Biomedical subjects

K Washiyama

Publications and source records attributed to K Washiyama.

At least 19 recordsLinked to original sources

PubMed2002

Gene expression profiles in the brain from phencyclidine-treated mouse by using DNA microarray.

Recently DNA microarray technology has been introduced into analyses of comprehensive biological functions. This DNA microarray is a new technology for simultaneous analysis to examine expression patterns of thousands of genes. It was thought that this technique should be very useful for examination of cellular and molecular mechanisms of drugs of abuse: cocaine, amphetamine, and others. This technology was therefore applied for the rapid analysis of gene expression in the brain from phencyclidine-treated mice. Mainly mouse DNA microarray was examined by using labeled cDNAs produced from a control mouse brain mRNA and from brain mRNA of mouse exposed to drugs as probes. Some changes in a probe from brain mRNA of drug-treated mouse could be observed, but it was necessary to examine another DNA microarray, including more samples from the brain.

Animals↗

PubMed2001

Primary malignant lymphoma of the brain: analysis of MMAC1 (PTEN) tumor suppressor gene.

With the use of RT-PCR (reverse transcriptase-polymerase chain reaction), Northern blot analysis, and Western blot analysis, seven primary brain lymphomas were examined for the state of the MMACI tumor suppressor gene. Nucleotide analysis of RT-PCR clones revealed no abnormality in the MMAC1 coding sequence in each case. Although Northern blot revealed variation among cases in the signal intensities for MMAC1 mRNA, Western blot revealed a distinct MMAC1 protein band in all cases, suggesting that the actual MMAC1 expressions were similar. In Western blot analysis of phosphorylated Akt (p-Akt), which is regulated positively by PI3K (phosphoinositide-3 kinase) and negatively by MMAC1, all the lymphomas revealed an Akt band but not a p-Akt band, suggesting that the MMAC1 phosphatase activity was maintained in each case. These findings suggest that the MMAC1 gene is normal in its coding sequence, gene expression, and phosphatase activity in the lymphomas. Thus, unlike the p16 and p15 tumor suppressor genes, which are frequently deleted and inactivated in brain lymphoma and represent a striking contrast to systemic lymphoma, MMAC1 may not play an important role in carcinogenesis in this tumor, as in the systemic counterpart.

Aged↗

PubMed2000

Demonstration of hydroxyindole-O-methyltransferase (HIOMT) mRNA expression in pineal parenchymal tumors: histochemical in situ hybridization.

The expression of hydroxyindole-O-methyltransferase (HIOMT), an enzyme catalyzing the final step of melatonin biosynthesis, was examined in three pineoblastomas and five pineocytomas by in situ hybridization analysis. Distinct hybridization signals for HIOMT mRNA, though weaker than in normal pineal gland pinealocytes, were detected in two of the three pineoblastoma and three of the five pineocytoma cases. Of the pineoblastomas, hybridization signals were observed in most tumor cells of one case, while in another, signals were detected in occasional cells clustered or scattered throughout the neoplastic field. Of the pineocytomas, signals were detected in most tumor cells of two cases, while in one case, signals were detected only in occasional cells. Among these specimens, one pineoblastoma and one pineocytoma were also analyzed using northern blot and reverse transcription polymerase chain reaction (RT-PCR) analyses. In the northern blot analysis, an apparently single band corresponding to the size of HIOMT mRNA was detected in both pineoblastoma and pineocytoma RNA blots. In the RT-PCR analysis, three species of HIOMT mRNA generated via alternative splicing were detected in both tumors. These results suggest that the neoplastic cells of pineoblastomas and pineocytomas often retain the ability to express HIOMT mRNA, as in normal pinealocytes, and that HIOMT is a useful tumor marker for the diagnosis of pineal parenchymal tumors.

Acetylserotonin O-Methyltransferase↗

PubMed2000

The effects of DCG-IV and L-CCG-1 upon phencyclidine (PCP)-induced locomotion and behavioral changes in mice.

The behavioral changes of mice induced by acute and repeated i.p. injection of phencyclidine (PCP) were observed by measuring locomotor activity and stereotyped behavior. Then, the effects of metabotropic glutamate receptor (mGluR) agonists, DCG-IV and L-CCG-1, on the above behavioral changes induced by PCP were found. The effects of DCG-IV were very strong and completely depressed the PCP-induced hyperlocomotion. The effects of L-CCG-1 were not so strong. Repeated injection of PCP for 20 days into mice induced lower locomotor activity than that in acutely injected mice. These behavioral changes may be related with the negative symptoms of schizophrenia. In order to examine some molecular mechanisms of PCP-induced behavioral changes, Northern blot analysis of total RNA from prefrontal cortical tissues of mice treated with PCP, DCG-IV, and L-CCG-1 was carried out.

