[Roles of phospholipids in protein translocation across membranes].
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Cell Membrane Permeability↗
Biomedical subjects
T Endo
Publications and source records attributed to T Endo.
[Sick sinus syndrome--indication of pacemaker implantation].
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Atropine↗
[Regulation of muscle cell differentiation by oncogenes and growth suppressor genes].
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Animals↗
[Non-vascular interventional radiology: percutaneous needle biopsy under imaging].
Recent advances in diagnostic imaging increase the opportunity to detect small lesions. Percutaneous needle biopsy (PNB) under imaging is a part of interventional radiology and very useful to diagnose such small lesions. PNB of lung tumors is performed by 21G-screw needle under CT imaging of fluoroscopy. In 78 cases, accuracy was about 80% and no major complications were observed except one (bleeding). PNB of the liver can be performed by 15-17G cutting type needle. Bleeding is easily avoided by using the trans-normal liver tissue route. PNB of the pancreas and percutaneous pancreatic ductography also can be done using trans-hepatic or trans-gastric route under US imaging without major complications. PNB by 21G needle can obtain a block material for pathological diagnosis. There is much gas in the intrapelvic space, so PNB to this area is usually performed under CT imaging. Image-guiding PNB to subcutaneous organs like thyroid gland and breast is more useful than so-called blind biopsy because of its exactitude. Calcified non-palpable breast cancer is demonstrated by only this method. Image-guiding PNB is non-invasive, exact and a safe method to diagnose small lesions, and the popularization of the method is required.
Biopsy, Needle↗
Novel methods to determine the epitopes on the asparagine-linked oligosaccharides of glycoproteins.
Oligosaccharides obtained from glycoproteins by hydrazinolysis followed by N-acetylation were covalently coupled to an amino-bonded thin-layer plate or to a poly-L-lysine coating on the microtiter plate. Bound oligosaccharides can be directly detected by immunostaining or by enzyme-linked immunosorbent assay (ELISA) using a mouse monoclonal antibody. These methods are simple and require small amounts of oligosaccharides and antibodies. The methods were successfully applied to determine the epitope of F48-60, a monoclonal antibody which reacts with the N-linked sugar chains of nonspecific cross-reacting antigen 2, but not with those of carcinoembryonic antigen. The results revealed that the antibody recognizes the Gal beta 1----3GlcNAc beta 1----group.
Animals↗
Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains.
Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.
Antigen-Antibody Complex↗
Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein.
Prion proteins from humans and rodents contain two consensus sites for asparagine-linked glycosylation near their C-termini. The asparagine-linked oligosaccharides of the scrapie isoform of the hamster prion protein (PrP 27-30) were released quantitatively from the purified molecule by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The radioactive oligosaccharides were fractionated into one neutral and three acidic oligosaccharide fractions by anion-exchange column chromatography. All oligosaccharides in the acidic fractions could be converted to neutral oligosaccharides by sialidase digestion. Structural studies on these oligosaccharides including sequential exoglycosidase digestion in combination with methylation analysis revealed that PrP 27-30 contains a mixture of bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6(GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4-(Fuc alpha 1----6)GlcNAc as their core. Variation is produced by the different combination of the oligosaccharides Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, GlcNAc beta 1----, Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Sia alpha 2----6Gal beta 1----4GlcNAc beta 1---- in their outer chain moieties. When both asparagine-linked consensus sites are glycosylated, the diversity of oligosaccharide structures yields over 400 different forms of the scrapie prion protein. Whether these diverse asparagine-linked oligosaccharides participate in scrapie prion infectivity or modify the function of the cellular prion protein remains to be established.
Acetylation↗
Conformational changes of a mitochondrial precursor protein on binding to phospholipid vesicles and SDS micelles. A circular dichroism and fluorescence spectroscopy study.
Conformations of an artificial mitochondrial precursor protein pCox IV-DHFR have been analyzed by CD and fluorescence spectroscopy in the presence of (cardiolipin-rich) phospholipid vesicles or SDS micelles. Binding of pCox IV-DHFR to phospholipid vesicles involves a conformational change, which is presequence-dependent, accompanies alteration in the secondary structure of the DHFR moiety, but is different from total unfolding of the polypeptide chain. On the other hand, a conformational change of the fusion protein on binding to the micelles of a positively charged detergent, SDS, is not presequence-dependent.
Circular Dichroism↗
Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma.
