Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells

Although induced pluripotent stem (iPS) cells are indistinguishable from ES cells in their expression of pluripotent markers, their differentiation into targeted cells is often limited. Here, we examined whether the limited capacity of iPS cells to differentiate into neural lineage cells could be mitigated by improving their base-line level of pluripotency, i.e. by converting them into the so-called “naive” state. In this study, we used rabbit iPS and ES cells because of the easy availability of both cell types and their typical primed state characters. Repeated passages of the iPS cells permitted their differentiation into early neural cell types (neural stem cells, neurons, and glial astrocytes) with efficiencies similar to ES cells. However, unlike ES cells, their ability to differentiate later into neural cells (oligodendrocytes) was severely compromised. In contrast, after these iPS cells had been converted to a naive-like state, they readily differentiated into mature oligodendrocytes developing characteristic ramified branches, which could not be attained even with ES cells. These results suggest that the naive-like conversion of iPS cells might endow them with a higher differentiation capacity.     

FGF15/19 protein levels in the portal blood do not reflect changes in the ileal FGF15/19 or hepatic CYP7A1 mRNA levels

It has been proposed that bile acid suppression of CYP7A1 gene expression is mediated through a gut-liver signaling pathway fibroblast growth factor (FGF)15/19-fibroblast growth factor receptor 4 which is initiated by activation of farnesoid X receptor in the ileum but not in the liver. This study evaluated whether FGF15/19 protein levels in the portal blood reflected changes in FGF15/19 mRNA in the ileum. Studies were conducted in Sprague Dawley rats and New Zealand white rabbits fed regular chow (controls), supplemented with cholesterol (Ch) or cholic acid (CA). After feeding CA, ileal FGF15 mRNA increased 8.5-fold in rats and FGF19 rose 16-fold in rabbits associated with 62 and 75% reduction of CYP7A1 mRNA, respectively. Neither FGF15 nor FGF19 protein levels changed in the portal blood to correspond with the marked increase of FGF15/19 mRNA levels in the ileum or inhibited CYP7A1 expression in the liver. Further, in Ch-fed rats, CYP7A1 mRNA increased 1.9-fold (P < 0.001) although FGF15 mRNA levels in the ileum and portal blood FGF15 protein levels were not decreased. In Ch-fed rabbits, although FGF19 mRNA levels in the ileum and liver did not increase significantly, CYP7A1 mRNA declined 49% (P < 0.05). We were unable to find corresponding changes of FGF15/19 protein levels in the portal blood in rats and rabbits where the mRNA levels of FGF15/19 in the ileum and CYP7A1 in the liver change significantly.     

Organelle acidification is important for localisation of vacuolar proteins in Saccharomyces cerevisiae

The acidic environments in the vacuole and other acidic organelles are important for many cellular processes in eukaryotic cells. In this study, we comprehensively investigated the roles of organelle acidification in vacuolar protein localisation in Saccharomyces cerevisiae. After repressing the acidification of acidic compartments by treatment with concanamycin A, a specific inhibitor of vacuolar H⁺-ATPase (V-ATPase), we examined the localisation of GFP-fused proteins that were predicted to localise in the vacuolar lumen or on the vacuolar membrane. Of the 73 proteins examined, 19 changed their localisation to the cytoplasmic region. Localisation changes were evaluated quantitatively using the image processing programme CalMorph. The delocalised proteins included vacuolar hydrolases, V-ATPase subunits, transporters and enzymes for membrane biogenesis, as well as proteins required for protein transport. These results suggest that many alterations in the localisation of vacuolar proteins occur after loss of the acidification of acidic compartments.     

An MSH4 Homolog,stpp1, from Pleurotus pulmonarius Is a “Silver Bullet” for Resolving Problems Caused by Spores in Cultivated Mushrooms

The enormous number of spores produced by fruiting bodies during cultivation of mushrooms can lead to allergic reactions of workers, reduction of commercial value, spread of mushroom disease, pollution of facilities, and depletion of genetic diversity in natural populations. A cultivar harboring a sporulation-deficient (sporeless) mutation would be very useful for preventing these problems, but sporeless commercial cultivars are very limited in usefulness because sporeless traits are often linked with traits that are unfavorable for commercial cultivation. Thus, identifying a causal gene of a sporeless phenotype not linked to the adverse traits in breeding and cultivation is crucial for the establishment of sporeless breeding using a strategy employing targeting induced local lesions in genomes (TILLING) in cultivated mushrooms. We used a Pleurotus pulmonarius (Fr.) Quél. sporeless strain to identify and characterize the single recessive gene controlling the mutation. The 3,853-bp stpp1 gene encodes a protein of 854 amino acids and belongs to the MutS homolog (MSH) family associated with mismatch repair in DNA synthesis or recombination in meiosis. Gene expression analysis of the fruiting body showed that this gene is strongly expressed in the gills. Phenotypic analysis of disruptants formed by gene targeting suggested a reproducible sporeless phenotype. Mutants deficient in a functional copy of this gene have no unfavorable traits for sporeless cultivar breeding, so this gene will be an extremely useful target for efficient and versatile sporeless breeding in P. pulmonarius and various other cultivated mushrooms.     

