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M Neubauer

Publications and source records attributed to M Neubauer.

At least 19 recordsLinked to original sources
PubMed1995

Cloning, mapping, and characterization of activated leukocyte-cell adhesion molecule (ALCAM), a CD6 ligand.

Antibody-blocking studies have demonstrated the role of CD6 in thymocyte-thymic epithelial (TE) cell adhesion. Here we report that CD6 expressed by COS cells mediates adhesion to TE cells and that this interaction is specifically blocked with an anti-CD6 monoclonal antibody (mAb) or with a mAb (J4-81) that recognized a TE cell antigen. We isolated and expressed a cDNA clone encoding this antigen and show that COS cells transfected with this cDNA bind a CD6 immunoglobulin fusion protein (CD6-Rg). This antigen, which we named ALCAM (activated leukocyte-cell adhesion molecule) because of its expression on activated leukocytes, appears to be the human homologue of the chicken neural adhesion molecule BEN/SC-1/DM-GRASP. The gene was mapped to human chromosome 3q13.1-q13.2 by fluorescence in situ hybridization of cDNA probes to metaphase chromosomes. We prepared an ALCAM-Rg fusion protein and showed that it binds to COS cell transfectants expressing CD6, demonstrating that ALCAM is a CD6 ligand. The observations that ALCAM is also expressed by activated leukocytes and that both ALCAM and CD6 are expressed in the brain suggest that ALCAM-CD6 interactions may play a role in the binding of T and B cells to activated leukocytes, as well as in interactions between cells of the nervous system.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1995

Cloning and characterization of murine CD6.

The lymphocyte antigen CD6 has been extensively studied in humans, but has not been reported in mouse. Here we describe the isolation and characterization of a cDNA clone encoding the murine homologue of the human lymphocyte antigen CD6. Comparison of the predicted amino acid sequence of human and murine CD6 shows that the cytoplasmic domain of murine CD6 is 160 residues longer than that of the human protein. Inspection of the 3' untranslated sequence of the human CD6 clone and the genomic organization of the murine CD6 gene suggests the expression of differentially spliced CD6 polypeptides in both species.

Amino Acid Sequence↗
PubMed1995

Cloning and expression of an endothelin receptor subtype B from human prostate that mediates contraction.

Recent evidence suggests a role for endothelin (ET) in contraction of human prostate [J. Urol. 149:495-499 (1993)]. Although both ETA and ETB receptors have been shown to mediate contraction of smooth muscle, the molecular identity of the contractile ETB receptor is controversial. The aim of this study was to examine the receptor subtype that mediates ET-induced contraction in prostate from patients with benign prostatic hyperplasia. Saturation binding with 125I-ET-1 and 125I-ET-3 in prostate stromal cells (PSC) indicated the presence of receptors with subnanomolar affinity for these radioligands, with equivalent receptor densities. Inhibition of specific 125I-ET-1 or 125I-ET-3 binding in PSC revealed a rank order of potency of ET-1 - ET-3 = sarafotoxin S6c >> BQ-123. These data are consistent with a predominance of ETB receptors in PSC. The functional effects of ET stimulation of PSC were examined in a collagen gel contraction assay. ET-1 and ET-3 caused contraction of underlying collagen gel matrices with EC50 values of 0.4 +/- 0.04 and 0.7 +/- 0.2 nM, respectively. To determine the molecular nature of the contractile ETB receptor in PSC, reverse transcription-polymerase chain reactions were conducted with oligonucleotide primers to the 5' and 3' ends of the coding sequence of the full length human ETB receptor. DNA sequence analysis of the 1.3-kilobase DNA product showed 99% homology to other human ETB receptor cDNAs. The encoded protein has a deduced amino acid sequence identical to that of other human ETB receptors, with the exception of two conservative substitutions. Expression of the PSC ETB cDNA in COS-7 cells resulted in a binding profile similar to that observed in parent cells. Polymerase chain reaction analysis revealed the presence of prepro-ET-1 mRNA in PSC. Collectively, these data indicate that PSC from patients with benign prostatic hyperplasia express ETB receptors that mediate ET-induced contraction.