Amino Acids, Dicarboxylic↗

PubMed1999

Clinicopathological study of pineal parenchymal tumors: correlation between histopathological features, proliferative potential, and prognosis.

This study describes the clinicopathologic features of 13 cases with pineal parenchymal tumors. Based on the histopathologic findings, especially the extent of atypia and pineocytic differentiation as determined by Bodian's staining, we classified these tumors into pineocytomas (4), pineocytomas with anaplasia (4) and pineoblastomas (5). All the cases with pineocytoma and pineocytoma with anaplasia were adults, and all the cases with pineoblastoma were younger children. One patient with pineocytoma died of other disease 7 months after initial treatment. One patient with pineocytoma with anaplasia died 168 months after initial treatment. The other patients with pineocytoma and pineocytoma with anaplasia survived between 9 and 179 months after surgery. However, all of the five pineoblastoma patients died within 14 months after initial treatment. The mean MIB-1 index in pineoblastomas was significantly higher than that in other types of pineal parenchymal tumors, but there were no differences between pineocytomas and pineocytomas with anaplasia with respect to the MIB-1 index. The mean MIB-1 index in neurofilament protein-immunopositive cases was significantly lower than that in immunonegative cases. With regard to the malignant potential, we emphasize that a clear distinction should be made between pineoblastomas in children and other types of pineal parenchymal tumors in adults.

Adult↗

PubMed1999

Rare-type mutations of MMAC1 tumor suppressor gene in human glioma cell lines and their tumors of origin.

A total of 10 glioma cell lines were examined to evaluate the status of the MMAC1 gene, a candidate tumor suppressor gene. Six cell lines showed mutations with presumed loss of heterozygosity and 1 cell line showed no mRNA expression. The 6 mutations consisted of 3 3-bp deletions (codons 17, 101 or 199), 1 missense mutation (codon 252) and 2 truncation mutations (1 nonsense mutation at codon 233 and 12-bp insertion at codon 241). Among them, the 3-bp deletions, which are a rare type of mutation in MMAC1 gene, were located in the N-terminal half (codons 1-212) of the coding region, which is considered important in MMAC1 function. The missense mutation was located unusually in the C-terminal half (codons 212-403), but it was in a small region in which some other reported missense mutations are clustered. Thus, these 4 mutations were suggested to have functional effects on the MMAC1 activity, like the other 2 mutations with predicted protein truncations. By sequence analysis of cDNA clones, we confirmed that all the mutations including these 4 rare ones were in the MMAC1 gene, not in the PTH2 pseudogene. In 2 cases, we also examined the primary glioma tissues from which the cell lines had been derived and found the same mutations as in the cell lines in both cases. This suggested that the mutations in these cell lines were derived from the primary glioma tissues, but not from artifacts arising during long-term in vitro cultivation.

Adult↗

PubMed1998

Frequent deletion and 5' CpG island methylation of the p16 gene in primary malignant lymphoma of the brain.

A total of 10 primary malignant lymphomas of the brain were examined for deletion, mutation, and 5' CpG island methylation of the p16 gene, which is a candidate tumor suppressor gene with CDK-inhibitory function. In Southern blot analysis, p16 gene deletion was suggested in nine cases, homozygously (five cases) or hemizygously (four cases). In the remaining one case, p16 gene deletion was not suggested. Although single-strand conformation polymorphism and nucleotide analyses suggested no mutations of the p16 gene in these cases, methylation analyses revealed 5' CpG island methylation in three cases, of which two were those with presumed hemizygous deletion and one was that without deletion in Southern blot analysis. Thus, p16 gene abnormality was detected in all 10 of the brain lymphomas examined, and in 8 of them, actual p16 gene inactivation was suggested by their homozygous deletion (5 cases) or 5' CpG island methylation (3 cases). These findings suggest that p16 gene abnormality and inactivation are closely related to carcinogenesis in primary malignant lymphoma of the brain. The p15 gene, another candidate tumor suppressor gene located in the vicinity of the p16 gene, to which it shows structural and functional similarity, was also presumed to be deleted similarly in most cases. Its methylation was seen in one case, the case without the methylated p16 gene.

