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Biomedical subjects

S Ichimiya

Publications and source records attributed to S Ichimiya.

At least 19 recordsLinked to original sources
PubMed1999

p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but its mutation is infrequent.

p73, a novel p53 family member, is a recently identified candidate neuroblastoma (NBL) suppressor gene mapped at chromosome 1p36.33 and was found to inhibit growth and induce apoptosis in cell lines. To test the hypothesis that p73 is a NBL suppressor gene, we analysed the p73 gene in primary human NBLs. Loss of heterozygosity (LOH) for p73 was observed in 19% (28/151) of informative cases which included 92 mass-screening (MS) tumors. The high frequency of p73 LOH was significantly associated with sporadic NBLs (9% vs 34%, P<0.001), N-myc amplification (10% vs 71%, P<0.001), and advanced stage (14% vs 28%, P<0.05). Both p73alpha and p73beta transcripts were detectable in only 46 of 134 (34%) NBLs at low levels by RT-PCR methods, while they were easily detectable in most breast cancers and colorectal cancers under the same conditions. They found no correlation between p73 LOH and its expression levels (P>0.1). We found two mutations out of 140 NBLs, one somatic and one germline, which result in amino acid substitutions in the C-terminal region of p73 which may affect transactivation functions, though, in the same tumor samples, no mutation of the p53 gene was observed as reported previously. These results suggest that allelic loss of the p73 gene may be a later event in NBL tumorigenesis. However, p73 is infrequently mutated in primary NBLs and may hardly function as a tumor suppressor in a classic Knudson's manner.

Chromosome Mapping↗
PubMed1998

p73, a gene related to p53, is not mutated in esophageal carcinomas.

A novel gene, termed p73, encodes a protein with a significant homology to p53 and has been mapped at chromosome 1p36.3, which is a locus of multiple suppressor genes for tumors including neuroblastoma and other cancers. Since the 1p36 locus is reported to be deleted and p53 is frequently mutated in esophageal carcinomas, we examined loss of heterozygosity (LOH) and mutation of the p73 gene in 48 untreated esophageal tumors, as well as mRNA expression in 8 tumors. We screened the P1 genomic library to obtain a P1 clone containing the p73 gene and found a polymorphic short tandem CT repeat site at intron 9. Intragenic sequences for 14 PCR primer sets and a primer pair flanking the repeat were also determined for the analysis of PCR single-strand conformation polymorphism (SSCP) and LOH studies, respectively. Expression of p73 mRNA was detectable but at low levels in all 8 tumor tissues by reverse transcriptase PCR. We did not find any type of mutation other than polymorphisms in the 48 esophageal carcinomas, though aberration of the p53 gene on the PCR-SSCP gels was observed in 15 of 38 (39%) tumors of the same set. In addition, LOH for p73 was found in only 2 of 25 (8%) tumors. These results suggest that, at least in esophageal carcinomas, allelic loss or mutation of p73 may not be a main genetic event for the tumorigenesis as it is with p53.

Adenocarcinoma↗
PubMed1998

Mutation, allelotyping, and transcription analyses of the p73 gene in prostatic carcinoma.

A novel gene, p73, encoding a protein with significant homology to p53, was recently identified at 1p36. To investigate penetrance of p73 in prostatic carcinogenesis, mutation, allelotyping, and transcription analyses of p73 were performed in prostatic carcinoma. No types of mutation causing amino acid substitutions or frameshifts were found in 106 cases examined. Loss of heterozygosity in the gene was found in 2 of 38 cases (5.3%). Various expression levels of p73 alpha variant were observed in tumor compared with those in normal tissue. These data suggest that the p73 gene is not playing an essential role, but expression of p73 may associate with tumor growth in prostatic carcinogenesis.

Aged↗
PubMed1998

Glial cell line-derived neurotrophic factor/neurturin-induced differentiation and its enhancement by retinoic acid in primary human neuroblastomas expressing c-Ret, GFR alpha-1, and GFR alpha-2.