Amino Acid Sequence↗
PubMed1994

beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice.

beta ig-h3 is a novel gene first discovered by differential screening of a cDNA library made from A549 human lung adenocarcinoma cells treated with transforming growth factor-beta 1 (TGF-beta 1). It encodes a 683-amino-acid protein containing a secretory signal sequence and four homologous internal domains. Here we show that treatment of several types of cells, including human melanoma cells, human mammary epithelial cells, human keratinocytes, and human fibroblasts, with TGF-beta resulted in a significant increase in beta ig-h3 RNA. A portion of the beta ig-h3 coding sequence was expressed in bacteria, and antisera against the bacterially produced protein was raised in rabbits. This antisera was used to demonstrate that several cell lines secreted a 68-kD beta IG-H3 protein after treatment with TGF-beta. Transfection of beta IG-H3 expression plasmids into Chinese hamster ovary (CHO) cells led to a marked decrease in the ability of these cells to form tumors in nude mice. The beta IG-H3 protein was purified from media conditioned by recombinant CHO cells, characterized by immunoblotting and protein sequencing and shown to function in an anti-adhesion assay in that it inhibited the attachment of A549, HeLa, and WI-38 cells to plastic in serum-free media. Sequencing of cDNA clones encoding murine beta ig-H3 indicated 90.6% conservation at the amino acid level between the murine and human proteins. Finally, the beta ig-h3 gene was localized to human chromosome 5q31, a region frequently deleted in preleukemic myelodysplasia and leukemia. The corresponding mouse beta ig-h3 gene was mapped to mouse chromosome 13 region B to C1, which confirms a region of conservation on human chromosome 5 and mouse chromosome 13. We suggest that this protein be named p68 beta ig-h3.

Amino Acid Sequence↗
PubMed1994

Cloning and expression of the gene for the melanoma-associated ME20 antigen.

Human melanoma cells, but not tumor cells of other histological origin, express a unique membrane-associated glycoprotein, designated ME20-M, and secrete a soluble glycoprotein, designated ME20-S, defined by monoclonal antibody ME20. Here we report the isolation and characterization of a cDNA clone that when transfected into COS cells directs the expression of ME20-M and ME20-S. This cDNA contains an open reading frame which encodes a 661-amino-acid-long precursor that contains a 23-amino-acid signal peptide and a 26-amino-acid transmembrane domain, separated by a hydrophilic region containing 5 potential Asn-linked and 14 predicted Pro-associated, Thr-linked glycosylation sites. The transmembrane domain is followed by a carboxy-terminal 45-amino-acid putative intracellular domain rich in Ser residues. Analysis of ME20-M by amino acid sequencing identified the proteolytic processing site. Signal peptide cleavage occurs at the Thr-24-Lys-25 peptide bond of the precursor and results in the 637-amino-acid ME20-M with a calculated molecular weight of 67,782. ME20-M is derived from a single 3.3- to 3.4-kb mRNA transcript that is expressed at varying levels in melanoma cell lines, correlating with immunofluorescence determination of protein expression. The amino acid sequence of the ME20 antigen deduced from the cDNA differs from the human neonatal melanocyte-specific Pmel 17 gene product by a single amino acid substitution and deletion of 7 amino acid residues, and it is 80% homologous with the bovine retinal pigment RPE1 cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Suppression of chronic lymphocytic leukaemia by megakaryocytic myelosis.

In a 77-year-old Caucasian female B-cell chronic lymphocytic leukaemia was diagnosed and classified as stage 1 according to the Rai classification. The disease remained stable and therefore no antileukaemic therapy had to be initiated. Four years after the initial diagnosis the patient developed hepatosplenomegaly, anaemia and leukopenia. Bone marrow biopsy revealed megakaryocytic myelosis supervening upon the pre-existing chronic lymphocytic leukaemia.

Aged↗
PubMed1993

The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome.

The prominent role of the CD40 receptor in B cell responses led us to investigate the role of the gp39-CD40 interaction in a group of primary immunodeficient patients with defective antibody production. Here we report that patients with hyper-IgM syndrome (HIM) have a defective gp39-CD40 interaction. B cells from HIM patients express functional CD40, but their T cells do not bind CD40-Ig. These patients expressed normal levels of gp39 mRNA, but these mRNAs encode defective gp39 proteins owing to mutations in the extracellular domain of gp39. Soluble recombinant forms of gp39 containing these mutations were unable to bind CD40 and drive normal B cell proliferation. The gene encoding gp39 was mapped to Xq26, the X chromosome region where the gene responsible for HIM had previously been mapped. These data suggest that a defect in gp39 is the basis of X-linked HIM.

Adolescent↗
PubMed1992

Successful treatment of primary adrenal insufficiency due to malignant non-Hodgkin's lymphoma.

