especially during the G1-phase of the cell cycle. Using a flow cytometric cell sorter, we fractionated G1- and S/G2-phase cells based on size to assess the DSB-repair activity in NHEJ factor-deficient DT40 and Nalm-6 cell lines. Colony formation assays revealed that the X-ray sensitivities of the G1-enriched populations correctly reflected the DSB-repair activities of both the DT40 and Nalm-6 cell lines. Furthermore, as assessed by γ-H2AX foci formation, the sorted cells exhibited less DNA damage than chemically synchronized cells. Given that it does not use fluorescent labeling or chemical agents, this method of cell sorting is simpler and less toxic than other methods, making it applicable to a variety of cell lines, including those that cannot be synchronized by standard chemical treatments.     

Protein kinase C is involved in cardioprotective effects of ischemic preconditioning on infarct size and ventricular arrhythmia in rats in vivo

Protein kinase C (PKC) has been known to play an important role in ischemic preconditioning (IP). This study was designed to examine whether the translocation of PKC is associated with the cardioprotective effects of IP in vivo on infarct size and ventricular arrhythmias in a rat model.Using anesthetized rats, heart rate, systolic blood pressure, infarct size and ventricular arrhythmias during 45 min of coronary occlusion were measured. PKC activity was assayed in both the cytosolic and cell membrane fraction . Brief 3-min periods of ischemia followed by 10 min of reperfusion were used to precondition the myocardium. Calphostin C was used to inhibit PKC.Infarct size was significantly reduced by IP (68.1 (2.5)%, mean (S.E.) vs. 45.2 (3.4)%, p < 0.01). The reduction in infarct size by IP was abolished by pretreatment with calphostin C. The total number of ventricular premature complex (VPC) during 45 min of coronary occlusion was reduced by IP (1474 (169) beats/45 min vs. 256 (82) beats/45 min, p < 0.05). The reduction the total number of VPC induced by IP was abolished by the administration of calphostin C before the episode of brief ischemia. The same tendency was observed in the duration of ventricular tachycardia and the incidence of ventricular fibrillation. PKC activity in the cell membrane fraction transiently increased immediately after IP (100 vs. 142%, p < 0.01) and returned to baseline 15 min after IP. Pretreatment with calphostin C prevented the translocation of PKC.The translocation of PKC plays an important role in the cardioprotective effect of IP on infarct size and ventricular arrhythmias in anesthetized rats.