Biomedical subjects

A Henschen

Publications and source records attributed to A Henschen.

At least 73 records · Page 4Linked to original sources

PubMed1988

Rat sperm protamine. Isolation and sequence analysis.

Rat protamine was extracted from S-(pyridylethylated) epididymal sperm cell nuclei with dilute hydrochloric acid. The final purification was achieved by reversed-phase high-performance liquid chromatography. The primary structure was determined by N-terminal sequencing of the total S-(pyridylethylated) protein, and of endoproteinase Lys-C- and thermolysin-derived fragments. Rat protamine consists of 50 amino-acid residues. It is a typical type 1 protamine and differs in two and ten positions from the corresponding mouse and rabbit protamine, respectively. Only 26 positions are invariant in all type 1 mammalian protamines.

Amino Acid Sequence↗

PubMed1988

Evidence for the identity of hirudin isolated after kidney passage with the starting material.

Natural, sulfated and recombinant, non-sulfated hirudin preparations re-isolated from urine after kidney passage were compared with the corresponding administered preparations by reversed-phase high-performance liquid chromatography, and by amino acid sequence and composition analysis. It could be demonstrated that the hirudins were excreted in unmodified form. Natural hirudin could be fractionated by the chromatographic system used into a large number of isoinhibitor forms, some of which were shown to differ from each other in their amino acid sequence.

Amino Acid Sequence↗

PubMed1987

Acrosin shows zona and fucose binding, novel properties for a serine proteinase.

The major fucose-binding protein of 53 kDa from boar spermatozoa was isolated to apparent homogeneity using a two-step procedure including high-performance gel filtration and reversed-phase chromatography. The N-terminal sequence of the protein revealed that it is identical with the sperm proteinase acrosin. By means of a solid-phase zona-binding assay based on the avidin-biotin system it was demonstrated that acrosin also interacts strongly with porcine zona pellucida. Thus, the acrosin molecule combines specific proteolytic activity with zona- and carbohydrate-affinity properties, i.e. previously unrecognized properties of a serine proteinase. It seems likely that this special affinity of acrosin directs the proteolytic activity to its structural target in the vivo situation.

Acrosin↗

PubMed1987

The innervation of intraocular spinal cord transplants by cografts of locus ceruleus and substantia nigra neurons.

The nucleus locus ceruleus and the substantia nigra were sequentially cografted with spinal cord in the anterior chamber of the eye of adult rats. The double grafts were allowed to mature in oculo. After a survival time of 2 months, half of the number of rats were sacrificed, the grafts were dissected out and prepared for tyrosine hydroxylase immunohistochemistry and Falck-Hillarp monoamine histochemistry. The remaining animals were sacrificed after 12-14 months and processed according to the same protocol. No ingrowth from the sympathetic ground plexus in the host iris was seen in single spinal cord grafts at any of the survival times. At the shorter survival time there were no or very few catecholaminergic nerve fibers from the locus ceruleus or substantia nigra that were innervating the spinal cord cograft. In double grafts that were allowed to remain in oculo for 12-14 months, the entire spinal cord graft was innervated with varicose, catecholamine-containing nerve fibers. The locus grafts were able to innervate the spinal cord cografts much more densely than the substantia nigra grafts. In conclusion, the results demonstrate that isolated intraocular spinal cord grafts can be innervated by adjacent central catecholamine neurons. However, this is done in a delayed manner as compared to other models. The isolated replicas of descending adrenergic pathways to the spinal cord thus formed should provide useful information about the function of descending spinal catecholamine pathways.

Animals↗

PubMed1987

Arrangement of the disulphide bridges in human low-Mr kininogen.

The arrangement of the disulphide bridges in human low-Mr kininogen has been elucidated. Low-Mr kininogen contains 18 half-cystine residues forming nine disulphide bridges. The first and the last half-cystine residues of the amino acid sequence form a disulphide loop which spans the heavy- and the light-chain portion of the kininogen molecule. The other 16 half-cystine residues are linked consecutively to form eight loops of 4-20 amino acids; these loops are lined up in the heavy-chain portion of the kininogen molecule. In this way, a particular pattern of disulphide loops is formed which seems to be of critical importance for the inhibitor function of human kininogen.

Amino Acid Sequence↗

PubMed1987

Fibrinogen Haifa: fibrinogen variant with absence of protective effect of calcium on plasmin degradation of gamma chains.

