Biomedical subjects

F Oyama

Publications and source records attributed to F Oyama.

At least 37 records · Page 2Linked to original sources

PubMed1993

Improved method for expression of Kunitz-type serine proteinase inhibitor domain of beta-amyloid protein precursor in Escherichia coli and characterization of disulfide bonds of the product.

Kunitz-type serine proteinase inhibitor (KPI) domain of Alzheimer's disease-related beta-amyloid protein precursor (APP) was expressed in Escherichia coli as a fusion protein with a truncated form of Staphylococcus protein A. The fusion protein was purified from the cell culture medium using an IgG Sepharose column. The KPI domain was separated from the protein A portion by cleavage with human alpha-thrombin at the engineered recognition sequence, followed by purification on IgG Sepharose and reversed-phase HPLC columns. The recombinant KPI domain strongly inhibited trypsin; the inhibition constant (Ki) for bovine trypsin was 2.5 x 10(-11) M, comparable to those of the secreted forms of APP with the KPI domain. The recombinant protein contained three intramolecular disulfide bonds, which were determined to be located between Cys-6 (C1) and Cys-56 (C6), Cys-15 (C2) and Cys-39 (C4), and Cys-31 (C3) and Cys-52 (C5) of the recombinant KPI domain, respectively. These positions are highly homologous to those of disulfide bonds in bovine pancreatic trypsin inhibitor. The trypsin-inhibitory activity of the recombinant protein was abolished by preincubation with 0.4 mM dithiothreitol under non-denaturing conditions. By this mild reduction, all the disulfide bonds were completely cleaved. These results clearly indicate that the disulfide bonds play an important role in the function of the KPI domain of APP.

Alzheimer Disease↗

PubMed1993

Beta-amyloid protein precursor and tau mRNA levels versus beta-amyloid plaque and neurofibrillary tangles in the aged human brain.

To learn whether or not the levels of beta-amyloid protein precursor (APP) and tau mRNAs are related to the formation of beta-amyloid and neurofibrillary tangles, we quantified these mRNA levels in three cortical regions of 38 aged human brains, which were examined immunocytochemically for beta-amyloid and tangles. Marked individual variabilities were noted in APP and tau mRNA levels among elderly individuals. The mean APP mRNA level was slightly reduced in the beta-amyloid plaque (+2) group, but not in the plaque (+) group, compared to the plaque (-) group. Some brains in the plaque (-) group showed increased APP expression, the extent of which was not seen in the plaque (+) or (+2) group. The differences in the mean tau mRNA levels were not statistically significant among the tangle (-), (+), and (+2) groups. These results show that beta-protein and tau deposition do not accompany increased expression of the APP and tau genes, respectively, and thus suggest that factors other than gene expression may be at work in the progression of beta-amyloid and/or tangle formation in the aged human brain.

Aged↗

PubMed1992

Absence of ras family point mutations at codons 12, 13 and 61 in N-ethyl-N-hydroxyethylnitrosamine- or N-nitrosomorpholine-induced renal cell tumors in rats.

The prevalence of Ki-ras, Ha-ras and N-ras point mutation within exons 1 and 2 was studied in 17 cases of renal cell tumors (8 carcinomas and 9 adenomas) induced by N-ethyl-N-hydroxyethyl-nitrosamine or N-nitrosomorpholine. DNA samples prepared from acetone-fixed, paraffin-embedded tissues were amplified by means of the polymerase chain reaction, and point mutations at codons 12, 13 and 61 were analyzed by direct sequence methods with oligonucleotide primers. No mutations were detected in any of the renal tumors. The results thus indicated that ras family point mutation is not necessary for kidney tumor development in rats, supporting the view that ras mutations may not be generally relevant to neoplastic development in various organs in different species.

Adenoma↗

PubMed1992

A novel correlation between the levels of beta-amyloid protein precursor and tau transcripts in the aged human brain.

beta-Amyloid protein precursor (APP) and tau are implicated in the pathogenesis of Alzheimer's disease. We quantified the levels of APP and tau transcripts in the three cortical regions of 38 aged human brains obtained from consecutive autopsied patients. The level of APP mRNA was directly proportional to that of tau mRNA to a remarkable extent, suggesting coordinate expression of the APP and tau genes, whereas much weaker correlations were noted among mRNAs encoding other neuronal proteins. From the previous data on the differential expression of APP and tau mRNAs, the levels of APP-751 and -695 mRNAs were calculated and found to be proportional to those of four-repeat and three-repeat tau mRNAs, respectively, whereas that of APP-770 mRNA was rather constant. These results suggest that the mRNA concentrations of APP isoforms are linked to those of tau isoforms in the aged human brain.

Aged↗

PubMed1991

Qualitative and quantitative changes of human tenascin expression in transformed lung fibroblast and lung tumor tissues: comparison with fibronectin.

