Biomedical subjects

Y Inoue

Publications and source records attributed to Y Inoue.

At least 1,045 records · Page 58Linked to original sources

PubMed1994

Purification and enzymatic properties of peptide:N-glycanase from C3H mouse-derived L-929 fibroblast cells. Possible widespread occurrence of post-translational remodification of proteins by N-deglycosylation.

Recently, we found the occurrence of N-deglycosylating enzyme, peptide:N-glycanase (PNGase), in mammalian cells and observed that PNGase is a rather common enzyme involved in post-translational remodification of proteins (Suzuki, T., Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1993) Biochem. Biophys. Res. Commun. 194, 1124-1130). We report here a 460-fold purification to homogeneity with 11.5% yield of PNGase from crude extract of C3H mouse-derived L-929 fibroblast cells. The purified enzyme, designated as L-929 PNGase, had the apparent molecular weight of 212,000 and was composed of two 105,000 subunits. Although this enzyme was capable of hydrolyzing structurally diverse natural glycopeptide substrates bearing high mannose, hybrid, and complex-type glycan units, the activity was completely inhibited by the presence of the fucose residue either alpha-1-->3- or alpha-1-->6-linked to the proximal GlcNAc residue. The enzyme showed maximal activity at pH near 7. This and the inability to act on glycoasparagine strongly support our view that this enzyme would not be involved in lysosomal degradation pathway. L-929 PNGase was characterized by having distinctly a low Km value, which may be of physiological significance. Possible wide occurrence of N-deglycosylation of glycoproteins was shown by a data bank survey of the protein sequences showing discrepancies between those determined directly (-D-X-(S/T)-) and those deduced from cDNA sequencing (-N-X-(S/T)-). We propose here that PNGase-catalyzed N-deglycosylation is a functionally important universal feature in living cells.

Amidohydrolases↗

PubMed1994

Enhancement of lysolecithin acyltransferase activity by LDL in thrombin-stimulated porcine-cultured endothelial cells.

Changes of intracellular activity of lysolecithin acyltransferase (LAT) during an interaction between endothelial cells (EC) and low-density lipoprotein (LDL) were investigated. Following an incubation of EC with LDL, endothelial LAT activity was assayed using [3H]lysophosphatidylcholine as the substrate. Stimulation of EC with either thrombin (0.01-1 U/ml) or Ca(2+)-ionophore A23187 (10(-10)-10(-7) M) dose- and time-dependently enhanced LAT activity in the presence of LDL (1 mg protein/ml), but no enhancement was observed in quiescent cells. Ionomycin together with 1-oleoyl-2-acetyl glycerol, a synthetic analog of diacylglycerols enhanced LAT activity in a similar degree to thrombin in the presence of LDL. Either staurosporine, a protein kinase C inhibitor or neomycin, a phospholipase C inhibitor completely blocked an increase of LAT activity in stimulated EC. Stimulation of EC with various agonists including 12-o-tetradecanoylphorbol-13-acetate, an activator of protein kinase C caused a marked increase in cellular uptake of LDL, and staurosporine inhibited the uptake. These results suggest that the transport of LDL into EC is facilitated by stimulation with thrombin and other agonists, and LDL subsequently activates intracellular LAT. Protein kinase C seems to mediate LDL uptake into EC. Intracellular regulatory roles of LDL in the presence of vasoactive substances were discussed.

1-Acylglycerophosphocholine O-Acyltransferase↗

PubMed1994

Identification and structural determination of the KDN-containing N-linked glycan chains consisting of bi- and triantennary complex-type units of KDN-glycoprotein previously isolated from rainbow trout vitelline envelopes.

KDN-gp, which is the unique glycoprotein of the rainbow trout egg envelope, was shown to have a small amount of N-linked oligosaccharide units in addition to a large number of O-linked glycan units. Structural analysis based on chemical analysis in combination with 400 MHz 1H NMR spectroscopy revealed the presence of fully KDNosylated bi- and triantennary complex-type oligosaccharide chains, mostly fucosylated at the innermost GlcNAc residue and bisected by the GlcNAc residue linked beta 1-->4 to the beta-Man residue. The structures thus determined represent the first demonstration of N-linked glycan unit containing the KDN residues in the KDN-containing glycoproteins (see Chart 1). The KDN-gp of the rainbow trout egg envelope is a molecule that is present in the second layer of the vitelline envelope but is exposed to the outer surface around the micropyle through which sperm can get in at fertilization. Like human hematopoietic cell surface glycoproteins such as glycophorin A and leukosialin, KDN-gp, which is now characterized to contain N-linked complex-type glycan chains as minor components, is heavily O-glycosylated with alpha 2-->8-linked oligo/polyKDN-containing glycan units attached O-glycosidically to Ser/Thr residues. Although little is known about the functional roles of these glycan chains, KDN-gp appears to form a model for further study on the function of cell surface receptor for sperm in fertilization.

