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Biomedical subjects

M Fukuda

Publications and source records attributed to M Fukuda.

At least 775 records · Page 43Linked to original sources

Primate genes for glycophorins carrying MN blood group antigens.

Glycophorin A, B, and E genes were derived from a common ancestral gene and this gene family appeared during primate evolution, probably between orangutan and gorilla divergences. Based on the study of genomic structures of these human glycophorins and the genetic and immunological study of primate glycophorins, we hypothesize that chimpanzee and gorilla glycophorin B could possess a longer extracellular region and carry a stronger N blood group antigenicity compared with that of the human.

Animals↗

[A prospective study on necessary and sufficient retinal photocoagulation for diabetic retinopathy].

The present studies were performed to determine whether slight photocoagulation was better than heavy photocoagulation for the early stage of progressive proliferative diabetic retinopathy (PDR). The authors selected 17 patients who had bilateral PDR of equal severity (BI or BII stage according to Fukuda's classification). In every case, we randomly performed heavy photocoagulation (laser burns were 1142 +/- 179) to one eye and slight photocoagulation (405 +/- 166) to the other eye. More than 6 months after the last photocoagulation, the effects of these treatments were compared in both eyes by fundus pictures, visual acuity and posterior vitreous fluorophotometric values. The results of judgement by fundus pictures and by vitreous fluorophotometric values were in perfect agreement. Eight cases (47%) in whom eyes received slight photocoagulation showed result better than the other eye. Two cases (12%) which received heavy photocoagulation were better than the other eye. Seven cases (41%) showed the same level of severity. No significant differences were found between slight and heavy photocoagulation.

Adult↗

[Dumbbell cervical neurinomas with subcutaneous extension: report of two cases].

Two cases of dumbbell cervical neurinomas with massive subcutaneous extension were reported. The first case was A 30-year-old woman who was admitted to our hospital because she had been aware of a left lateral cervical subcutaneous mass and was suffering from shoulder dullness. On admission, neurological examination revealed hypesthesia to touch and pain in the segmental area of C4, and hyperreflexia in the left biceps and patellar tendon reflexes. Plain X-ray showed enlargement of the left C3/4 intervertebral foramen. CT scan, post-myelogram CT and MRI demonstrated a dumbbell shaped tumor at the level of C3-4. Angiogram showed an anterior shift of the left vertebral artery (VA) and tumor stain. Temporary occlusion of the left VA by a balloon catheter was performed leaving no neurological deficits. The second case was a 36-year-old woman who had been aware of a left lateral cervical subcutaneous mass. She complained of shoulder pain and finger clumsiness. On admission, neurological examination revealed weakness of the left deltoid muscle, hypesthesia in the segmental area of C3-4 and exaggeration of all deep tendon reflexes in the left-side extremities. Plain X-ray showed enlargement of the C2/3, C3/4 and C4/5 intervertebral foramina. CT scan, post myelogram CT and MRI demonstrated a dumbbell shaped tumor at the level of C2-5. Angiogram showed an anteromedial shift of the left VA and tumor stain. Temporary occlusion test of the left VA by a balloon catheter was performed with negative results. In each case two-stage operations were undertaken with excellent results.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Poly-N-acetyllactosaminyl O-glycans attached to leukosialin. The presence of sialyl Le(x) structures in O-glycans.

