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Y Akahori

Publications and source records attributed to Y Akahori.

At least 19 recordsLinked to original sources
PubMed2005

Two-step models to predict binding affinity of chemicals to the human estrogen receptor alpha by three-dimensional quantitative structure-activity relationships (3D-QSARs) using receptor-ligand docking simulation.

Binding of chemicals to the estrogen receptor (ER) is known to be a key mode of action of endocrine disruption effects. In this study, combined quantitative structure-activity relationship (QSAR) models from discriminant and multilinear regression (MLR) analyses, termed a two-step model, were developed. These were used to predict the binding potency to human ERalpha of four chemical groups, namely alkylphenols, phthalates, diphenylethanes and benzophenones. These groups are considered to be important chemical classes of ER-binders. The descriptors investigated were calculated following the simulation of docking between the receptor and ligand. Discriminant analysis in the first step of a two-step model was applied to distinguish binders from non-binders. It had a concordance, following leave-one-out (LOO), of greater than 87% for all chemical classes. Binders were defined as chemicals whose IC50 was reliably measured in a competitive binding assay. The MLR analysis in the second step was performed for the quantitative prediction of the binding affinity of chemicals that were previously discriminated as binders. The q2 values for alkylphenols and diphenylethanes were 0.75 and 0.74, respectively. However good MLR relationships were not obtained for phthalates and benzophenones as the observed binding affinities of chemicals in these categories were weak and in a too narrow range.

Benzene Derivatives↗
PubMed2001

Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression.

We developed a system by which antibodies, fused to fluorescent proteins with different wavelengths, can be prepared within a month against various antigens. An antibody library composed of a large number of single-chain Fv-CL fragment was constructed by means of a phage-display system. The constructs were designed to facilitate changing of the protein forms by simple enzyme manipulation. In the present study, we adopted a molecular form of antibody in which a single-chain Fv-CL fragment is fused with a green fluorescent protein (GFP) or red fluorescent protein (RFP). In addition, a His-tag was inserted between CL and GFP (or RFP). We describe the utility of this system using Caenorhabditis elegans embryo as a model.

Amino Acid Sequence↗
PubMed2000

Unusual core histones specifically expressed in male gametic cells of Lilium longiflorum.

We have cloned three novel histone genes using antibodies that recognize only nuclei of the male gametic (generative and sperm) cells of Lilium longiflorum. The deduced amino acid sequence of each clone shows only between 40% and 50% identity with the H2A, H2B and H3 somatic core histones of other plant species. Transcripts of these genes were first detected in bicellular pollen soon after microspore mitosis, and their mRNAs, as revealed by in situ hybridization, were observed only in the cytoplasm of the generative cells. As expression of these three genes was specific to generative cells within the bicellular pollen, we designated the clones gH2A, gH2B and gH3. Immunocytochemistry further revealed that the proteins encoded by these genes accumulated in the elongating and condensing generative nucleus during development of bicellular pollen, and were most abundant in the two sperm nuclei within an elongated pollen tube. We therefore propose that these male gamete-specific core histones contribute to chromatin condensation of male gametes or to chromatin remodeling, and result in the repression of gene expression in male gametes.

Amino Acid Sequence↗
PubMed1999

A novel histone variant localized in nucleoli of higher plant cells.

Immunofluorescence staining with antisera raised against p35, a basic nuclear protein that accumulates in the pollen nuclei of Lilium longiflorum, specifically stained the nucleoli in interphase nuclei of somatic tissues, including root and leaf, and in pachytene nuclei during meiotic division, whereas antisera raised against histone H1 uniformly stained the entire chromatin domain with the exception of the nucleoli in these nuclei. Further, p35-specific antisera stained the nucleoli in root and leaf nuclei of the monocotyledonous plants Tulipa gesneriana, Allium cepa and Triticum aestivum and of the dicotyledonous plants Vicia faba and Nicotiana tabacum. Thus, these novel antisera stained the nucleoli in cells of all higher plants examined, although the staining patterns within nucleoli were somewhat different among plant species and tissues. The full-length cDNA of p35 was cloned on the basis of the partial amino acid sequence. The deduced amino acid composition and amino acid sequence of p35 indicate that this nucleolar protein is a novel variant of histone Hl. Further, p35 was strongly bound to ribosomal DNA in vitro. The results of immunoblotting of histones extracted from each tissue of the various plant species with the nucleolus-specific antibodies also suggested the conservation of similar epitope(s) in both mono- and dicotyledonous plants. From these results, it is suggested that similar variants of histone Hl are specifically distributed in the nucleoli of all plant species and help to organize the nucleolar chromatin.

