Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.     

Postnatal changes in the rheological properties of the aorta in Sprague-Dawley rats.

Postnatal changes in the rheological properties of the aortic wall were investigated in relation to morphological development of the wall in Sprague-Dawley (SD) rats at 3, 8 and 20 weeks old. The mechanical tensile characteristics of the longitudinal wall strip excised from the proximal thoracic aorta were assessed with stress-strain and stress-relaxation tests. Wall tension in the low and medium strain ranges was significantly lower in 3-week-old rats than in 8-week-old rats and in 8-week-old rats than in 20-week-old rats. Wall stress was significantly lower in 3-week-old rats than in 8- and 20-week-old rats mainly in the medium strain range, but was significantly greater in 3-week-old rats than in 8- and 20-week-old rats in the high strain range. The value of incremental elastic modulus at 3 weeks old was significantly smaller than that at 8 and 20 weeks old at a strain of 0.25 and significantly larger than that at 8 and 20 weeks old at a strain of 0.50. The value of relaxation strength at 5 min after the stretching was significantly greater at 3 weeks old than that at 8 and 20 weeks old. The wall was viscoelastic in the low and medium strain ranges at 3 weeks though large wall stress was generated in the high strain range. Histological investigation revealed that the smooth muscle layer, fine elastin fiber connecting thick elastin fibers and wall thickness were thin at 3 weeks old in comparison with those at 8 and 20 weeks old, though there was no significant difference in number of nuclei of the smooth muscle cells among the three age groups. Changes in the tensile characteristics of the wall reflected well those of the microstructure of the wall with growth. The rheological properties and microstructure of the aortic wall were close to maturation at 8 weeks in SD rats.     

Steady-state force–velocity relation of ATP-dependent sliding between slime mold myosin, arranged on paramyosin filaments, and algal cell actin cables

To investigate characteristics of ATP-dependent sliding of a non-muscle cell myosin, obtained from a cellular slime mold Dictyostelium discoideum, on actin filament, we prepared hybrid thick filaments, in which Dictyostelium myosin was regularly arranged around paramyosin filaments obtained from a molluscan smooth muscle. A single to a few hybrid filaments were attached to a polystyrene bead (diameter, 4.5 μm; specific gravity, 1.5), and the filaments were made to slide on actin filament arrays (actin cables) in the internodal cell of an alga Chara corallina, mounted on the rotor of a centrifuge microscope. The filament-attached bead was observed to move with a constant velocity under a constant external load for many seconds. The steady-state force–velocity relation of Dictyostelium myosin sliding on actin cables was hyperbolic in shape except for large loads ≤0.7–0.8 P₀, being qualitatively similar to that of skeletal muscle fibres, despite a considerable variation in the number of myosin molecules interacting with actin cables. Comparison of the P–V curves between Dictyostelium myosin and muscle myosins sliding on actin cables suggests that the time of attachment to actin in a single attachment–detachment cycle is much longer in Dictyostelium myosin than in muscle myosins.     

Differential effects of various growth factors and cytokines on the syntheses of DNA,type I collagen,laminin, fibronectin,osteonectin/secreted protein,acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture

The purpose of this study is to differentiate roles of several growth factors and cytokines in proliferation and differentiation of pulp cells during development and repair. In human pulp cell cultures, laminin and type I collagen levels per cell remained almost constant during the whole culture period (22 days). On the other hand, secreted protein, acidic and rich in cysteine (SPARC/osteonectin) and alkaline phosphatase (ALPase) levels markedly increased after the cultures reached confluence. Laminin and type I collagen, as well as fibronectin, stimulated the spreading of pulp cells within 1 h. Adding transforming growth factor-β (TGF-β) decreased laminin and ALPase levels, whereas it increased SPARC and fibronectin levels 3- to 10-fold. Western and Northern blots showed that TGF-β enhanced SPARC synthesis at the protein and mRNA levels. Basic fibroblast growth factor (bFGF) decreased type I collagen, laminin, SPARC, and ALPase levels without changing the fibronectin level. Platelet-derived growth factor (PDGF) selectively decreased laminin, SPARC, and ALPase levels. Epidermal growth factor (EGF) also decreased SPARC and ALPase levels. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) decreased type I collagen and laminin levels, and abolished SPARC and ALPase syntheses. Of these peptides, bFGF and PDGF showed the greatest stimulation of [³H]thymidine incorporation into DNA. TGF-β, EGF, and TNF-α had less effect on DNA synthesis, whereas IL-1β inhibited DNA synthesis. These findings demonstrated that TGF-β, bFGF, EGF, PDGF, TNF-α, and IL-1β have characteristically different patterns of actions on DNA, laminin, type I collagen, fibronectin, ALPase, and SPARC syntheses by pulp cells. J. Cell. Physiol. 174:194–205, 1998. © 1998 Wiley-Liss, Inc.     