Qualitative and quantitative alterations of human tenascin (TN) expression in virally transformed lung fibroblasts and in lung tumor tissues were investigated using S1 nuclease protection analysis in comparison with those of fibronectin (FN). Transformed fibroblasts and fetal lung tissues expressed more TN mRNA with an extra sequence encoding the sixth FN type III repeat than did normal cells and adult tissues. The splicing pattern of TN mRNA was also altered in many lung cancer tissues, showing increased or sometimes decreased expression of the TN mRNA with the extra sequence when compared with their surrounding normal tissues. These results provide additional evidence for the oncodevelopmental regulation of alternative RNA splicing in human lung tissues, first observed with FN mRNA (F. Oyama, et al., Cancer Res., 50: 1075-1078, 1990). Quantitative analysis of the levels of TN and FN mRNAs showed that the ratio of TN mRNA to FN mRNA was significantly increased in transformed fibroblasts and in some lung tumor tissues, when compared with their normal counterparts. Among different types of lung tumors, a significant increase of the TN/FN ratio was observed with most squamous cell carcinoma but with only a small fraction of adenocarcinoma. Since TN has been shown to inhibit cell adhesion to FN, the altered ratio of TN mRNA to FN mRNA may well affect the adhesive and migratory properties of tumor cells in lung cancer tissues.

Base Sequence↗

PubMed1991

Comparative base specificity, stability, and lectin activity of two lectins from eggs of Rana catesbeiana and R. japonica and liver ribonuclease from R. catesbeiana.

Two lectins with RNase activity obtained from eggs of Rana catesbeiana and R. japonica and RNase obtained from R. catesbeiana liver show 65-83% protein homology. The base specificity of these frog proteins was studied with 8 dinucleoside phosphates as substrates and 8 nucleotides as inhibitors. The base specificities of the B1 and B2 sites of these proteins are U greater than C and G greater than U greater than A, C, respectively. The three frog proteins are more resistant than RNase A to heat treatment, guanidine-HCl and pH-induced denaturation; i.e., they retain their native conformation up to at least 70 degrees C at pH 7.5. Differences in stability and base specificity among RNase A and the three frog proteins are discussed in relation to the primary structures. Although the two lectins agglutinate tumor cells (e.g., Ehrlich, S-180 and AH109A ascites carcinoma cells), the liver RNase has no such activity. Agglutination of AH109A cells by the two lectins is inhibited by nucleotides. Our results indicate that the agglutination sites are not identical with, but are related to, the active sites of the three frog proteins.

Amino Acid Sequence↗

PubMed1991

Differential expression of beta amyloid protein precursor (APP) and tau mRNA in the aged human brain: individual variability and correlation between APP-751 and four-repeat tau.

We investigated the relationship between the differential expression of beta amyloid protein precursor (APP) and tau mRNA, and the extent of beta and tau deposition in three regions from each of the 38 aged brains obtained from consecutive autopsied cases. Remarkable variabilities were noted in the ratios of APP-770/-751/-695 and four-repeat tau among elderly individuals. There was no consistent alteration in the APP differential expression among beta plaque (-), (+), and (++(-) ) groups. Also, no differences in the four-repeat tau ratios were noted among tangle (-), (+), and (++) groups. Despite these great individual variabilities, APP-751 was found to be well-correlated with four-repeat tau. It is possible that APP-751 and four-repeat tau are increasing during aging, while APP-695 and three-repeat tau are decreasing.

Aged↗

PubMed1990

Oncodevelopmental regulation of the alternative splicing of fibronectin pre-messenger RNA in human lung tissues.

Alternative splicing of fibronectin pre-mRNA at the ED-A region has been shown to be deregulated in malignant human liver tumors (F. Oyama et al., J. Biol. Chem., 264: 10331-10334, 1989). In order to extend this observation to other human cancers, we investigated the splicing patterns of fibronectin pre-mRNA at both ED-A and ED-B regions in normal, fetal, and cancerous lung tissues. Unlike in the liver, the ED-A+ mRNA was constitutively expressed in the lung irrespective of ontogenic or oncogenic stages. Although fetal tissues expressed the ED-A+ mRNA slightly more than did adult tissues, there was virtually no significant difference between malignant and nonmalignant tissues in the level of the ED-A+ mRNA. In contrast, significant expression of the ED-B+ mRNA was observed with fetal and cancerous tissues but not with normal adult tissues. Increased expression of the ED-B+ mRNA was associated with all types of lung cancer including adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. These results indicate that it is the ED-B, but not the ED-A, region where the alternative splicing of fibronectin pre-mRNA is oncodevelopmentally regulated in the lung. Our results also suggest that deregulation of the tissue-specific alternative splicing of fibronectin pre-mRNA is not a unique phenotype of liver cancer but rather a general feature of naturally occurring human cancer.

