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M L Hartmann

Publications and source records attributed to M L Hartmann.

6 recordsLinked to original sources
PubMed1996

Detection of a secreted MUC1/SEC protein by MUC1 isoform specific monoclonal antibodies.

Although MUC1 proteins are known to be secreted by breast cancer cells, the mechanism of their release from the cell is still obscure. Our previously reported MUC1 cDNA sequences suggested the existence of a secreted MUC1 isoform, MUC1/SEC, that includes a sequence of intron 2, and terminates prematurely at a stop codon within this intron. It is thus devoid of a transmembrane domain. As no formal evidence for MUC1/SEC expression at the protein level had been provided, we generated monoclonal antibodies (mAbs) against a peptide sequence (sec peptide) that is unique for the MUC1/SEC protein. Two anti-sec peptide mAbs were obtained which reacted strongly with (a) the immunizing peptide, (b) recombinant MUC1/SEC protein, and (c) MUC1 proteins secreted from breast cancer cells. The immunoreactivity of the anti-sec peptide mAbs with MUC1 proteins secreted by breast cancer cells was specifically inhibited by the sec peptide-it was completely unaffected by a peptide sequence that represents a MUC1 repeat motif. Significantly, the anti-sec peptide mAbs also detected MUC1/SEC protein in sera of breast cancer patients. We have established here that these mAbs recognize the MUC1/SEC isoform via a peptide sequence which is unique for the MUC1/SEC protein. Our studies thus demonstrate that the MUC1/SEC protein is a bona-fide MUC1 isoform and that its expression may contribute to the secretion of MUC1 proteins by secretory epithelial cells in general and breast cancer cells in particular.

Animals↗
PubMed1993

The Arg-4 mutant factor IX Strasbourg 2 shows a delayed activation by factor XIa.

We have characterized at the DNA and protein levels a mutant factor IX, factor IX Strasbourg 2, which is responsible for a severe form (< 0.01 U/ml) of haemophilia B. Factor IX Strasbourg 2 has a higher molecular weight than normal factor IX. A mutation G-->A at position 6365 of the gene was demonstrated by DNA sequencing and confirmed by restriction mapping which showed absence of a Hae III site. This leads to the substitution of glutamine for arginine at position -4 of the propeptide. Factor IX Strasbourg 2 was purified from plasma by DEAE Sepharose chromatography and immunoaffinity and relative to normal factor IX, binding of calcium to the mutant protein was clearly reduced in calcium lactate agarose gel. Quantification of gamma-carboxyglutamic acid residues gave about 50% carboxylation as compared to normal factor IX. Microsequencing of the NH2-terminal part of factor IX Strasbourg 2 confirmed the attachment of the propeptide and the mutation Arg-->Gln. Activation of factor IX Strasbourg 2 by purified factor XIa was found to be retarded as compared to normal factor IX, but after activation the mutant factor IXa was able to activate factor X. In conclusion, factor IX Strasbourg 2 circulates with the attached propeptide and shows reduced gamma-carboxylation and delayed activation by factor XIa but a normal capacity to activate factor X after total cleavage by factor XIa.

1-Carboxyglutamic Acid↗
PubMed1992

Antibodies to thrombomodulin induce receptor-mediated endocytosis in human saphenous vein endothelial cells.

The membrane glycoprotein thrombomodulin (TM) is an essential endothelial cell (EC) cofactor, which forms a 1:1 stoichiometric complex with thrombin. Binding of thrombin to the high affinity TM receptor transforms its procoagulant activity into an anticoagulant potential, by activating protein C. The fate of TM in the presence of thrombin is still unclear: some authors claim that the thrombin-TM complex is internalized in EC, while others find this complex to be stable for at least 2 h at 37 degrees C on the EC surface. In the present study, we investigated the interactions of thrombin and Fab-fragments of anti-TM antibodies, coupled to 5 or 15 nm gold particles with saphenous vein endothelial cells. Our results demonstrate that TM can be observed both on the plasma membrane and in coated structures only in the presence of anti-TM antibodies. Addition of thrombin decreased the extent of this labeling, while in double labeling experiments, where cells were incubated with 5 nm gold coupled thrombin and 15 nm gold coupled Fab fragments of anti-TM antibodies, thrombin was cointernalized only when anti-TM antibodies were present. These results show that thrombin-TM complex is not significantly internalized in EC. The internalization of this complex induced by anti-TM antibodies could play an important role in the thrombotic complications induced by anti-EC autoantibodies.

Amino Acid Sequence↗
PubMed1987

Antigenic regions of ribosomal protein S1 as defined by monoclonal antibodies.

Analysis of the antigenic structure of the E. coli ribosomal protein S1 was undertaken using a set of 13 monoclonal antibodies (MAbs) directed against the isolated S1. The location of the epitopes was mapped using a series of large fragments and truncated forms of S1. Most of the epitopes were localized in the C-terminal half of the molecule, while only one antibody bound to the N-terminal region. Two MAbs were able to bind to more than one region of S1, suggesting the presence of repeated epitopes related to internal sequence homologies. Six distinct antigenic domains were identified by competitive binding assays. Competition between some antibodies suggested that the C-terminal region of S1 might be in spatial proximity with the N-terminal domain in the tertiary structure of the protein. The binding of a few MAbs induced conformational changes in the protein which resulted in the complete inhibition of antibody binding at non-adjacent sites. All the MAbs reacted with the isolated form of S1 or with the protein bound to the small ribosomal subunit. This indicated that the same epitopes were expressed in the two forms of the antigen and that they were accessible to antibody binding when S1 was part of the ribosomal subunit.

Animals↗
PubMed1986

Rapid separation of Escherichia coli 30S ribosomal proteins by fast protein liquid chromatography (FPLC).

The proteins of the 30S ribosomal subunit from Escherichia coli have been separated by reverse-phase high-performance liquid chromatography on a short alkyl chain (C1/C8)-coated phase. The reverse-phase column was connected to a fast protein liquid chromatography (FPLC) system. The 21 proteins of the 30S ribosomal subunit were resolved into 16 peaks. Eleven proteins were isolated in purified form in a single chromatographic run as shown by polyacrylamide gel electrophoresis and amino acid analysis. Interestingly, the retention times of some proteins differed from the retention times observed on other reversed-phase support materials. The results show the speed and resolution of reverse-phase FPLC for both analytical and semi-preparative separations of 30S ribosomal proteins.

Amino Acids↗