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Biomedical subjects

R Blacher

Publications and source records attributed to R Blacher.

At least 19 recordsLinked to original sources
PubMed2004

MEPE has the properties of an osteoblastic phosphatonin and minhibin.

Matrix extracellular phosphoglycoprotein (MEPE) is expressed exclusively in osteoblasts, osteocytes and odontoblasts with markedly elevated expression found in X-linked hypophosphatemic rickets (Hyp) osteoblasts and in oncogenic hypophosphatemic osteomalacia (OHO) tumors. Because these syndromes are associated with abnormalities in mineralization and renal phosphate excretion, we examined the effects of insect-expressed full-length human-MEPE (Hu-MEPE) on serum and urinary phosphate in vivo, (33)PO(4) uptake in renal proximal tubule cultures and mineralization of osteoblast cultures. Dose-dependent hypophosphatemia and hyperphosphaturia occurred in mice following intraperitoneal (IP) administration of Hu-MEPE (up to 400 microg kg(-1) 31 h(-1)), similar to mice given the phosphaturic hormone PTH (80 microg kg(-1) 31 h(-1)). Also the fractional excretion of phosphate (FEP) was stimulated by MEPE [65.0% (P < 0.001)] and PTH groups [53.3% (P < 0.001)] relative to the vehicle group [28.7% (SEM 3.97)]. In addition, Hu-MEPE significantly inhibited (33)PO(4) uptake in primary human proximal tubule renal cells (RPTEC) and a human renal cell line (Hu-CL8) in vitro (V(max) 53.4% inhibition; K(m) 27.4 ng/ml, and V(max) 9.1% inhibition; K(m) 23.8 ng/ml, respectively). Moreover, Hu-MEPE dose dependently (50-800 ng/ml) inhibited BMP2-mediated mineralization of a murine osteoblast cell line (2T3) in vitro. Inhibition of mineralization was localized to a small (2 kDa) cathepsin B released carboxy-terminal MEPE peptide (protease-resistant) containing the acidic serine-aspartate-rich motif (ASARM peptide). We conclude that MEPE promotes renal phosphate excretion and modulates mineralization.

Amino Acid Sequence↗
PubMed1999

Analysis of hamster protamines: primary sequence and species distribution.

Basic nuclear proteins were isolated from the sperm of the Syrian hamster Mesocricetus auratus and characterized by gel electrophoresis, amino acid analysis, and sequencing. Analyses of the proteins by gel electrophoresis show that sperm of this species contain both protamines 1 and 2. The two proteins were purified by HPLC and the complete primary sequence of hamster protamine 1 was determined by automated amino acid sequence analysis. The protein sequence was subsequently confirmed by sequencing the PCR-amplified protamine 1 gene. The first forty-two residues of the hamster protamine 2 sequence were obtained by amino acid sequence analysis of the isolated protein, and this sequence was also confirmed and extended by sequencing the gene. Total basic nuclear protein was also isolated from sperm of six other species of hamsters, the protamines were identified by HPLC and amino acid analysis, and the proportion of protamines 1 and 2 in each species was determined. Marked differences in the protamine 2 content of sperm were observed among the different species of hamster. This variation and the high level of sequence similarity between mouse and hamster protamines provide insight into how the two protamines may be organized in sperm chromatin. Mol. Reprod. Dev. 54:273-282, 1999. Published 1999 Wiley-Liss, Inc.

Amino Acid Sequence↗
PubMed1993

Molecular cloning, expression, and chromosomal localization of the human earliest lymphocyte activation antigen AIM/CD69, a new member of the C-type animal lectin superfamily of signal-transmitting receptors.

