Biomedical subjects

T Endo

Publications and source records attributed to T Endo.

At least 649 records · Page 36Linked to original sources

PubMed1993

The bcl-2 protein is inserted into the outer membrane but not into the inner membrane of rat liver mitochondria in vitro.

The bcl-2 gene encodes a 26 kDa protein, overexpression of which blocks cell apoptosis. Since conflicting data have been reported concerning the localization of the Bcl-2 protein with mitochondrial inner and outer membranes, we analyzed in vitro import of the human Bcl-2 protein into isolated rat liver mitochondria. The Bcl-2 protein translated in rabbit reticulocyte lysate was efficiently inserted into the mitochondrial outer rather than the inner membrane in a membrane potential-independent manner. The carboxyl-terminal hydrophobic stretch is essential for the Bcl-2 protein to integrate into the outer membrane. Binding of the Bcl-2 protein to the mitochondria was not affected by pretreatment of the mitochondria with trypsin, indicating that the Bcl-2 protein does not require protease-sensitive mitochondrial surface components for its membrane insertion.

Animals↗

PubMed1993

JunD mutants with spontaneously acquired transforming potential have enhanced transactivating activity in combination with Fra-2.

Although a replication-competent retrovirus that carries junD has no transforming activity in chicken embryo fibroblasts, we have isolated mutant viruses that have spontaneously acquired transforming activity. The molecularly cloned junD genes of three such mutant viruses (T1, T2, and T3) were shown to be responsible for the cellular transformation. DNA sequence analysis indicated that a specific polynucleotide in the junD sequence was tandemly multiplied three times of five times in T1 and T2, respectively. The repeated polynucleotide encodes 16 amino acid residues that are located in a highly conserved region among Jun family proteins. The junD mutation in T3 involved an inversion, a translocation, and nucleotide substitutions that caused drastic amino acid exchanges in another well-conserved region among Jun family proteins. The transcriptional activity of these mutants was analyzed by means of transient expression experiments in F9 cells using a reporter gene containing a single AP-1 binding site. Compared with the wild-type JunD, none of them showed enhanced transactivating activity in the forms of homodimers or of heterodimers with c-Fos or Fra-1. However, they did exhibit much higher transactivating activity than the wild type when they formed heterodimers with Fra-2, indicating that the mutated regions function as transactivation domains in a partner-specific manner. Since we have previously reported that there is a basal level of Fra-2 expression in chicken embryo fibroblasts, the results may indicate that protein complexes between JunD mutants and Fra-2 play a crucial role in the cellular transforming activity.

Amino Acid Sequence↗

PubMed1993

Induction of cytochrome P450 1A2 by musk analogues and other inducing agents in rat liver.

We characterized the inducing effects of two musk analogues, musk xylene and musk ambrette, on phase I and phase II drug-metabolizing enzymes in rat liver and compared their effects with 3-methylcholanthrene, isosafrole and 2(3)-tertbutylhydroxyanisole (BHA) at 0.1 mmol/kg dose level. Musk xylene and isosafrole increased more efficiently the metabolic activation of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) to mutagen than that of benzo(a)pyrene. Musk ambrette increased both the activation of Glu-P-1 and benzo(a)pyrene to the same extent. Western blot analyses revealed that musk xylene, musk ambrette, isosafrole and BHA induced more strongly cytochrome P450 1A2 (CYP1A2) in microsomes than CYP1A1. 3-Methylcholanthrene induced CYP1A1 in preference to CYP1A2. On the other hand, all drugs except for 3-methylcholanthrene did not show remarkable increases in phase II enzyme activities, such as DT-diaphorase, glutathione S-transferase and UDP-glucuronyltransferase, at 0.1 mmol/kg dose level. These results show that musk xylene, musk ambrette, isosafrole and BHA at the dose level used in this study possess the potency to induce CYP1A2 without remarkable induction of CYP1A1 and phase II enzyme activities as observed for 3-methylcholanthrene, although they have been considered to induce both phase I and phase II drug-metabolizing enzymes at higher doses.

