Biomedical subjects

J Roth

Publications and source records attributed to J Roth.

At least 127 records · Page 7Linked to original sources

PubMed1995

[Expression of CD44 isoforms in normal human liver and also in regenerative and neoplastic liver changes].

CD44 is a transmembrane glycoprotein of which a large number of isoforms exist. There is evidence, mostly from experimental systems, that isoforms of CD44 generated by alternative splicing of ten variant exons are involved in tumor invasion and metastasis formation. We have evaluated the expression of CD44 standard (CD44s) and variant exons (CD44v) encoded gene products in formalin-fixed and paraffin-embedded human liver specimens, using an immunohistochemical protocol with microwave-based antigen-retrieval and signal amplification. Tissue sections from normal, regenerative and neoplastic liver were studied. Our results indicate that: 1. in normal liver, both, hepatocytes and bile duct epithelia lacked detectable CD44s and CD44v containing isoforms; 2. most cirrhotic liver specimens were unreactive. Some regenerative nodules showed a focal weak positivity for CD44v5 and v9. Most proliferating bile ductules were weakly stained for CD44v9 and some of them also for v5 and v6; 3. most hepatocellular carcinomas displayed a heterogeneous staining of varying intensity for CD44v5, v6, v9 and CD44s. In a few tumors a weak staining for CD44v3 and v4 was also present. Furthermore, there was a tendency to an increased staining intensity of CD44 isoforms with decreasing differentiation; 4. cholangiocarcinomas showed a high expression of CD44s, v3, v5, v6 and v9 containing isoforms. We conclude that neoplastic transformation of hepatocytes and bile duct epithelia is associated with qualitative and quantitative changes in the expression of some CD44 variant exons encoded products. The clinical implications of these findings remain to be determined in a large series of patients.

Adenoma↗

PubMed1995

Expression of murine VCAM-1 in vitro and in different models of inflammation in vivo: correlation with immigration of monocytes.

VCAM-1 (vascular cell adhesion molecule-1) is a cytokine-inducible adhesion molecule which is known to mediate adhesion of mononuclear cells to endothelial cells in vitro via binding to the integrin VLA-4 (very late antigen-4). To further elucidate the role and regulation of VCAM-1 in vitro, we compared in vitro and in vivo expression of VCAM-1 in response to cytokines and investigated immunohistochemically the expression of VCAM-1 in three murine models of experimental inflammation. These models differed with regard to the pathogenetic mechanism, and the subsesquent infiltrate: allergic contact dermatitis (ACD) to DNFB as a T cell-controlled, DTH type of inflammation, cutaneous infection with Leishmania major as a chronic granulomatous inflammation and the cauterized cornea as a model for acute inflammation. VCAM-1 was found to be markedly enhanced on vascular endothelia in all types of inflammation and after subcutaneous administration of LPS and TNF-alpha. Administration of IL-4, however, failed to induce VCAM-1 both in vivo and in vitro. The increased VCAM-1 expression in the inflammatory models correlated with the appearance of infiltrating monocytes/macrophages. A concomitant influx of CD4-positive/CD8-positive lymphocytes was only observed in ACD and Leishmaniasis.

Animals↗

PubMed1994

Differential expression of cell surface sialoglycoconjugates on wild-type and cultured Ehrlich tumor cells as revealed by quantitative lectin-gold ultrastructural cytochemistry.

Three variants of the classical Ehrlich ascites tumor (EAT) cell have been studied by quantitative, sialic acid-specific, lectin-gold ultrastructural cytochemistry. Electron microscopic examination revealed pronounced differences in the surface morphology of the three cell variants. The wild-type Ehrlich cells (EAT-wt), grown in the peritoneal cavity of mice, exhibited a smooth surface profile. A variant form selected for growth as monolayer on basement membrane (EAT-c) showed a complex surface profile with numerous microvilli. The third variant (EAT-c/m), the cultured cells reinoculated into mice and passaged 20-25 times as ascites, presented a smooth surface profile similar to the EAT-wt cells. Quantitative single as well as double lectin-gold labeling revealed significant differences in the nature of cell surface sialoglycoproteins. The most significant finding was the presence of cell surface Neu5Ac alpha 2-6Gal residues as detected with the Sambucus nigra lectin on EAT-c and EAT-c/m cells, whereas EAT-wt cells contained little or none of such carbohydrate sequences. On the contrary, labeling by Maackia amurensis lectin, which recognizes the Neu5Ac alpha 2-3Gal beta 1-4GlcNAc sequence, was intense on all three Ehrlich cell variants; it was 20-60 times greater than alpha-2,6-linked sialic acid-containing glycoconjugates. Specific cell surface lectin binding combined with morphologic study appears to have identified a small subpopulation of cells within the ascites tumor that are capable of attaching to and growing on a basement membrane.