Brain Neoplasms↗

PubMed1998

Postnatal developmental changes in NSE and NNE mRNA expression in the rat pineal gland: in situ hybridization histochemistry.

By in situ hybridization, neuron-specific enolase (NSE) and non-neuronal enolase (NNE) mRNAs were examined in the rat pineal gland at the postnatal developmental and adult stages. The distributions of hybridized signals were analyzed in comparison with immunohistochemical staining of synaptophysin (SYN), which is a marker for pinealocytes. In SYN-positive areas that were observed throughout postnatal developmental and adult stages, we detected both NSE and NNE signals, which increased simultaneously during early postnatal development and thereafter became stationary. Quantitative analysis revealed that NNE signals were 2- to 3-fold greater in number than NSE signals at any given stage. This predominant expression pattern of NNE differed from that in neurons, which usually showed both signals at similar levels and seemed to reflect the difference in physiological function from neurons. During the early postnatal stages, a cord-like arrangement of cells without distinct SYN staining was observed. This arrangement was the thickest at postnatal day 0 and became dispersed and thinner with development, showing a relationship with formation of vascularized connective tissue stroma. By in situ hybridization, many of the constituent cells showed weak NNE signals but no distinct NSE signals. However, some cells also showed weak NSE signals, suggesting heterogeneity of these cells. The characteristic NSE and NNE expression patterns in the pineal gland cells clarified in this study might provide a basis for further studies of the differentiation and function of the pineal gland.

Animals↗

PubMed1997

cDNA cloning and mRNA expression analysis of the human neuronatin. High level expression in human pituitary gland and pituitary adenomas.

The authors cloned the nearly complete cDNA of human neuronatin with the aid of an expressed sequence tag (EST) database, and analyzed its expression in various human tissues by Northern blot analysis. The nucleotide and deduced amino acid sequences of the human neuronatin showed a high similarity to those of rodents. The Northern blot analysis revealed that the human neuronatin message was expressed predominantly in the fetal brain in the brain-specific manner, but only faintly in the adult brain. Among the various adult human tissues examined, the anterior pituitary gland was shown to be the only place where the neuronatin mRNA was strongly expressed. Intense neuronatin expression was also observed in several human pituitary adenomas, including ACTH-producing, GH-producing, and nonfunctioning adenomas, but hardly detected in other brain tumors.

Adenoma↗

PubMed1997

Infrequent mutation of Waf1/p21 gene, a CDK inhibitor gene, in brain tumors.

Possible mutations of the Waf1/p21 gene, a cyclin-dependent kinase (CDK) inhibitor gene, were investigated in biopsy specimens of 28 brain tumors using polymerase chain reaction (PCR)-single strand conformation polymorphism and nucleotide sequence analysis. There were 15 astrocytic tumors, six medulloblastomas, five pineal parenchymal tumors, and two neuroblastomas. A mutation was detected in exon 2, which comprises 90% of the cording region including the CDK inhibitory domain, in only two samples. A missense mutation was detected at codon 33 in rare PCR clones from an astrocytic tumor and a silent mutation was detected at codon 35 in a medulloblastoma. Therefore, mutation of the Waf1/ p21 gene is infrequent in these brain tumors. Examination of deoxyribonucleic acid (DNA) from normal subjects and paired DNA of brain tumor patients observed a polymorphism at codon 31, which encodes either Ser (AGC) or Arg (AGA).

Adolescent↗

PubMed1996

Primary malignant lymphoma of the brain: demonstration of frequent p16 and p15 gene deletions.

The p16 (MTS-1) gene, a candidate tumor suppressor gene, was examined by means of Southern blot, PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and nucleotide analyses in 5 cases of primary malignant lymphoma of the brain. By Southern blot analysis, the p16 gene was found to be deleted in at least 4 cases, homozygously (3 cases) or hemizygously (1 case). The p15 (MTS-2) gene, another candidate tumor suppressor gene located in the vicinity of the p16 gene, to which it shows structural and functional similarity, was also found to be deleted in 4 cases. Our frequent detection (80%) of p16 and p15 gene deletions might suggest that these deletions are closely related to carcinogenesis in primary malignant lymphoma of the brain. SSCP and nucleotide analyses revealed no mutations of the p16 gene in any of the cases.