Poly-N-acetyllactosamine extension has been found in O-glycans in addition to N-glycans and glycosphingolipids. Attempts were made in HL-60 and K562 cells to determine the amount of poly-N-acetyllactosaminyl O-glycans in the major sialoglycoprotein, leukosialin. Leukosialin was immunoprecipitated from [3H]glucosamine-labeled HL-60 and K562 cells. Glycopeptides were prepared by Pronase digestion, and O-glycan-containing glycopeptides were isolated by affinity chromatography using Jacalin-agarose. The glycopeptides bound to Jacalin-agarose and those unbound were treated with alkaline borohydride, and the released O-glycans were fractionated by Bio-Gel P-4 filtration. Sequential glycosidase digestion of the O-glycans, with or without pretreatment by fucosidase or neuraminidase, revealed the following conclusions. 1) Leukosialin from HL-60 cells contains about 1-2 poly-N-acetyllactosaminyl O-glycan chains/molecule. 2) About 50% of these poly-N-acetyllactosaminyl O-glycans contain sialyl Le(x) termini, NeuNAc alpha 2-->3Gal beta 1-->4 (Fuc alpha 1-->3)GlcNAc beta 1-->R. The amount of sialyl Le(x) structure in leukosialin is roughly equivalent to that on cell surfaces of HL-60 cells. 3) Leukosialin from K562 cells, on the other hand, contains no detectable amount of poly-N-acetyllactosaminyl O-glycans. 4) The presence of poly-N-acetyllactosamine in O-glycans is dependent on the core 2 beta 1,6-N-acetylglucosaminyl transferase. 5) Jacalin-agarose binds to sialylated small oligosaccharides such as NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->6) GalNAc but not the hexasaccharide NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->6) GalNAc. These results indicate that the formation of polylactosaminyl O-glycans and sialyl Le(x) structure in O-glycans is dependent on the core 2 formation.

Antigens, CD↗

O-linked oligosaccharides of glycophorins A and B in erythrocytes of two individuals with the Tn polyagglutinability syndrome.

Tn polyagglutinability syndrome is an acquired condition where erythrocytes express Tn neo-antigen and become susceptible to hemagglutination by the naturally occurring anti-Tn present in normal sera. Early studies had indicated that O-linked N-acetyl galactosamine was the sole serologic Tn determinant, but more recently O-linked NeuNAc alpha 2, 6GalNAc also has been implicated as a Tn antigen (sialosyl-Tn). However, none of these studies were performed on purified glycoproteins. In this report we examine oligosaccharides of glycophorins A and B purified from Tn erythrocytes of two affected individuals to establish how N- and O-linked saccharides differ from normal. Analysis of carbohydrate composition and treatment with N-glycanase showed that the Asn-linked unit of glycophorin A was not affected. O-linked oligosaccharides were obtained by beta-elimination in the presence of tritiated sodium borohydride. The reduced radiolabeled products were fractionated by Bio-Gel P-2 chromatography, and their structures were investigated by comparison with standards, by monosaccharide quantification, and by neuraminidases of known specificities. The results show that Tn glycophorins from both donors contain intact and truncated forms of trisaccharide and tetrasaccharide NeuNAc alpha 2,3Gal beta 1,3GalNAc and NeuNAc alpha 2,3Gal beta 1,3- (NeuNAc alpha 2,6)GalNAc usually present in glycophorins A and B. The truncated forms include the protein O-linked monosaccharide, GalNAc and disaccharide, NeuNAc alpha 2,6GalNAc (major isomer). The presence of intact glycans in the total population of Tn erythrocytes was confirmed by their susceptibility to T activation after treatment with neuraminidase. The proportion of the four species was not identical in glycophorins of these two donors but, in both, the truncated units predominated and the amount of the disaccharide was approximately one half of that of the monosaccharide. The data are consistent with alterations in UDPGal:GalNAc beta 1,3galactosyl transferase that may have multiple molecular origins and with induction of a specific GalNAc protein alpha 2,6 sialosyl transferase in Tn hematopoietic precursor cells. The molecular basis for these alterations awaits further study.

Antibodies, Monoclonal↗

Activation of mitogen-activated protein kinase and its activator by ras in intact cells and in a cell-free system.

Mitogen-activated protein (MAP) kinase is a serine/threonine kinase whose function is thought to be essential for the transduction of mitogenic signals. MAP kinase is activated by phosphorylation induced by a variety of extracellular stimuli, and its direct upstream activator has been identified. Using amphibian and mammalian systems, we show here that ras can activate MAP kinase and its activator. Injection of v-Ha-ras p21 into Xenopus immature oocytes activated both MAP kinase and maturation-promoting factor (MPF) activities. The activation of MAP kinase preceded that of MPF, demonstrating that ras activates MAP kinase in an MPF-independent pathway. Moreover, we found that the MAP kinase activator is also activated in ras-injected oocytes. Activation of MAP kinase and its activator occurred also when the v-Ki-ras gene was conditionally induced in rat fibroblastic 3Y1 cells. Furthermore, we observed that ras activated MAP kinase and its activator in a cell-free system prepared from Xenopus oocytes. Using an antibody against the Xenopus 45-kDa MAP kinase activator, we demonstrated that the 45-kDa activator molecule was activated by ras. These findings suggest that the MAP kinase activator/MAP kinase system may be the downstream components of ras signal transduction pathways.