Animals↗

PubMed1993

Cloning of the YAP19 gene encoding a putative yeast homolog of AP19, the mammalian small chain of the clathrin-assembly proteins.

Mouse brain AP19 is the smallest polypeptide chain component of AP-1, the clathrin-assembly protein complex located at the Golgi complex. We isolated a gene, termed YAP19, potentially encoding a homolog of AP19 from the genomic DNA of the yeast Saccharomyces cerevisiae. The deduced amino acid sequence, 156 amino acids long, shows 53% identity with the mouse brain AP19 protein, but 37% with Yap17p, a yeast homolog of the mammalian AP17 in AP-2, another clathrin-assembly protein complex located at the plasma membrane. The identification of YAP19 supports the proposal that yeast cells also contain the Golgi-associated clathrin-assembly protein complex.

Adaptor Protein Complex 1↗

PubMed1993

The structure of N-linked oligosaccharides of human pancreatic bile-salt-dependent lipase.

This study describes the structure of the N-linked oligosaccharide chains of bile-salt-dependent lipase isolated from the pancreatic juice of a normal donor. After hydrazinolysis, neutral sugar chains were separated from acidic chains by paper electrophoresis and were fractionated using serial column chromatography with immobilized lectins and Bio-Gel P-4 filtration. Structural analysis was performed by means of sequential glycosidase digestion and revealed that the neutral sugar chains are mainly of the biantennary complex type. Fucose residues were identified for some trimannosyl core structures and were alpha(1-6) or alpha(1-2) linked to the innermost GlcNAc residue and a terminal Gal residue, respectively. Sialyl residues were also involved in the oligosaccharide structures. Most of these structures have no linear N-acetyllactosamine repeats. Evidence from several approaches suggests that the sugar chains of the human pancreatic bile-salt-dependent lipase possess a blood-group-related antigenic determinant.

Bile Acids and Salts↗

PubMed1993

Inositol 1,4,5-trisphosphate 3-kinase highest levels in the dendritic spines of cerebellar Purkinje cells and hippocampal CA1 pyramidal cells. A pre- and post-embedding immunoelectron microscopic study.

Inositol 1,4,5-trisphosphate 3-kinase (InsP3 3-kinase) plays a crucial role in calcium homeostasis by regulating InsP3 levels. We have reported the highest concentrations of InsP3 3-kinase in the dendrites of cerebellar Purkinje cells and hippocampal pyramidal cells of the CA1 sector of the Ammon's horn. We here investigate its subcellular localization by pre- and post-embedding immunoelectron microscopic study. In both populations of neurons, the major structure expressing a high level of InsP3 3-kinase is the dendritic spines.

Animals↗

PubMed1993

A novel domain sequence of connectin localized at the I band of skeletal muscle sarcomeres: homology to neurofilament subunits.

A cDNA of 4.0 kb was cloned from a chicken embryo skeletal muscle cDNA library, using an antibody to muscle elastic protein connectin (titin), the molecular mass of which is estimated to be 3,000 kDa. Immunoelectron microscopy revealed that the antiserum raised against the product of the cDNA expressed in E. coli bound to the epitopes of the connectin filament near the N2 line of chicken breast muscle sarcomeres. The predicted amino acid sequence contains eight immunoglobulin C2 motifs and regions highly homologous with the high and medium molecular weight subunits of neurofilament. In addition, there are regions homologous with desmoplakin, calsequestrin, and calpastatin.

Amino Acid Sequence↗

PubMed1993

Establishment and characterization of an erythropoietin-dependent subline, UT-7/Epo, derived from human leukemia cell line, UT-7.

