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U Neumann

Publications and source records attributed to U Neumann.

At least 19 recordsLinked to original sources
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↗
PubMed1991

Interleukin-4 promotes the expansion of skin-infiltrating lymphocytes from atopic dermatitis in vitro.

Functional studies of lymphocytes in atopic dermatitis (AD) have so far focused on peripheral blood mononuclear cells (PBMC), whereas cells at the involved site, the skin, have not been examined. Accordingly, we have developed methods to generate lymphocyte cultures from biopsies of inflammatory skin areas. Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of 6 patients with severe AD and expanded in vitro in the presence of interleukin-2 (IL-2) without additional antigens. After 6-10 d in culture, outgrowth of mononuclear cells from biopsy tissue was observed in all cases. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T-helper/inducer phenotype in SIL populations. Parallel cultures of SIL and PBMC showed an increase and expansion of CD8+ T cells in cultured PBMC, whereas the CD4+ phenotype was predominant in SIL cultures. As indicated by their expression of HLA-DR and CD25 antigens, most of the SIL were activated and the cells mainly expressed T-cell receptors (TCR) composed of alpha and beta chains. Different strategies for expansion of SIL in vitro were examined. High levels of IL-4 (1,000 U/ml) in combination with IL-2 (50 U/ml or 1,000 U/ml) preferentially promoted growth of SIL derived from AD and were much more effective than IL-2 alone. No cells expanded in cultures with IL-4 alone. SIL grown with high concentrations of IL-4 contained a significant proportion of double-positive CD4+8+ cells. No other marked differences were observed in the distribution of T cell subsets in cultures propagated under different conditions for 21 d. Our results demonstrate the feasibility of growing infiltrating T lymphocytes from inflammatory skin of AD patients. The use of high concentrations of IL-2 in combination with high levels of IL-4 allows a large expansion of these cells and thus represents a useful strategy to expand cells for further functional and molecular biologic studies.

Adult↗
PubMed1991

Functional characterization of skin-infiltrating lymphocytes in atopic dermatitis.

Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of patients with hyperimmunoglobulin E (IgE) atopic dermatitis (AD) and expanded in vitro in the presence of IL-2 in combination with IL-4. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T helper/inducer phenotype in SIL populations. In 3H-thymidine incorporation assays, SIL showed proliferation in response to IL-2, IL-3, IL-4, ionomycin (Io) + 12-o-tetradecanoyl-phorbol-13-acetate (TPA) and OKT3 + TPA. OKT4 with and without TPA did not induce proliferation. Tumour necrosis factor alpha (TNF-alpha) did not block proliferative responses of SIL to IL-2 and IL-4. Cultured SIL showed no cytotoxic activity against K562 and Jurkat target cells. Expanded skin-derived T cells were tested for their capacity to secrete several cytokines in vitro. SIL secreted significant amounts of IL-4, GM-CSF and TNF-alpha upon stimulation with mitogens but failed to secrete IFN-gamma. Io in combination with phorbol-ester induced the secretion of larger amounts of IL-4, GM-CSF, TNF-alpha and low amounts of IFN-gamma. The data indicate that SIL derived from AD lesions were defective in their capacity to secrete IFN-gamma but were enriched in T cells capable of producing IL-4 upon stimulation. The results support the possibility of a predominant 'TH2-like' cell-mediated immune response in lesional skin of AD patients.

Antibodies, Monoclonal↗
PubMed1991

Reaction of dipeptidylpeptidase IV with substrate-analogous azapeptides.

The reaction of dipeptidyl peptidase IV (EC 3.4.14.5.) with azapeptide substrates containing azaalanine or azaproline in the P1-position was investigated. Accumulation of a fairly stable acyl-enzyme could be shown for ester substrates. Ala-AzaPro-pNA is a very poor substrate of DP IV and does not accumulate an acyl-enzyme. DP IV does not react with active-site titrants for trypsin-like serine proteases.

Amino Acid Sequence↗
PubMed1991

Inhibition of chymotrypsin and pancreatic elastase by 4H-3,1-benzoxazin-4-ones.

Eight 3,1-Benzoxazin-4-ones have been used to inactivate chymotrypsin and pancreatic elastase. Whereas 6,7-dimethoxy substitution only slightly decreased the acylation rate constant, the deacylation reaction was nearly unaffected. Bulky alkoxy groups in position 2 of the heterocyclic moiety were shown to increase enormously the acylation rate of chymotrypsin, but not that of elastase.