Animals↗

Expression cloning of a cDNA encoding UDP-GlcNAc:Gal beta 1-3-GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen.

A cDNA encoding UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase (EC 2.4.1.102), which forms critical branches in O-glycans, has been isolated by an expression cloning approach using Chinese hamster ovary (CHO) cells. Increased activity of this enzyme and the concomitant occurrence of the O-glycan core 2 structure [Gal beta 1-3(GlcNAc beta 1-6)GalNAc] has been observed in a variety of biological processes, such as T-cell activation and immunodeficiency due to the Wiskott-Aldrich syndrome and AIDS. Since CHO cells do not express this enzyme, CHO cell lines were established to stably express polyoma large tumor (T) antigen, which enables transient expression cloning. Because the antibody used was found to detect most efficiently the oligosaccharide products attached to leukosialin, the CHO cells were also stably transfected with leukosialin cDNA. By using this particular CHO cell line, a cDNA that encodes a protein determining the formation of the core 2 structure was isolated from an HL-60 cDNA library. The cDNA sequence predicts a protein with type II membrane topology, as has been found for all other mammalian glycosyltransferases cloned to date. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate unequivocally that the cDNA encodes the core 2 beta-1,6-N-acetylglucosaminyltransferase, the enzyme responsible for the formation of Gal beta 1-3(GlcNAc beta 1-6)GalNAc structures. No activity with this enzyme was detected toward the acceptors for other beta 1-6GlcNAc transferases.

Amino Acid Sequence↗

The lysosomal membrane glycoprotein lamp-1 is transported to lysosomes by two alternative pathways.

It has been demonstrated that lysosomal membrane proteins are directed to lysosomes by a tyrosine-containing structural motif in their cytoplasmic tails. It is presently unclear whether lysosomal membrane proteins are directly transported to lysosomes or first taken to the plasma membrane and then directed to the lysosomes via the endocytic pathway. In the present study, the transport pathways taken by one of the members of highly glycosylated lysosomal membrane proteins, lamp-1, were examined in human HL-60 cells. Pulse-chase labeling, combined with cell surface biotinylation and Percoll density gradient fractionation, was used to measure the kinetics of transport to the cell surface and lysosomes. The results show that the majority of lamp-1 is directly transported to lysosomes by a fast pathway (half-time 60 min), which involves sorting at an intracellular site, presumably in the trans-Golgi network. A minor part of lamp-1 is transported out to the cell surface, where it is internalized and eventually delivered to lysosomes. Transport by this pathway requires a long transit time (half-time greater than 2 h). After granulocytic differentiation of HL-60 cells by dimethyl sulfoxide, the synthesis of lamp-1 was increased approximately twofold. In these cells, the sorting in the Golgi apparatus is more effective, leaving only a minute fraction of lamp-1 for the bulk flow to the cell surface. This study establishes that the majority of lamp-1 is directly transported to lysosomes and that, in certain cells, the minority of the molecules is transported to lysosomes via the cell surface.

Antigens, CD↗

Differential glycosylation and cell surface expression of lysosomal membrane glycoproteins in sublines of a human colon cancer exhibiting distinct metastatic potentials.

