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Biomedical subjects

M Shoji

Publications and source records attributed to M Shoji.

At least 19 recordsLinked to original sources
PubMed1996

Intracellular generation of amyloid beta-protein from amyloid beta-protein precursor fragment by direct cleavage with beta- and gamma-secretase.

Two amyloid beta protein precursor (beta APP) fragments involving Met and 103 amino acids of C-terminus of beta APP (delta NOR-beta) and its KM-NL substitution (delta NL-beta) were expressed in COS-7 cells to clarify the proteolytic cleavages to generate amyloid beta protein (A beta). The 4.5-kD protein, A beta with additional N-terminal amino acids, and 4-kD A beta were directly produced and released from 12.5-kD expression proteins without any production of 11.4-kD C-terminal fragment starting at N-terminus of A beta and 3-kD "p3" A beta derivative. Intracellular 4-kD A beta was also detected. The substitution of KM-NL of beta APP found in Swedish familial Alzheimer's disease (AD) promoted the production of intracellular A beta and its release with no increase in level of 11.4-kD C-terminal fragment. These results suggested the presence of a distinct pathway in which A beta is directly cleaved at both N- and C-termini from beta APP fragment intracellularly to release A beta. Since KM-NL substitution enhanced intracellular A beta generation, this pathway may be associated with amyloidogenesis in AD.

Alzheimer Disease↗
PubMed1996

A reproducible assay of polymerase chain reaction to detect trinucleotide repeat expansion of Huntington's disease and senile chorea.

A simple and reproducible method of polymerase chain reaction (PCR) assay was established to detect trinucleotide repeat expansion for Huntington's disease (HD) using a new DNA polymerase and buffer system. The system consists of an extremely heat stable DNA polymerase (Pfu), and a buffer supplemented with ammonium sulfate and dimethyl sulfoxide. Previous methods to amplify expanded alleles for HD have been very complex in PCR conditions, but the reproducibility was sometimes very low because of repetitive sequences around the primer sequences. With the present method, strong bands for the disease alleles were reproducibly visible in a conventional agarose gel stained with ethidium bromide without using isotopes. Three cases with sporadic HD and a case with senile chorea showed expanded alleles for HD with smaller sizes of the expansion than cases with typical HD. These results showed that the present method provides a simple and reproducible way to detect HD allele, and some cases with sporadic HD and senile chorea had expanded HD alleles.

Adult↗
PubMed1996

Production of recombinant human monoclonal antibody using ras-amplified BHK-21 cells in a protein-free medium.

A ras oncogene-amplified recombinant BHK-21 cell line (ras-rBHK-IgG) has been established, and was shown to hyperproduce the recombinant IgG chimeric human monoclonal antibody (hMAb) AE6F4, which recognizes lung cancer cells. We found that the ras-rBHK-IgG cell could be easily cultured in a protein-free ERDF medium supplemented with iron(III) nitrate, hydroxyethyliminodiacetic acid, and non-protein synthetic attachment factor as well as in a serum-free ERDF medium supplemented with insulin, transferrin, ethanolamine, and sodium selenite. The productivity of recombinant hMAb from the cells cultured in dishes at high cell densities was higher in protein-free medium than in serum-containing medium. True high density culture of the ras-rBHK-IgG cells was done in protein-free medium using the Tecnomouse, which is a novel hollow fiber bioreactor system. After culture for 30 days in protein-free culture, a total amount of about 14 mg of the recombinant hMAb AE6F4 was obtained, and was shown to be reactive against lung cancer cells in tissues.

Animals↗
PubMed1995

Selective inhibition of DNA polymerase-alpha family with chemically synthesized derivatives of PHYLPA, a unique Physarum lysophosphatidic acid.

PHYLPA, a unique Physarum lysophosphatidic acid (LPA), showed selective inhibition of a family of DNA polymerase alpha, including DNA polymerases alpha, delta and epsilon; but no inhibition of DNA polymerase beta or gamma was observed. To reveal the molecular mechanism of inhibition of DNA polymerases by PHYLPA, four stereoisomers and some other derivatives were synthesized and their effects on DNA polymerases were studied. Among eight derivatives synthesized, PHYLPA-1 (the natural PHYLPA; sodium 1-O-[(9'S,10'R)-9',10'-methanohexadecanoyl]-sn-glycerol 2,3-cyclic phosphate) and PHYLPA-2 (sodium 3-O-[9'S,10'R)-9',10'-methanohexadecanoyl]-sn-glycerol 1,2-cyclic phosphate) were strong and specific inhibitors of a family of DNA polymerase alpha. But their stereoisomers PHYLPA-3 (sodium 1-O-[9'R,10'S)-9',10'-methanohexadecanoyl]-sn-glycerol 2,3-cyclic phosphate) and PHYLPA-4 (sodium 3-O-[9'R,10'S)-9',10'-methanohexadecanoyl-sn-glycerol 1,2 cyclic phosphate) were weak inhibitors, showing the critical importance of stereochemistry of a cyclopropane-containing fatty acid for the inhibitory activity. Some derivatives having no cyclopropane-containing fatty acids--palmitoyl-, oleoyl-, and palmitoleoyl-PHYLPA--showed inhibition to some extent; but 1-palmytoyl and 1-oleoyl lysophosphatidic acid, which has no cyclic phosphate, did not show an apparent inhibitor activity on DNA polymerases. Hence, the extent of the inhibition apparently depends on the stereochemistry of both the fatty acid moiety and the cyclic phosphate.