The common ancestral core of vertebrate and fungal telomerase RNAs

Telomerase is a ribonucleoprotein with an intrinsic telomerase RNA (TER) component. Within yeasts, TER is remarkably large and presents little similarity in secondary structure to vertebrate or ciliate TERs. To better understand the evolution of fungal telomerase, we identified 74 TERs from Pezizomycotina and Taphrinomycotina subphyla, sister clades to budding yeasts. We initially identified TER from Neurospora crassa using a novel deep-sequencing–based approach, and homologous TER sequences from available fungal genome databases by computational searches. Remarkably, TERs from these non-yeast fungi have many attributes in common with vertebrate TERs. Comparative phylogenetic analysis of highly conserved regions within Pezizomycotina TERs revealed two core domains nearly identical in secondary structure to the pseudoknot and CR4/5 within vertebrate TERs. We then analyzed N. crassa and Schizosaccharomyces pombe telomerase reconstituted in vitro, and showed that the two RNA core domains in both systems can reconstitute activity in trans as two separate RNA fragments. Furthermore, the primer-extension pulse-chase analysis affirmed that the reconstituted N. crassa telomerase synthesizes TTAGGG repeats with high processivity, a common attribute of vertebrate telomerase. Overall, this study reveals the common ancestral cores of vertebrate and fungal TERs, and provides insights into the molecular evolution of fungal TER structure and function.     

Monoenyl hydrocarbons in female body wax of the yellow peach moth as synergists of aldehyde pheromone components

The non-polar components of female body wax and pheromone gland extracts of the yellow peach moth synergistically enhanced male behavioral responses from close to pheromone sources in wind tunnel tests when mixed with an aldehyde pheromone blend. When the non-polar fractions (NPFs) of female body wax were further separated by column chromatography, synergistic activities were found in the 3 and 50% ether in hexane fractions, and they additively increased male responses. The main components of the first fraction were (Z)-9-tricosene, (Z)-9-pentacosene, (Z)-9-heptacosene, (Z)-9-nonacosene and (Z)-9-hentriacontene. Only (Z)-9-heptacosene showed a significant synergistic effect in enhancing male responses, but the other components had no effect. A mixture of the five monoenyl hydrocarbons lost activity at lower doses than 5 ng. Natural ratios of these hydrocarbons in the female body wax and pheromone gland extracts were similar, but the amount of (Z)-9-heptacosene in the female body wax was significantly higher than in the pheromone gland extracts. We conclude that (Z)-9-heptacosene increases male responses to aldehyde pheromones, and unknown component(s) in the 50% ether in the hexane fraction are required for full synergistic enhancement by the NPFs of the female body wax and the pheromone gland extracts.     

Thermostability of Rhodopseudomonas viridis and Rhodospirillum rubrum chromatophores reflecting physiological conditions

Relationships between growth conditions and thermostability were examined for photosynthetic inner membranes (chromatophores) from Rhodopseudomonas viridis and Rhodospirillum rubrum of which morphology, lipid composition, and protein/lipid rate are rather mutually different. Signals observed by differential scanning calorimetry of the chromatophores were correlated with thermal state transitions of the membrane components by reference to temperature dependencies of circular dichroism and absorption spectra of the purified supramolecule comprising a photoreaction center and surrounding light-harvesting pigment-protein complexes that are the prominent proteins in both membranes. The differential scanning calorimetry curves of those chromatophores exhibited different dependencies on growth stages and environmental temperatures. The obtained result appeared to reflect the differences in the protein/lipid rate and protein-lipid specificity between the two chromatophores.     

Epoxy ceriporic acid produced by selective lignin-degrading fungus Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a selective white rot basidiomycete which degrades lignin in wood at a distance far from enzymes. Low molecular mass metabolites play a central role in the oxidative degradation of lignin. To understand the unique wood-decaying mechanism, we surveyed the oxidized derivatives of ceriporic acids (alk(en)ylitaconic acids) produced by C. subvermispora using high-resolution liquid chromatography multiple-stage mass spectrometry (HR-LC/MSⁿ). The analysis of the precursor and product ions from the extract suggested that an epoxidized derivative of ceriporic acid is produced by the fungus. To identify the new metabolite, an authentic compound of ceriporic acid epoxide was synthesized in vitro by reacting (R)-3-[(Z)-hexadec-7-enyl]-itaconic acid (ceriporic acid C) with m-chloroperbenzoic acid. The precursor and product ions from the natural metabolite and authentic epoxide were identical and distinguishable from those of hydroxy and hydroperoxy derivatives after reduction with NaBD₄. Feeding experiments with [U-¹³C]-glucose, 99% and the subsequent analyses of the first and second generation product ions demonstrated that the oxidized ceriporic acid was (R)-3-(7,8-epoxy-hexadecyl)-itaconic acid. To our knowledge, this study is the first to report that natural alkylitaconic acid bears an epoxy group on its side chain.