Animals↗

PubMed1994

[The apomorphine test in the differential diagnosis of Parkinsonism. Initial experience].

BACKGROUND: The reactivity of postsynaptic dopamine receptors in the striatum is preserved in Parkinson's disease (PD) but not in parkinsonian syndromes of various origin (PS). Hence, the effect of dopaminergic stimulation can serve as one of the diagnostic criteria allowing to distinguish between PD and PS. The present study aimed to ascertain the clinical utility of Apomorphine (APO), a powerful short acting direct D1 and D2 receptors agonist drug, in the differential diagnosis of PD and PS. METHODS AND RESULTS: We studied 39 patients presenting parkinsonian signs. Based on clinical examination, 23 subjects (7 f, 16 m, mean age (SD) 59.2 (7.8) years were diagnosed as having probable PD, and 16 patients (5 f, 11 m, mean age 72 (7.6) yrs, p < 0.001) as having probable PS. In the PD group, mean Hoehn and Yahr degree of severity was 2.7 (1.1), mean duration of the disease 9.5 (6.2) yrs, mean duration of L-DOPA treatment (20 patients) 6.7 (5.5) yrs. In the PS group, the respective values of Hoehn and Yahr were 3.3 (0.9), mean duration of the disease 6.2 (4.1) yrs and mean duration of L-DOPA treatment (10 patients) 1.6 (1.1) yrs. After an overnight withdrawal of all other dopaminergic medication, a single subcutaneous dose of Apomorphine hydrochloride solution corresponding to 0.05 mg per kg of weight was administered. Domperidone was given 60 mg daily prior to the testing to avoid undesirable peripheral effects of APO. 20 minutes after APO administration, we noted a marked clinical improvement i.e. at least 30% decrease of pre-treatment motor score values on Columbina University Rating Scale (CURS) in 19 of 23 PD and in one of 16 PS patients. In the PD group, the difference between mean CURS values, 30.7 (19.5) before and 14.7 (10.3) after APO was highly significant (p < 0.001). In the PS group, only a slight posttreatment CURS decrease was found, 39.8 (17.5) before and 37.8 (17.5) after APO (p < 0.05). CONCLUSIONS: In individual patients, these results mostly confirm the presumed diagnosis of PD or PS. Even in the most serious cases from the PD group, the testing showed that the function of dopamine receptors in the striatum was still preserved. Thus, the Apomorphine test appears to be a helpful tool for differential diagnosis of PD and PS.

Aged↗

PubMed1994

Cellular site of synthesis and dynamics of cell surface re-expression of polysialic acid of the neural cell adhesion molecule.

Homopolymers of alpha-2,8-ketosidically linked sialic acid (polysialic acid) represent a posttranslational modification which, in mammals, appears to be unique for the neural cell adhesion molecule and the alpha subunit of sodium channels in brain. Under steady-state conditions, polysialic acid is detectable in the plasma membrane of different cell types but not in the cytoplasm. We have studied the site of synthesis and the cell surface re-expression of polysialic acid in a clonal subline of small cell lung carcinoma using the monoclonal antibody 735 and bacteriophage endosialidase, both specific reagents for polysialic acid. After enzymic removal, cell surface polysialic acid re-expression reached control levels only after 5 days. When Golgi to plasma membrane transport of endosialidase-treated cells was blocked by culture at 20 degrees C or in the presence of monensin at 37 degrees C, de-novo-synthesized polysialic acid became detectable in the Golgi apparatus. Our data show that synthesis of polysialic acid of the neural cell adhesion molecule with a degree of polymerization of at least nine occurs intracellular in the Golgi apparatus of a human small cell lung carcinoma cell line.

Animals↗

PubMed1994

A synthetic tris-sulfotyrosyl dodecapeptide analogue of the insulin receptor 1146-kinase domain inhibits tyrosine dephosphorylation of the insulin receptor in situ.