Animals↗

PubMed1994

Distinct spatiotemporal expressions of five NMDA receptor channel subunit mRNAs in the cerebellum.

The distribution of five NMDA receptor channel subunit mRNAs was examined in the mouse cerebellum from embryonic day 13 through postnatal day 56, by in situ hybridization with subunit-specific oligonucleotide probes. At postnatal days 21 and 56, each cerebellar neuron displayed differential expressions of the epsilon subunit mRNAs. The granule cells showed hybridizing signals for the epsilon 1 and epsilon 3 subunit mRNAs, the molecular layer neurons for the epsilon 4 subunit mRNA, and the cerebellar nucleus neurons for the epsilon 1 and epsilon 4 subunit mRNAs, whereas the Purkinje cells did not express any epsilon subunit mRNAs. At early postmitotic stages of development, the epsilon 2 subunit mRNA appeared in each cerebellar neuron, including the Purkinje cells, and the epsilon 4 subunit mRNA appeared in neurons of the molecular layer and the cerebellar nuclei. The expression patterns in the cerebellum altered drastically during the first 2 postnatal weeks; the epsilon 1 and epsilon 3 subunit mRNAs appeared in the granule cells and the cerebellar nucleus neurons, whereas the epsilon 2 subunit mRNA disappeared from each neuron and the signal levels of the epsilon 4 subunit mRNA decreased remarkably. In contrast to the differential expressions of the four epsilon subunit mRNAs, intense signals for the zeta 1 subunit mRNA were observed in each cerebellar neuron from early postmitotic stages through the mature stage. These findings suggest that anatomical organization of the epsilon subunits is heterogeneous in the cerebellum both spatially and temporally, which would give rise to functional diversity of the NMDA receptor channel.

Aging↗

PubMed1994

Distinct distributions of five NMDA receptor channel subunit mRNAs in the brainstem.

The distribution of five NMDA receptor channel subunit mRNAs in the mouse brainstem at postnatal day 21 was semiquantitatively examined by in situ hybridization with subunit-specific oligonucleotide probes. The epsilon 1 subunit mRNA was observed in various brainstem nuclei. On the other hand, the epsilon 2 and epsilon 3 subunit mRNAs were restricted to particular nuclei, and the epsilon 4 subunit mRNA was detected in several nuclei at very low levels. The dorsal cochlear nucleus exhibited differential expression of the epsilon subunit mRNAs in distinct neuron types: the epsilon 2 subunit mRNA in the pyramidal cells, the epsilon 3 subunit mRNA in the granule cells, and the epsilon 1 subunit mRNA in other neurons. In the vestibular nuclei, the medial nucleus expressed the epsilon 1, epsilon 3, and epsilon 4 subunit mRNAs, whereas the lateral and superior nuclei expressed the epsilon 1 subunit mRNA. Such region-specific expressions were also discerned in the central gray, the superior and inferior colliculi, the medial accessory oculomotor nucleus, the locus ceruleus, the parabrachial nucleus, nucleus of the solitary tract, the caudal subnucleus of the trigeminal spinal tract nucleus, and the inferior olive. In contrast to the differential distributions of the four epsilon subunit mRNAs, intense signals for the zeta 1 subunit mRNA were distributed throughout the brainstem. These findings suggest that anatomical organization of the epsilon subunits is heterogeneous in various regions of the brainstem, which would give rise to functional diversity of the NMDA receptor channel in these regions.

Animals↗

PubMed1994

Developmental expression of trout egg polysialoglycoproteins and the prerequisite alpha 2,6-, and alpha 2,8-sialyl and alpha 2,8-polysialyltransferase activities required for their synthesis during oogenesis.

