Recombination of the GFP gene to the BFP gene using a man-made site-selective DNA cutter

By using the recently developed man-made DNA cutter [a combination of Ce(IV)/EDTA and two DNA additives], green fluorescent protein (GFP) was converted to closely related blue fluorescent protein (BFP). The phosphodiester linkages at T196-A200 in the sense strand of GFP were hydrolyzed by the cutter, and the A1-T196 fragment in the product was selectively connected with the downstream fragment (C197-A720) of BFP by T4 DNA ligase. This recombination changed three codons in the GFP gene (TGC at 196–198, TAT at 199–201, and ACC at 502–504) to TCT, CAT, and ATC in BFP, and accordingly three amino acids in GFP (Cys65, Tyr66, and Thr167) were altered to Ser65, His66, and Ile167. The recombinant gene was successfully expressed in Escherichia coli and emitted blue fluorescence, confirming the absence of undesired side reactions (mutation, deletion, insertion, depurination, etc.) in the DNA manipulation. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Detection of β-glucosidase as saprotrophic ability from an ectomycorrhizal mushroom, Tricholoma matsutake

We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects. We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized. The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca²⁺ and Mn²⁺ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction.     

Isolation and characterization of acidic glycosphingolipids from the gill of the Pacific Salmon (Oncorhynchus keta): A novel hybrid-type ganglioside with isoglobo- and neolacto-Series

Monosialosyl gangliosides and sulfoglycolipids in the gill of pacific salmon, Oncorhynchus keta, have been prepared by solvent extraction and DEAE-Sephadex column chromatography. Acidic glycolipid bands (M1-M13) detected by thin layer chromatography were separated by Iatrobeads column chromatography and 13 components were characterized by TLC, compositional analysis, methylation analysis, chemical and enzymatic degradation, liquid secondary ion mass spectrometry and (1)H nuclear magnetic resonance spectroscopy. In addition to the acidic glycolipids with known structures (SM4s, SM3, GM3, LM1, GM1b and V(3)alphaFuc,IV(3)betaGalNAc-GM1a), two fractions (M11 and M13) of unknown monosialosyl gangliosides with TLC mobility slower than GM1a were isolated and characterized as having the following structure with a hybrid of isoglobo- and neolacto-series. [formula: see text] Analysis of fatty acid indicated predominance of C24:1 fatty acid in the upper band (M11) and shorter chain saturated fatty acids in the lower band (M13). The tissue concentrations of M11 and M13 were 1.15 and 0.96 mumol/kg wet weight, respectively.     

Unique disialosyl gangliosides from salmon kidney: Characterization of V3αFuc, IV3βGalNAc, II3(αNeuAc)2-Gg4Cer and its analogue with 4-O-acetyl-N-acetylneuraminic acid

Four unidentified acidic glycolipids (X3-X6) were isolated from the kidney of the Pacific salmon on an anion exchange column and by high performance liquid chromatography using a silica bead (Iatrobeads) column. Based on methylation analysis, chemical and enzymatic degradation, proton nuclear magnetic resonance spectroscopy and mass spectrometry, the glycon structure of X5 and X6 was identified as a unique disialosyl fucosyl-N-acetylgalactosaminyl ganglio-N-tetraose: Fucα3GalNAcβ3Galβ3GalNAcβ4[NeuAcα8NeuAcα3] Galβ4Glcβ1Cer. NMR showed that X3 and X4 were analogues of X5 and X6 and contained O-acetyl groups on C4 of the outer N-acetylneuraminic acid, first disialosyl gangliosides containing 4-O-acetyl-N-acetylneuraminic acid. The ceramides of X3 and X5 contained predominantly C24: 1, and X4 and X6 contained saturated fatty acids (C14: 0, C16: 0 and C18: 0), whereas the long chain base was exclusively sphingenine. The concentrations of X3 and X4 were 0.13 and 0.16 nmol/g of kidney respectively and those of X5 and X6, were 0.07 nmol/g each.     

Characteristics of rat round spermatids differentiated from spermatogonial cells during co-culture with Sertoli cells, assessed by flow cytometry, microinsemination and RT-PCR

The present study was undertaken to investigate whether rat spermatogonial stem cells can differentiate into developmentally competent round spermatids during co-culture with Sertoli cells. Type-A spermatogonia and Sertoli cells were prepared from 7-d-old Wistar-strain male rats, and seeded at 4 x 10(6) cells/ 4 mL/35-mm dish (Day 0). They were co-cultured at 37 degrees C for 3 d and at 34 degrees C for the subsequent 7d in 5% CO(2)/air. Round spermatid-like cells (approximately 15 microm in diameter) were first observed on Day 5. A flow cytometric analysis showed that a single peak of haploid cells was detected in the cell populations harvested on Day 10. The participation of the spermatid-like cells to full-term development was examined by microinjection into activated oocytes. The oviductal transfer of 143 microinseminated oocytes resulted in only 8 implantation sites (6%), but no viable offspring. The expression of the round spermatid-specific marker gene, PRM-2, was confirmed in the Day 10 cell population by RT-PCR; however, no mRNA of two other haploid makers, TP1 or TP2, was detected. These results suggested that rat type-A spermatogonial cells underwent meiosis during the primary co-culture with the Sertoli cells, based on morphology, flow cytometry and PRM-2 expression, but the normality of the spermatid-like cells was not supported by microinsemination and TP1/2 expression.     