Although the adrenal glands are frequently the site of tumor metastases, adrenal insufficiency is exceedingly rare. We report on a patient with high-grade B-cell centroblastic lymphoma who initially presented with right axillary lymphadenopathy and bilateral adrenal masses. Four months after axillary lymphadenectomy the patient developed overt signs of Addison's disease. He recovered promptly after initiation of hormone replacement therapy and bilateral adrenalectomy. At present, 16 months after additional chemo- and radiation therapy the patient is considered free of tumor. To our knowledge this is the first report on a patient who presented with adrenal insufficiency in the course of non-Hodgkin's lymphoma and who was successfully treated. Demonstrating this case, we would also like to stress that the development of adrenal insufficiency does not necessarily indicate widespread tumor manifestation in patients with non-Hodgkin's lymphoma.

Adrenal Cortex Hormones↗
PubMed1992

cDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) is capable of affecting the proliferation of many cell types. To identify novel genes whose protein products may mediate cellular responses to this factor, a cDNA library was made from mRNA isolated from a human lung adenocarcinoma cell line (A549) that had been treated for 3 days with TGF-beta. The library was screened by differential hybridization and a cDNA clone, beta ig-h3, was isolated. This gene was induced up to 20-fold in A549 cells after 2 days of treatment with TGF-beta 1. It was also induced in several other cell lines, including PC-3 and H2981. DNA sequence analysis of beta ig-h3 indicated that it encoded a novel protein, beta IG-H3, of 683 amino acids, which contained an amino-terminal secretory sequence and a carboxy-terminal Arg-Gly-Asp (RGD) sequence that can serve as a ligand recognition site for several integrins. beta IG-H3 also contained short amino acid regions homologous to similar regions in Drosophila fasciclin-I and four homologous internal domains, which can be folded into a potential bivalent structure and could act as a bridge between cells expressing the appropriate ligand. beta ig-h3 RNA was detected in several cell lines and tissues. COS cells transfected with plasmids encoding beta IG-H3 secreted a major 68-kD protein that was detected by immunoblotting using antipeptide antibodies. Since beta ig-h3 is induced in several cell lines whose proliferation is affected by TGF-beta 1, it may be involved in mediating some of the signals of this multifunctional growth modulator.

Adenocarcinoma↗
PubMed1992

Site-directed mutagenesis of glycosylation sites in the transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 (414) precursors and of cysteine residues within mature TGF beta 1: effects on secretion and bioactivity.

The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.

Amino Acid Sequence↗
PubMed1991

Identification of a gene family regulated by transforming growth factor-beta.

We have identified two related genes whose mRNAs are increased after treatment with transforming growth factor-beta (TGF-beta 1). Mouse AKR-2B cells were treated with TGF-beta 1 in the presence of cyclohexamide and a cDNA library was subjected to differential screening. Several TGF-beta-induced genes (beta IG) were isolated and two of these, beta IG-M1 and beta IG-M2, were characterized. beta IG-M1 and beta IG-M2 RNAs were significantly increased after TGF-beta 1 treatment and both were superinduced in the presence of cyclohexamide. cDNA sequence analysis of beta IG-M1 showed that it encoded a 379-amino-acid protein which was 81% homologous to CEF-10, a v-src and TPA-inducible gene, and identical to cyr61, a gene induced by serum in growth-arrested BALB-3T3 cells. cDNA sequence analysis of beta IG-M2 showed that it encoded a 348-amino-acid protein that was 50% homologous to beta IG-M1. Thirty-eight cysteine residues are conserved between beta IG-M1 and beta IG-M2, which are clustered at the amino and carboxy ends: The middle regions of the two proteins are cysteine free and display the highest degree of nonhomology. Both proteins contain an amino-terminal cysteine-rich motif common to insulin-like growth factor binding proteins and a carboxy-terminal domain with strong homology to a motif found near the carboxy-terminal of the malarial circumsporozoite protein which may be involved in cell adhesion. The regulation of mRNA encoding these proteins by TGF-beta 1 suggests that they may be involved in mediating some of the pleiotropic effects of this multipotent modulator of cell growth and differentiation.

Amino Acid Sequence↗
PubMed1990

Peripheral blood lymphocytes of nonleukemic lymphoma patients exhibit aberrant expression of T-cell activation markers after polyclonal stimulation in vitro.