The abnormal fibrinogen Haifa is characterized by the fact that calcium present during enzymatic digestion by plasmin does not protect the Haifa D gamma chain against further plasmin attack as it does in normal molecules. Since calcium binding to fibrinogen, ADP--platelet aggregation cofactor activity and gamma dimerization process induced by factor XIIIa are normal for fibrinogen Haifa, the corresponding sequences in the gamma chain are not involved. It seems rather that the anomaly resides near the gamma 302 plasmin cleavage site that is protected when calcium is bound to the gamma chain and that this affects the availability of the polymerization site located in the C terminal part of the chain.

Adult↗

PubMed1987

Plasminogen activator inhibitors from placenta and fibrosarcoma cells are antigenically different as evaluated with monoclonal and polyclonal antibodies.

Plasminogen activator inhibitor (PAI) purified from human placenta was compared to PAI purified from conditioned cell culture fluid of the human fibrosarcoma cell line HT-1080. The two inhibitors had a similar mobility (Mr approximately 50,000) in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Purified placental inhibitor revealed 2 major and 1 minor Coomassie blue stainable bands, while the fibrosarcoma inhibitor appeared as one band. By immunoblotting analysis both monoclonal and polyclonal antibodies against each of the inhibitors showed reaction with the inhibitor against which they were raised, but not cross reaction with the other inhibitor. Similar results were obtained, when antibody binding was tested by ELISA with the inhibitors coated on the solid phase. HPLC fingerprint patterns of cyanogen bromide fragments of the two inhibitors were different. The inhibitory activity of the placental PAI was decreased by a factor of 3 after incubation with SDS, while that of the fibrosarcoma PAI was increased by a factor of 30. It is concluded that the two inhibitors show no detectable common antigenic determinants and most likely are products of different genes.

Antibodies↗

PubMed1987

Heavy riboflavin synthase of Bacillus subtilis. Primary structure of the beta subunit.

Heavy riboflavin synthase is a 1,000,000-Da protein catalyzing the last two reactions of riboflavin biosynthesis. The enzyme complex consists of 60 beta subunits (Mr = 16,200) and approximately three alpha subunits (Mr = 23,000). beta subunits were isolated and cleaved with cyanogen bromide. Fragments were isolated and further digested with trypsin and staphylococcal protease. Peptides were isolated by high performance liquid chromatography. Sequences were determined by automated liquid-phase Edman degradation. The complete sequence of the beta subunit (154 amino acids) was established by direct sequencing of the NH2 terminus, sequencing of overlapping peptides, and carboxypeptidase degradation of the COOH terminus. The sequence shows no detectable homologies to other proteins. A computer prediction of secondary structure elements indicates 34% alpha helix and 30% beta sheet.

Amino Acid Sequence↗

PubMed1987

The major protamine from stallion sperm. Isolation and amino-acid sequence.

The major stallion protamine was isolated from sperm cell nuclei by extraction with 6M guanidine/5% mercaptoethanol, alkylation with 4-vinylpyridine and subsequent reversed-phase high-performance liquid chromatography. The primary structure of stallion protamine was determined by N-terminal sequencing of the intact protein and of the fragments obtained from thermolysin cleavage of the S-pyridylethylated and from endoproteinase Lys-C cleavage of the S-aminoethylated protein. Stallion protamine consists of 49 amino-acid residues and shows 49% identity with all other sequenced mammalian type 1 protamines.

Acetates↗

PubMed1986

Three abnormal fibrinogen variants with the same amino acid substitution (gamma 275 Arg----His): fibrinogens Bergamo II, Essen and Perugia.

We report on three unrelated individuals with the same uncommon type of dysfibrinogenemia, originating from Bergamo, Essen and Perugia. None of them showed bleeding symptoms while the Bergamo patient and members of her family presented with a thrombotic tendency. The presence of a defective fibrinogen was suggested by prolonged thrombin and reptilase times. Furthermore, fibrinogen concentrations of less than 0.28 g/L were determined by the functional assay whereas values of 1.5-2.4 g/L were measured by heat precipitation or electroimmunoassay. Fibrinogen was isolated by affinity chromatography on insoluble fibrin monomer. The rate of fibrinopeptide release by thrombin was normal while the fibrin polymerization reaction was strongly delayed. An abnormal peptide (gamma 265-310) was isolated by high-performance liquid chromatography after cyanogen bromide cleavage of the purified gamma-chain of fibrinogen Bergamo II and Essen. The same peptide was also isolated following cyanogen bromide treatment of the intact fibrinogen Perugia. Sequence analyses of these peptides demonstrated the same amino acid exchange in all three fibrinogens: gamma 275 arginine----histidine. The described fibrinogen variants appear to possess a molecular defect which has thus far only been observed in fibrinogen Haifa.

