Biomedical subjects
Publications and source records attributed to Y Ichihara.
Taspine has been isolated as a cytotoxic substance from Sangre de Grado, sap of Croton palanostigma (Euphorbiaceae), by bioassay guided fractionation. The cytotoxicity (IC50) of taspine was found to be 0.39 microgram/ml against KB cells and 0.17 microgram/ml against V-79 cells.
Administration of hydrocortisone to infant rats caused a precocious increase in levels of mucosal pepsinogen and its mRNA together with morphological maturation of pepsinogen-producing cells. The increase in levels of pepsinogen mRNA was induced rapidly and was associated with increase in levels of its precursors, suggesting transcriptional regulation of pepsinogen genes by hydrocortisone. Methylation analysis with the methylation-sensitive restriction enzymes, HpaII and HhaI, revealed that hydrocortisone also induced sequential demethylation changes of CCGG and GCGC sites in and around pepsinogen genes. Most of these changes occurred after increases in transcription of the genes and did not appear to play a causal role in gene activation. Superficially, the observed demethylations corresponded to the sequential processes of morphological maturation of pepsinogen-producing cells. Thus, these changes in methylation are probably linked to hydrocortisone-induced differentiation of pepsinogen-producing cells and may reflect the mechanism in vivo for the maturation of pepsinogen genes.
The entire rat pepsinogen C gene has been isolated from a rat genomic library, using the rat pepsinogen C cDNA as a probe. Southern blot analysis showed that there exists at least two rat pepsinogen C genes. The nucleotide sequences of the coding regions and the 5'- and 3'-flanking regions of one of the rat pepsinogen C genes have been determined. This gene is split into 9 exons interrupted by eight intervening sequences. The 5'-flanking region is similar to that of the human pepsinogen C gene, but only the former has the core sequence of the Sp1 binding site. The amount of transcripts of the rat pepsinogen C genes was found to increase during development, and a similar increase was shown to be induced by injection of hydrocortisone. As a candidate of a factor which regulates the transcription, we found a 25-kDa protein by Southwestern blotting. It binds to a specific site in the 5'-flanking region of the gene only in the presence of Mg2+ ion, and it is present in the nuclear fraction of the gastric mucosa but not of the liver.
Both primates and rodents apparently originated from insectivores and then evolved separately. We isolated the immunoglobulin mu gene from DNA of the insectivore Suncus murinus and determined its nucleotide sequence. The gene organization was CH1 exon (318 bp)-intron (89 bp)-CH2 exon (345 bp)-intron (80 bp)-CH3 exon (318 bp)-intron (85 bp)-CH4 exon (392 bp)-coding sequence and 3'-untranslated region. Comparison of nucleotide sequences of mu genes between mouse, human and Suncus murinus indicated that the evolutionary distance between human and mouse is equal to that between Suncus murinus and human, and that mouse is deviated further from Suncus murinus than the two other combinations. This conclusion was further supported by sequence comparison of non-coding regions.
In mice, 12 germ-line DH genes belonging to three different families (DQ52, DSP2 and DFL16) have been identified. The DH genes other than DQ52 are clustered in the 60 kb-long region located between VH and JH genes. Since there are seven DH gene families (DHQ52, DXP, DA, DK, DN, DM and DLR) in humans, we tried to identify new DH gene families in the 60 kb-long region using human DH gene probes. Mouse and human DH genes showing the highest similarity were mouse DFL16 genes and human DA genes. Southern hybridization of the mouse clones covering the 60-kb region with human DH probes did not detect any other DH genes. Nucleotide sequence analysis of the 4.0-kb fragment containing the DFL16.1 gene confirmed this conclusion. Comparison of the 12 germ-line DH genes and more than 150 somatic DH sequences also indicated that there are not more germ-line DH genes in the mouse genome. Moreover, comparison of nucleotide sequences of DFL16.1 and DSP2.2 genes and their surrounding regions suggests that both DH gene families originate from the same primordial DH gene. Using the flanking sequences of both DH genes, the divergence date between DFL16 and DSP2 genes was estimated at around 37 million years ago.