Neuroblastomas often undergo spontaneous differentiation and/or regression in vivo, which is at least partly regulated by the signals through neurotrophins and their receptors. Recently, glial cell line-derived neurotrophic factor (GDNF) and a second family member, neurturin (NTN), have been found to mediate their signals by binding to a heterotetrameric complex of c-Ret tyrosine kinase receptors and glycosylphosphatidylinositol-linked proteins, GFR alpha-1 (GDNFR-alpha) or GFR alpha-2 (TrnR2/GDNFR-beta/NTNR-alpha/RETL2). Here, we studied the effect of GDNF and NTN on human neuroblastomas in the short-term primary culture system, as well as the expression of c-Ret, GFR alpha-1, GFR alpha-2, GDNF, and NTN. GDNF (1-100 ng/ml) induced morphological differentiation in 34 of 38 primary neuroblastomas and an accompanying increase in c-Fos induction. These effects were markedly enhanced by treatment with 5 microM all-trans-retinoic acid. Although GDNF alone induced a rather weak differentiation independent of the disease stages, the enhancement of neurite outgrowth induced by treatment with both GDNF and all-trans-retinoic acid was significantly correlated with younger age (less than 1 year; P = 0.0039), non-stage 4 diseases (P = 0.0023), a single copy of N-myc (P = 0.027), and high levels of TRK-A expression (P = 0.0062). To examine the expression levels of GFR alpha-1, we cloned a short form of the human GFR alpha-1 gene with a 15-bp deletion by screening a human adult substantia nigra cDNA library. Many primary neuroblastomas expressed c-Ret, GFR alpha-1, and GFR alpha-2 as well as their ligands, GDNF and NTN, suggesting the presence of a paracrine or autocrine signaling system within the tumor tissue. The effect of NTN on primary culture cells of neuroblastoma was similar to that of GDNF. These imply that the GDNF(NTN)/c-Ret/GFR alpha-1(GFR alpha-2) signaling may have an important role in regulating the growth, differentiation, and cell death of neuroblastomas.

Antineoplastic Agents↗
PubMed1998

Ectopic expression of DAN enhances the retinoic acid-induced neuronal differentiation in human neuroblastoma cell lines.

Retinoic acid (RA) plays a major role in neuronal cell differentiation. Neuroblastoma cells differentiate in vitro by extending neurites and forming ganglion-like aggregates in response to RA. In the present study, we have examined a biological role(s) of DAN in the regulation of RA-mediated cellular differentiation in neuroblastoma cells. RTBM1 and SH-SY5Y cells undergo marked morphological changes associated with a remarkable induction of DAN gene expression when exposed to RA. By transfecting an expression vector harboring a rat DAN cDNA into SH-SY5Y cells, we have obtained two independent transfectants which express a large amount of DAN. The forced expression of DAN gene enhanced the neurite extension in the presence of RA, suggesting that DAN gene product might contain some regulatory role(s) in the RA-induced cellular differentiation in neuroblastoma cells.

Animals↗
PubMed1998

Mutational analysis of the p73 gene localized at chromosome 1p36.3 in colorectal carcinomas.

Genetic alteration of p53, which monitors DNA damage and operates cellular checkpoints, is a major factor in the development of human colorectal carcinoma (CRC). Recently, p73, a novel family member of p53, has been identified and found, like p53, to activate p21Waf1/Cip1 and to induce apoptosis. The p73 gene was mapped at chromosome 1p36.3 which is a region frequently deleted in CRCs and other cancers including neuroblastoma. To assess whether or not p73 is a tumor suppressor gene of CRC, we performed mutational analysis of p73 in 82 colorectal tumor tissues paired with constitutional DNA. Using a microsatellite marker for p73, the loss of heterozygosity (LOH) study was performed and allelic loss of p73 was found in 17% of the CRCs. RT-PCR single strand conformation polymorphism analysis showed no mutation except three polymorphisms in the p73 coding region. In addition, p73 was expressed at higher levels in the CRC tissues than in the normal mucosa or neuroblastoma tissues, though the transcripts were detectable only by the RT-PCR method. Our results suggest that, in CRCs, p73 may not play a role as a tumor suppressor, at least not in a classic Knudson manner.

