Biomedical subjects

U Neumann

Publications and source records attributed to U Neumann.

At least 91 records · Page 5Linked to original sources

PubMed1994

N-(sulfonyloxy)phthalimides and analogues are potent inactivators of serine proteases.

A series of 2-(sulfonyloxy) and 2-(acyloxy)-1H-isoindole-1,3(2H)-diones and analogous 1H-benz[de]isoquinoline-1,3(2H)-diones was prepared, and their potential to inactivate chymotrypsin was investigated. The N-(sulfonyloxy) and N-(acyloxy)phthalimides were found to be potent inactivators of chymotrypsin and related serine proteinases. For the most active compounds, N-(dansyloxy)phthalimide and N-(tosyloxy)phthalimide, the second-order rate constant of chymotrypsin inactivation was in the range of 250,000 m-1 s-1. N-(Mesyloxy)-phthalimide was the most active compound for inactivation of leukocyte elastase. It was shown that these compounds act as true suicide substrates. Enzyme-catalyzed opening of the heterocyclic ring results in the formation of an acyl-enzyme with attached O-acyl or O-sulfonylhydroxamic acid moiety. Subsequent Lossen rearrangement leads to the formation of a highly reactive isocyanate, which irreversibly modifies the target protease.

Acylation↗

PubMed1994

A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization.

The synthesis and enzymatic characterization of DUPAAA, a novel fluorogenic substrate for RNases of the pancreatic type is described. It consists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a fluorophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, have been attached by means of phosphodiester linkages. Due to intramolecular quenching the intact substrate displayed very little fluorescence. Cleavage of the phosphodiester bond at the 3'-side of the uridylyl residue by RNase caused a 60-fold increase in fluorescence. This allowed the continuous and highly sensitive monitoring of enzyme activity. The substrate was turned over efficiently by RNases of the pancreatic type, but no cleavage was observed with the microbial RNase T1. Compared to the dinucleotide substrate UpA, the specificity constant with RNase A, RNase PL3 and RNase U(s) increased 6-, 18-, and 29-fold, respectively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in the binding of elongated substrates. Studies on the interactions of RNase inhibitor with RNase A using DUPAAA as a reporter substrate showed that it was well suited for monitoring this very tight protein-protein interaction using pre-steady-state kinetic methods.

Aminobenzoates↗

PubMed1994

Quantitation of electrophoretically separated proteins in the submicrogram range by dye elution.

A new method for the fast, reliable and reproducible submicrogram quantitation of proteins separated by different electrophoretic techniques is presented. The method is based on a modified sensitive staining technique using Coomassie Brilliant Blue G-250 in colloidal solution combined with an optimized elution procedure of the bound dye in a 3% w/v solution of sodium dodecyl sulfate followed by photometric determination of dye concentration in the eluate. In addition a new method is provided for background correction, even suitable for gels showing strong background staining. The staining procedure allows the detection of 20 ng depending on the nature of the protein and the separation technique used. Quantitation is linear at least in the range from 50 ng to 10 micrograms and highly reproducible even under non-optimized conditions. The presented method can be applied to sodium dodecyl sulfate-electrophoresis, isoelectric focusing and two-dimensional electrophoresis.

Electrophoresis, Polyacrylamide Gel↗

PubMed1994

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor.

Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamido-contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1, Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol-1. Modifications that introduced a positive charge in position 7 (S-aminoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-1 loss in binding energy. S-protein bound to RI with Ki = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol-1 difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.

Animals↗

PubMed1994

Expression of neutral glycosphingolipids and gangliosides in human skeletal and heart muscle determined by indirect immunofluorescence staining.

The expression of neutral glycosphingolipids and gangliosides has been studied in human skeletal and heart muscle using indirect immunofluorescence microscopy. Transversal and longitudinal cryosections were immunostained with specific monoclonal and polyclonal antibodies against the neural glycosphingolipids lactosylceramide, globoside, Forssman glycosphingolipid, gangliotetraosylceramide, lacto-N-neotetraosylceramide and against the gangliosides GM3(Neu5Ac) and GM1(Neu5Ac). To confirm the lipid nature of positive staining, control sections were treated with methanol and chloroform:methanol (1:1) before immunostaining. These controls were found to be either negative or strongly reduced in fluorescence intensity, suggesting that lipid bound oligosaccharides were detected. In human skeletal muscle, lactosylceramide was found to be the main neutral glycosphingolipid. Globoside was moderately expressed, lacto-N-neotetraosylceramide and gangliotetraosylceramide were minimally expressed and Forssman glycosphingolipid was not detected in human skeletal muscle. The intensities of the immunohistological stains of GM3 and GM1 correlated to the fact that GM3 is the major ganglioside in skeletal muscle whereas GM1 is expressed only weakly. In human heart muscle globoside was the major neutral glycosphingolipid. Lactosylceramide and lacto-N-neotetraosylceramide were moderately expressed, gangliotetraosylceramide was weakly expressed and the Forssman glycosphingolipid was not expressed at all in cardiac muscle. GM3 and GM1 were detected with almost identical intensity. All glycosphingolipids were present in plasma membranes as well as at the intracellular level.