Animals↗
PubMed1995

Modified amyloid beta protein ending at 42 or 40 with different solubility accumulates in the brain of Alzheimer's disease.

Serial extraction study of Alzheimer's disease (AD) and control brains revealed 4, 3.7 and 3 kD amyloid beta protein (A beta) species accumulated in AD brains. In the fractions extracted with TBS, 10% SDS and formic acid, considerable amounts of A beta species were recovered in SDS fractions besides TBS and formic acid fractions. Immunoblotting with several site-specific antibodies confirmed not only the presence of 4 kD A beta starting at the first amino acid of A beta but also 2 smaller A beta species with modification of their amino-termini in the highly resolutional Tris/Tricine gel system. A beta solubility using these solvents was associated with both modification of the amino-terminus and length of carboxyl-terminus of A beta. Especially, a large amount of modified A beta was found to be accumulated as forms with different solubility in AD brains.

Aged↗
PubMed1995

Tau in cerebrospinal fluids: establishment of the sandwich ELISA with antibody specific to the repeat sequence in tau.

Clinical diagnosis for Alzheimer's disease (AD) is provided by the criteria of DSMIV and clinical progress in addition to imaging analysis with MRI after negative screening. The final exclusive diagnosis is confirmed by the neuropathological findings of neurofibrillary tangles and senile plaques in autopsy brains. We developed a new ELISA system to measure the amount of tau in cerebrospinal fluids (CSF) using phosphorylation-independent and sequence-specific antibodies. The present ELISA was sensitive enough to detect tau in CSF of normal subjects. The amount of tau was significantly elevated in CSF of AD subjects compared with those of normal subjects and subjects with dementia of cerebrovascular disease, suggesting that tau in CSF reflects the massive and continuous neuronal cell death in the AD brain. In conclusion, we established an ELISA system which enabled us to detect tau in CSF and demonstrated that tau was significantly and specifically elevated in CSF of AD subjects. This assay system can provide us with a potent diagnostic tool for clinical AD.

Alzheimer Disease↗
PubMed1995

Molecular cloning of the 31 kDa cytosolic phospholipase A2, as an antigen recognized by the lung cancer-specific human monoclonal antibody, AE6F4.

The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A2 (cPLA2), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA2 expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA2 was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA2, but they also play a crucial role in antigen recognition by the monoclonal antibody.

14-3-3 Proteins↗
PubMed1995

Elevation of ratio of urinary N-acetylneuraminlactose to free sialic acid in some advanced cancer patients.

We estimated the levels of free sialic acid and sialylated oligosaccharides excreted in the urine of normal donors (n = 10) and patients with gastric cancer (n = 6) and colorectal cancer (n = 4). The total sialic acid level in cancer patients was similar to that in normal donors. However, the ratios of glycosidically bound sialic acids to free sialic acid were higher in some advanced cancer patients than in the normal donors. A major component of sialylated oligosaccharides was N-acetylneuraminyl alpha (2-->3) lactose. The elevation of the urinary ratio of this sialylated oligosaccharide to free sialic acid observed in some advanced cancer patients in this study may reflect the elevation of sialyltransferase activity in tumor tissues.

Adult↗
PubMed1995

Megakaryocytic differentiation of a leukemic cell line, MC3, by phorbol ester: induction of glycoprotein IIb/IIIa and effects on expression of IL-6, IL-6 receptor, mpl and GATA genes.

We investigated megakaryocytic differentiation in a newly-established Ph1-positive leukemic cell line, MC3, which showed tri-lineage immunophenotypes (myeloid antigens2+, CD19(1+) and CD41a1+) and was positive for CD34 and CD38. TPA induced MC3 cells to differentiate to an early stage of megakaryocyte lineage exhibiting an increase in the expression of platelet glycoproteins (GP) IIb/IIIa (CD41a), and an increase in cell size and nuclear ploidy. TPA treatment also enhanced the expression of GPIIb mRNA, and induced the expression of interleukin-6 (IL-6) and its receptor mRNAs, while it did not induce transcripts of the genes IL-11 and mpl ligand, and further decreased the transcript of the mpl gene. Consistent with these findings, MC3 cells treated with TPA showed an increased expression of GATA-1, but not GATA-3 transcripts, whereas those without TPA treatment expressed only the GATA-2 transcript. These results provide an insight into the study for the regulatory mechanism of megakaryocytopoiesis and leukemic cell differentiation.