A synthetic tris-sulfotyrosyl dodecapeptide (TRDIY(S)ETDY(S)Y(S)RK-amide), whose primary sequence is identical to the 1142-1153 sequence of the insulin proreceptor, inhibited insulin receptor dephosphorylation in solubilized membranes, and digitonin-permeabilized cells derived from Chinese hamster ovary (CHO) cells expressing high levels of human insulin receptors (CHO/HIRc). It also inhibited the dephosphorylation of a synthetic tyrosine phosphorylated substrate by recombinant PTP-1B, a protein tyrosine phosphatase (PTPase), indicating that it acted via interaction with PTPase(s). A N-stearyl derivative of the peptide caused an approximately 4.5-fold increase in insulin-stimulated receptor autophosphorylaction in intact CHO/HIRc cells. The peptide displayed specificity toward tyrosine-class phosphatases only, as it had no effect on the activities of the serine/threonine phosphatases PP-1 and PP-2A, or alkaline phosphatase. The tyrosine sulfate ester bonds of the peptide were stable when incubated with PTP-1B (1 h, 30 degrees C). These data suggest that the sulfotyrosyl peptide functions as a nonhydrolyzable phosphotyrosyl peptide analogue capable of direct interaction with PTPase catalytic domain.

Alkaline Phosphatase↗

PubMed1994

Changes in body temperature and circulating levels of interleukin-6 after intra-arterial injections or infusions of tumor necrosis factor alpha in guinea pigs.

Tumor necrosis factor alpha (TNF) is released systematically during the early phase of endotoxin induced fever. To study the effects of this cytokine in guinea pigs, 2 micrograms TNF were intra-arterially injected as a bolus or slowly infused within 60 min. Both modes of administration induced a biphasic elevation of the animals' abdominal temperature lasting 6 h and stimulated the release of endogenous interleukin-6 (IL-6)-like activity. The second phase of the thermal response and the release of endogenous IL-6-like activity were significantly higher, when TNF was slowly infused into the animals' circulation, in spite of a transiently higher TNF-like activity after the bolus injection of TNF. Both TNF and IL-6 may therefore be regarded as candidates to trigger the febrile response in guinea pigs.

Animals↗

PubMed1994

Subcellular localization of the UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation reaction in the submaxillary gland.

Addition of N-acetylgalactosamine to threonine and serine is the first step in the synthesis of O-glycosidically linked oligosaccharides. A UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (EC 2.4.1.41) from porcine submaxillary glands was recently purified to electrophoretic homogeneity, and polyclonal antibodies against the purified transferase were raised. Immunoblots of porcine, bovine, and ovine submaxillary gland extracts with the anti-transferase antibodies gave a single band and the antibodies reacted equally well with the purified glycosylated and N-glycanase-treated transferase. Immunoelectron microscopic localization of the transferase was achieved in Lowicryl K4M thin sections and frozen-thawed thin sections of porcine and bovine submaxillary gland by using the protein A-gold technique. Specific gold particle labeling was observed in the cis Golgi apparatus and smooth-membraned vesicular structures in close topological relation with it. Labeling was undetectable in the rough endoplasmic reticulum, its transitional elements, and smooth-membraned structures close to them, the trans Golgi apparatus, mucin droplets, and the plasma membrane. The onset of labeling for peptide-bound GalNAc as detected with Vicia villosa isolectin G4 mirrored the transferase immunolocalization as directly shown by double labeling and extended into the trans Golgi apparatus and mucous droplets. Apomucin immunolabeling was found throughout the endoplasmic reticulum and the intermediate compartment and partially overlapped the region of transferase labeling in the Golgi apparatus as demonstrated by double immunolabeling. Thus, the initial step of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation in porcine and bovine submaxillary gland cells occurs in the cis Golgi apparatus. The possible involvement of the intermediate compartment remains to be clarified.

Animals↗

PubMed1994

Expression and complex formation of S100-like proteins MRP8 and MRP14 by macrophages during renal allograft rejection.