The extract of seeds of Euphorbia lathyris L. showed antitumor activity against Sarcoma 180 ascites in mice. Systematic fractionation of the extract led to the characterization of ingenol-3-hexadecanoate as an active principle, together with an inactive diterpene ingenol-20-hexadecanoate.
cDNA of rat pepsinogen C (PGC) hybridizes to, among others, a 3.2-kb band in Southern blot analysis of BamHI-cleaved human genomic DNA. This property was employed to localize the human PGC gene. Use of flow-sorted human chromosomes and 12 human x mouse somatic cell hybrid lines demonstrated that the gene is located on chromosome 6.
The variable region of immunoglobulin heavy chain is encoded by three separate genes on the germline genome: variable (VH), diversity (DH) and joining (JH) genes. Most human DH genes are encoded in 9-kb repeating sequences. We determined the nucleotide sequence of a 15-kb DNA fragment containing more than one and a half of these repeating units, and identified 12 different DH genes. Based on the sequence similarities of DH coding and the surrounding regions, they can be classified into six different DH gene families (DXP, DA, DK, DN, DM and DLR). Nucleotide sequences of DH genes belonging to different families diverge greatly, while those belonging to the same families are well conserved. Since the 9-kb DNA containing the six DH genes are multiplied at least five times, the total number of DH genes must be approximately 30. These DH genes are sandwiched by 12-nucleotide spacer signals. Most of the somatic DH sequences found in the published VH-DH-JH structures (the somatic DH segment being defined as the region which is not encoded either by germline VH or JH gene) were assigned to one of the germline DH genes. Other than these typical DH genes, however, we found a new kind of DH gene (which we termed DIR) the spacer lengths of whose neighbouring signals were irregular. The DIR gene appears to be involved in DIR-DH or DH-DIR joining by inversion or deletion. Two of the somatic DH sequences were assigned to the DIR genes. Long N segments might, therefore, originate from DIR genes.
The pepsinogen gene is hypomethylated in the stomach, in which it is expressed. For demonstration that this hypomethylation of the pepsinogen gene in the stomach reflects pepsinogen-producing cells, we analyzed fractions of dispersed mucosal cells with various contents of pepsinogen-producing cells prepared from guinea pig stomach by centrifugal elutriation. mRNA expression and the extent of hypomethylation of the pepsinogen gene in each fraction was closely correlated with the content of pepsinogen-producing cells. These results suggested hypomethylation of the pepsinogen gene in pepsinogen-producing cells and differential pepsinogen gene methylation in cell subpopulations in the stomach.
A clone, pSPcA2, which carries the full-length swine pepsinogen cDNA was isolated. The coding sequence comprised the signal peptide [15 amino acids (aa)], the activation peptide segment (44 aa) and mature pepsin (327 aa). The deduced amino acid sequence agrees with the published sequence with two exceptions. Asparagine instead of aspartate is present at aa positions 19 and 308. Two types of plasmids, pAS and pUCtacSPc series, were constructed for expressing swine pepsinogen cDNA. These plasmids directed the synthesis of polypeptides which were detected by employing an antibody to swine pepsinogen. However, all the polypeptides formed aggregates and showed no acid protease activity. Only the protein directed by pAS5 regained the acid protease activity after renaturation procedures. The activity was completely inhibited by pepstatin. Furthermore, the renatured pAS5 protein was spontaneously converted to pepsin under acidic conditions. The presence of Arg-8 in the activation peptide segment appears important for the stabilization of the pepsinogen molecule.