The sugar chains of transferrin samples, purified from sera of patients with hepatocellular carcinoma and of healthy individuals, were released quantitatively as radioactive oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Comparative study of their structure by serial lectin column chromatography, by Bio-Gel P-4 column chromatography, and by sequential exoglycosidase digestion revealed that prominently altered glycosylation is commonly found in the hepatoma transferrins, although they all contain two complex-type asparagine-linked sugar chains in one molecule like in the case of normal transferrins. The alteration is quite various, including the increase of highly branched sugar chains, of those with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----groups in their outer chain moieties and of those with a fucosylated trimannosyl core. Many but not all of the hepatoma transferrin samples contained a small amount of a bisected biantennary sugar chain, which was not detected in the normal transferrin samples.
Biomarkers, Tumor↗
Binding of a tightly folded artificial mitochondrial precursor protein to the mitochondrial outer membrane involves a lipid-mediated conformational change.
An artificial mitochondrial precursor protein (the presequence of cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase) binds to isolated yeast mitochondrial outer membranes and to liposomes whose phospholipid composition resembles that of outer membranes. In both cases, binding is strongly inhibited by low temperature or methotrexate (which stabilizes the dihydrofolate reductase moiety) and partly inhibited by adriamycin (which binds to acidic phospholipids). Binding is accompanied by partial unfolding of the protein. Binding of the urea-denatured fusion protein to outer membranes or liposomes is insensitive to low temperature, methotrexate, or adriamycin. These results, and those reported in the accompanying paper (Eilers, M., Endo, T., and Schatz, G. (1989) J. Biol. Chem. 264, 2945-2950) suggest that import of this fusion protein into isolated mitochondria involves at least partial unfolding by acidic phospholipids on the mitochondrial surface.
Doxorubicin↗
Adriamycin, a drug interacting with acidic phospholipids, blocks import of precursor proteins by isolated yeast mitochondria.
Acidic phospholipids such as cardiolipin partially unfold an artificial precursor protein which consists of a mitochondrial presequence fused to mouse dihydrofolate reductase (Endo, T., and Schatz, G. (1988) EMBO J. 7, 1153-1158). We now show that import of this precursor protein into isolated yeast mitochondria is blocked by adriamycin, a drug binding to cardiolipin and other acidic phospholipids. This inhibition is lessened if the precursor's dihydrofolate reductase moiety is labilized by point mutations; inhibition is abolished altogether if the "wild-type" precursor is presented to mitochondria in a urea-denatured state. These and other observations suggest that adriamycin interferes with the generation of a translocation-competent, loose structure of the precursor protein. They imply that acidic phospholipids such as cardiolipin participate, directly or indirectly, in the translocation of this fusion protein into isolated mitochondria.
Doxorubicin↗
Nitrile hydratase of Rhodococcus sp. N-774. Purification and amino acid sequences.
The nitrile hydratase of Rhodococcus sp. N-774 was purified and crystallized. The enzyme is composed of two different subunits (molecular masses: subunit alpha, 28,500 Da; subunit beta, 29,000 Da). The amino-terminal amino acid sequence of each subunit was determined. There is no sequence homology between the two subunits, suggesting that the peptides originate from different cistrons. The activity of the purified enzyme did not decrease during incubation in the dark, whereas it gradually decreased in intact cells.
Amino Acid Sequence↗
Function and pathology of the sugar chains of human immunoglobulin G.
Human immunoglobulin G (IgG) is unique among serum glycoproteins because it contains more than 30 different biantennary complex-type asparagine-linked oligosaccharides. This extremely high microheterogeneity is probably produced because human individuals have a series of B cell clones equipped with different sets of glycosyltransferases. Despite this complex composition, IgG samples purified from whole human sera have the same mole ratios of oligosaccharides, indicating that the ratio of B cell clones synthesizing IgGs with different sugar chains is constant in healthy individuals. We found that the glycosylation patterns of whole serum IgGs obtained from patients with rheumatoid arthritis (RA) are quite different from those of whole serum from healthy individuals. Structural studies of the oligosaccharides revealed that the sugar chains of the IgGs obtained from patients with RA are depleted of the beta-galactose residues. The sugar chains of transferrin from patients with RA are fully galactosylated. Therefore the galactose deletion from IgG is probably brought about by a decrease in galactosyltransferase activity in B cells rather than by degradation by galactosidase during circulation. Enzymic study revealed that human B cells contain various beta-galactosyltransferases which form the Gal(beta 1-4)GlcNAc groups in the sugar chains of different glycoproteins. Among these enzymes, abnormality in patients with RA was found only in the one that transfers beta-galactose residues specifically to degalactosylated IgG. This enzyme showed lower affinity toward UDP-Gal in B cells of patients with RA than that in healthy individuals.