UT-7 is a human leukemic cell line capable of growing in interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo) (Komatsu et al, Cancer Res 51:341, 1991). To study the effect of Epo on proliferation and differentiation of UT-7, we maintained the UT-7 cell culture for more than 6 months in the presence of Epo. As a result, a subline, UT-7/Epo, was established. The growth of UT-7/Epo could be supported by Epo but not by GM-CSF or IL-3. UT-7/Epo showed a greater level of heme content and ratio of benzidine-positive staining cells than did UT-7. Butyric acid promoted the synthesis of hemoglobin in UT-7/Epo, but not UT-7. Further, the mRNA concentrations of the c-myb oncogene and GM-CSF receptor beta-subunit were decreased substantially in UT-7/Epo cells. These findings showed that UT-7/Epo cells had progressed further in erythroid development than UT-7 cells, and suggested that long-term culture in Epo had promoted this differentiation. Whereas availability of the Epo receptor (Epo-R) for binding of Epo was reduced in UT-7/Epo cells compared with UT-7 cells, the Epo-R showed a similar affinity for Epo. This observation suggested that change(s) in postreceptor signaling step might be involved in the establishment and maintenance of the UT-7/Epo phenotype.

Butyrates↗

PubMed1993

Detection of glycosylation abnormality in rheumatoid IgG using N-acetylglucosamine-specific Psathyrella velutina lectin.

Although the galactose deficiency in the Asn297-linked sugar chains of serum IgG from patients with rheumatoid arthritis (RA) has been established, structural analysis of sugar chains has not been readily available. Psathyrella velutina lectin (PVL) preferentially interacts with the N-acetylglucosamine beta 1-->2Man group, exposed at the termini of sugar chains in agalacto IgG. Biotinylated PVL reacted strongly in Western blotting with H chains of IgG derived from patients with RA. An ELISA-based assay for the detection of agalacto IgG was developed using recombinant protein G and biotinylated PVL in combination, and the screening of patients' sera was performed. PVL binding of serum IgG significantly correlated with percentage of galactose-deficient IgG determined by the structural analysis. Age-related slight increase in PVL binding was observed among normal controls. Patients with RA showed significantly higher PVL binding (37.90 +/- 42.25 U/ml, n = 93) as compared with normal controls (5.75 +/- 2.92 U/ml, n = 112) (p = 0.0001). Patients with SLE showed lower but still significant PVL binding (17.86 +/- 5.18 U/ml, n = 10, p = 0.0001). PVL binding correlated with C-reactive protein level in serial analysis of individual RA patients, and was significantly higher in the synovial fluid compared with paired serum samples. PVL binding assay may provide an ideal tool for the simple and sensitive detection of agalacto IgG.

Acetylglucosamine↗

PubMed1993

Sec-Y protein is localized in both the cytoplasmic and thylakoid membranes in the cyanobacterium Synechococcus PCC7942.

Members of the SecY protein family mediate protein export in bacterial cells. Southern analyses showed that secY is likely a single copy gene in the cyanobacterium Synechococcus PCC7942. Then the subcellular location of the cyanobacterial SecY protein was determined; i) antiserum raised against a fusion protein between the SecY fragment and maltose binding protein were used for immunoblotting of the membrane fractions, and ii) a modified SecY protein carrying the c-Myc peptide tag was expressed in the cyanobacterial cells, and the subcellular distribution of the SecY-c-Myc fusion protein was analyzed with the anti-c-Myc antibodies. The obtained results suggest that the SecY protein is localized in the thylakoid membrane as well as the cytoplasmic membrane; the SecY protein probably mediates protein translocation across both the cytoplasmic and thylakoid membranes in Synechococcus PCC7942.

Amino Acid Sequence↗

PubMed1993

Structural changes in the N-linked sugar chains of serum immunoglobulin G of HTLV-I transgenic mice.

IgGs were purified from the sera of HTLV-I transgenic and nontransgenic mice. Comparative studies of the N-linked sugar chains released by hydrazinolysis revealed that their structures of transgenic IgG are quite different from those of nontransgenic IgG. Although both IgGs contained biantennary complex-type oligosaccharides, transgenic IgG had more agalactosylated forms (45%) than those from nontransgenic IgG (28%), just as was found in patients with rheumatoid arthritis (RA). Since these transgenic mice express arthritis similar to RA, it will be a useful model to investigate the relationship between the galactosylation of IgG and the development of RA.