Amino Acid Sequence↗

PubMed1986

Identification and assay of phosphoserine and tyrosine-O-sulphate in fibrinopeptides by reversed-phase high-performance liquid chromatography.

A procedure utilizing reversed-phase high-performance liquid chromatography (HPLC) is described for the identification and quantitation of individual phosphorylated and sulphated fibrinopeptides present in fibrin clot supernatants. Fibrinopeptides from human, rabbit and canine fibrinogens, which have different structures and degrees of phosphorylation and sulphation, were used to demonstrate the applicability of these methods. The procedure relies on the increased peptide hydrophobicity following removal of highly charged phosphate or sulphate groups. Dephosphorylated or desulphated peptides are thus more strongly retained on the reversed-phase HPLC column and are eluted later than their corresponding phosphorylated or sulphated peptide counterparts. Dephosphorylation is achieved by treatment of fibrinopeptide-containing clot supernatants with alkaline phosphatase. Phosphorylated peptides are characterized by an increased retention time resulting from loss of phosphate, whereas non-phosphorylated peptides remain unaffected. Similarly, a prolongation of the peptide retention time resulting from desulphation by mild acid hydrolysis serves to verify sulphation of a peptide.

Amino Acids↗

PubMed1986

Characterization of human platelet basic protein, a precursor form of low-affinity platelet factor 4 and beta-thromboglobulin.

Platelet basic protein (PBP) was purified from the supernatant of thrombin-stimulated, washed human platelets by ion-exchange, affinity, molecular sieve, and high-performance liquid chromatography (HPLC). The NH2-terminal amino acid sequence was determined by automated Edman degradation, revealing 9 unique residues followed by 10 residues of the established low-affinity platelet factor 4/beta-thromboglobulin (LA-PF4/beta TG) sequence. Among the nine were three basic residues, accounting for the high isoelectric point of PBP. Additional evidence for precursor status includes the immunological cross-reactivity of all three species and the ability of plasmin and trypsin to produce from PBP a species resembling beta TG in charge, hydrophobicity, and size. Tryptic peptide maps of PBP and LA-PF4 obtained by reverse-phase HPLC were very similar, and from each protein, a peptide was isolated which showed the amino acid composition predicted for the COOH-terminal tryptic peptide of beta TG. Normal platelets contained predominantly LA-PF4, with PBP ranging from 10% to 30% of total beta TG antigen. This was true even when fresh platelets were lysed with trichloroacetic acid in order to provide the most complete and rapid inhibition of proteolytic activity. beta TG itself was never detected in this situation or in the release supernatant of stimulated platelets, and only rarely in unprotected lysates. In agreement with earlier results, crude preparations of PBP were mitogenic for 3T3 cells, but highly purified preparations of PBP and LA-PF4 were free of this activity.

Amino Acid Sequence↗

PubMed1986

Completion of the primary structure of human high-molecular-mass kininogen. The amino acid sequence of the entire heavy chain and evidence for its evolution by gene triplication.

The amino acid sequence of the heavy chain of human high-molecular-mass kininogen has been determined. It completes the primary structure of the high-Mr kininogen molecule. The heavy chain contains 362, the total kininogen molecule 626 amino acid residues. Three carbohydrate side chains were found in the heavy chain, all of them N-glycosidically linked to asparagine, which is present in the acceptor sequon Asn-Xaa-Thr (or -Ser); one additional potential glycosylation site devoid of a sugar side chain is found at position 30. There is a high degree of homology between the heavy chains of human high-Mr kininogen and bovine high-Mr kininogen (74% identity), or rat T-kininogen (61%). Comparison of the primary structure of human high-Mr kininogen with that of human low-Mr kininogen predicted from its cDNA sequence, reveals that the heavy chains of the two human kininogens are completely identical. Two heavy chain segments believed to contain the reactive sites for cysteine proteinase inhibition show an extensive sequence homology with other mammalian cysteine proteinase inhibitors. Within the heavy chain of human high-Mr kininogen are repetitive units strongly suggesting that the heavy chain of human kininogens has evolved from at least two ancestral units by a series of gene duplication and fusion events.