Antigens, CD↗
PubMed1995

Variable clinical symptoms in familial amyotrophic lateral sclerosis with a novel point mutation in the Cu/Zn superoxide dismutase gene.

We report a novel missense point mutation in exon 4 of the Cu/Zn superoxide dismutase (SOD) gene of affected members of a Japanese kindred segregating familial amyotrophic lateral sclerosis (FALS) through at least three successive generations. The mutation, which is predicted to cause the replacement of isoleucine at codon 104 by phenylalanine (I104F), is associated with a significant reduction in Cu/Zn SOD enzyme activity but results in a highly variable clinical phenotype. Age at onset varied from 6 to 55; the initial symptoms occurred in either the lower or upper extremities in different family members. The duration of the disease varied from 3 to 38 years. Two subjects, aged 59 and 34, remained asymptomatic until their death from other causes, although their offspring carrying the same mutation have already developed clinical evidence of the disease. These results suggest that FALS from this novel I104F mutation shows considerable clinical variation.

Adult↗
PubMed1995

Effects of V1- and V2-vasopressin (AVP) antagonists on the pressor, AVP and atrial natriuretic peptide responses to a hypertonic saline infusion in conscious anephric rats.

To examine the role of vasopressin (AVP) receptors in the regulation of the hemodynamics and release of atrial natriuretic peptide (ANP), and the participation of renal nerve inputs in the osmotic AVP release, hypertonic saline (HS) was infused into conscious, bilaterally nephrectomized rats with non-peptide, selective antagonists for the V1-receptor or V2-receptor of AVP. In the control group, HS alone increased mean arterial pressure, plasma ANP and AVP, plasma volume and plasma osmolality, and decreased the heart rate. In the V1-receptor antagonist group, an increase in the mean arterial pressure and a decrease in heart rate were completely abolished and an increase in plasma ANP was attenuated. In the V2-receptor antagonist group, increases in mean arterial pressure and plasma ANP and a decrease in heart rate were attenuated. However, the ratio of the changes in heart rate to the changes in mean arterial pressure in the V2-receptor antagonist group is significantly higher than that in the control group. In both experimental groups, increases in plasma AVP, plasma volume and plasma osmolality were not different from those in the control group. These results suggest that a HS-induced increase in mean arterial pressure is mediated by the pressor effect of AVP, mainly through V1-receptors, and that the depressor effect of AVP through V2-receptors may not influence tonically HS-induced hypertension. Moreover, HS-induced increase in plasma ANP is mediated mainly by increases in plasma volume and blood pressure, but may not be affected by a direct action of AVP to the heart. Renal afferent nerve inputs may not have effects on the regulation of osmotic AVP release.

Animals↗
PubMed1995

Changes in plasma vasopressin levels and cardiovascular function due to postural changes in diabetic neuropathy.

Decreases in blood pressure are well known to increase the release of vasopressin. Studies were carried out to investigate whether vasopressin responses to postural changes in blood pressure are maintained in diabetic patients with orthostatic hypotension [DM-OH(+)] as well as non-diabetic patients with orthostatic hypotension [nonDM-OH(+)] and these responses were compared with those observed in normal subjects and diabetic patients without orthostatic hypotension [DM-OH(-)]. After 30 min in the supine position, the upright posture for 40 min was maintained and then the supine for 10 min. Blood pressure and heart rate (HR) were measured every 5 min and plasma vasopressin levels (plasma AVP) were determined every 10 min. In normal subjects and DM-OH(-), mean arterial blood pressure (MABP) did not change, but HR increased significantly by the upright position. Plasma AVP did not change in these groups. On the other hand, in DM-OH(+) MABP fell abruptly and remained to decrease during the upright posture. The HR responses in this group, however, were similar to those in normal control and DM-OH(-). Plasma AVP in DM-OH(+) significantly increased only at 30 min during upright. These increases were significantly greater than those in normal and DM-OH(-). There were significant correlation in changes in MABP (delta MAP) and plasma AVP (delta AVP) in DM-OH(+) (delta AVP = -0.13 MABP + 1.5, r = -0.32, p < 0.01). Relationship between delta MABP and delta AVP in nonDM-OH(+) was similar to that in DM-OH(+). It is concluded that AVP responses to orthostatic hypotension in diabetic and non-diabetic neuropathies were attenuated, but heart rate responses in these patients ware well reserved.