Anisotropic nucleation growth of actin bundle: a model for determining the well-defined thickness of bundles

Biopolymers such as DNA, F-actins, and microtubules, which are highly charged, rodlike polyelectrolytes, are assembled into architectures with defined morphology and size by electrostatic interaction with multivalent cations (or polycations) in vivo and in vitro. The physical origin to determine their morphology and size is not clearly understood yet. Our results show that the actin bundle formation consists of two stages: the thickness of actin bundles is determined nearly at the initial stage, while the length of actin bundles is determined later on. It is also found that the thickness of actin bundles decreases with the increase of polycation-mediated attraction between F-actins. From these results, we propose the anisotropic nucleation-growth mechanism, in which the thickness of actin bundles is determined by critical nucleus size, whereas the length of actin bundles is determined by the concentration of free actins relative to nucleus concentration. Observing that polycations are concentrated in some sites of actin bundles, which are thought to be nucleation sites to initiate the formation of actin bundles, supports this model. This anisotropic nucleation-growth mechanism of actin bundles can be broadly applied to the self-assembly of rodlike polyelectrolytes.     

p53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias latipes)

Acid-base imbalance leads to pathological cognition and behaviors in the clinical practices. In the comparison with acidosis, the cellular mechanisms underlying alkalosis-induced brain dysfunction remain unclear. By using electrophysiological approach, we investigated the influences of high extracellular pH environment on cortical GABAergic neurons in terms of their responsiveness to synaptic inputs and their ability to produce action potentials. Artificial cerebral spinal fluid in high pH impairs excitatory synaptic transmission and spike initiation in cortical GABAergic neurons. The alkalosis-induced dysfunction of GABAergic neurons is associated with the decrease of receptor responsiveness and the increases of spike refractory periods and threshold potentials. Our studies reveal that alkalosis impairs cortical GABAergic neurons and subsequently deteriorate brain functions. The molecular targets for alkalosis action include glutamate receptor-channels and voltage-gated sodium channels on GABAergic neurons.     

Establishment and characterization of Roberts syndrome and SC phocomelia model medaka (Oryzias latipes)

Roberts syndrome and SC phocomelia (RBS/SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/SC by reverse genetics. The RBS/SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80S mutant. The R80S mutant is the animal model for RBS/SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/SC.     

Blood oxygenation using microbubble suspensions

Microbubbles have been used in a variety of fields and have unique properties, for example shrinking collapse, long lifetime, efficient gas solubility, a negatively charged surface, and the ability to produce free radicals. In medicine, microbubbles have been used mainly as diagnostic aids to scan various organs of the body, and they have recently been investigated for use in drug and gene delivery. However, there have been no reports of blood oxygenation by use of oxygen microbubble fluids without shell reagents. In this study, we demonstrated that nano or microbubbles can achieve oxygen supersaturation of fluids, and may be sufficiently small and safe for infusion into blood vessels. Although Po(2) increases in fluids resulting from use of microbubbles were inhibited by polar solvents, normal saline solution (NSS) was little affected. Thus, NSS is suitable for production of oxygen-rich fluid. In addition, oxygen microbubble NSS effectively improved hypoxic conditions in blood. Thus, use of oxygen microbubble (nanobubble) fluids is a potentially effective novel method for oxygenation of hypoxic tissues, for infection control, and for anticancer treatment.     

Single nucleotide mutation leading to an amino acid substitution in the variant <Emphasis Type="Italic">Tik</Emphasis> soybean Kunitz trypsin inhibitor (SKTI) identified in Chinese wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &; Zucc.)

The wild soybean (Glycine soja), which is the progenitor of cultivated soybean (Glycine max), is expected to offer more information about genetic variability and more useful mutants for evolutionary research and breeding applications. Here, a total of 1,600 wild soybean samples from China were investigated for genetic variation with regard to the soybean Kunitz trypsin inhibitor (SKTI). A new mutant SKTI, Tik, was identified. It was found to be a Tia-derived codominant allele caused by a transversion point mutation from C to G at nucleotide +171, leading to an alteration of one codon (AAC → AAG) and a corresponding amino acid substitution (Asn → Lys) at the ninth residue. Upon examination of this variant and others previously found in wild soybeans, it became clear that SKTI has undergone high-level evolutionary differentiation. There were more abundant polymorphisms in the wild than in the cultivated soybean.