MRP8 and MRP14, two S100-like calcium-binding proteins, are expressed during differentiation of monocytes/macrophages. Both assemble to different noncovalently associated complexes that are supposed to represent the biologically active states. The present study was intended to investigate the molecular basis of macrophage heterogeneity with respect to expression and complex formation of MRP8 and MRP14 during acute and chronic rejection of renal allografts. First, specificity of antisera and mAbs to be directed against MRP8, MRP14, or MRP8/MRP14 heterodimers was determined by immunocytochemical and Western blot analyses of L132 fibroblasts (co-)transfected with MRP8 and/or MRP14 cDNA. Then, immunohistochemical analysis of biopsy specimens obtained from kidney allografts after acute rejection was performed, revealing a parallel expression of MRP8 and MRP14 with coincident MRP8/MRP14 heterodimer formation in infiltrating monocytes. In contrast, chronic allograft rejection was characterized by a subpopulation of monocytes defined by the absence of MRP8/MRP14 complex formation despite expression of MRP8 and MRP14 monomers. Double-labeling experiments showed that this was due in part to differential expression of MRP8 and MRP14 in infiltrate macrophages of chronic rejection. The data presented demonstrate for the first time differences in MRP8/MRP14 complex assembly by infiltrating monocytes in situ. These seem to be of pathophysiological relevance since complex formation defines subpopulations of monocytes associated with distinct pathways of immunological reactions. Differences in the mode of calcium-dependent signaling may, therefore, be of importance for understanding the molecular basis of macrophage heterogeneity during acute and chronic allograft rejection.

Adolescent↗

PubMed1994

Expression of the calcium-binding proteins MRP8 and MRP14 in monocytes is regulated by a calcium-induced suppressor mechanism.

MRP8 and MRP14 are two calcium-binding proteins of the S-100 family the expression of which is restricted to distinct stages of monocytic differentiation. Heteromeric MRP8/MRP14 complexes have been shown to represent their biologically active forms. However, it is not as yet clear whether biochemical modification of complexes, or regulation on the transcriptional level, are responsible for the control of MRP8/MRP14 expression. Employing Western-blot analysis and metabolic labelling we have demonstrated that patterns and metabolism of MRP8/MRP14 complexes do not change during up- or down-regulation of MRP8/MRP14. By Northern-blot analysis it was shown that MRP8/MRP14 are regulated at the transcriptional level rather than by biochemical modification of the complexes. Elevation of intracellular calcium levels by A23187, as well as by thapsigargin, was found to lead to specific down-regulation of MRP8/MRP14 mRNA which is in contrast with data reported for inflammatory factors such as interleukin-1 or tumour necrosis factor alpha. Concomitant application of actinomycin D and calcium ionophore indicated that this suppressive effect is mediated by decreased synthesis rather than increased degradation of MRP8/MRP14 mRNA. Finally, we demonstrated that calcium-mediated down-regulation of MRP8-MRP14 can be antagonized by cycloheximide, suggesting that a calcium-induced repressor protein is responsible for suppression of MRP8-MRP14 at the transcriptional level. Our data indicate that the function of MRP8-MRP14 is restricted to events associated with early stages of myelomonocytic activation.

Antigens, Differentiation↗

PubMed1994

Detection of immunoglobulin heavy-chain gene rearrangement at the single-cell level in malignant lymphomas: no rearrangement is found in Hodgkin and Reed-Sternberg cells.

Hodgkin and Reed-Sternberg (HRS) cells, the neoplastic cells of Hodgkin's disease (HD), represent only a minority of the cellular infiltrate in affected tissue. Therefore, rearrangements of the immunoglobulin heavy-chain (IgH) gene detected in DNA extracted from an entire Hodgkin's lymph node cannot be attributed to the HRS cells and cannot be used as an argument for the B-cell origin of HRS cells. We developed a new method for the amplification of rearranged DNA of the IgH gene from single cells. Using 6 "forward primers" which were constructed corresponding to consensus sequences of the 6 known families of the IgH variable (V) region (framework region I) and a mix of 2 "reverse primers" corresponding to consensus sequences of the different joining (J) segments, rearrangements of all 6 V-families were detected in human peripheral blood lymphocytes. Rearranged IgH DNA could be amplified from single cells of B-cell lymphoma-cell lines and from 13 patients with B-cell non-Hodgkin's lymphomas. However, analysis of HRS cells isolated from lymph nodes of 13 patients with Hodgkin's disease did not show any rearrangement of the IgH gene locus. These findings, obtained by polymerase chain reaction (PCR) on isolated single HRS cells, contrast with previous studies that used Southern-blot analysis of entire tissues affected by Hodgkin's disease. We conclude that the neoplastic HRS cells in Hodgkin's disease--with the possible exception of the nodular paragranuloma subtype--are probably not derived from B cells.

B-Lymphocytes↗

PubMed1994

Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): rapid detection of deletions and duplications of gene sequences.