The developmental expression of the alpha 2,6- and alpha 2,8-linked sialic acid (Sia) residues in trout egg polysialoglycoproteins (PSGPs) was studied by correlating the temporal expression of these sugar residues, and the prerequisite sialyltransferases responsible for their synthesis, during oogenesis. The following new findings are reported. 1) Disialylated glycoproteins were identified in ovaries 4-6 months prior to ovulation. Three months prior to ovulation, a second more highly sialylated glycoprotein appeared. Structural studies confirmed that the two glycoproteins were discrete molecular species, designated PSGP(low Sia) and PSGP(high Sia), which differed only in their Sia content. PSGP(low Sia) contained mostly disialyl (Sia alpha 2,8-Sia alpha 2,6-) side chains, whereas PSGP(high Sia) contained alpha 2,8-linked oligo/polySia side chains ranging in length from 2 to over 20 Sia residues. The average degree of polymerization ([DP]av) was 6. 2) Biosynthetic studies using CMP-[14C]Neu5Ac indicated that three sialyltransferase activities were responsible for synthesis of the polysialyl residues of PSGPs: (i) alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase (alpha 2,6-ST), which catalyzed formation of the Sia residues alpha 2,6-linked to the proximal GalNAc residues in asialo-PSGP; (ii) alpha 2,6-sialoside alpha 2,8-sialyltransferase (alpha 2,8-ST or "initiase"), which catalyzed transfer of the first alpha 2,8-Sia residue to the alpha 2,6-linked Sia residue; and (iii) an alpha 2,8-polysialyltransferase (alpha 2,8-polyST or "polymerase"), responsible for synthesis of the alpha 2,8-linked poly/oligo Sia chains in PSGP(high Sia). Expression of these enzyme activities increased in accordance with the developmental appearance of each PSGP. 3) Structural characterization of the [14C]Sia-labeled side chains of each PSGP at different stages of development confirmed that synthesis of the disialyl unit containing a single alpha 2,8-Sia residue occurred before alpha 2,8-polysialylation. 4) In ovaries, 96% of the sialyltransferase activities were found in the Golgi-derived immature cortical vesicles or as soluble enzymes released from the fragile vesicles. Less than 4% of the activities were localized in the membrane (Golgi) fraction. In mature eggs, the sialyltransferases were also detected as soluble enzymes, and within the cortical vesicles.

Amino Acid Sequence↗

PubMed1994

Structural studies of a novel type of pentaantennary large glycan unit in the fertilization-associated carbohydrate-rich glycopeptide isolated from the fertilized eggs of Oryzias latipes.

In a previous report (Kitajima, K., Inoue, S., and Inoue, Y. (1989) Dev. Biol. 132, 544-553), we found the presence of a heavily glycosylated polyprotein, "H-hyosophorin," isolated from the unfertilized eggs of Oryzias latipes. We now report our detailed analysis of the structure of the N-glycan chain in L-hyosophorin, the smallest repeating unit of H-hyosophorin, which was isolated from the fertilized eggs of O. latipes and formed from H-hyosophorin upon fertilization. The N-glycan structures were defined by a combination of compositional analysis, methylation analysis, selective chemical degradation (i.e. mild methanolysis, periodate-Smith degradation, and hydrazinolysis-nitrous acid deamination), enzymatic (endo-beta-galactosidase, peptide:N-glycanase, and Newcastle disease virus sialidase) digestion, and instrumental analyses (one- and two-dimensional proton nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry) which revealed novel and unique features: (a) the presence of highly branched poly-N-acetylactosamino pentaantennary structures; (b) the presence of a beta-galactosylated Lewis X antigenic epitope, Gal beta 1-->4 Gal beta 1-->4 (Fuc alpha 1-->3) GlcNAc beta 1-->; (c) the presence of a beta-galactosylated sialyl Lewis X structure, Gal beta 1-->4 (Neu5Ac alpha 2-->3) Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->; (d) the presence of Gal beta 1-->4 Gal beta 1--> and Gal beta 1--> 4Gal beta 1-->4Gal beta 1--> as the major and minor groupings, respectively; and (e) the presence of the branched Gal residues, -->4GlcNAc beta 1-->3(Gal beta 1-->4) Gal beta 1-->. This study represents the first detailed investigation regarding the nature of highly branched complex asparagine-linked pentaantennary glycans in glycoproteins. The unique expression of such bulky multiantennary glycan units on proteins could be essential during early embryogenesis.