Adult↗
PubMed1995

Role of atrial natriuretic peptide in interleukin 1 beta-induced natriuresis in conscious rats.

To assess whether atrial natriuretic peptide (ANP) plays a role in the natriuresis induced by interleukin 1 beta (IL-1 beta), the following experiments were carried out. Experiment (Ex) I: IL-1 beta (7.5 micrograms/kg BW) was given intravenously (i.v.) in conscious hydrated rats (n = 6). Plasma ANP, vasopressin (AVP) osmolality (Posm), Na and K, urine Na (UNa V) and K excretion (UK V), osmolality and flow (UF), and mean arterial blood pressure (MABP) and heart rate were simultaneously determined. In the control group (n = 6), the drug was omitted, and the same protocols were carried out. Ex II: Three mg/kg BW of the specific ANP antagonist, HS-142-1 (HS), was administered i.v. and then, IL-1 beta (7.5 micrograms/kg BW) was given i.v. (n = 6). In the HS alone group (n = 6), IL-1 beta was omitted. The experimental protocols were the same as those in Ex I. IL-1 beta increased significantly plasma ANP and AVP and UNa V, but not UF, accompanied by decreases in Posm and UKV and increases in MABP (ExI). HS inhibited the natriuresis mediated by IL-1 beta, despite increases in plasma ANP and had no influence on plasma AVP and MABP. In the control (ExI) and HS alone (ExII) groups, these parameters did not change, except for decreases in Posm in both groups and increased plasma ANP in the latter. These results suggest that plasma ANP may play an essential role in the IL-1 beta-mediated natriuresis.

Animals↗
PubMed1995

Alpha 1-antichymotrypsin level in cerebrospinal fluid is closely associated with late onset Alzheimer's disease.

The levels of alpha 1-antichymotrypsin (ACT) in cerebrospinal fluid (CSF) from 66 sporadic Alzheimer's disease (AD) patients and 54 normal controls were measured by enzyme immunoassay and compared. There was no correlation (r = 0.259, n = 54) between the ACT level and normal aging. The levels of ACT were significantly higher in the total AD group (p < 0.01) than in the normal control group. Dividing AD patients into early onset AD (n = 27) and late onset AD groups (n = 39), the mean level of CSF ACT in the late onset AD group was significantly higher than that in the normal control group (p < 0.001) and that in the early onset AD group (p < 0.01). Thus, the level of ACT in CSF is closely associated with late onset AD.

Adolescent↗
PubMed1995

Establishment and characterization of a new Ph1-positive chronic myeloid leukemia cell line MC3 with trilineage phenotype and an altered p53 gene.

A new Ph1-positive leukemic cell line (MC3) expressing the P210bcr/abl oncoprotein was established from a patient with CML in blast crisis. The MC3 cells showed the trilineage phenotype of myeloid, lymphoid (CD19) and megakaryocytoid lineages, and had a proliferative response to rhIL-1 and rhIL-3 in the serum-free culture. These results and the expression of CD34 indicated that the MC3 cells have characteristics of hematopoietic progenitor cells. Recently, it has been documented that alterations of the p53 gene in leukemic cells are frequently detected during the blast crisis of CML. The MC3 cells contained the altered p53 gene. In addition, the original leukemic cells showed the point-mutational activation of the N-ras gene and an additional chromosomal abnormality inv(3q), but the MC3 cells contained no such abnormalities, indicating that not all of the original leukemic cells had these abnormalities. Thus, the MC3 cell line may provide several insights into investigations of the blast crisis in CML as well as hematopoietic progenitor cells.

3T3 Cells↗
PubMed1995

Immunocytochemical detection of lung cancer cells with monoclonal antibodies to 14-3-3 proteins.

Murine monoclonal antibodies were raised against 14-3-3 proteins, the antigen of human monoclonal antibody AE6F4 which had been shown potentially useful for the immunochemical diagnosis of lung cancer via sputum cytology. Enzyme-linked immunosorbent assays of the murine anti-14-3-3 monoclonal antibodies with isolated bovine brain 14-3-3 isoforms showed that the antibodies were classified into four different profiles of isoform reactivity. The comparison of 14-3-3 isoform and lung cancer tissue on the reactivity with murine monoclonal antibodies indicated that beta isoform can be responsible for cancer recognition, whereas human monoclonal antibody AE6F4 showed preferential binding to zeta isoform. No murine monoclonal antibody of the same isoform specificity as human monoclonal antibody AE6F4 was obtained. Since murine monoclonal antibodies with different isoform specificities could immunostain lung cancer cells in sputum successfully, the combination use of murine monoclonal anti-14-3-3 antibodies with human monoclonal antibody AE6F4 is potentially useful for facilitating the sputum cytodiagnosis of lung cancer.

14-3-3 Proteins↗