Screening methods based on the polymerase chain reaction (PCR), such as denaturing gradient gel electrophoresis, single-stranded conformational polymorphism, and heteroduplex analysis, are powerful tools for the detection of point mutations as well as small deletions and insertions, but are unable to detect heterozygous deletions or duplications of exons, genes, or chromosomes. We now report a PCR-based approach, designated gene dosage-PCR (gd-PCR), that allows rapid screening for heterozygous deletions and duplications of genes or exons. Gene dosage-PCR is a quantitative method in which two in vitro synthesized DNA internal standards are coamplified with the genomic DNA sample, one corresponding to the gene of interest (test sequence) and the other to a reference (disomic) gene (reference sequence). Both internal standards are designed to be amplified with the same primer pairs and with efficiencies similar to those of their genomic DNA counterparts, yielding PCR products slightly smaller than those derived from genomic DNA. Amplification of approximately equimolar amounts of the two internal standards and genomic DNA, in the presence of [32P]dCTP, results in four radiolabeled PCR products; after electrophoresis and quantification of the products, gene dosage is easily calculated. For validation, genomic DNA from 56 subjects, 28 with cytogenetically documented Down syndrome (trisomy 21) and 28 controls that were disomic for chromosome 21, was assayed. Using the beta-amyloid precursor protein gene (APP: chromosome 21q21) as the test sequence, control subjects had an adjusted mean gene dose of 2.00 +/- 0.29, while subjects with Down syndrome had a mean gene dose of 3.05 +/- 0.27.(ABSTRACT TRUNCATED AT 250 WORDS)

Amnion↗

PubMed1994

Attenuation of fever and release of cytokines after repeated injections of lipopolysaccharide in guinea-pigs.

1. The effects of repeated injections of bacterial lipopolysaccharide (LPS) at 3 day intervals on abdominal temperature and systemic release of tumour necrosis factor alpha (TNF)-like and interleukin-6 (IL-6)-like activity were measured in guinea-pigs. 2. After the third injection of LPS the fever response was significantly attenuated. 3. TNF-like activity (peak 1 h after LPS injection) and IL-6-like activity (peak 3 h after LPS injection) in plasma changed correspondingly, both being significantly reduced after the third and subsequent injections of LPS. 4. The increase of IL-6-like activity in plasma after LPS injection correlated to the febrile change in body temperature. This correlation remained manifest throughout the whole time course of the development of endotoxin tolerance. 5. The reduced production of TNF-like activity after repeated injections of LPS correlated to the attenuation of the fever index, the integration of the thermal response after LPS application. 6. The results support the hypothesis that one component of the development of endotoxin tolerance is reduced production and release of cytokines in response to repeated injections of the same amount of LPS.

Animals↗

PubMed1994

Dynamic regulation of intact and C-terminal truncated insulin receptor phosphorylation in permeabilized cells.

Using digitonin-permeabilized Chinese hamster ovary (CHO) cells that were transfected with intact human insulin receptors (CHO/HIRc cells), we examined insulin receptor phosphorylation and dephosphorylation using pulse-chase techniques. Insulin activated receptor autophosphorylation on tyrosyl residues to a level severalfold over basal, reaching maximal levels after 2, 5, and 10 min of stimulation at 34, 18, and 6 degrees C, respectively. Phosphopeptide analysis revealed that the triply phosphorylated form of the 1146-kinase domain of the insulin receptor was the major species, which is characteristic of the fully active tyrosine kinase function. The dephosphorylation reaction was time- and temperature-dependent with t1/2 values of 0.67 and 2 min at 18 and 6 degrees C, respectively. Vanadate completely inhibited dephosphorylation. Under similar permeabilization conditions when compared with CHO/HIRc cells, CHO/delta CT cells (CHO cells overexpressing a mutated form of the receptor with a 43 amino acid deletion at the C-terminus) stimulated with insulin exhibited larger increases in receptor autophosphorylation levels and in tyrosine kinase activity toward a synthetic peptide substrate; the rate of CHO/delta CT receptor dephosphorylation was not reduced. There was near-complete absence of insulin receptor substrate 1 (IRS-1) in the cell ghosts after permeabilization. We therefore examined the pattern of tyrosine phosphorylation and dephosphorylation of residual cellular proteins in permeabilized CHO/HIRc cells by Western blot analysis. In addition to the 95-kDa receptor beta-subunit, we detected the phosphorylation of two glycoproteins which included the commonly found 120-kDa protein and a novel 195-kDa protein whose dephosphorylation rate is slower than that of receptor beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Two mutations in a conserved structural motif in the insulin receptor inhibit normal folding and intracellular transport of the receptor.

