Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.     

Involvement of ASK1–p38 pathway in the pathogenesis of diabetes triggered by pancreatic ß cell exhaustion

Background

Diabetes mellitus is characterized by high blood glucose levels. Pancreatic ß cell death contributes to type 1 and type 2 diabetes. Akita mice, which harbor a human permanent neonatal diabetes-linked mutation (Cys96Tyr) in the insulin gene, are well established as an animal model of diabetes caused by pancreatic ß cell exhaustion. Mutant Insulin 2 protein (Ins2^C96Y) induces endoplasmic reticulum (ER) stress and pancreatic ß cell death in Akita mice, although the molecular mechanism of Ins^C96Y-induced cell death remains unclear.

Methods

We investigate the mechanisms of Ins2^C96Y-induced pancreatic ß cell death in vitro and in vivo, using p38 inhibitor (SB203580), MIN6 cell (pancreatic ß cell line), Akita mice and apoptosis signal-regulating kinase 1 (ASK1) knockout mice.

Results

The expression of Ins^C96Y activated the ASK1–p38 pathway. Deletion of ASK1 mitigated Ins^C96Y-induced pancreatic ß cell death and delayed the onset of diabetes in Akita mice. Moreover, p38 inhibitor suppressed Ins^C96Y-induced MIN6 cell death.

Conclusions

These findings suggest that ER stress-induced ASK1–p38 activation, which is triggered by the accumulation of Ins^C96Y, plays an important role in the pathogenesis of diabetes.

General significance

Pancreatic ß cell death caused by insulin overload appears to be involved in the pathogenesis of type 1 and type 2 diabetes. Inhibition of the ASK1–p38 pathway may be an effective therapy for various types of diabetes.     

Morphology and taxonomy of chain-forming species of the genus Cochlodinium (Dinophyceae)

The morphology of an unarmored chain-forming harmful dinoflagellate Cochlodinium polykrikoides and its similar species such as Cochlodinium catenatum, Cochlodinium fulvescens, and Cochlodinium convolutum was carefully observed, emphasizing the single cell stage for clarifying taxonomically important morphological features. To differentiate C. polykrikoides from C. convolutum, the shape and the position of the nucleus are useful characters. C. polykrikoides also differs from C. fulvescens in being smaller in size, possessing many rod-shaped chloroplasts and having the sulcus running just below the cingulum on the dorsal surface. Careful observation of the ichnotype of C. catenatum suggests that C. catenatum sensu Kofoid and Swezy collected from off La Jolla, CA, USA, is not identical to C. catenatum sensu Okamura and is probably a different species, in having no chloroplasts and a nucleus positioned at the center of the cell. In addition, C. polykrikoides has many morphological features in common with C. catenatum sensu Okamura except for slightly elongate cells and is probably a junior synonym of this species.     

Spleen-Derived Interleukin-10 Downregulates the Severity of High-Fat Diet-Induced Non-Alcoholic Fatty Pancreas Disease

Obesity is associated with systemic low-grade inflammation and is a risk factor for non-alcoholic fatty pancreas disease (NAFPD), but the molecular mechanisms of these associations are not clear. Interleukin (IL)-10, a potent anti-inflammatory cytokine, is released during acute pancreatitis and is known to limit inflammatory responses by downregulating the release of proinflammatory mediators. The origin of IL-10 that suppresses pancreatitis has not been investigated. Since obesity is known to reduce expression of proinflammatory cytokines in the spleen, we examined whether spleen-derived IL-10 regulates NAFPD caused by high-fat (HF) diet-induced obesity. The following investigations were performed: 1) IL-10 induction from spleen was examined in male mice fed a HF diet; 2) triglyceride content, expression of pro- and anti-inflammatory cytokines and infiltration of M1 and M2 macrophages were determined to evaluate ectopic fat accumulation and inflammatory responses in the pancreas of splenectomy (SPX)-treated mice fed HF diet; 3) exogenous IL-10 was systemically administered to SPX-treated obese mice and the resulting pathogenesis caused by SPX was assessed; and 4) IL-10 knockout (IL-10KO) mice were treated with SPX and ectopic fat deposition and inflammatory conditions in the pancreas were investigated. Obesity impaired the ability of the spleen to synthesize cytokines, including IL-10. SPX aggravated fat accumulation and inflammatory responses in the pancreas of HF diet-induced obese mice and these effects were inhibited by systemic administration of IL-10. Moreover, SPX had little effect on fat deposition and inflammatory responses in the pancreas of IL-10KO mice. Our findings indicate that obesity reduces IL-10 production by the spleen and that spleen-derived IL-10 may protect against the development of NAFPD.     