Animals↗

PubMed1990

Amino acid sequence of a lectin from Japanese frog (Rana japonica) eggs.

The complete amino acid sequence and the location of disulfide bonds of a lectin from Japanese frog (Rana japonica) eggs, which specifically agglutinates transformed cells, are presented. The sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with Achromobacter protease I, or chymotrypsin, and by chemical cleavage with BNPS-skatole or cyanogen bromide. The lectin is a single-chain protein consisting of 111 residues, with a pyroglutamyl residue at the amino terminus. Four disulfide bonds link half-cystinyl residue 19 to 72, 34 to 82, 52 to 97, and 94 to 111. The sequence and the location of the disulfide bonds are highly homologous to those of bull frog (Rana catesbeiana) egg S-lectin. They are also homologous to human angiogenin, a tumor angiogenesis factor, and a family of pancreatic ribonucleases.

Amino Acid Sequence↗

PubMed1989

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis.

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.

Amidohydrolases↗

PubMed1989

Intrinsic properties of reverse transcriptase in reverse transcription. Associated RNase H is essentially regarded as an endonuclease.

The intrinsic properties of reverse transcriptase in reverse transcription were studied using a synthetic, partial ovalbumin mRNA with a synthetic DNA oligonucleotide annealed to the 3'-end of the RNA as a model substrate. With or without concomitant cDNA synthesis, the RNase H activity of avian myeloblastosis virus (AMV)-reverse transcriptase cleaved the substrate at a site which would leave a hybrid of between 7 and 14 base pairs between the 3' termini of the RNA and DNA oligonucleotide. Variability in the exact size of the hybrid probably reflects some weak base preference for cleavage by the enzyme. These short hybrids can be recognized as substrates by Escherichia coli RNase H and can be utilized by reverse transcriptase as sites for continuation of cDNA synthesis. Substrates with 5'-triphosphorylated termini, 3'-OH, 3'-phosphate, 3'-end hairpin structures and 20 base pair hybrids on the middle region of long RNA more than 300 bases or on circular RNA were all cleaved by AMV-reverse transcriptase-associated RNase H, indicating that the RNase H activity is essentially regarded as an endonuclease degrading RNA moiety in RNA-DNA hybrid. The modes of action of reverse transcriptase from murine leukemia virus and Rous-associated virus 2 were the same as that of AMV-reverse transcriptase, except that the size of the remaining hybrid and the specificity for cleavage depended on the reverse transcriptase. We propose a possible model to explain the mode of action of RNase H and RNA-dependent DNA polymerase activities in reverse transcription.

Avian Leukosis Virus↗

PubMed1989

Deregulation of alternative splicing of fibronectin pre-mRNA in malignant human liver tumors.

Alternative splicing of fibronectin pre-mRNA at the ED-A region has been shown to be regulated in a tissue- and developmental stage-specific manner. We investigated the splicing pattern at this region in malignant and nonmalignant human liver tissues and found that the relative population of the fibronectin mRNA containing the ED-A sequence is markedly increased in malignant liver tumors. Nontumorous liver tissues including those with chronic hepatitis and cirrhosis did not show any significant change in the alternative splicing at the ED-A region. It was also found that the increased expression of the ED-A-containing fibronectin mRNA closely correlates with the occurrence of portal vein tumor thrombus and intrahepatic metastasis, which are two characteristic features of invasive liver tumors. These results indicate that tissue-specific alternative splicing of fibronectin mRNA is modified in human liver cancer and raise a possibility that the putative molecular machinery governing alternative RNA splicing of not only fibronectin but also other cellular proteins is deregulated in malignant human tumors.

Carcinoma, Hepatocellular↗

PubMed1989

Patterns of alternative splicing of fibronectin pre-mRNA in human adult and fetal tissues.

Alternative splicing of fibronectin pre-mRNA at two distinct regions, termed ED-A and IIICS, was investigated with human adult and fetal tissues by the nuclease S1 protection assay. A clear tissue specificity was observed in the splicing pattern at the ED-A region. More ED-A+ than ED-A- mRNAs were identified in lung, whereas ED-A- mRNAs were predominantly expressed in liver. Endometrium contained nearly equal amounts of ED-A+ and ED-A- mRNAs. The splicing pattern at the ED-A region was also different between adult and fetal liver but not between adult and fetal lung. Tissue type specific splicing was also observed at the IIICS region. Although the mRNA species containing the complete IIICS sequence comprised 40-65% of the total fibronectin mRNAs irrespective of tissue types, expression of the mRNA species lacking a part or all of the IIICS sequence was more pronounced in adult liver than in other tissues including fetal liver. These results strongly suggest that the alternative splicing of fibronectin pre-mRNA in vivo is regulated in a tissue type specific manner at both the ED-A and IIICS regions and that it is developmentally regulated in liver but not in lung. On the basis of these and other observations reported previously, a possibility that a part of the fibronectins synthesized and secreted by hepatocytes is deposited in the tissue matrix is discussed.