Adolescent↗
PubMed1997

Identification and chromosome assignment of a human gene encoding a novel phosphatidylinositol-3 kinase.

We identified a novel phosphatidylinositol (PI) 3-kinase by screening human brain cDNA libraries with probes designed from the conserved kinase-domain sequence. Analysis of cDNAs indicated that two different forms of transcripts are present: one is the full-length form composed of 1,044 amino acid residues and the other is the short form that the N-terminal 216 amino acid residues including a putative p85 binding domain has been truncated (828 amino acid residues). Database search revealed the sequence of the full-length form to be identical to that recently registered by D. Chantry et al. (Accession No. U86453 in GenBank release, August 1997). Northern blot analysis showed this mRNA to be ubiquitously expressed in various tissues, with relatively higher expression was observed in spleen, thymus and leukocytes. Based on fluorescence in situ hybridization and PCR-based analyses with both human/rodent mono-chromosomal hybrid cell panels and radiation hybrid mapping panels, this gene was localized to chromosome region 1p36.2. This region is frequently lost in a variety of human malignancies, including neuroblastoma. The novel PI3K could be a candidate target of the 1p36 alteration that occurs in neuroendocrine tumors.

Animals↗
PubMed1997

Interaction of DA41, a DAN-binding protein, with the epidermal growth factor-like protein, S(1-5).

Recently, we have identified a new protein (DA41) which can associate with candidate tumor-suppressor DAN protein. In the present study, we have searched for DA41-interacting protein(s), using a yeast two-hybrid system. An adult rat lung cDNA library was screened by using a truncated form of DA41 (1-308) which lacks a DAN-binding region as bait. One of the positive clones, T16, contained a cDNA sequence of 1934 nucleotides with a single open reading frame of 493 amino acids. A data base search revealed that T16 exhibited a strong sequence similarity to the human epidermal growth factor (EGF)-like protein, S(1-5). The region encoding amino acids 155-232 of DA41 was identified for the interaction with T16. Since DAN and S(1-5) proteins are known to suppress and stimulate DNA synthesis, respectively, it is possible that functional interaction of DAN with S(1-5) through DA41 might play an important role(s) in the regulation of cell growth.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Identification of a structural constituent and one possible site of postembryonic formation of a teleost otolithic membrane.

A gelatinous otolithic membrane (OM) couples a single calcified otolith to the sensory epithelium in the bluegill sunfish (Lepomis macrochirus) saccule, one of the otolithic organs in the inner ear. Though the OM is an integral part of the anatomic network of endorgan structures that result in vestibular function in the inner ear, the identity of the proteins that make up this sensory accessory membrane in teleosts, or in any vertebrate, is not fully known. Previously, we identified a cDNA from the sunfish saccular otolithic organ that encoded a new member of the collagen family of structural proteins. In this study, we examined biochemical features and the localization of the saccular collagen (SC) protein in vivo using polyclonal antisera that recognize the noncollagenous domains of the SC protein. The SC protein, in vivo, was identified as a 95-kDa glycoprotein in sunfish whole-saccule lysate and in homogenates of microdissected saccular OMs. Immunohistochemical analyses demonstrated that the SC protein was localized within one of the two distinct layers of the sunfish saccular OM. The SC protein was also detected within the cytoplasm of supporting cells at the edges of the saccular sensory epithelium, indicating that these cells are a primary site for the synthesis of this structural protein. Further studies of the organization of this matrix molecule in the OM may help clarify the role of this sensory accessory membrane in vestibular sensory function.

Animals↗
PubMed1997

RT1.P, rat class Ib genes related to mouse TL: evidence that CD1 molecules but not authentic TL antigens are expressed by rat thymus.