Changes in the glycosylation of asparagine-linked oligosaccharides have been shown in various tumor cells, including human colon cancer. Attempts were made to elucidate the difference in Asn-linked oligo-saccharides attached to lysosomal membrane glycoproteins isolated from sublines of human colon carcinoma exhibiting high and low metastatic potentials in nude mice. Lysosomal membrane glycoproteins (lamp) 1 and 2 were immunoprecipitated from the cells after labeling with radioactive sugars, and the glycopeptides prepared were fractionated by serial lectin affinity chromatography employing immobilized concanavalin A, Datura stramonium agglutinin, and tomato lectin. Comparison of Asn-linked oligosaccharides from the different colonic carcinoma cells revealed the following features. First, the highly metastatic carcinoma cells express more poly-N-acetyllactosaminyl side chains with branched galactose residues than cells with low metastatic potential. Second, sialylation is more significant in the highly metastatic carcinoma cells than in the poorly metastatic ones. Conversely, N-acetyllactosamine units are less fucosylated in the highly metastatic cells than in poorly metastatic cells. These structural changes were apparently caused by the increase in sialyltransferase and the decrease in alpha 1----3 fucosyltransferase in the highly metastatic cells. The results also suggest that highly metastatic carcinoma cells express more sialyl Lex structures at the termini of poly-N-acetyllactosaminyl side chains than poorly metastatic carcinoma cells. Further, highly metastatic cells were found to express more lamp-1 and lamp-2 on the cell surface. These results were found to be correlated to the increased expression of sialyl Lex structures with high affinity binding of anti-sialyl Lex antibody on highly metastatic cells. Increased expression of sialyl Lex in the poly-N-acetyllactosamines of the cell surface may contribute to the metastatic behavior of the cells, assuming that this structure can serve as a better ligand for selectins present on endothelial cells and platelets.

Antibodies↗

Microanalysis of beta-cyclodextrin and glucosyl-beta-cyclodextrin in human plasma by high-performance liquid chromatography with pulsed amperometric detection.

High-performance liquid chromatography with pulsed amperometric detection was applied to the determination of beta-cyclodextrin (beta-CD) and glucosyl (G)-beta-CD in human plasma. They were well resolved from each other and from background components of plasma on a polymer-based reversed-phase column with 0.6% acetonitrile aqueous solution containing 1 mM sodium hydroxide as an eluent. The samples in the effluent were detected with a pulsed amperometric detector after postcolumn alkalization. The detection limits of beta-CD and G-beta-CD in plasma at a signal-to-noise ratio of 3 were 11 and 5 pmol, respectively.

Chromatography, High Pressure Liquid↗

Monkey hippocampal neuron responses to complex sensory stimulation during object discrimination.

The purpose of this study was to investigate, during the performance of an object discrimination task, responses of neurons in the monkey hippocampal formation to the sight of several objects that have biological meaning, and compare these responses with those of amygdalar neurons studied previously using the same task. Neuronal activity in the hippocampal formation of conscious monkeys was recorded during performance of a task that led to presentation of familiar rewarding, familiar aversive, or unfamiliar objects. Of 864 neurons recorded in the hippocampal formation and adjacent cortices, 160 (18.5%) responded to the sight of a certain object(s). Responses to the sight of different kinds of objects were analyzed in detail. Nondifferential neurons (n = 73) responded to different objects with no significant difference in response magnitudes, and differential neurons (n = 87) responded to different objects with different response magnitudes. Of the differential neurons, 23 responded more strongly to rewarding objects than to other objects (rewarding-object-dominant neurons), but the magnitude of responses to objects did not necessarily correlates with the order of preferences to the objects as determined from observation of animal behavior. Aversive-object-dominant neurons (n = 13) responded more to aversive objects than to other objects. Unfamiliar-object-dominant neurons (n = 7) responded more to unfamiliar objects than to familiar objects. Selective neurons (n = 10) responded selectively to only one object or one category of objects. Fourteen of the rewarding- or averse-object-dominant neurons were tested in extinction or reversal trials. In 12 of 14 neurons, responses to a rewarding or aversive object did not change, or slightly weakened, in extinction or reversal trials. The results suggest the following. (1) Responses of rewarding- or aversive-object-dominant neurons may be involved in object-reward or object-aversion association. However, responses of many of these neurons might reflect past inputs to reinforcement rather than extant emotional processing. (2) Responses of unfamiliar-object-dominant neurons may be involved in recognition of objects based on their familiar or unfamiliar aspects. These results are further discussed and compared with responsiveness of amygdalar neurons.