Animals↗

PubMed1994

CS-045, a new oral antidiabetic agent, stimulates fructose-2,6-bisphosphate production in rat hepatocytes.

Fructose-2,6-bisphosphate is a potent activator of 6-phosphofructo-1-kinase, a key enzyme in glycolysis. We previously revealed that sulfonylureas stimulate fructose-2,6-bisphosphate production in the rat liver by activating 6-phosphofructo-2-kinase. In the present study, we show that CS-045, a new antidiabetic agent, activated 6-phosphofructo-2-kinase and raised fructose-2,6-bisphosphate levels in dispersed rat hepatocytes. This action was time- and dose-dependent. Ten micromolar CS-045 raised the fructose-2,6-bisphosphate content linearly to the submaximal level in 20 min. Dose dependency was observed in the range of 1-30 microM. Thirty micromolar CS-045 completely reversed the inhibitory effect of 0.1 nM glucagon on fructose-2,6-bisphosphate production. CS-045 activated 6-phosphofructo-2-kinase by decreasing the Km value for the substrate (fructose-6-phosphate) without affecting the Vmax. The combination of suboptimal doses of CS-045 and tolbutamide increased fructose-2,6-bisphosphate content more than that induced by each agent alone. These results indicate that CS-045 may reduce plasma glucose by facilitating glycolysis in the liver.

Animals↗

PubMed1994

Synthesis and antibacterial activity of thiazolopyrazine-incorporated tetracyclic quinolone antibacterials.

A novel series of 8-substituted-9,1-[(N-methylimino)methano]- 7-fluoro-5-oxo-5H-thiazolo[3,2-alpha]-quinoline-4-carboxylic acids 5a-q having a unique thiazolopyrazine-incorporated tetracyclic structure were synthesized, and the in vitro and in vivo activities were determined against Gram-positive and Gram-negative bacteria. All compounds 5a-q had more potent activity than ofloxacin (6), which is one of the most popular quinolones, against Gram-positive and Gram-negative bacteria. The 8-pyrrolidinyl, 5a-e, and 8-morpholino, 5p, derivatives showed the most potent activity against Gram-positive bacteria. It is also significant that these compounds, 5a-q, showed more potent antibacterial activity against methicillin-resistant Staphylococcus aureus isolates (MRSA) than ofloxacin (6). The combination of the morpholino group and this unique tetracyclic thiazolopyrazine skeleton contributes to the enhancement of the antibacterial activity against MRSA isolates. The in vivo antibacterial activities of these compounds, 5a-q, were limited and depended on the structure of the 8-substituent. The 8-(4-alkyl-1-piperazinyl) derivatives 5g, 5h, 5j, and 5n provided good oral efficacy and exhibited more potent activity than ofloxacin (6) against the systematic infection with S. aureus IID 803 in mice.

Animals↗

PubMed1994

Calcium ion binding of three different types of oligo/polysialic acids as studied by equilibrium dialysis and circular dichroic methods.

Ca2+ binding properties of three different types of oligo/polysialic acid chains, i.e., oligo/poly(Neu5Ac), oligo/poly(Neu5Gc), and oligo/poly(KDN), were studied by equilibrium dialysis and circular dichroism. Colominic acid, high molecular weight polysialoglycoprotein (H-PSGP), low molecular weight polysialoglycoprotein (L-PSGP), and 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN) glycoprotein were found to bind calcium ions with about 8-100 times the affinity of sialic acid monomer. Analysis by equilibrium dialysis of the binding of Ca2+ to colominic acid was biphasic, and the high-affinity interaction was shown to change with the degree of polymerization. Specific binding of Ca2+ to polysialic acid (polySia) caused characteristic effects in the circular dichroism spectrum. A pronounced decrease in the circular dichroism of polySia at 205 nm was observed upon addition of calcium. H-PSGP was found to bind calcium ions with 3-fold higher affinity than L-PSGP.

Animals↗

PubMed1994

Differential distributions of the NMDA receptor channel subunit mRNAs in the mouse retina.