The authors evaluated suppressed in vitro functions of peripheral blood lymphocytes (PBL) as a possible tool in the early diagnosis of human lymphoma. In 13 of 22 patients with recent onset of various types of nonleukemic lymphomas (Mb. Hodgkin and non-Hodgkin's lymphomas of B-cell and T-cell origin) the mitogen response of PBL against phytohemagglutinin (PHA) and concanavalin A (Con A), as measured by 3H-thymidine (3HTdR) uptake, was found to be significantly suppressed, whereas the response to pokeweed mitogen (PWM) was normal in 18 cases. In parallel, cytofluorimetric analysis was done with PBL after 72 hours in culture with and without PHA, using antibodies against the differentiation antigens: CD3, CD8, CD4, CD19, and CDw14 and the activation antigens: interleukin-2 (IL-2) receptor (IL-2R, CD25), human leukocyte antigen DR (HLA-DR), and transferrin receptor (TR). Compared with healthy controls and patients with other diseases, a significant reduction of the total T-cell blast response, i.e., the percentage of large T-cells bearing activation markers, was found in all lymphoma cases including those with a normal 3HTdR uptake. Furthermore, a pronounced inhibition in the expression of the activation markers Il-2R and TR, but not of HLA-DR, was detected on CD3+ cells in PHA-stimulated PBL of all lymphoma cases. Thus, polyclonal activation combined with activation antigens seems to give more accurate information about the functional defect(s) of PBL in an early state of lymphoma; these parameters may therefore be valuable diagnostically. The abnormal pattern in the expression of T-cell activation antigens after polyclonal stimulation may help in the understanding the cellular immune defects associated with lymphoma.

Adolescent↗
PubMed1990

[Sex disorders in male diabetic patients].

Sexual disorders occur in about 50% of male diabetics, appearing in two different forms: (1) temporary disorders caused mainly be general disturbances due to metabolic derangements; (2) chronic disorders characteristic of diabetes, although their symptomatology is not typical. Here, erectile dysfunction predominates. The etiology has not yet been definitely clarified. Psychological, neurogenic, vascular and endocrine factors are all involved. In the individual case, it is necessary to identify the major causes by employing subtile investigative measures, in order to be able to plan rational treatment.

Aged↗
PubMed1990

Molecular cloning of a novel bone-forming compound: osteoinductive factor.

cDNA clones encoding osteoinductive factor (OIF) have been isolated from a bovine osteoblast library. Sequence analysis of these clones indicated that the 105-amino-acid OIF is synthesized as a larger 299-amino-acid precursor, the carboxyl terminus of which is cleaved to yield the mature protein. Northern blot analysis of bovine osteoblast mRNA revealed two OIF-specific transcripts of 1.9 and 2.4 kb. The polymerase chain reaction was used to obtain clones coding for human OIF from the osteosarcoma cell line, MG-63. The human OIF cDNA encodes a precursor of 298 amino acids that exhibits 94% identity to the bovine protein. Northern blot analysis of various cell lines and tissues indicated that expression of OIF transcripts is limited and may be restricted to cells of bone lineage.

Amino Acid Sequence↗
PubMed1990

Cleavage of a hydrophilic C-terminal domain increases growth-inhibitory activity of oncostatin M.

Oncostatin M is a polypeptide cytokine, produced by normal and malignant hematopoietic cells, that has several in vitro activities, including the ability to inhibit growth of cultured carcinoma cells. Here we present a structural and functional comparison of two oncostatin M-related proteins (Mr 36,000 and 32,000) secreted by COS cells transfected with oncostatin M cDNA. The smaller of these forms lacked a hydrophilic C-terminal domain comprising predominantly basic amino acids. This domain was also absent from native oncostatin M produced by U937 cells. The 32,000-Mr form of oncostatin M was not produced by cells transfected with plasmids (G195 and G196) in which a potential trypsinlike cleavage site within the hydrophilic C-terminal domain was altered by site-directed mutagenesis. A 32,000-Mr fragment was produced by trypsin treatment of the 36,000-Mr form of oncostatin M. These observations suggest that the 32,000-Mr form of oncostatin M was derived from the 227-amino-acid propeptide by proteolytic cleavage at or near the paired basic residues at positions 195 and 196. Pro-oncostatin M was equally active in radioreceptor assays as the processed form but was 5- to 60-fold less active in growth inhibition assays. Likewise, nonprocessed mutant protein encoded by plasmid G196 was equally active in the radioreceptor assays as the processed form but was five- to ninefold less active in growth inhibition assays. Thus, the highly charged C-terminal domain of pro-oncostatin M is not required for receptor binding or growth-inhibitory activity but may alter the functional properties of the molecule. Propeptide processing of oncostatin M may be important for regulating in vivo activities of this cytokine.

Amino Acid Sequence↗