Not only light quality but also mechanical stimuli are involved in height convergence in crowded Chenopodium album stands

? In crowded stands, height is often similar among dominant plants, as plants adjust their height to that of their neighbours (height convergence). We investigated which of the factors, light quality, light quantity and mechanical stimuli, is primarily responsible for stem elongation and height convergence in crowded stands. ? We established stands of potted Chenopodium album plants. In one stand, target plants were surrounded by artificial plants that were painted black to ensure that the light quality was not modified by their neighbours. In a second stand, target plants were surrounded by real plants. In both stands, one-half of the target plants were anchored to stakes to prevent flexing by wind. The target plants were lifted or lowered by 10?cm to test whether height convergence was affected by the different treatments. ? Stem length was affected by being surrounded by artificial plants, anchoring and pot elevation, indicating that light quality, light quantity and mechanical stimuli all influenced stem elongation. Height convergence did not occur in the stand with artificial plants or in anchored plants. ? We conclude that light quality and mechanical stimuli are important factors for the regulation of stem growth and height convergence in crowded stands.     

Magnaporthe oryzae endopolygalacturonase homolog correlates with density-dependent conidial germination.

Endopolygalacturonases (endoPGs) of some phytopathogens are virulent factors for dicots. To investigate the function of the endoPG of Magnaporthe oryzae, a disruption mutant of MGG_08938, the homolog of endoPG found in the genome database of this fungus, was generated. The pathogenicity, mycelial growth, and appressorium formation of this mutant were comparable with those of the wild-type strain; however, the germination of conidia in a highly concentrated suspension of conidia was affected by the mutation. Whereas the germination of the wild-type strain was inhibited at high concentrations, this effect was canceled out by disruption by the endoPG homolog gene. The authors named the gene MDG1 (M. oryzae density-dependent germination), which delineates this new function in the fungus.     

Crystallographic and mutational analyses of cystathionine β‐synthase in the H2S‐synthetic gene cluster in Lactobacillus plantarum

Cystathionine β‐synthase (CBS) catalyzes the formation of l ‐cystathionine from l ‐serine and l ‐homocysteine. The resulting l ‐cystathionine is decomposed into l ‐cysteine, ammonia, and α‐ketobutylic acid by cystathionine γ‐lyase (CGL). This reverse transsulfuration pathway, which is catalyzed by both enzymes, mainly occurs in eukaryotic cells. The eukaryotic CBS and CGL have recently been recognized as major physiological enzymes for the generation of hydrogen sulfide (H₂S). In some bacteria, including the plant‐derived lactic acid bacterium Lactobacillus plantarum, the CBS‐ and CGL‐encoding genes form a cluster in their genomes. Inactivation of these enzymes has been reported to suppress H₂S production in bacteria; interestingly, it has been shown that H₂S suppression increases their susceptibility to various antibiotics. In the present study, we characterized the enzymatic properties of the L. plantarum CBS, whose amino acid sequence displays a similarity with those of O‐acetyl‐l ‐serine sulfhydrylase (OASS) that catalyzes the generation of l ‐cysteine from O‐acetyl‐l ‐serine (l ‐OAS) and H₂S. The L. plantarum CBS shows l ‐OAS‐ and l ‐cysteine‐dependent CBS activities together with OASS activity. Especially, it catalyzes the formation of H₂S in the presence of l ‐cysteine and l ‐homocysteine, together with the formation of l ‐cystathionine. The high affinity toward l ‐cysteine as a first substrate and tendency to use l ‐homocysteine as a second substrate might be associated with its enzymatic ability to generate H₂S. Crystallographic and mutational analyses of CBS indicate that the Ala70 and Glu223 residues at the substrate binding pocket are important for the H₂S‐generating activity.     