Adult↗

PubMed1996

A comparative study of glioma cell lines for p16, p15, p53 and p21 gene alterations.

A total of 10 glioma cell lines were examined for alterations of the p16, p15, p53 and p21 genes, which are tumor suppressor genes or candidates with direct or indirect CDK-inhibitory functions. Genetic alterations (deletions or mutations) were frequently seen in the p16, p15 and p53 genes in these cell lines, but not in the p21 gene. When the states of the p16, p15 and p53 genes were compared among cell lines, all the cell lines showed abnormalities in at least 1 gene, often in 2 or 3 genes coincidentally, suggesting that dysfunction of these genes is closely related to glioma cell growth. Although alteration of all 3 genes was most frequent, there were cell lines having either p16/p15 or p53 or pl6 and p53 gene alterations, suggesting that the time order of these genetic alterations was variable depending on the cell line. Among cell lines examined, one with homozygous p53 gene deletion seemed of particular practical value, since such a cell line might be useful in various studies, including investigation of the functions of various mutant p53 genes in the absence of heteromeric protein formation. On examination of the primary tumor tissues, the same alterations of the p16/p15 and p53 genes as detected in the cell lines were demonstrated in all 6 cases examined: p16/p15 gene deletion in 1, p16 gene mutation in 1 and p53 gene mutations in 5 cases. This suggested that the p16/p15 and the p53 gene alterations and their combinations in at least some glioma cell lines reflected those in the primary glioma tissues.

Adult↗

PubMed1996

Neurotrophin and neurotrophin receptor proteins in medulloblastomas and other primitive neuroectodermal tumors of the pediatric central nervous system.

Primitive neuroectodermal tumors (PNETs) of the central nervous system (CNS) are poorly understood childhood neoplasms, and medulloblastomas are the most common pediatric PNETs. Neoplastic cells in medulloblastomas and other PNETs resemble progenitor cells of the developing central nervous system, but they also may exhibit the molecular phenotype of immature neurons or glia. As neurotrophins play a role in regulating differentiation, proliferation, and cell death in the normal developing central nervous system, and recent evidence suggests that neurotrophins may influence the behavior of medulloblastomas, we studied 29 PNET biopsy samples (27 of which were posterior fossa medulloblastomas) by immunobistochemistry using antibodies specific for each of the major high affinity neurotrophin receptor proteins, ie, TrkA, TrkB, and TrkC. A subset of these tumors also was examined by Western blot. Immunoreactive TrkA, TrkB, and TrkC were observed in neoplastic cells in 8 (27%), 18 (62%), and 14 (48%) of these PNETs, respectively. Additional immunohistochemical studies of a subset of these PNETs using antibodies to neurotrophins that primarily activate TrkB and TrkC, ie, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5, showed that immunoreactive brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 were detected in 22, 9, and 19% of these PNET biopsies, respectively. Finally, 19 pediatric brain tumors other than these PNETs also were studied here, and they expressed these neurotrophins and their receptors to a variable extent. The demonstration here that neurotrophins and their cognate receptor proteins are expressed in PNETs as well as in other pediatric brain tumors may imply that signal transduction pathways mediated by neurotrophins and/or their receptors influence the induction or progression of these common childhood neoplasms.

Adolescent↗

PubMed1996

Analysis of p53 gene mutations in human glioma cell lines.

Seven human glioma cell lines were examined for mutations of the p53 gene and their mRNA and protein expressions. Five cell lines revealed a missense mutation at, codons 237, 245, or 273. Their p53 mRNA expression was variably distinct and not always comparable to p53 protein expression, suggesting difference in the transcriptional and posttranscriptional regulation. One cell line had a splicing mutation in intron 9 and abnormal splicing was actually demonstrated by RT-PCR analysis. The remaining 1 cell line showed no PCR-amplification of the p53 gene sequence. In an examination of the original tumor tissues, the same mutations were demonstrated in the 5 tumors examined, strongly suggesting that the mutations in the glioma cell lines were derived from their original tumor tissues.

Brain Neoplasms↗

PubMed1995

Intracranial germ cell tumors: detection of p53 gene mutations by single-strand conformation polymorphism analysis.