Insulin initiates its biological response by binding to the extracellular domain of the insulin receptor. The N-terminal half of the alpha-subunit contains several repeats of a loosely conserved motif consisting of a central glycine plus several hydrophobic amino acid residues upstream from the glycine, Hy phi-Xaa-Xaa-Hy phi-Xaa-Hy phi-Hy phi-Xaa-Gly (where Hy phi represents a hydrophobic amino acid residue). This structural motif has been proposed to be important in determining the three-dimensional structure of the insulin binding domain. We have identified two naturally occurring mutant alleles of the insulin receptor gene in an insulin-resistant patient, substitution of Ala for Val28 and Arg for Gly366. The mutations alter conserved amino acid residues in two distinct repeats of the structural motif described above. When mutant cDNAs were expressed in NIH-3T3 cells, both mutations severely impaired proteolytic processing of the proreceptor to mature alpha- and beta-subunits. Transport of mutant receptors to the plasma membrane was also impaired. However, the minority (< 10%) of receptors that were eventually transported to the plasma membrane retained the ability to bind insulin with normal affinity and to undergo insulin-stimulated phosphorylation. In conclusion, the effects of these naturally occurring mutations provide experimental support for the importance of the conserved glycine-containing structural motifs described above. By interrupting these structural motifs, the Ala28 and Arg366 mutations prevent normal folding of the insulin receptor alpha-subunit, thereby inhibiting post-translational processing and intracellular transport of the mutant receptors.

3T3 Cells↗

PubMed1994

The monoclonal antibody MAC387 detects an epitope on the calcium-binding protein MRP14.

The present study was initiated to identify the antigen recognized by the monoclonal antibody (MAb) MAC387 which is widely used for phenotypical characterization of myelomonocytic cells in situ. MAC387 has been described to show a similar reaction pattern as antisera to a complex formed by the calcium-binding proteins MRP8 and MRP14. However, the exact nature of the molecule recognized by MAC387 has been controversial. Using Western blot analysis, MAC387 was found to detect a single protein band of 14 kDa in lysates of human monocytes and granulocytes. Transfection of embryonic lung fibroblasts L132 with either MRP8 or MRP14 cDNA revealed that MAC387 reacts with MRP14 but not MRP8. This finding was confirmed by dot blot analysis of recombinant MRP8 and MRP14. Our data thus provide unequivocal evidence that MAC387 is a monoclonal antibody directed against the calcium-binding protein MRP14.

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↗

PubMed1994

Ultrastructural localization of the S-100-like proteins MRP8 and MRP14 in monocytes is calcium-dependent.

MRP8 and MRP14 are members of the S-100 family of Ca(2+)-binding proteins and are expressed by granulocytes and monocytes. Members of this family have been described to be involved in membrane and cytoskeleton interactions; we therefore studied the subcellular distribution of MRP8/MRP14 in cultured human monocytes at the ultrastructural level. Monospecific rabbit antisera against MRP8 and MRP14 and a monoclonal antibody (moAb 27E10), which exclusively recognizes the MRP8/MRP14 heterodimer but not the monomers, were used in both immunoperoxidase/preembedding- and immunogold/cryotechniques. Comparing non-stimulated monocytes with Ca2+ ionophore A23187-treated cells, we could demonstrate that MRP8 and MRP14 associate with membrane and cytoskeletal structures in a Ca(2+)-dependent manner. Employing moAb 27E10, MRP8/MRP14 complexes were shown to be translocated to these cellular components. In addition, immunogold double-labelling experiments revealed a clear co-localization of MRP8/MRP14 complexes with the type III intermediate filament vimentin. Analysis of immunogold-labelled cryosections of renal allografts after acute vascular rejection demonstrated that a subpopulation of infiltrating macrophages showed a similar association of MRP8/MRP14 to the cytoskeleton in situ; this finding emphasizes the in vivo relevance of our observations. We conclude that Ca(2+)-dependent translocation of MRP8/MRP14 occurs to distinct subcellular components suggesting a role of these proteins for the modulation of cytoskeletal and membrane interactions.

Antigens, Differentiation↗