CD1 and TL were once thought to be genetic homologues because of their thymus-specific expression. We investigated their equivalents in the rat to clarify whether their structure and pattern of expression are conserved in rodents. Two rat class Ib genes, containing 3' sequences very similar to mouse TL, were identified and designated RT1.P. Neither of them, however, can encode ordinary class I molecules due to the accumulation of harmful mutations in the 5' regions that are unique to RT1.P, while the 3' TL-like regions still retain protein-coding capacity. Comparison of the structural organization of three types of TL family genes, which include mouse T3/T18-encoding TL antigens, mouse T1/T16, and rat RT1. P1/P2 pseudogenes, revealed the presence of a clear demarcation between the type-specific and TL-specific sequences at intron 3. This finding suggests that recombination plays an important role in creating the TL family genes in rodents. Characteristic features of TL, such as a low level of polymorphism and linkage to the major histocompatibility complex, were also observed in the rat. On the other hand, rat CD1 molecules were expressed at a high level on the surface of thymocytes. Absence of authentic TL antigens and thymic expression of CD1d molecules in the rat suggest the plasticity and conservation of class Ib genes in rodent evolution. Functions of TL may be substituted with CD1 or other class Ib molecules expressed by rat thymus.

Amino Acid Sequence↗
PubMed1997

Localization of rat CD1 transcripts and protein in rat tissues--an analysis of rat CD1 expression by in situ hybridization and immunohistochemistry.

CD1 molecules are cell surface glycoproteins non-covalently associated with beta2-microglobulin. Recently, functional features of the CD1 family such as a target ligand and an antigen-presenting structure for T cells have been reported. In the current study, tissue distribution of rat CD1 was analysed by in situ hybridization (ISH) in conjunction with immunohistochemistry to clarify the precise localization of both transcripts and proteins. CD1 transcripts were detected by ISH in a variety of organs: spleen, thymus, liver, lung, heart, kidney, small intestine and skin. In most organs, CD1 immunoreactivity paralleled the amount of CD1 mRNA expression and was localized in the same regions as its gene transcripts. However, there was a clear difference between the level of rat CD1 transcription and protein expression in the small intestine. CD1 mRNA was detected in the enterocytes of crypts of Lieberkuhn, but not in those of the intestinal villi, while immunoreactivity of CD1 protein was observed in the cells of the intestinal villi but not in those of crypts of Lieberkuhn. This suggests that CD1 gene transcription occurs in the enterocytes of intestinal crypts, and that as the cells of intestinal crypts migrate from the crypts to the intestinal villi, CD1 proteins are synthesized and accumulated in the intestinal villi. Such CD1 expression in the enterocytes appears to be consistent with migration-associated differentiation, and suggests that rat CD1 may take part in mucosal immunity as a first line of defence. In addition, clear cell membrane CD1 immunoreactivity on lymphoid cells raises the possibility that intercellular interaction via rat CD1 and T cell receptors may be involved in both lymphoid cell differentiation and immunoregulation.

Animals↗
PubMed1997

Murine thioredoxin peroxidase delays neuronal apoptosis and is expressed in areas of the brain most susceptible to hypoxic and ischemic injury.

Thioredoxin peroxidase (TPx) is an antioxidant protein that limits the activity of reactive oxygen species (ROS). We cloned the cDNA encoding the mouse homolog of TPx from an E14.5 brain cDNA library and analyzed its distribution and function in murine tissues. Comparison of the amino acid sequence of mouse TPx with those of other species revealed that TPx was highly conserved across all species. Mouse TPx had broad tissue distribution, but its expression was especially marked in cells that metabolize oxygen molecules at high levels such as erythroid cells, renal tubular cells, cardiac and skeletal muscle cells, and certain types of neurons. Levels of increased expression of TPx in the brain were coincident with regions known to be especially sensitive to hypoxic and ischemic injury in humans. Models of erythroid differentiation and neuronal survival were employed to study the function of TPx. Murine erythroleukemia cells (MEL cells) increased TPx transcription when in a chemically differentiated state. Furthermore, expression of mouse TPx in PC12 pheochromocytoma cells prolonged their survival in the absence of nerve growth factor (NGF) and serum, indicating that TPx could promote neuronal cell survival. We propose that TPx contributes to antioxidant defense in erythrocytes and neuronal cells by limiting the destructive capacity of oxygen radicals. These findings identify a novel gene that appears to be relevant to hypoxic brain injury and may be of importance in development of new approaches to abrogate the effects of ischemic- and hypoxic-related injury in the central nervous system (CNS).