Amino Acid Sequence↗
PubMed1997

IGF-I enhances neurite regeneration but is not required for its survival in adult DRG explant.

We clarified the roles of insulin-like growth factor (IGF) I and II in peripheral neural regeneration after axotomy using cultured adult DRG explants either with or without associated nerve bundles. When IGF-I was applied at concentrations of 1-10 nM to DRG explants without associated nerve bundles in a serum-free medium, clear enhancement of neural regeneration from both central and peripheral transected nerve terminals was seen. When the same concentrations of IGF-I were applied to DRG explants with associated nerve bundles this enhancement was reduced at central sites and increased at peripheral sites. Neural survival was not affected by IGF-I in any of these culture systems. In comparison with IGF-I, there was no specific effect of IGF-II on neurite regeneration.

Animals↗
PubMed1997

IL-1 beta enhances neurite regeneration from transected-nerve terminals of adult rat DRG.

We clarified the roles of IL-1 beta in peripheral neural regeneration after axotomy in a three-dimensional collagen gel culture system ranging from a single neurone to a dorsal root ganglion (DRG) explant with its associated nerve bundles. Application of 30 U/ml IL-1 beta to the culture systems clearly enhanced neural regeneration. This regeneration was evident in transected nerve terminals of DRG explants with or without associated nerve bundles, but not in dissociated single neurones. Neural survival was not affected by IL-1 beta in any of these culture systems. These results suggest that IL-1 beta stimulates surrounding non-neuronal cells to secrete neurotrophic factors, thus enhancing neurite regeneration from transected nerve terminals in cultured adult DRG explants.

Animals↗
PubMed1997

Nucleotide sequences of all the gamma gene loci of murine immunoglobulin heavy chains.

The nucleotide sequence of the entire region that encodes the gamma chains of mouse immunoglobulin was determined, namely, 11,942 bp that include the whole gamma 3 gene and 59,647 bp from the gamma 1 to the gamma 2a gene. The sequence of 21,012 bp that includes the entire human gamma 3 gene was also determined. The present analysis revealed three new findings. (1) The S regions are composed of repetitive sequences. A 26-bp repeating unit, in addition to a 49-bp repeating unit, is present in the S gamma 2b and S gamma 2a regions. In the case of the gamma 2b gene, the 26-bp unit is repeated 5 times and the 49-bp unit is repeated 69 times. In the case of the gamma 2a gene, by contrast, the 26-bp unit is repeated 63 times and the 49-bp unit is repeated 14 times. (2) Two pseudo-gamma-genes were identified between the gamma 1 and the gamma 2b genes as well as between the gamma 2b genes and the gamma 2a genes. (3) A possible functional region was identified in the region downstream of each gamma gene. This region, designated the 3' conserved region (3'CR), was also found in the region downstream of the human gamma 3 gene. Duplication of a primordial gamma 2 gene seems likely to have occurred by homologous recombination that involved the 3'CR sequences.

Animals↗
PubMed1997

Highly sensitive mutation assay for mutagenicity monitoring of indoor air using Salmonella typhimurium YG1041 and a microsuspension method.

A highly sensitive mutation assay for indoor mutagenicity monitoring was investigated by a combination of Salmonella typhimurium YG strains and the microsuspension method. Tester strains were YG1024, YG1029, YG1041 and YG1042. YG1041 gave the highest sensitivity in the mutagenicity test for the extracts of airborne particulates. The sensitivity of the microsuspension assay using S.typhimurium YG1041 in the absence of S9 mix was approximately 200 times higher than that of the preincubation assay using strain TA98, which has been widely used for mutagenicity monitoring of airborne particulates outdoors. Furthermore, a significant correlation was observed between mutagenicities determined by the microsuspension assay using S.typhimurium YG1041 and TA98, where mutagenicity assay was carried out for airborne particulates collected by a high volume sampler for 24 h every 12 days for 1 year. This new method was also useful for indoor mutagenicity monitoring in which a small amount of airborne particulates was collected by a low noise sampler for 12 h each of 6 consecutive days. The monitoring showed that mutagenicity in the daytime is generally higher than that in the night and that smoking is an important factor in increasing mutagenicity in indoor air.

