Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.     

Enhancing effect of dietary vinegar on the intestinal absorption of calcium in ovariectomized rats.

We studied the effect of dietary vinegar on calcium absorption by using ovariectomized rats fed on a low-calcium diet. The apparent absorption of calcium was higher when the rats were fed on a diet containing 1.6% vinegar for 32 days than when fed on a diet without vinegar (P < 0.05). The calcium content in the femur of the rats given diets containing 0.4% and 1.6% vinegar were also higher (P < 0.05). The serum parathyroid hormone level was lower and the crypt depth of the duodenum thicker in the rats fed on a diet containing 1.6% vinegar (P < 0.05). These results suggest that dietary vinegar enhanced intestinal calcium absorption by improving calcium solubility and by the trophic effect of the acetic acid contained in vinegar, which would reduce the bone turnover caused by ovariectomy and be helpful in preventing osteoporosis.     

Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status

Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30-38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.     

Roles of integrins and CD44 on the adhesion and migration of fetal liver cells to the fetal thymus.

Adhesion and migration of mouse fetal liver (FL) cells to the thymus were investigated using cells from green fluorescent protein transgenic (GFP+) mice. FL cells from GFP+ embryos at 12 gestational days (E12) of mice were incubated with 2'-deoxyguanosine-treated fetal thymus lobe (from E14) by thymic repopulation (hanging drop) culture methods. GFP+ cells were observed in the thymus lobe at the end of the repopulation culture period. A large part of the infiltrated cells expressed CD44 until day 2 of culture on a permeable membrane, then lost the expression. CD25 expression was observed from day 1 to day 4. Around day 8, GFP+ cells became both CD4+ and CD8+. The results support the early observation of the sequential expression of CD44, CD25, and CD4/8 during the early stages of thymocyte development. When anti-CD44 mAb was added at the beginning of the repopulation culture period, GFP+ FL cells adhered to the surface of the thymus lobe but did not migrate into the thymus. Pretreatment of the thymus with hyaluronidase or hyaluronate produced results similar to the results of anti-CD44 treatment. On the other hand, the addition of anti-integrin alpha4 mAb inhibited adhesion to the thymus, and almost no GFP+ cells were seen on the surface of the thymus lobe. The data suggest that integrin alpha4 and CD44 play different roles, i.e., integrin alpha4 is required for the adhesion of FL cells to the thymus lobe and CD44 is required for the migration of the cells into the thymus.     

Micropatterning contractile C2C12 myotubes embedded in a fibrin gel

Contractile C₂C₁₂ myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C₂C₁₂ myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish. Biotechnol. Bioeng. 2010;105: 1161–1167. © 2009 Wiley Periodicals, Inc.     

Detection and mapping of six miniF-encoded proteins by cloning analysis of dissected miniF segments

Summary Various DNA subfragments were derived from miniF DNA by complete or partial PstI cleavage, and cloned in the plasmid vectors pBR322 or dv1. The recombinant plasmids obtained were introduced into an Escherichia coli minicell-producing strain, and the plasmid-coded proteins were radiolabeled and analyzed by gel electrophoresis. Six miniF-encoded proteins, larger than 11 000 daltons, were detected and their coding regions were mapped on the F plasmid genome. Three of them were assigned by taking into account the known nucleotide sequences (Murotsu et al. 1981; K. Yoshioka, personal communication). The coding directions of some proteins were determined by inserting the lac promotor into one of the recombinant plasmids and analyzing the increase in production of the proteins. The coding direction of the five proteins analyzed so far was uniform. Comparison of these results with a functional map of miniF suggested possible roles of the proteins.     

GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers

GAMYB is a component of gibberellin (GA) signaling in cereal aleurone cells, and has an important role in flower development. However, it is unclear how GAMYB function is regulated. We examined the involvement of a microRNA, miR159, in the regulation of GAMYB expression in cereal aleurone cells and flower development. In aleurone cells, no miR159 expression was observed with or without GA treatment, suggesting that miR159 is not involved in the regulation of GAMYB and GAMYB-like genes in this tissue. miR159 was expressed in tissues other than aleurone, and miR159 over-expressors showed similar but more severe phenotypes than the gamyb mutant. GAMYB and GAMYB-like genes are co-expressed with miR159 in anthers, and the mRNA levels for GAMYB and GAMYB-like genes are negatively correlated with miR159 levels during anther development. Thus, OsGAMYB and OsGAMYB-like genes are regulated by miR159 in flowers. A microarray analysis revealed that OsGAMYB and its upstream regulator SLR1 are involved in the regulation of almost all GA-mediated gene expression in rice aleurone cells. Moreover, different sets of genes are regulated by GAMYB in aleurone cells and anthers. GAMYB binds directly to promoter regions of its target genes in anthers as well as aleurone cells. Based on these observations, we suggest that the regulation of GAMYB expression and GAMYB function are different in aleurone cells and flowers in rice.     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.