Adult↗

PubMed1994

Different distributions of glycosphingolipids in mouse and rabbit skeletal muscle demonstrated by biochemical and immunohistological analyses.

The expression of neutral glycosphingolipids and gangliosides was investigated in mouse and rabbit skeletal muscle by means of biochemical and immunochemical techniques. Neutral glycosphingolipids from muscle of the inbred rabbit strain used in this study showed a simple TLC pattern, comprising mainly monohexosylceramide. In addition to this compound, lactosylceramide, lacto-N-neotetraosylceramide, globoside and Forssman GSL were detected in mouse muscle. The major ganglioside in both species was GM3; GM3 (Neu5Ac) and GM3(Neu5Gc) were found in a 3:1 ratio in mouse muscle, whereas the absence of GM3(Neu5Gc) is characteristic of rabbit muscle. As a general structural feature of all muscle GM3 gangliosides investigated, a C18 fatty acid and C18 sphingosine were the major components besides minor C22 and C24:1 fatty acids of the respective ceramide portions, as revealed by positive and negative ion FAB-MS. alpha 2-3 sialylated lacto-N-neotetraosyl-ceramide (sialylparagloboside) was expressed in both species, whereas the alpha 2-6 sialylated isomeric compound was found only in mouse muscle. Minute quantities of ganglio-series GM1, GD1a, GD1b, and GT1b were detected in muscles from both species. Glycosphingolipid expression could be confirmed immunohistochemically by examining transverse and longitudinal cryosections of skeletal muscle samples. The results provide the basis for the investigation of muscle specific glycosphingolipids that might modulate membrane protein functions in muscle.

Animals↗

PubMed1994

Expression of gangliosides GM3 (NeuAc) and GM3 (NeuGc) in myelomas and hybridomas of mouse, rat, and human origin.

In this study gangliosides from various myelomas and hybridomas of mouse, rat, and human origin were characterized by thin-layer and high-performance liquid chromatography, immunological methods (overlay technique) and fast atom bombardment mass spectrometry. Exclusively GM3 substituted with C24:1- and C16:0-fatty acid, was found in all B cell-derived cell lines. C18 sphingosine was the single long chain base in each GM3 ceramide portion. The mouse myeloma (NS-1) and all hybridomas, obtained by fusion of mouse, rat, or human B lymphocytes with murine myelomas, showed high GM3 (NeuGc) content (> 75%) and low GM3 (NeuAc) expression. Absolute amounts of GM3 ranged from 0.2 up to 0.8 mg x 10(-9) cells. Normally, human cells do not express NeuGc, and an Epstein-Barr virus-transformed human B lymphocyte line analyzed in this study retained this sialylation status, expressing exclusively GM3 (NeuAc) (100%). The fusion of human B lymphocytes with mouse myelomas led to high GM3 (NeuGc) expression (average about 85%) in all mouse/human heterohybridomas examined. Our results indicate the chromosomal gene "transfer" and/or the activation of enzymes involved in NeuGc-biosynthesis due to the somatic cell fusion process, which might explain the mouse dominance in the manifestation of the NeuGc-phenotype in hybridomas of human origin.

Animals↗

PubMed1994

[Pilot study of the occurrence of Chlamydia psittaci infections in commercial turkey flocks in Niedersachsen].

Turkeys from 8 commercial flocks, of which were 6 clinically healthy, were examined by use of enzyme-linked immunosorbent assay systems for genus-specific Chlamydia antigens and also for antibodies against Chlamydia psittaci in 4 flocks. Results of the tests indicate the presence of Chlamydia psittaci infections in all 8 flocks. The percentage of combined cloacal and conjunctival swabs positive for Chlamydia antigen ranged from 20-91% [20-70%] whereas the percentage of antibody-positive serum samples ranged from 81-100%. Our findings suggest, that the prevalence of Chlamydia psittaci infections in clinically healthy turkey flocks is higher than hitherto suspected.

Animals↗

PubMed1993

Residues 287-301 of human ribonuclease inhibitor do not affect ribonuclease activity and inhibitor binding; a reply.