Air Pollution↗
PubMed1995

Relation between micellar structure of model bile and activity of esterase.

In a model bile solution composed of lecithin (L)-bile salt (B), the solubilization of lipid and the accessibility of enzyme to the lipid were examined by observation of EPR spectra and measurement of enzyme activity. The lifetime of the spin probe in the micellar phase was estimated to be approx. 1 microsecond by means of line shape analysis. Both population and lifetime increased with temperature and the molar ratio of lecithin to bile salt (L/B). The EPR data indicated that simple micelle of bile salt, mixed disk micelle of bile salt-lecithin, and multi-lamellar mixed disk micelle can exist in a model bile solution, depending on the L/B molar ratio across a range from 0 to 1.5. The maximal power of the mixed disk micelle to solubilize cholesteryl ester in the model bile at a L/B molar ratio of 1:1 was confirmed by EPR measurement of cholesteryl 12-DOXYL-stearate. Observation of the enzyme activity on a mixture of model bile and substrate at 37 degrees C revealed selective accessibility of cholesterol esterase (bovine pancreas) to mixed disk micelle, of cholesterol oxidase (Streptomyces cinnamomeus) to both simple and mixed disk micelle, and of pancreatic lipase (porcine pancreas) to both simple micelle and an oil droplet of substrate. The temperature-dependent activity of cholesterol oxidase to cholesterol in mixed disk micelle can be explained in terms of mesomorphic phase transition of lecithin side chains followed with fluidity of liquid crystal phase. Regarding phospholipase C from Bacillus cereus, though the selective accessibility to the micelles was not observed at 37 degrees C, a decrease in activity for mixed disk micelle could be found at lower temperatures.

Bile↗
PubMed1995

Induction of apoptotic DNA fragmentation and cell death by natural ceramide.

We have found that a solvent mixture of ethanol and dodecane was able to disperse natural ceramide (Cer) into aqueous solution. In U937 cells, addition of natural Cer to medium caused a characteristic pattern of DNA fragmentation, which is indistinguishable from that caused by TNF alpha. The effective concentration of Cer is as low as 25 nM. The present study provides direct evidence that natural Cer functions as a second messenger mediating TNF alpha-induced DNA fragmentation. The use of this solvent to deliver hydrophobic natural Cer to cells will contribute to the elucidation of the biological function of Cer.

Apoptosis↗
PubMed1994

Three-dimensional cell aggregation enhances growth-promoting activity of NGF in adult DRG.

After removal of the connective tissues with collagenase-trypsin treatment, DRG explants without nerve fibres from mature rodents regenerated numerous neurites all around the explants in response to NGF. To observe the response in the absence of any non-neuronal cells in the ganglia, we introduced three-dimensional cell aggregates consisting of variable numbers of highly purified isolated adult DRG neurones. In this culture system, the addition of NGF to the medium increased the maximum neurite length, the number of neurites, and the cell survival rate. Furthermore, the magnitude of this effect increased with cell aggregate size. Our results suggest that the addition of NGF produces extensive neurite outgrowth in three-dimensional aggregates of matured sensory neurones in the absence of any non-neuronal cells.

Animals↗
PubMed1994

Evidence for defects in V(D)J rearrangements in patients with severe combined immunodeficiency.

We investigated the patterns of DNA rearrangements at loci for Ig JH genes in patients with severe combined immunodeficiency (SCID). Four SCID patients without B cells (B- SCID) and four SCID patients with B cells (B+ SCID) were examined. Bone marrow cells of these patients were transformed with EBV. The majority of the transformed cells from three B- SCID patients had the germline configuration at their JH gene loci. The rearranged fragments from one patient were analyzed extensively. The rearranged regions in all of the fragments had a common structure wherein two fragments derived from the JH-S mu region were connected inversely. The possible presence of rearranged forms of VHDJH and DHQ52JH sequences in bone marrow cells of two B- SCID patients were examined directly by the polymerase chain reaction (PCR) method. In one patient, we found neither a VHDJH sequence nor a DHQ52JH sequence within the range of sensitivity of the PCR method. In another patient, we found a VHDJH sequence at an extremely low level and DHQ52JH sequences at a relatively low level. Either RAG-1 or RAG-2 gene was not expressed in the B- SCID-derived cell lines. B+ SCID patients did not show any abnormalities in terms of VHDJH rearrangements. These results indicate that B- SCID may be caused by defects in factors involved in V(D)J rearrangements.