Role of the CD47-SHPS-1 system in regulation of cell migration

SHPS-1 is a transmembrane protein whose extracellular region interacts with CD47 and whose cytoplasmic region undergoes tyrosine phosphorylation and there by binds the protein tyrosine phosphatase SHP-2. Formation of this complex is implicated in regulation of cell migration by an unknown mechanism. A CD47-Fc fusion protein or antibodies to SHPS-1 inhibited migration of human melanoma cells or of CHO cells overexpressing SHPS-1. Overexpression of wild-type SHPS-1 promoted CHO cell migration, whereas expression of the SHPS-1-4F mutant, which lacks the phosphorylation sites required for SHP-2 binding, had no effect. Antibodies to SHPS-1 failed to inhibit migration of CHO cells expressing SHPS-1-4F. SHPS-1 ligands induced the dephosphorylation of SHPS-1 and dissociation of SHP-2. Antibodies to SHPS-1 also enhanced Rho activity and induced both formation of stress fibers and adoption of a less polarized morphology in melanoma cells. Our results suggest that engagement of SHPS-1 by CD47 prevents the positive regulation of cell migration by this protein. The CD47- SHPS-1 system and SHP-2 might thus contribute to the inhibition of cell migration by cell-cell contact.     

Hydration regulates the thermodynamic stability of DNA structures under molecular crowding conditions

Although water is an integral part of DNA structures, the effects of water molecules on various DNA structures which are formed by not only Watson-Crick but also Hoogsteen base pairs are still unclear. Here, we studied quantitatively the effects of molecular crowding on the thermodynamics of a parallel G-quadruplex formation of [d(TG 4)2]4 with Hoogsteen base pairs. It was demonstrated that molecular crowding conditions stabilized the parallel G-quadruplex. Moreover, the plot of stability of the parallel G-quadruplex structure versus water activity suggested that water molecules were released through the G-quadruplex formation. The stabilization of the DNA structures consisting of Hoogsteen base pairs under cell-like conditions may lead to a structural polymorphism of various DNA sequences regulated by water molecules.     

Brefeldin A effects on the trans-Golgi network and Golgi bodies inBotryococcus braunii are not uniform during the cell cycle

Summary Using cryo-fixation and freeze-substitution electron microscopy, the effects of brefeldin A (BFA) on the structure of the trans-Golgi network (TGN), the endoplasmic reticulum (ER), and Golgi bodies in the unicellular green algaBotryococcus braunii were examined at various stages of the cell cycle. In the presence of BFA, all the TGNs of interphase and dividing cells aggregated to form a single tubular mass. In contrast, the TGNs decomposed just after cell division and disappeared during cell wall formation. Throughout the cell cycle, the TGN produced at least six kinds of vesicles, of which two were not formed in the presence of BFA: vesicles with a diameter of 200 nm and fibrillar substances, which formed in interphase cells; and vesicles with a diameter of 180–240 nm, which may participate in septum formation. In addition, the number of clathrin-coated vesicles attaching to the TGN decreased. In interphase cells, BFA induced the disassembly of Golgi bodies and an increase in the smooth-ER cisternae at the cis-side of Golgi bodies. This result may suggest the existence of retrograde transport from the Golgi bodies to the ER in the presence of BFA. These drastic structural changes in the Golgi bodies and the ER of interphase cells were not observed in BFA-treated dividing cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TGN trans-Golgi network