Residues 287-301 of human placental ribonuclease inhibitor have been reported to inhibit pancreatic ribonuclease A in a similar way as the entire inhibitor (Crevel-Thieffry, I., Cotterill, S. and Schuller, E. (1992) Biochim. Biophys. Acta 1122, 107-112). Using three different assays, we were unable to observe inhibition by the synthetic peptide. Moreover, the peptide did not compete with the entire inhibitor for binding to RNAase A.

Amino Acid Sequence↗

PubMed1993

Novel N-peptidyl-O-acyl hydroxamates: selective inhibitors of cysteine proteinases.

A series of N-peptidyl-O-acyl hydroxamates with a lysine in P1 was synthesized and tested as inactivators of lysosomal cysteine proteinases (cathepsins S, L, B and H) and trypsin-like serine proteinases (trypsin, thrombin, plasmin, t-PA). N-peptidyl-O-acyl hydroxamates were shown to be selective inhibitors of cysteine proteinases. With the exception of cathepsin H, the lysosomal cysteine proteinases were inactivated 2-5 orders of magnitude more rapidly than serine proteinases with a comparable primary substrate specificity. The highest second-order rate constants of inactivation for the cysteine proteinases are in the range of 10(5)-10(6) M-1 s-1. The order of inhibitor specificity for the cysteine proteinases is comparable to the enzyme's substrate specificity.

Amino Acid Sequence↗

PubMed1993

Crystallization of porcine liver ribonuclease inhibitor a member of the family of proteins containing leucine-rich repeats.

Single crystals of the RNase inhibitor from porcine liver have been obtained from 30 to 34% saturated ammonium sulphate solutions at pH 6.0 to 7.2, containing 20 mM dithiothreitol, at room temperature over a period of two to three weeks. Because the inhibitor contains 30 1/2-cystinyl residues, all of which occur in the free thiol form, crystallization experiments were carried out in a desiccator under a nitrogen atmosphere. The crystals belong to the tetragonal space group I4, with cell dimensions a = b = 134.76 A and c = 83.65 A. The asymmetric part of the unit cell contains two molecules with a molecular mass of 49,093 Da, as could be shown with a self-rotation function calculated in the resolution range 10.0 to 3.2 A. The crystals diffract to at least 3.2 A resolution and are suitable for an X-ray structure determination.

Amino Acid Sequence↗

PubMed1992

Mapping of the catalytic site of CHO-t-PA and the t-PA variant BM 06.022 by synthetic inhibitors and substrates.

BM 06.022 is a t-PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process. Until now, no X-ray and NMR structures of BM 06.022 were available. Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine-derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t-PA. Our data reveal that the single-chain as well as the two-chain form of BM 06.022 and native t-PA are similar in catalytic and in inhibitor binding properties. This indicates that the active site and the highly complex rearrangement of t-PA upon cleavage of the Arg275-Ile276 bond are maintained in BM 06.022.

Amino Acid Sequence↗

PubMed1992

Quantitation of proteins separated in N, N'-1,2-dihydroxyethylenebisacrylamide-crosslinked polyacrylamide gels.

A simple and rapid method for the quantitation of proteins separated either by sodium dodecyl sulfate-electrophoresis or by isoelectric focusing in slab gels is presented. The method is based on the solubility of polyacrylamide gels crosslinked with N, N'-1, 2-dihydroxyethylenebisacrylamide (DHEBA) in periodic acid. After electrophoretic separation proteins are stained with Coomassie brilliant blue G-250. DHEBA gels show considerable swelling during the staining and destaining process but can be shrunk to their normal size in a 10% (w/v) solution of ammonium sulfate. Stained bands are cut from the gel and solubilized in periodic acid. During dissolution the dye decolorizes. Protein concentration in the solution is determined by a modified Coomassie dye-binding assay. Quantitation is linear in the range of 100 ng to 5 micrograms and not disturbed by dissolved gel. Separations in N, N'-1, 2-dihydroxyethylenebisacrylamide-crosslinked gels show qualities similar to those in normal crosslinked gels.

Acrylamides↗

PubMed1992

Occurrence of Campylobacter spp. in young gulls, duration of Campylobacter infection and reinfection by contact.

Two groups of three week old Herring Gulls (Larus argentatus) were held to observe the carrier state with Campylobacter. All 27 birds of group I excreted Campylobacter jejuni biotype III when they were caught from their colony. Four weeks later all but one were negative, indicating that the carrier state lasts until about the seventh week of life, with self-elimination if infection with another Campylobacter species is prevented by housing in a closed environment as in this study. Only one bird became reinfected one year later when gulls from group I were brought into contact with gulls from group II, consisting of ten freshly caught gulls, four of which were infected with the same biotype of Campylobacter, indicating that there might be some kind of immunity protection against infection with the same biotype of Campylobacter spp.