The relationship between methylation and expression of rat pepsinogen 1 (Pg1) genes was investigated in various tissues. On Northern blotting with a Pg1 complementary DNA probe, Pg1 mRNA was detected only in the glandular stomach of normal rats. Methylation analysis with Msp1/HpaII and Hha1 revealed tissue specific methylation patterns of Pg1 genes with less methylated in the stomach than in other normal tissues not expressing the genes. During stomach development, there was a progressive increase in the Pg1 mRNA level that almost coincided with change in the mucosal pepsinogen level and progressive demethylation after the onset of transcription. Thus, there was an inverse correlation between methylation and expression of Pg1 genes, suggesting a role of DNA methylation in Pg1 gene regulation during normal differentiation, although not its primary role in gene activation. There was no detectable Pg1 mRNA in either primary or transplanted stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine. The methylation patterns of Pg1 genes were different from those of normal tissues that expressed the gene and of those that did not and no simple correlation was observed between methylation and expression of Pg1 genes. This result is consistent with a previous finding that DNA methylation is deranged in tumor cells.
We have examined the correlation between the extents of methylation and expression of pepsinogen A genes in normal human tissues. Expression of pepsinogen A mRNA was detected only in the fundic mucosa of the stomach and both CCGG and GCGC sites in the genes region were less methylated in the fundic mucosa than in other non-expressing tissues. Thus, there was an inverse correlation between the extents of methylation and expression of pepsinogen A genes and the role of DNA methylation in the regulation of pepsinogen A genes expression during normal differentiation was suggested.
The entire human pepsinogen C gene has been isolated from a cosmid genomic library. The nucleotide sequences of all the exons and the 5'- and 3'-flanking regions of the gene are presented. The organization of the gene is fundamentally compatible with those of other aspartic proteinases, allowing us to conclude that the genes of these aspartic proteinases including pepsinogen C are derived from a common ancestral gene. The predicted 388-residue amino acid sequence of human pepsinogen C consists of a signal sequence of 16-amino acid residues, an activation peptide of 43 residues, and the mature pepsin of 329 residues containing the two active-site aspartic acids. In the light of present notions about eukaryotic gene expression, possible regulatory roles of the oligonucleotide DNA sequences in the promoter region of the gene are discussed.
The variable region of immunoglobulin (Ig) heavy chain is encoded by three separate genes: variable (VH), diversity (DH) and joining (JH) genes on the germ-line genome. In mice, most complementarity determining region (CDR) III of the heavy chains of myelomas and hybridomas sequenced so far can be assigned to one of the 12 already identified germ-line DH genes by the homology of nucleotide sequences of DH gene-coding regions although extranucleotides, the so-called N segments, are found at the boundaries between DH and JH as well as VH and DH. On the other hand, Siebenlist et al. (Nature 1981. 294:631) identified two DH gene families in human genome: DHQ52, located at 45 bp upstream of the JH gene cluster, and another family encoded at 9-kb regular intervals possibly between VH and JH gene clusters. However, the somatic DH sequences found in VH-DH-JH structure (the somatic DH segment being defined as the region which is not encoded either by germ-line VH or JH gene) are relatively long and apparently random, and do not seem to have the homology to any of the germ-line DH sequences. To explain the origin of high diversity in the CDR III of human Ig heavy chains, Siebenlist et al. predicted the presence of another mechanism, namely DH-DH joinings. In the present study, we identified five DH genes in one of the above 9-kb repeats. This suggests that the total number of germ-line DH genes is much higher in man than in mouse. The comparison between somatic DH sequences and germ-line DH sequences indicates that most somatic DH sequences in human Ig heavy chains are also produced by VH-DH and DH-JH joinings without the joining of multiple DH gene segments.
In an attempt to characterize the genes that cause immunodeficiencies such as X-linked agammaglobulinemia (XLA) and severe combined immunodeficiency (SCID) we established precursor B-cell lines by transforming the patients' bone marrow cells with Epstein-Barr viruses. DNA rearrangements of immunoglobulin JH gene loci were observed on both chromosomes in pre-B cells derived from an XLA patient. We cloned and characterized both rearranged bands from one cell line. Both of the rearrangements occurred between DH and JH gene loci without the VH-DH structure. On the other hand, JH gene loci retained the germline configuration on both chromosomes in almost all the transformants derived from a SCID patient that had been determined according to their surface markers, to be in an early precursor B-cell stage. The implications of the observations are discussed.
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