Animals↗

PubMed1993

Isolation and influenza virus receptor activity of glycophorins B, C and D from human erythrocyte membranes.

(1) Glycophorins (GPs) AM, AN, B, C and D were each isolated into a high state of purity from human erythrocyte membranes by a combination of lithium diiodosalicylate (LIS)-phenol extraction, gel-filtration with Bio-Gel A1.5m and HPLC with LiChrospher 1000 TMAE. (2) GPs-B, -C and -D reacted with influenza A and B viruses as well as GPs-AM and -AN and the order of reactivities against two viruses of the glycophorins was as follows: GP-B > GP-C > GP-AM = GP-AN >> GP-D for the former virus and GP-C > GP-B > GP-AM = GP-AN >> GP-D for the latter virus.

Amino Acids↗

PubMed1993

Interindividual variability of carbonyl reductase levels in human livers.

Interindividual variability of carbonyl reductase levels in human livers (N = 11) was examined by measuring reductase activity toward various substrates and by western blot analysis using anti-rat ovarian carbonyl reductase CR2 antibody. The carbonyl reductase activity toward p-nitrobenzaldehyde (PNBA) (58.1 +/- 5.4 nmol/mg protein/min, mean +/- SE) was highest among the substrates examined, followed by 4-benzoylpyridine (4BP) (14.4 +/- 2.0 nmol/mg protein/min) and p-nitroacetophenone (PNAP) (2.00 +/- 0.37 nmol/mg protein/min). The reductase activity (6.33 +/- 0.56 pmol/mg protein/min) toward 13,14-dihydro-15-keto-prostaglandin F2 alpha (15KD-PGF2 alpha), which is a diagnostic substrate for rat ovarian carbonyl reductases, was relatively high compared to that in other species. Western blot analysis revealed that each human liver contained several immunoreactive proteins to anti-CR2 antibody. The activities toward 15KD-PGF2 alpha (r = 0.85, P < 0.01) and 4BP (r = 0.84, P < 0.01), but not PNBA (r = 0.53, not significant) or PNAP (r = 0.52, not significant), were closely correlated with the relative amounts of the high molecular weight immunoreactive proteins determined with a densitometer. Thus, the major carbonyl reductases in human liver are similar to those of rat ovarian enzymes.

Acetophenones↗

PubMed1993

Ascidian entactin/nidogen. Implication of evolution by shuffling two kinds of cysteine-rich motifs.

Entactin/nidogen, a major component of the basement membrane, has a domain structure comprising three globular domains, and thread-like and rod-like domains connecting them. It contains six epidermal-growth-factor-(EGF)-like motifs and one thyroglobulin-like motif. In the present study, ascidian entactin/nidogen has been identified by a monoclonal antibody technique. We prepared anti-(ascidian entactin/nidogen)IgG, named anti-AsEnt1, then cloned the cDNA of ascidian entactin/nidogen using anti-AsEnt1 as a probe, and determined its entire sequence. Mainly because the deduced amino acid sequence exhibited high similarity to mouse entactin and human nidogen, and because the antigen localized in basement membrane of ascidian body-wall muscle, we have concluded that the antigen anti-AsEnt1 corresponds to the ascidian entactin/nidogen homologue. The deduced amino acid sequence of ascidian entactin/nidogen clearly showed that the ascidian homologue also has a domain structure. However, the ascidian homologue lacked the thread-like domain, and the rod-like domain differed from that of mouse entactin in composition, consisting of two kinds of cysteine-rich motifs, that is, the EGF-like motif and the thyroglobulin-like motif. These results suggest that entactin/nidogen have evolved by modifying the domains, especially by shuffling the two kinds of cysteine-rich motifs.

Amino Acid Sequence↗

PubMed1993

Thyrotropin receptor in non-thyroid tissues.