Animals↗

PubMed1992

Avian paramyxovirus serotype 1 (Newcastle disease virus)--infections in falcons.

From eight falcons and one pigeon which died from NDV over a period of 15 months in Dubai, United Arab Emirates, PMV-1 viruses were isolated on quail embryo cell cultures. The identification of all 9 strains were achieved with the haemagglutination inhibition test against polyclonal chicken PMV-1 antiserum, against mouse monoclonal antibodies as well as with the immunoperoxidase test. Intracerebral pathogenicity index and intravenous pathogenicity index tests were also carried out. Although the virus isolates in this study fell into two distinct groups, the overall clinical symptoms displayed by the falcons tailed to demonstrate any trends or specificity unique to a group. The isolate obtained from a pigeon was similar to the isolates from one group of the falcons and showed no identity with the pigeon variant virus.

Animals↗

PubMed1991

Tumor-infiltrating lymphocytes isolated from a Ki-1-positive large cell lymphoma of the skin. Phenotypic characterization and analysis of cytokine secretion.

Tumor-infiltrating lymphocytes (TIL) were obtained from a biopsy of a patient with a Ki-1-positive large cell lymphoma of the skin. Immunohistologic studies of the large anaplastic tumor cells showed an "aberrant" T "helper/inducer" phenotype (CD30 + CD3-CD4+ CD8-IL-2R + HLA-DR+). Using cDNA probe for the constant region of the T-cell receptor (TCR) beta gene, the cells were identified by their distinct monoclonal rearrangement of T-cell receptor (TCR)-beta DNA. Tumor cells isolated from biopsies were cultured in the presence of interleukin-2 (IL-2). Outgrowing lymphocytes were cloned, expanded in vitro, and 11 clones were subjected to phenotypic analysis: ten clones showed a predominantly CD4-positive T "helper/inducer" phenotype whereas one clone expressed CD8 T "cytotoxic/suppressor" antigens. In contrast to the tumor cells, cells of all clones grown in vitro expressed the TCR-associated CD3 complex. Furthermore, cells from all clones analyzed expressed CD5, CD7, CD45RO (UCHL1), CD11a (LFA-1), CD25, and HLA-DR antigens. Cells of two of ten CD4-positive clones expressed CD45RA (2H4) in addition to UCHL1. T-cell clones isolated from the tumor and grown in vitro exhibited individual DNA restriction band patterns different from that of a DNA tumor biopsy specimen. Therefore, the authors conclude that these T-cell clones represent presumably nonmalignant TIL. All clones tested secreted interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha in vitro. Nine of 11 clones were found to secrete additionally IL-2 and IL-4 upon stimulation with phytohemagglutinin (PHA) whereas two clones did not secrete detectable amounts of IL-4. Selective growth of TIL in the presence of IL-2 opens the possibility to use these cells in adoptive immunotherapy of cutaneous T-cell lymphoma (CTCL). Cytokines secreted by TIL cells in vitro (IL-2, IL-4, IFN-gamma, TNF-alpha) may be involved in their antitumorigenic activity. Moreover, these data implicate that CD4-positive TIL derived from CTCL cannot be grouped into different subsets based on the production of IL-2, IL-4, IFN-gamma, and TNF-alpha.

Aged↗

PubMed1991

Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry.

YAC-1 cells were propagated in bioreactors in 1 l and 7.5 l volumes. The cells were metabolically labelled with D-[1-14C]galactose and D-[1-14C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between GM1 and GD1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of GM1b ganglioside. As well as small amounts of GM2 and GM1, the major gangliosides found in the complex mixture were GM1b and GalNAc-GM1b. The structural heterogeneity of these gangliosides was caused by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0, C24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse5Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse5Cer backbone. No cross-reaction with GM1b or GgOse4Cer was observed.

Acetylgalactosamine↗

PubMed1991

Inhibition of the protein C activator Protac, a serine proteinase from the venom of the southern copperhead snake Agkistrodon contortrix contortrix.

ProtacR, the single chain glycoprotein in the venom of Agkistrodon contortrix snake subspecies and related pit vipers has found broad application as a protein C activator in blood coagulation research and diagnosis. Inhibition studies were performed to further elucidate the nature of this serine proteinase. Benzamidine derivatives are poor inhibitors of ProtacR. Only a few 4-amidinoanilides of N alpha-substituted omega-phenyl-alpha-aminoalkylcarboxylic acids exert potent inhibitory activities. The active site of ProtacR appears to be accessible only for inhibitors with well-fitting structural features.

Anilides↗