Efficient establishment of pure cultures of yellow chanterelle Cantharellus anzutake from ectomycorrhizal root tips,and morphological characteristics of ectomycorrhizae and cultured mycelium

Species of fleshy yellow Cantharellus are known as chanterelles, which are among the most popular wild edible mycorrhizal mushrooms in the world. However, pure culture isolates of Cantharellus are rare. We report an efficient isolation technique of the Japanese golden chanterelle, Cantharellus anzutake, from its ectomycorrhizal root tips. Field-sampled fresh ectomycorrhizal root tips of C. anzutake on various hosts such as pines, spruce, and oaks were vortexed with 0.005% Tween 80 solution, surface sterilized with 1% calcium hypochlorite solution, rinsed with sterilized distilled water, and placed on modified Norkrans’ C (MNC) agar plate medium. Most ectomycorrhizal root tips of C. anzutake produced yellowish mycelial colonies within a few months. In contrast, tissue isolation from basidiomata provided limited cultures of C. anzutake but much contamination of bacteria and molds, even on media that contained antibiotics. The established C. anzutake cultures had clamp connections on the hyphae and contained intracellular oily droplets. These cultured isolates were identified as C. anzutake by sequence analysis of the rRNA internal transcribed spacer (ITS) region and translation elongation factor EF1-alpha (tef-1) genes.     

Sequence of an intestinal cDNA encoding human motilin precursor

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is identical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Syntheses of ( + )-Phrymarolin II and Its Stereoisomers

Phrymarolin II, a unique naturally-occurring lignan having a 1,2-dioxygenated 3,7-dioxabicyclo[3.3.0]octane skeleton, was synthesized by the reaction of sesamol, in the presence of cadmium carbonate, with 1-acetoxy-2-chloro-6-(2′-methoxy-4′,5′-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane. The latter was prepared through 13 steps starting from an aldol condensation of β-vinyl-γ-butyrolactone with 2-methoxy-4,5-methylenedioxybenzaldehyde. Three diastereomers of phrymarolin II were also obtained.     

Protein kinase A determines timing of early differentiation through epigenetic regulation with G9a

Timing of cell differentiation is strictly controlled and is crucial for normal development and stem cell differentiation. However, underlying mechanisms regulating differentiation timing are fully unknown. Here, we show a molecular mechanism determining differentiation timing from mouse embryonic stem cells (ESCs). Activation of protein kinase A (PKA) modulates differentiation timing to accelerate the appearance of mesoderm and other germ layer cells, reciprocally correlated with the earlier disappearance of pluripotent markers after ESC differentiation.?PKA activation increases protein expression of G9a, an H3K9 methyltransferase, along with earlier H3K9 dimethylation and DNA methylation in Oct3/4 and Nanog gene promoters. Deletion of G9a completely abolishes PKA-elicited acceleration of differentiation and epigenetic modification. Furthermore, G9a knockout mice show prolonged expressions of?Oct3/4 and Nanog at embryonic day 7.5 and delayed development. In this study, we demonstrate molecular machinery that regulates timing of multilineage differentiation by linking signaling with epigenetics.     

Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer

In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.     

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.