Base Sequence↗
PubMed1990

Presence of immunoglobulin (Ig) M and IgG double isotype-bearing cells and defect of switch recombination in hyper IgM immunodeficiency.

We established a transformed B cell line expressing both IgM and IgG on the cell surface from a patient with hyper IgM immunodeficiency using Epstein-Barr viruses. DNA and RNA of the cells were analyzed. DNA rearrangements of Ig JH gene loci were observed on both chromosomes. Cloning and DNA sequence analyses showed that one has a VHDHJH structure while the other has a DHJH structure. Southern hybridization with 5'-S mu and S gamma region-containing probes indicated germline configuration in the switch regions of mu and gamma genes on both chromosomes. To test expression of mu and gamma chains in the transformed cells at the mRNA-level, we used the polymerase chain reaction with three kinds of synthetic oligonucleotides as primers, one of which was part of the VH gene, while the other two were complementary to parts of C mu and C gamma genes. Sequence analysis of the amplified products showed that the same VHDHJH sequence is directly connected with either the C mu or the C gamma sequence in the mRNAs. This is direct evidence showing that in double isotype-bearing cells one VHDHJH exon in the transcript is alternatively spliced to C mu or C gamma without DNA rearrangement. The defect in this disease could be at the S-S recombination stage.

Amino Acid Sequence↗
PubMed1989

Class switch from mu to delta is mediated by homologous recombination between sigma mu and sigma mu sequences in human immunoglobulin gene loci.

Class switch of immunoglobulin from mu to gamma occurs by recombination between two repetitive switch sequences: S mu and S gamma. However, there are no such sequences in the mu-delta introns of human and mouse genomes. Although the frequency of IgD-secreting cells is extremely low in mouse about 1% of patients with myeloma produce IgD in human. In a previous report (Nucleic Acids Res. 1988. 16: 9497) we reported that a 442-bp DNA sequence located in the JH-mu intron (defined as sigma mu) was inserted into the mu-delta intron (defined as sigma mu) in human genome. There is no such insertion in mouse. We analyzed Ig H chain gene loci of two human IgD myelomas: one was analyzed by cloning and sequencing and the other by Southern hybridization. We found that recombination had occurred between these two homologous DNA sequences, resulting in loss of the DNA segment from sigma mu to sigma mu. On the other hand, in a Burkitt lymphoma, Daudi, the DNA fragment from sigma mu to sigma mu was duplicated. These results suggest that homologous recombination between sigma mu and sigma mu sequences mediates class switch from mu to delta in human and that it occurs via unequal crossing-over between sister chromatids or daughter chromosomes.

Amino Acid Sequence↗
PubMed1988

Sigma region located between C mu and C delta genes of human immunoglobulin heavy chain: possible involvement of tRNA-like structure in RNA splicing.

Noncoding regions within the cluster of immunoglobulin heavy chain constant genes in the human genome contained a number of repeats. In the mu-delta intron, two repeating units were contained. One 442-base-long fragment located JH-mu intron (defined as "sigma mu(sigma mu)") occupied the position in the mu-delta intron. The other 1166-base-long fragment located somewhere in front of S (class switch) region of C gamma gene was also found in the mu-delta intron. We defined the repeats in the mu-delta intron as "SIGMA (sigma)". The polarities of the longer repeats in the genome were opposite between the mu-delta intron and the upstreams of C gamma genes. These inverted copies (defined as sigma gamma 3 and sigma gamma 4), located 6 kb upstream of their respective C gamma's, were apparently transcribed in vitro, via RNA polymerase III and transcripts should have contained tRNA-like structures. Small DNA fragments capable of encoding tRNA-like structures were also found in corresponding regions of mouse Ig C gamma cluster.

Animals↗