Purification and characterization of eugenol dehydrogenase from Pseudomonas fluorescens E118

Pseudomonas fluorescens E118 was isolated from soil as an effective eugenol-degrading organism by a screening using eugenol as enrichment substrate. The first enzyme involved in the degradation of eugenol in this organism, eugenol dehydrogenase, was purified after induction by eugenol, and the purity of the enzyme was shown by SDS-PAGE and gel-permeation HLPC. The enzyme is a heterodimer that consists of a 10-kDa cytochrome c and a 58-kDa subunit. The larger subunit presumably contains flavin, suggesting a flavocytochrome c structure and an electron transfer via flavin and cytochrome c during dehydrogenation. The activity of the purified enzyme depended on the addition of a final electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, cytochrome c, or potassium ferricyanide. The enzyme catalyzed the dehydrogenation of three different 4-hydroxybenzylic structures including the conversion of eugenol to coniferyl alcohol, 4-alkylphenols to 1-(4-hydroxyphenyl)alcohols, and 4-hydroxybenzylalcohols to the corresponding aldehydes. The catalytic and structural similarity between this enzyme and a Penicillium vanillyl-alcohol oxidase and 4-alkylphenol methylhydroxylases from several Pseudomonas species is discussed. Received: 17 June 1998 / Accepted: 12 October 1998     

Development of a series of gateway binary vectors possessing a tunicamycin resistance gene as a marker for the transformation of Arabidopsis thaliana

We made two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing a UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene driven by the nopaline synthase promoter (Pnos) as a tunicamycin resistance marker for the transformation of Arabidopsis thaliana. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, and TAP. Selection of transformants was successful on plates containing 0.15 mg/L of tunicamycin. These vectors were compatible with existing pGWB and R4pGWB vectors for kanamycin, hygromycin B, and BASTA? selection, and are useful new tools for making transgenic Arabidopsis.     

Calmodulin-dependent activation of MAP kinase for ROS homeostasis in Arabidopsis

Rapid recognition and signal transduction of mechanical wounding through various signaling molecules, including calcium (Ca2+), protein phosphorylation, and reactive oxygen species (ROS), are necessary early events leading to stress resistance in plants. Here we report that an Arabidopsis mitogen-activated protein kinase 8 (MPK8) connects protein phosphorylation, Ca2+, and ROS in the wound-signaling pathway. MPK8 is activated through mechanical wounding, and this activation requires direct binding of calmodulins (CaMs) in a Ca2+-dependent manner. MPK8 is also phosphorylated and activated by a MAPKK MKK3 in the prototypic kinase cascade, and full activation of MPK8 needs both CaMs and MKK3 in planta. The MPK8 pathway negatively regulates ROS accumulation through controlling expression of the Rboh D gene. These findings suggest that two major activation modes in eukaryotes, Ca2+/CaMs and the MAP kinase phosphorylation cascade, converge at MPK8 to monitor or maintain an essential part of ROS homeostasis.     

Endothelin-1 activates p38 mitogen-activated protein kinase via endothelin-A receptor in rat myocardial cells

In myocardial cells (MCs), endothelin-1 (ET-1) exerts various effects such as hypertrophy, and causes cellular injury. Long-term treatment with an endothelin-A (ET_A) receptor antagonist improves the survival of rats with heart failure, suggesting that myocardial endothelin system contributes to the progression of heart failure. p38 mitogen-activated kinase (MAPK) is a member of the MAPK family and activated by several forms of environmental stresses. We show here the effect of ET-1 on p38 MAPK activation and the role of ET-1-activated p38 MAPK on morphological changes in MCs. ET-1-stimulated p38 MAPK phosphorylation was detectable within 2 min and maximal at 5 min and was concentration dependent. The maximum effect was obtained at 10 nM. An ET_A receptor antagonist, BQ-123, but not an endothelin-B receptor antagonist, BQ-788, inhibited these reactions. A p38 MAPK inhibitor, SB203580, failed to inhibit the morphological changes associated with ET-1-induced myocardial cell hypertrophy. These results indicate that p38 MAPK is activated by ET-1 but does not contribute to the development of ET-1-induced myocardial cell hypertrophy.     

Growing and differentiating characterization of aortic smooth muscle cell line, p53LMAC01 obtained from p53 knock out mice

Recently we have established an aortic smooth muscle cell line, p53LMAC01 obtained from p53 knockout mice. This cell line showed some differentiated properties which were accelerated by 5-azacytidine treatment [1]. In this study, further characterization of p53LMAC01 cell line was investigated according to cell growth and differentiation, and especially focused into the changes of cell feature, actin filaments' formation, and changes of intracellular calcium concentrations to sympathetic nerve transmitter, norepinephrine. While the cell feature was changed from flattened shape to extended form during 4 days, actin filaments were developing, arranging in parallel to longitudinal direction, and gathering under the surface membrane. In 11 days many cells died and detached from substrate, while actin filaments became poor except for the surface membrane in the remained cells. Appearance of calcium response to noradrenalin needed several days after passage as well as a morphological change of the cells for the extended form and development of actin filaments. The calcium response was maintained on 11 days, which coincided with the result that the cells hold actin filaments under the surface membrane. These results suggest that p53LMAC01 cell line maintains several differentiated characters of adult smooth muscle cell and that their expression needs several days after passage.     

Establishment and characterization of a new human glioblastoma cells line, NYGM

A cell line designated NYGM was established from a human cerebral glioblastoma multiforme (GBM) obtained from a 75-year-old Japanese woman. The cell line has grown slowly without interruption and has been propagated continuously by serial passages (more than 80 passage) during the past 3 years. The cultured cells were fusiform or polyhedral in shape. The population doubling time was 24 hours. The chromosomal number varied between 77 and 88, with modal chromosomal number of 84. NYGM cells concomitantly expressed MET receptor tyrosine kinase (a product of c-met protooncogene) and its ligand HGF/SF (hepatocyte growth factor/scatter factor), as well as HGF activator and HGF activator inhibitors. The cells might be useful for the study of pericellular regulation of HGF/SF-MET signaling and HGF activation of GBM cells.