Amino Acid Sequence↗

PubMed1986

Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins.

A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75-kbp plasmid from a toxigenic non-sporulating strain of Clostridium tetani. The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol. wt of 150,700. In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol. wt light chain and the 98,300 mol. wt heavy chain, respectively. Cysteine (467) was involved in the disulfide linkage between the two subchains. The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C. tetani and C. botulinum are derived from a common ancestral gene. Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies. The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E. coli.

Amino Acid Sequence↗

PubMed1986

Amino acid sequence of the light chain of human high molecular mass kininogen.

The light chain of human high molecular mass kininogen consists of 255 amino acid residues. The half-cystine residue which forms the single disulfide bridge to the heavy chain is located in position 225. The light chain contains 9 O-glycosidically linked carbohydrate side chains. A comparison of the human high molecular mass kininogen light chain with the bovine high molecular mass kininogen light chain reveals a pronounced homology. However, in position 88 of the human sequence an insertion of 22 amino acid residues was found. This insertion and an amino acid exchange in position 131 may explain the different behaviour of human and bovine high molecular mass kininogen during plasma kallikrein digestion.

Amino Acid Sequence↗

PubMed1986

Amino acid sequence of the light chain of human low molecular mass kininogen.

The light chain of human low molecular mass kininogen consists of 38 amino acid residues. The half-cystine residue which forms the disulfide bridge to the heavy chain is located in the position 18. Alignment of the low molecular mass kininogen light chain with corresponding sections of other kininogens revealed that the N-terminal part of it is species specific and the C-terminal part is function specific. Furthermore, some internal homologies between various sections of the total molecule were found. A statistically significant sequence homology between the low molecular mass kininogen light chain and the C-terminal part of the ribonucleases was observed.

Amino Acid Sequence↗

PubMed1986

Limited proteolysis of HMW kininogen by plasma kallikrein in man--evidence for a processing mechanism different from the bovine system.

The limited proteolysis of human HMW kininogen by plasma kallikrein has been studied. Kallikrein liberated bradykinin from HMW kininogen (Mr 114 kDa) and generated a two-chain molecule with a heavy chain of Mr 63 kDa and a light chain of Mr 58 kDa interconnected via a single disulfide bridge. As proteolysis proceeded, a step-wise processing of the initially formed light chain occurred giving rise to modified light chains of Mr 45 and 41 kDa. Sequence analysis indicated that two polypeptides had been cleaved from the amino- and carboxy-terminal parts of the 58 kDa light chain. Major part of the histidine-rich peptide which is critical to surface binding of HMW kininogen was kept in the shortened light chains. These findings are consistent with the observation that trimming of the human HMW kininogen does not abolish its procoagulant activity. By contrast, the bovine HMW kininogen is inactivated due to removal of the entire histidine-rich peptide. Hence, the proteolytic processing mechanisms for HMW kininogen are distinct in the human and the bovine contact phase activation systems.

Animals↗

PubMed1986

Raphe dorsalis-spinal cord cografts in oculo: electrophysiological evidence for an excitatory serotonergic innervation of transplanted spinal neurons.

Intraocular replicas of descending serotonergic bulbospinal pathways were constructed by means of sequential intraocular grafting of nucleus raphe dorsalis and spinal cord. Using extracellular recordings we have studied the functional connections between such double grafts. Superfusion of single spinal cord grafts with serotonin causes an increase in spontaneous activity. This excitation is reversibly blocked by the specific 5-hydroxytryptamine (5-HT) antagonist metergoline. Stimulation of the raphe part of nucleus raphe dorsalis-spinal cord double grafts causes a long-lasting excitation of the spinal neurons similar to that seen in single spinal cord grafts given serotonin. The electrically induced excitation could also be reversibly blocked with metergoline. It is concluded that serotonin-containing nerves from grafts of nucleus raphe dorsalis are not only morphologically organotypic, but also form functional contacts with neurons in cografted spinal cord. The results further support an excitatory or modulatory role of the descending spinal serotonergic pathways and demonstrate that functional contacts can be established between isolated CNS grafts when 5-HT fibers invade immature or mature spinal cord tissue.

Animals↗