Acoustic Stimulation↗

Spatial responsiveness of monkey hippocampal neurons to various visual and auditory stimuli.

To investigate involvement of the hippocampal formation in spatial information processing, activity of neurons in the hippocampal formation of the conscious monkey was recorded during presentation of various visual and auditory stimuli from several directions around the monkey. Of 1,047 neurons recorded, 106 (10.1%) responded to some stimuli from one or more directions. Of these 106 neurons with directionally differentiating responsiveness, 49 responded to visual stimulation, 35 to auditory stimulation, and 22 to both. Among 81 neurons, each tested with more than 10 different stimuli, one type responded independent of the nature of the stimulus (nonselective, n = 39), and responses of the other type depended on the nature of the stimulus (selective, n = 42). To investigate effects of change in spatial relations between test stimuli and background stimuli fixed on the monkey or fixed in the environment, 59 of 106 neurons were tested while the experimental apparatus holding the stimulus was moved relative to the monkey. Of these 59 neurons, 36 changed their responsiveness; 7 maintained the magnitude of their responses but changed the response direction with the movement of the apparatus, 5 changed direction regardless of the movement, and 24 did not change direction, but decreased or extinguished responses from the preferred direction. Thirty-two of 106 neurons were also tested by rotating the monkey. The directionally differentiating responsiveness of 11 neurons followed the monkey (egocentric neurons), that of 9 remained in place in the environment (allocentric neurons), and responses of 12 were reversibly extinguished when the monkey was rotated. The results suggest that these hippocampal neurons may be involved in identification of relations among various kinds of stimuli in different spatial frameworks (egocentric or allocentric) and this identification may be developed from multiple sensory modalities.

Acoustic Stimulation↗

Changes in plasma catecholamine levels following injection of prostaglandin F2alpha into the basal cistern in rabbits.

We measured plasma epinephrine and norepinephrine concentrations in a rabbit model simulating subarachnoid hemorrhage (SAH), following the injection of prostaglandin F(2alpha) (PGF(2alpha)) into the basal cistern. In this model, plasma epinephrine values increased significantly (to 4.2-fold those before injection), substantially more than norepinephrine (which increased 1.3-fold) at 5 minutes (min) after PGF(2alpha) injection. Dissection of autonomic outflow from the cervical spinal cord or ligation of the suprarenal veins reduced the changes in plasma catecholamine concentrations associated with PGF(2alpha) injection. These results suggest that the sympathetic discharge seen after PGF(2alpha) injection into the basal cistern in rabbits occurred through the sympatho-adrenal pathways.

Journal Article↗

Chediak-Higashi lymphoblastoid cell lines: granule characteristics and expression of lysosome-associated membrane proteins.

Chediak-Higashi syndrome (CHS) is characterized morphologically by the presence of giant lysosomal granules resulting from the dysregulated fusion of primary lysosomes. Lysosome-associated membrane proteins comprise a family of highly glycosylated proteins which are postulated to facilitate many aspects of normal lysosomal function. In this study, Epstein-Barr virus-transformed lymphoblastoid cell lines derived from a patient with CHS were analyzed for the presence of giant granules and the expression of the lysosome-associated membrane proteins lamp1 and lamp2. Giant myeloperoxidase positive granules typical of CHS, which had a complex structure when examined by electron microscopy, could be demonstrated in the lymphoblastoid cell lines. In situ immunofluorescence with antibodies directed against lamp1 and lamp2 demonstrated abundant expression of each of these proteins in the giant CHS granules. Lack of expression of lysosomal cathepsin G in these granules was also noted. These observations suggest that the lymphoblastoid cell lines provide a convenient model for the study of Chediak-Higashi granules and the lysosome-associated membrane proteins and provide additional evidence that CHS is a "lysosomal" disease. Further study will be necessary to delineate whether the function of these membrane proteins is altered in Chediak-Higashi syndrome.

Antigens, CD↗