In the retina, the epsilon 2 and zeta 1 subunit mRNAs of the NMDA receptor channel were expressed from embryonic stages and found in ganglion cell layer and whole layer of inner nuclear layer at postnatal day 21 (P21). The epsilon 1 subunit mRNA appeared postnatally and was distributed in ganglion cell layer and an inner third of inner nuclear layer at P21. These findings suggest that molecular organization of the NMDA receptor channel may alter during the retinal development.

Animals↗

PubMed1994

Studies on antiulcer drugs. 7. 2-Guanidino-4-pyridylthiazoles as histamine H2-receptor antagonists with potent gastroprotective effects against nonsteroidal antiinflammatory drug-induced injury.

A series of 2-guanidino-4-pyridylthiazole derivatives were synthesized and evaluated for anti-aspirin-ulcer, gastric antisecretory, and histamine-H2-receptor-antagonist activities. Several compounds showed superior anti-aspirin-ulcer activity to that of clinically used H2-antagonists in the rat. Among them, 4-[6-(acetamidomethyl)pyridin-2-yl]-2-guanidinothiazole (8) demonstrated potent inhibitory activities against gastric lesions caused by two kinds of nonsteroidal antiinflammatory drugs, aspirin and indomethacin, respectively, in addition to strong antisecretory activity. Compound 8 possessed a preventable ability for the aspirin-induced reduction of the gastric mucosal blood flow at an intragastric administration of 32 mg/kg in the rat. On the other hand, famotidine (32 mg/kg) exhibited no significant effect and ranitidine (100 mg/kg) aggravated the blood flow in this system.

Animals↗

PubMed1994

Distinct gene expression of the N-methyl-D-aspartate receptor channel subunit in peripheral neurons of the mouse sensory ganglia and adrenal gland.

The distribution of five N-methyl-D-aspartate (NMDA) receptor channel subunit mRNAs was examined in the mouse peripheral nervous tissues by in situ hybridization. The trigeminal and dorsal root ganglion cells showed signals for the zeta 1 subunit mRNA from embryonic stages through postnatal day 21, but not for the epsilon 1, epsilon 2, epsilon 3, and epsilon 4 subunit mRNAs at any developmental stages examined. The adrenal medulla also expressed the zeta 1 subunit mRNA alone. These findings suggest that molecular organization of the NMDA receptor channel in peripheral neurons are distinct from those in central neurons, which may result in different functional properties of the channel between them.

Adrenal Glands↗

PubMed1994

Difference in sero-diagnostic values among KL-6-associated mucins classified as cluster 9.

KL-6 classified as Cluster 9 (MUC-I) is a circulating high-molecular-weight mucin-like molecule. Serum level of KL-6 was measured by a sandwich assay using KL-6 antibody as not only a catcher but also as a tracer. We established 2 additional monoclonal antibodies (MAbs), LISA 101 and EH-123, reacting with KL-6 epitopes different from the epitope recognized by KL-6 antibody. The KL-6-associated mucins detected by the sandwich assay using LISA 101 or EH-123 antibody as a catcher and KL-6 antibody as a tracer were designated as LISA 1-6 and CAM 123-6 respectively. The diagnostic values as the serum markers of KL-6, LISA 1-6 and CAM 123-6 were evaluated measuring their levels in the same serum from healthy individuals and from patients with pulmonary, pancreatic and breast adenocarcinomas. KL-6 was increased abnormally at high rates of more than 50% in pancreatic cancer and in benign lung diseases, LISA 1-6 only in pancreatic cancer, and CAM 123-6 only in pulmonary adenocarcinoma. In benign lung diseases, however, LISA 1-6 and CAM 123-6 were increased abnormally at the rates of only 5.3% and 0% respectively. These observations clearly indicate that LISA 1-6 and CAM 123-6 constitute a part of KL-6, but that they are superior to KL-6 as tumor markers for pancreatic cancer and for pulmonary adenocarcinoma respectively, because of their much lower false-positive rates.

Adenocarcinoma↗

PubMed1994

Conservative surgical treatment in cervical cancer with 3 to 5 mm stromal invasion in the absence of confluent invasion and lymph-vascular space involvement.