Using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis, p53 gene mutation was examined in 12 intracranial germ cell tumors (5 yolk sac carcinomas and 7 germinomas), many of which were derived from young patients in the first to the second decade. A total of 10 mutations were detected in 4 of the 12 cases and, in 3 of them, the mutations were multiple or tandem. Among the 10 mutations, 7 were missense, 1 was splicing and 2 were silent. The 7 missense mutations were located at previously proposed hot spot codons or in their vicinity or, when outside the hot spots, at a codon encoding an amino acid conserved in most vertebrates. These findings suggested that all 7 missense mutations may actually give rise to functional alteration of the p53 protein. The splicing mutation was considered to be a germ-line mutation, though its biological effect was equivocal, since the neoplastic tissue contained an additional mutation. The pattern of the mutations was predominancy of G:C-A:T transition with frequent involvement of the CpG site. These mutations were more frequently detected in yolk sac carcinomas (60%; 3/5 cases) than in germinomas (14%; 1/7 cases), suggesting that the contribution of the p53 mutation to carcinogenesis differed with the histological type of the intracranial germ cell tumor.

Adolescent↗

PubMed1995

The analysis of p53 tumor suppressor gene in pineal parenchymal tumors.

p53 gene mutation was examined in 9 pineal parenchymal tumors, 4 pineoblastomas and 5 pineocytomas, by the immunohistochemical and the polymerase chain reaction-mediated single strand conformation polymorphism (PCR-SSCP) analyses. In each case, immunohistochemical analysis revealed no positive staining for p53 protein with either PAb1801 or DO-1 antibody and PCR-SSCP analysis revealed no abnormal migration in exons 5 to 8 of the p53 gene. These findings suggested that p53 gene mutation is rarely related with the tumorigenesis of pineal parenchymal tumors.

Adult↗

PubMed1994

Analysis of p53 gene mutations in low- and high-grade astrocytomas by polymerase chain reaction-assisted single-strand conformation polymorphism and immunohistochemistry.

Using polymerase chain reaction-assisted single-strand conformation polymorphism (PCR-SSCP) and immunohistochemical analyses, mutations in the p53 tumor suppressor gene were examined in 19 low- and high-grade gliomas. By PCR-SSCP and nucleotide analyses, p53 gene mutation was seen in 7 gliomas. Out of the 7 mutations, 3 were located at the CpG site of the previously proposed hot-spot codons 248 and 273, 2 were at codons 171 and 214 and the other 2 were in intron 5, 1 at the splice acceptor site and the other in the vicinity of the splice donor site. The latter 4 mutations have not, or only rarely, been observed in gliomas or in other tumors. However, their effect on the structural and functional alteration of the p53 protein was suggested by positive intranuclear p53 immunostaining in neoplastic cells in 3 mutations including the 1 at the splice acceptor site. In connection with glioma grading, the p53 gene mutation was shown to have occurred in both low- and high-grade gliomas, often in most of the neoplastic cells, as suggested by lack of distinct normal bands and ladders in SSCP and direct sequencing, respectively. The absence of recurrence and malignant transformation over a considerably long postoperative time in our low-grade glioma cases suggested that the p53 gene mutation might not be sufficient for the progression from low- to high-grade gliomas. The frequency of detection of mutation was 7/19(37%) by PCR-SSCP, 8/19(42%) by immunohistochemistry and 10/19(53%) by both methods. The results of PCR-SSCP and immunohistochemistry were consistent in 14 cases (73.7%), but not in 5 cases(26.3%).(ABSTRACT TRUNCATED AT 250 WORDS)

Astrocytoma↗

PubMed1994

Primary malignant lymphoma of the brain: demonstration of the p53 gene mutations by PCR-SSCP analysis and immunohistochemistry.

Using PCR-SSCP and immunohistochemical analyses, mutations of the p53 tumor suppressor gene were examined in 5 cases of primary malignant lymphoma of the brain (diffuse large cell type). By PCR-SSCP and nucleotide analyses, p53 gene mutations were seen in 2 of the 5 cases. The mutation in one case was a missense G: C-T:A transversion at codon 176 (TGC-TTC; Cys-Phe) which was located in the highly conserved domains and adjoined a previously proposed hot spot codon (codon 175) in various tumors. p53 immunoreactivity was also shown in this case. The mutation in another case was a nonsense G:C-A:T transition at codon 52 (TGG-TGA; Trp-stop codon) leading to a truncated p53 peptide. Thus, these mutations may have actually given rise to serious structural and functional alterations of the p53 protein. These findings suggested that the p53 gene mutation was related with oncogenesis in the primary malignant lymphoma of the brain.

Adult↗