The activation of T lymphocytes, both in vivo and in vitro, induces the expression of CD69. This molecule, which appears to be the earliest inducible cell surface glycoprotein acquired during lymphoid activation, is involved in lymphocyte proliferation and functions as a signal transmitting receptor in lymphocytes, natural killer (NK) cells, and platelets. To determine the structural basis for CD69 function, the cDNA coding for CD69 was isolated by a polymerase chain reaction-based strategy using oligonucleotides deduced from peptide sequences of the purified protein. The isolated cDNA exhibited a single open reading frame of 597 bp coding for CD69, and predicted a 199-amino acid protein of type II membrane topology, with extracellular (COOH-terminal), transmembrane, and intracellular domains. The CD69 clone hybridized to a 1.7-kb mRNA species, which was rapidly induced and degraded after lymphocyte stimulation, consistent with the presence of rapid degradation signals at the 3' untranslated region. Transient expression of the polypeptide encoded by CD69 cDNA in COS-7 cells demonstrated that it presented properties comparable to native CD69 protein. The CD69 gene was regionally mapped to chromosome 12 p13-p12 by both somatic cell hybrid DNA analysis and fluorescence in situ hybridization coupled with GTG banding (G bands by trypsin using Giemsa). Protein sequence homology search revealed that CD69 is a new member of the Ca(2+)-dependent (C-type) lectin superfamily of type II transmembrane receptors, which includes the human NKG2, the rat NKR-P1, and the mouse NKR-P1 families of NK cell-specific genes. CD69 also has a structural homology with other type II lectin cell surface receptors, such as the T cell antigen Ly49, the low avidity immunoglobulin E receptor (CD23), and the hepatic asialoglycoprotein receptors. The CD69 protein also shares functional characteristics with most members of this superfamily, which act as transmembrane signaling receptors in early phases of cellular activation.

Amino Acid Sequence↗
PubMed1990

Alpha 1 beta 1 integrin heterodimer functions as a dual laminin/collagen receptor in neural cells.

A monoclonal antibody (3A3) raised against a rat neural cell line (PC12) was shown previously to bind to the surfaces of these cells, inhibiting substratum adhesion. Immunochemical and other data indicated that the heterodimer recognized by 3A3 was a member of the integrin family of adhesive receptors and had a beta 1 subunit. The relationship of the alpha subunit to other integrins was unknown. Here we show that 3A3 recognizes in rat tissues a heterodimer (approximately 185 kDa, approximately 110 kDa; unreduced) that is electrophoretically and immunochemically indistinguishable from the antigen in PC12 cells. Immunoaffinity purification of the heterodimer from neonatal rats and protein microsequencing indicate that the alpha subunit is identical at 11 or 13 N-terminal residues with VLA-1, an integrin on human hematopoietic cells. Monoclonal antibody 3A3 inhibits the attachment of rat astrocytes to laminin or collagen but not to fibronectin or polylysine. These data suggest strongly that the integrin recognized by 3A3 is the rat homologue of VLA-1, i.e., alpha 1 beta 1, and that alpha 1 beta 1 is a dual laminin/collagen receptor.

Amino Acid Sequence↗
PubMed1990

Isolation from fetal bovine serum of an apolipoprotein-H-like protein which inhibits thymidine incorporation in fetal calf erythroid cells.

A 46 kDa heparin-binding protein which inhibits thymidine incorporation in cultures of fetal calf liver erythroid cells was isolated from fetal bovine serum by affinity chromatography on heparin-Sepharose, ion-exchange chromatography, gel filtration and reversed-phase h.p.l.c. The N-terminal sequence of the first 22 amino acids showed 81% identity with the published sequence of human apolipoprotein H. The isolated protein inhibited thymidine incorporation with an ED50 (concn. producing 50% of maximal effect) of 36 nM. A 100% inhibition of thymidine incorporation and a 40% decrease in cell numbers in cultures of fetal calf erythroid cells were observed at a protein concentration of 840 nM. No effects could be seen in cultures of 3T3 cells used as controls. Human apolipoprotein H had no inhibitory activity in any of the cell cultures tested, suggesting a species-specificity or a different structure or function for the bovine heparin-binding protein.

Amino Acid Sequence↗
PubMed1990

A heparin-binding erythroid cell stimulating factor from fetal bovine serum has the N-terminal sequence of insulin-like growth factor II.

An 8 kd heparin-binding peptide which stimulates thymidine incorporation in cultures of fetal calf liver erythroid cells was isolated from fetal bovine serum by affinity chromatography on Heparin-Sepharose, ion exchange chromatography, gel filtration and reversed-phase HPLC. The N-terminal sequence of the isolated peptide was identical to the N-terminal sequence of bovine erythrotropin or insulin-like growth factor II (IGF II). The potential heparin-binding site of IGF II is probably situated in the arginine-rich C-peptide region. The affinities of human recombinant IGF I and II were compared with those of apolipoprotein H (a plasma heparin-binding protein) and bovine insulin in a heparin-affinity column. The retention times were in the order: Apolipoprotein H greater than hrIGF II greater than hrIGF I greater than insulin (no retention). This unusual property of IGF II suggests that it may be captured in the extracellular matrix in a similar way to fibroblast growth factor, interleukin 3 or granulocyte/macrophage colony-stimulating factor.