We have previously reported that simple hysterectomy is adequate therapy for microinvasive carcinoma of the cervix with less than 3 mm stromal invasion (Yajima, A., et al, Am. J. Obstet. Gynecol. 135, 685-688, 1979). However, whether it is possible to effectively treat patients with deeper stromal invasion remained in question. A policy for surgical treatment in patients with 3 to 5 mm invasion has not been standardized. Although radical hysterectomy is usually performed, some authors insist that patients with 3 to 5 mm invasion can be safely treated conservatively. Patients treated with conservative surgery should be carefully assessed because cases with confluent invasion or lymph-vascular space invasion have a greater potential for lymph node metastasis and carry a worse prognosis. Conservative treatment in this study was limited to patients with microinvasive carcinoma with 3 to 5 mm invasion, stage IB occult patients (classified by the FIGO 1973 staging system). The patients were preoperatively diagnosed with either carcinoma in situ or microinvasive carcinoma, and treated by conservative surgery (conization, simple hysterectomy, or extended hysterectomy). Twenty-two patients with 3 to 5 mm invasion but without additional risk factors, i.e., confluent invasion or lymph-vascular space involvement, were followed without postoperative radiation therapy. All patients survived except one (who died of an intercurrent disease) and there were no signs of recurrence of serious surgical complications. The present study with stage IB "occ" subjects suggests that conservative therapy rather than radical hysterectomy can be used in a very carefully selected subset of patients, i.e., in cases with 3 to 5 mm microinvasive carcinoma without additional risk factors, such as confluent invasion and lymph-vascular space invasion.

Carcinoma in Situ↗

PubMed1994

Distribution of acetylcholinesterase activity in the rat embryonic heart with reference to HNK-1 immunoreactivity in the conduction tissue.

Acetylcholinesterase (AChE) activity was topographically investigated in the presumptive cardiac conduction tissue regions visualized by HNK-1 immunoreactivity in rat embryos, and AChE-positive cells were examined with the electron microscope. On embryonic day (ED) 14.5, when HNK-1 was most intensely visualized, AChE activity could not be detected enzyme-histochemically in the conduction tissue regions, except in the ventricular trabeculae and part of the AV node. On ED 16.5, however, the AChE activity was clearly demonstrated in some parts of the developing conduction tissue. One exception was the AV node region, where an AChE-positive area was in close proximity to an area showing HNK-1 immunoreactivity but did not overlap. Furthermore, AChE activity was demonstrated predominantly in the ventricular trabeculae, including cardiac myocytes, but was rather weak in the atrium. With the electron microscope, AChE reaction products were observed predominantly intracellularly in both developing conduction tissue cells and developing ordinary myocytes, and no reactivity was found in neuronal components. From ED 18.5 until birth, both AChE activity and HNK-1 immunoreactivity faded away in the conduction tissue. Thus, transient AChE activity in the embryonic heart seems to be different from the developing adult form and may be related to a morphogenetic function in embryonic tissues, as proposed by other authors.

Acetylcholinesterase↗

PubMed1994

Expression of platelet-derived growth factor and c-myc in atherosclerotic lesions in cholesterol-fed chickens: immunohistochemical and in situ hybridization study.

Immunohistochemical examination showed no significant expression of platelet-derived growth factor-A (PDGF-A), PDGF-B, PDGF receptors, or of c-myc in the thoracic and abdominal aortas of normal roosters. In cholesterol-fed roosters, intense immunohistochemical reaction for PDGF-B, PDGF receptor, and c-myc was seen in the lipid-rich thickened intimal lesions of the thoracic and abdominal aortas while no significant immunoreaction for PDGF-A was demonstrated in the same lesions. In accordance with immunohistochemical findings, in situ hybridization demonstrated a significant level of expression of PDGF-B, PDGF-A receptor, PDGF-B receptor, and c-myc genes in proliferating intimal cells of the thoracic and abdominal aortas. These results suggest that coordinate actions of PDGF-B and c-myc play an important role in proliferation of intimal cells in the developing atherosclerotic lesions in chickens.

Animals↗

PubMed1994

Monoclonal antibody specific for alpha 2-->8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry.

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing alpha 2-->8-linked polysialic acid (-->8Neu5Gc alpha 2-->)n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (-->8KDN alpha 2-->)n-->6(KDN alpha 2-->3Gal beta 1-->3G alpha lNA-c alpha 1-->3) GalNAc alpha 1-->residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