Arthritis, Rheumatoid↗
Identification of cell types containing S-100b protein-like immunoreactivity in the islets of Langerhans of the guinea pig pancreas with light and electron microscopy.
Cell types containing S-100b protein-like immunoreactivity in the islets of Langerhans of the guinea pig were studied by light- and electron-microscopic immunocytochemistry using antisera to S-100b protein, insulin, glicentin, somatostatin, and pancreatic polypeptide. Two types of S-100b-immunoreactive cells were identified. The first type was stellate and characterized by thin cytoplasmic processes sheathing endocrine-type cells, especially pancreatic A-cells. It was located predominantly in the neuro-insular complex and in large islets, both of which were located near the main pancreatic duct. Intense immunoreactivity was found in the cytoplasmic matrix as well as in the nucleoplasm. Nerve fibers or endings were occasionally ensheathed by its cytoplasmic processes. The second type, whose immunoreactivity was rather weak and varied from one cell to another, was oval to polygonal in shape and located randomly throughout the islets. It was an endocrine cell-type and its immunoreactivity was located in the secretory granule. With the use of immunostained consecutive sections for demonstrating pancreatic endocrine cell-types, it was found that a portion of the pancreatic B-cell population expressed S-100b-like immunoreactivity.
Animals↗
A proton nuclear magnetic resonance study on the solution structure of crotamine.
Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. The pH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidic pH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for Ile-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution.
Amino Acid Sequence↗
Step-wise thermal denaturation of cobrotoxin, a snake venom neurotoxin from Naja naja atra: a proton nuclear magnetic resonance study.
Temperature dependence of proton nuclear magnetic resonance spectra has been followed for cobrotoxin, a postsynaptic neurotoxin from Naja naja atra venom. Several aromatic amino-acid residues, including the functionally essential Trp-29 located at the tip of the central loop of the molecule, have been found to undergo a thermal structural transition above the global thermal denaturation temperature. It is suggested that a local structure around these residues behaves somehow independently of the rest of the molecule, and that such structural organization may be favorable for a conformational change of a neurotoxin molecule on binding to acetylcholine receptor.
Cobra Neurotoxin Proteins↗
State of functionally essential Trp-29 in snake venom neurotoxins: a proton nuclear magnetic resonance study.
Proton nuclear magnetic resonance (NMR) spectra have been recorded of various neurotoxins from snake venoms. pH dependence of the chemical shifts and resonance intensity has been followed for the functionally essential Trp-29. The indole N-1 proton of Trp-29 in alpha-bungarotoxin, toxin B, and cobrotoxin exhibits appreciably large upfield shifts as the pH is lowered and the suppressed exchange with the solvent hydrogen at pH 3-4, but not in Naja haje annulifera 10 where Asp-31 is replaced with Gly-31. This observation strongly suggests the presence of a hydrogen bond between Trp-29 and Asp-31 that is probably important in stabilizing the arrangement of the functionally essential residues to form a distinct binding region for the receptor.
Elapid Venoms↗
Structural study of the carbohydrate moieties of two human immunoglobulin subclasses (IgG2 and IgG4).
Asparagine-linked sugar chains were quantitatively released as oligosaccharides from human IgG2 and IgG4 myeloma proteins by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Each oligosaccharide was isolated by serial lectin column chromatography. Study of their structures by sequential exoglycosidase digestion and methylation analysis, revealed that all of them were of the bi-antennary complex-type containing Man alpha 1-6(+/- GlcNAc beta 1-4)(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAc as core structures, and GlcNAc beta 1-, Gal beta 1-4GlcNAc beta 1- and Sia alpha 2-6Gal beta 1- in their outer chain moieties. However, the molar ratio of each oligosaccharide was different in each IgG sample, indicating that clonal variation is included in the sugar chain moieties of IgG molecules. One of the IgG2 contained four asparagine-linked sugar chains in one molecule, two on the Fc fragment and the remainder on the Fab fragment. The sugar chains in the Fc fragment contained much less galactose as compared with the Fab fragment.
Asparagine↗