Amino Acid Sequence↗
PubMed1988

cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors (AChRs) immunoaffinity-purified from brains are composed of only two kinds of subunits rather than the four kinds present in muscle-type AChRs. Here we report the N-terminal protein sequences of the structural subunits of AChRs from rat and chicken brains and the cloning of full-length cDNAs for the chicken brain AChR structural subunit. Previously, the N-terminal amino acid sequence of the ACh-binding subunit of AChR immunoaffinity-purified from rat brain was shown to correspond to the cDNA alpha 4. Thus, cDNA sequences are now known for both of the subunits that form one AChR subtype in vivo.

Amino Acid Sequence↗
PubMed1988

Evidence for the transneuronal regulation of cerebellin biosynthesis in developing Purkinje cells.

We have investigated the expression of a unique class of neuropeptides, the cerebellins, in normal and neurodevelopmentally mutant mice. Employing HPLC separation, gas-phase Edman sequencing, and immunocytochemistry, the normal Balb/c mouse cerebellum is shown to contain 2 Purkinje cell-specific neuropeptides, cerebellin and des-Ser1-cerebellin. In this strain of mouse the cerebellins appear during early postnatal development and their subsequent levels parallel the most dramatic period of cerebellar development: granule cell migration and parallel fiber formation, synaptogenesis, Purkinje cell dendritic maturation, and establishment of adult cytoarchitecture. In mutant mice (reeler, weaver, and staggerer), in which these early developmental events are markedly disrupted, Purkinje cells contain much lower levels of cerebellin and des-Ser1-cerebellin. In general there is a correlation between the formation and number of parallel fiber-Purkinje cell synapses and cerebellin levels. For example, the staggerer mutant, which totally lacks these synapses, is essentially devoid of cerebellin, whereas in reeler, cerebellin seems to be dependent upon the position of individual Purkinje cells and their ability to form contacts with granule cells. These results indicate that granule cells can modulate the level of cerebellin in Purkinje cells. A number of models to explain these data are discussed.

Animals↗
PubMed1986

Amino acid sequence of a unique neuronal protein: rat olfactory marker protein.

A neuron-specific protein, the olfactory marker protein (OMP), has been sequenced. This was achieved by gas phase sequencing of peptides isolated by HPLC following chemical and enzymatic cleavages of the intact rat protein. The amino terminus of the intact protein is acetylated. This has been determined by fast atom bombardment mass spectrometry of the amino terminal dodecapeptide isolated following BrCN cleavage of the OMP. Comparison of the sequence reported here with over 3000 protein sequences stored in the NBRF protein data base indicates no significant homology with any previously sequenced protein. This, coupled with the occurrence of OMP only in mature olfactory neurons of many vertebrate species, suggests that this protein has a olfactory neurons of many vertebrate species, suggests that this protein has a unique function in the metabolism of these neurons.

Acetylation↗
PubMed1985

Primary structure of rat thymus prothymosin alpha.

The primary structure of prothymosin alpha from rat thymus, containing 113 amino acid residues, is reported as follows: (formula; see text) The sequence of the first 28 amino acids at the NH2 terminus is identical to that of calf thymosin alpha 1. The dicarboxylic amino acids, which account for nearly half of the total residues in prothymosin alpha, are largely clustered in the central portion of the polypeptide chain. The polypeptide contains no aromatic or sulfur-containing amino acids. A computer analysis of the three-dimensional structure based on the primary sequence suggests that the molecule is composed of at least five alpha-helical regions interrupted by one short extended chain and three short random coils.

Amino Acid Sequence↗
PubMed1985

Parathymosin alpha: a peptide from rat tissues with structural homology to prothymosin alpha.

A peptide, parathymosin alpha, containing approximately equal to 105 amino acid residues, has been isolated from rat thymus, and the sequence of the first 30 residues at the NH2 terminus has been determined. In this region, it shows 43% structural identity with thymosin alpha 1 and prothymosin alpha. The common sequences do not include residues 2-9, which accounts for the poor reactivity of parathymosin alpha with an antibody directed against this epitope in thymosin alpha 1. Parathymosin alpha appears to modulate the action of prothymosin alpha in protecting sensitive strains of mice against opportunistic infection with Candida albicans.

Adjuvants, Immunologic↗
PubMed1985

Characterization of the polypeptide composition of human factor VIII:C and the nucleotide sequence and expression of the human kidney cDNA.