Thyrotropin receptor (TSH-R), the main target for the autoantibody in Graves' disease, has been thought to be a thyroid-specific protein. However, we successfully obtained the cDNA fragments of TSH-R from rat retro-orbital tissues and adipose tissues by using polymerase chain reaction methods. Sequencing has revealed that the nucleotide sequence of the cDNAs from these non-thyroid tissues was identical to that from the thyroid. TSH-R peptide antibody, which recognizes rat TSH-R, stained a 104 kDa protein from the retro-orbital tissues and the adipose tissues. The band was not detected with the antibody preabsorbed with the peptide. These results suggest that the message is translated to make a TSH-R protein even in these non-thyroid tissues.

Animals↗

PubMed1993

Role of sugar chains in the in-vitro activity of recombinant human interleukin 5.

The structures of O-linked sugar chains of recombinant human interleukin 5 (rhIL-5) produced by Chinese hamster ovary cells were determined employing high-performance anion-exchange column chromatography under high pH conditions. The core sequence was elucidated to be Gal beta 1-->3-GalNAc by its susceptibility to endo-alpha-N-acetylgalactosaminidase and sialic acid linkages were determined using sialidases of different specificities. To investigate the role of sugar chains in the in-vitro activity of rhIL-5, it was digested with various glycosidases. While removal of N-linked sugar chains resulted in 2.8-fold increase of the activity, de-O-glycosylated rhIL-5 showed 10-fold higher activity than intact rhIL-5, suggesting that the presence of O-linked sugar chains suppresses the activity more effectively than that of N-linked chains. While incubation of de-N-glycosylated rhIL-5 at 70 degrees C for 30 min decreased the activity, intact and de-O-glycosylated rhIL-5 lost little activity, suggesting that N-linked sugar chains contributed to the thermostability of the molecule.

Borohydrides↗

PubMed1993

Decreased expression of DCC mRNA in human colorectal cancers.

The mRNA expression levels of DCC gene, which is cloned from the deleted region of chromosome 18q in colorectal cancers and thought to be a tumor-suppressor gene, was evaluated in tissue specimens surgically resected from patients with colorectal cancer by RT-PCR. This method was chosen as the expression level of DCC mRNA is below the detectable level for Northern-blot analysis. Semi-quantitative measurements of DCC mRNA were performed based on a standard curve defined by serial dilution of DCC cDNA. As a result, the expression level of DCC mRNA was found to be lower in 17 out of 30 colorectal cancers than in adjacent non-cancerous tissues. Inclusion of smooth muscle in tissue specimens was observed to have little disturbing effect on comparisons between cancerous and non-cancerous regions. In addition, all 4 specimens of colorectal cancer with liver metastasis showed the decreased expression level of DCC mRNA, suggesting that functional loss of DCC in cancerous tissues may play an important role in metastatic events.

Actins↗

PubMed1993

Purification and characterization of an alpha-L-fucosidase from Pomacea canaliculata.

An alpha-L-fucosidase (EC 3.2.1.51) was isolated from the hepatopancreas of Pomacea canaliculata. The enzyme was purified 285-fold from the crude enzyme extract by procedures involving first heat treatment, ammonium sulfate fractionation, second heat treatment, and chromatography on DEAE-Sepharose, hydroxylapatite, and L-fucosylamine-CH-Sepharose. When assayed by using p-nitrophenyl glycosides as substrates, the final preparation was free from other glycosidase activities and gave a single protein band which corresponded to alpha-L-fucosidase activity on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be 260,000 by Sephacryl S-300 column chromatography. The enzyme has two optimum pH values, 2.5 and 5.0, and the apparent Km value and the maximum velocity for p-nitrophenyl alpha-L-fucoside at both pH were calculated to be 0.45 mM and 1.46 mumol/min/mg of protein, respectively. The enzyme was shown to hydrolyze the Fuc alpha 1-->2Gal, the Fuc alpha 1-->4GlcNAc, and the Fuc alpha 1-->6GlcNAc linkages, but hardly acts on the Fuc alpha 1-->3GlcNAc linkage in various oligosaccharides.

Ammonium Sulfate↗