Adult↗

PubMed1988

Avian myeloblastosis virus reverse transcriptase is easier to use than the Klenow fragment of DNA polymerase I for labeling the 3'-end of a DNA fragment.

The 3'----5' exonuclease activity of the Klenow fragment operates in 3'-end labeling of DNA fragments. In the presence of excess deoxyribonucleoside 5'-triphosphates (dNTPs), the 5'----3' polymerase activity is dominant over the exonuclease activity. However, in the presence of a small amount of dNTPs, the exonuclease activity removed deoxyribonucleoside 5'-monophosphate (dNMP) incorporated in the 3'-end of a DNA strand by the polymerase activity. We found that the radioactivity of incorporated dNMP decreased remarkably in the course of 3'-end labeling by the Klenow fragment. On the other hand avian myeloblastosis virus (AMV) reverse transcriptase also possesses the polymerase activity. The decline of the incorporated radioactivity was not observed, indicating that the enzyme has neither exo- nor endonuclease activities. Furthermore, the level of the incorporated radioactivity was the same as that obtained by the Klenow fragment. We conclude that AMV reverse transcriptase is easier to use than the Klenow fragment for labeling the 3'-end of a DNA fragment.

Affinity Labels↗

PubMed1988

A synthetic, partial pre-mRNA for ovalbumin primes its own complementary DNA with reverse transcriptase.

Synthetic, partial pre-mRNA for ovalbumin was found to prime its own cDNA with reverse transcriptase. Initiation of the cDNA occurred 36 bases upstream of the 3'-end of the RNA, probably as a result of intramolecular base pairing at this end. Inhibition of self-priming occurs following ligation of pCp or poly-adenylation at the 3'-terminus of the synthetic RNA. The secondary structure of 3'-end region of the template RNA strongly affected the ability of self-priming.

Base Sequence↗

PubMed1988

3'-end labeling of DNA fragments by AMV-reverse transcriptase.

The efficiency on 3'-end labeling of DNA fragments was compared between Klenow fragment of DNA polymerase I from E. coli and AMV-reverse transcriptase. In the case of Klenow fragment, the exonuclease activity of this enzyme rapidly removed the labeled dNMP incorporated in 3'-end of DNA fragment by the polymerase activity of it. AMV-reverse transcriptase caused no decrease of the labeled dNMP incorporated by the polymerase activity because it has no exonuclease activity. Therefore, AMV-reverse transcriptase is easier to use than Klenow fragment for labeling the 3'-end of DNA fragment.

Avian Myeloblastosis Virus↗

PubMed1985

Molecular cloning of the fibroin light chain complementary DNA and its use in the study of the expression of the light chain gene in the posterior silk gland of Bombyx mori.

Fibroin light chain (L-chain) mRNA (mol. wt 4.0 X 10(5) daltons) was purified from the posterior silk gland of the silkworm, Bombyx mori (J-131 strain). Double-stranded complementary DNA was synthesized and inserted into the PstI site of pBR322 employing the oligo(dC)-oligo(dG) tailing method. Several recombinant plasmids containing the inserts of about 800 base pairs were isolated. Hybridization-translation assay demonstrated that these clones hybridized specifically with the fibroin L-chain mRNA. One of these clones (pLA23) was used as a probe to investigate relative concentrations of the fibroin L-chain gene and mRNA in the posterior silk glands at different stages of late larval development.

Animals↗

PubMed1984

Reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm, Nd(2).

Fibroin is normally composed of one H chain (350 kd) and one L chain (25 kd) which are connected by disulfide bond(s). However, the small amount of fibroin secreted into the lumen of the posterior silk gland of the Nd(2) (naked pupa) mutant does not contain L chain, although L chain mRNA is present and L chain is synthesized in the posterior silk gland cells of the mutant. In a hybrid silkworm, Nd(2)/Tamanashikasuri, where Tamanashikasuri is a normal producer of fibroin, L chain from the two alleles are distinguishable electrophoretically. It is demonstrated using this system that the L chain from the Nd(2) allele can combine normally with the H chain from Tamanashikasuri and the H-L complex is secreted normally. In another hybrid system, Nd(2)/J-131, where J-131 is a normal producer of fibroin, fibroin derived from the two alleles are distinguishable due to the different electrophoretic mobility of H chain. The fibroin derived from the J-131 allele is composed of H chain and L chain, while the fibroin derived from the Nd(2) allele is devoid of L chain, and its secretion is greatly reduced. We present evidence suggesting that the H chain derived from the Nd(2) allele is structurally abnormal and discuss how the H-L subunit structure is advantageous in the secretion of fibroin.

Alleles↗