Human coagulation factor VIII:C has been purified approximately 5000-fold from commercial preparations with an average activity yield of 35%. Proteins of 92 kD and 77-80 kD enriched during purification are precipitated by a human serum polyclonal antibody which inhibits factor VIII:C activity. Evidence suggests that these polypeptides are linked by a calcium ion bridge. Partial amino acid sequence information from these proteins has been obtained from the intact polypeptides and from products of digestion with thrombin, endoproteinase lysC, or trypsin after citraconylation. An oligonucleotide probe designed from one of the amino acid sequences was used to isolate a partial genomic clone from a human 4X chromosome library in bacteriophage lambda. The genomic segment was used to isolate two cDNA molecules encompassing the entire human kidney factor VIII:C mRNA. Biologically active factor VIII:C has been produced in a mammalian cell line utilizing a complete cDNA construction.

Amino Acid Sequence↗
PubMed1984

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site.

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A1, has been determined as (sequence; see text) This is the largest fragment obtained by BrCN cleavage of the subunit A1 (Mr 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A1 polypeptide. The site of self ADP-ribosylation in the A1 subunit [C. Y. Lai, Q.-C. Xia, and P.T. Salotra (1983) Biochem. Biophys. Res. Commun. 116, 341-348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A2 subunit in the holotoxin is at position 91.

Adenosine Diphosphate Ribose↗
PubMed1984

Use of high-performance liquid chromatography for preparing samples for microsequencing.

A method has been developed for the microsequencing of protein at subnanomole levels. The protein is carboxymethylated and freed of salts and reagents by reversed-phase chromatography prior to automated Edman degradation on a gas-phase sequencer. The carboxymethylated protein can also be fragmented chemically or enzymatically for further sequence analysis. The analytical techniques used to monitor the progress of the reactions all have picomole level sensitivity.

Amino Acid Sequence↗
PubMed1984

Adrenal medullary chromaffin granule peptides: two major ovine peptides and their precursor.

An octapeptide and decapeptide which are not derived from proenkephalin were isolated from ovine adrenal chromaffin granules. Their sequences are Asn-Leu-Asp -Pro-Lys-Leu-Asp-Leu and Val-Ala-Glu-Leu-Asp-Gln-Leu-Leu-His-Tyr. These two peptides were found to be derived from a single precursor peptide which has also been isolated and sequenced. The proteolytic cleavage occurs at a Lys-Arg site typical of prohormone to hormone cleavages.

Adrenal Medulla↗
PubMed1984

Isolation and sequencing of two cerebellum-specific peptides.

By comparing the HPLC elution profiles of peptides isolated from different brain regions, two cerebellum-specific species have been identified. The peptides were isolated by sequential chromatography on reverse phase, followed by ion-pairing HPLC using alkane sulfates as pairing reagents. The sequences of both peptides have been determined by gasphase Edman degradation and carboxypeptidase Y analysis and were confirmed by synthesis. The larger peptide, termed cerebellin, is a hexadecamer of primary amino acid sequence NH(2)-Ser-Gly-Ser-Ala-Lys-Val-Ala-Phe-Ser-Ala-Ile-Arg-Ser-Thr-Asn-His- OH. The second peptide is a pentadecamer with a primary sequence identical to residues 2-16 of cerebellin, the nomenclature of which is des-Ser(1)-cerebellin. Subcellular fractionation of cerebellum followed by ion-pairing HPLC analysis shows that both cerebellin and des-Ser(1)-cerebellin are enriched between 2.5- and 4-fold in the P2 crude synaptosomal fraction, suggesting their sequestration in some subcellular particle in vivo.

Journal Article↗
PubMed1984

Purification to homogeneity and partial characterization of interleukin 2 from a human T-cell leukemia.

A method utilizing reversed-phase high-performance liquid chromatography has been developed for the purification to homogeneity of interleukin 2 (IL-2) isolated from a human T-cell leukemia. A final purification of 500,000-fold was obtained with a specific activity of pure IL-2 of 10(9) units/mg. The amino acid analysis of natural IL-2 is strikingly similar to the composition deduced from sequence analysis of a cDNA coding for human IL-2. Protein sequence analysis of CNBr-derived peptides yields data consistent with the sequence proposed from cloned cDNA. The availability of homogeneous IL-2 will allow accurate biological studies of its activity free from the contamination of the numerous lymphokine species that are known to be co-produced with IL-2 during the induction procedure.

Amino Acid Sequence↗