Amino Acid Sequence↗
PubMed1996

Cloning of m-ehk2 from the murine inner ear, an eph family receptor tyrosine kinase expressed in the developing and adult cochlea.

To identify receptor tyrosine kinases (RTKs) present in the murine inner ear, a degenerate polymerase chain reaction (PCR) methodology was employed to clone partial cDNAs encoding RTKs from embryonic day-17.5 mouse whole inner ear RNA. At least 20 distinct TKs were identified within the first 50 subcloned PCR products obtained by this analysis (Davis/Lee et al., 1996). One of the receptor RTKs identified encoded an eph-related kinase not previously described in the mouse. Analysis of full-length cDNAs revealed that this RTK is the mouse homolog of the rat ehk-2 gene product (Maisonpierre et al., 1993). Differences in the carboxyl terminal of the mouse and rat ehk2 RTKs suggest that differential splicing of this gene may occur resulting in transcripts encoding truncated and nontruncated forms of the ehk2 RTK. Multiple transcripts corresponding to this RTK were detected by Northern blot analysis only in the mouse brain. RT-PCR analysis revealed the presence of transcripts encoding this kinase in adult mouse brain, inner ear, testes, ovary, thymus, and spleen. Transcripts encoding this kinase were localized using in situ hybridization to the postembryonic day 1 cochlear ganglion neurons in the inner ear and to neurons in discrete regions of the nervous system. This is the first report of eph-related RTK in inner ear tissue that is present in both the developing and adult inner ear tissue. Because this is a member of a family of RTKs that is implicated in establishing the specificity of neuron-target cell interactions (Garrity and Zipursky, 1995), additional studies to determine if the ehk-2 gene product is involved in such processes in the murine cochlea are warranted.

Amino Acid Sequence↗
PubMed1996

Veno-occlusive disease of the liver following bone marrow transplantation: a clinical-pathological study of autopsy cases.

A total of 28 patients with leukemia underwent intensive therapy with megadose chemotherapy and stem cell transplantation. Of 16 patients who had busulfan therapy, 2 developed the clinical syndrome of veno-occlusive disease of the liver (VOD) characterized by progressive abnormalities in liver function, abdominal pain, and ascites. Postmortem findings showed VOD of the central and sublobular hepatic vein. Immunohistochemical analysis using anti-actin and CD34 mAb was employed in an attempt to clarify the process of fibrosis. Actin positive cells had accumulated around the central vein. Ito cell activation might be responsible for secondary fibrosis following endothelial injury. We report autopsy findings and immunohistochemical results.

Adolescent↗
PubMed1994

Structural analysis of the rat homologue of CD1. Evidence for evolutionary conservation of the CD1D class and widespread transcription by rat cells.

The cDNA encoding the rat homologue of CD1 was isolated and the complete nucleotide sequence was determined. It contained an open reading frame of 1008 bp that was capable of encoding a polypeptide with 336 amino acids composed of hydrophobic leader and transmembrane sequences, three extracellular domains, and 5' and 3' untranslated sequences. Comparison of the amino acid sequence of rat CD1 with those of other species revealed that it showed the highest similarity to mouse CD1, which belongs to the CD1D class of the CD1 system and is distinct from the classic CD1 class including CD1a, CD1b, and CD1c expressed primarily on human thymocytes and some dendritic cells. Widespread transcription of rat CD1 was readily detected by Northern blot analysis in nonlymphoid organs, including the liver, kidney, and heart, as well as in lymphoid organs, including the thymus, lymph node, and spleen. Intestinal expression was also demonstrated by the more sensitive reverse transcription-PCR method. Immunoprecipitation with a rabbit anti-rat CD1 Ab showed that rat CD1 was expressed on the cell surface as a beta 2-microglobulin-associated heterodimer. Southern blot analysis of inbred rat strains suggested that rat CD1 shows limited polymorphism and that only one CD1 gene is detectable in the F344 rat genome. These results provide evidence for the conservation of CD1D class through mammalian evolution and an apparent lack of the classic CD1 class genes in rodents. Functional similarity of rodent CD1 is implied.

Amino Acid Sequence↗