Acylation↗
PubMed1991

["Ledderhose" disease. Plantar fibromatosis--clinical aspects].

The Ledderhose's diseases is a relatively rare and not well known clinical picture. Even there are tight pathomorphological and clinical relations to the Dupuytren's contracture, the genesis is also here quite unknown. Because of inefficiency of conventional therapy the surgical treatment is the only alternative. On the sample of 12 operations in 7 patients from 1979 to 1989 surgical procedure and long-term results are discussed.

Adult↗
PubMed1991

[The occurrence of Campylobacter spp. and Salmonella spp. in gulls in northern Germany].

Out of a total of 207 gulls--20 Black headed gulls, 185 Herring gulls and 2 Common gulls--128 (62%) and 23 (11%) birds, respectively, were infected with Campylobacter spp. (C.) and Salmonella spp. C. jejuni was predominant in gulls less than two years old (89%) and C. coli in older birds (75%). Furthermore, the infection rate with Campylobacters was depending on the habitats of the birds. The rate for C. jejuni and C. coli, respectively, was in gulls from regional garbage dumps 78% and 4%, from the coast 58% and 21%, and from islands 47% and 47% of the isolations in the corresponding area. Salmonellae were mainly isolated in the period from September to February from gulls less than one year old and from birds from the coast.

Animals↗
PubMed1990

Effect of antipyrine and phenobarbital on renal gamma-glutamyltransferase excretion in human urine.

Serum gamma-glutamyltransferase is used as a marker of hepatic enzyme induction. The kidney contains high activities of gamma-glutamyltransferase in the brush border membrane of the proximal tubule, from which it is released into urine. This study investigated the effect of phenobarbital and antipyrine, two inducers of hepatic monoxygenases and gamma-glutamyltransferase, on the urinary excretion of renal gamma-glutamyltransferase. Three groups (n = 6) of healthy male volunteers received 100 mg phenobarbital for 7 and 14 days and 1200 mg antipyrine for 7 days, respectively. Antipyrine and phenobarbital increased antipyrine elimination, serum gamma-glutamyltransferase, and the urinary excretion of renal gamma-glutamyltransferase, whereas urinary beta-N-acetylglucosaminidase, beta-glucuronidase, and total protein and glucose excretion were unchanged. No correlation was found between serum and urinary gamma-glutamyltransferase or both enzymes and antipyrine elimination. Increases in antipyrine elimination were positively correlated to increases in serum, but not urinary gamma-glutamyltransferase. The findings suggest that antipyrine and phenobarbital increase urinary gamma-glutamyltransferase excretion. However, the increase in urinary gamma-glutamyltransferase does not reflect the magnitude of hepatic enzyme induction.

Adolescent↗
PubMed1989

Modified photometric method for the determination of phospholipase A activities.

A modified photometric method for the determination of phospholipase A activities which is based on a previously published reaction principle is described. The modified assay uses a lyophilized substrate emulsion rather than a freshly prepared phospholipid emulsion and a new chromogen (tribromohydroxybenzoic acid and 4-amino-antipyrine) with a high molar absorption coefficient (1,930 m2/mol at 546 nm, 2,900 m2/mol at 512 nm wavelength). The new test is more practicable with respect to pipetting volumes and incubation times. Preliminary results of a method evaluation indicate that the modified assay fulfills the usual criteria for clinical chemical enzyme measurements.

Humans↗
PubMed1989

Reactions between dipeptidyl peptidase IV and diacyl hydroxylamines: mechanistic investigations.

Kinetics of inactivation of dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) by N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines and their enzyme-catalyzed hydrolysis were followed using independent monitoring methods, all giving similar efficiency ratios of Kcat/Kinact. Different temperature dependences of the DP IV-inactivation and enzyme-catalyzed hydrolysis provide evidence of independent rate determining steps for both reactions. Activation parameters of inactivation are similar to those of spontaneous decomposition of the compounds, suggesting a mechanistic relationship. Investigation of DP IV-inactivation, DP IV-catalyzed hydrolysis of N-Ala-Pro-O-Bz(4-NO2) and the decomposition of the suicide substrate in H2O and D2O gave solvent isotope effects of 4.65, 2.54 and 1.02, respectively. A proton inventory of the inactivation reaction indicates involvement of more than one proton in the formation or breakdown of its transition state. The linear proton inventory found for the hydrolytic reaction is consistent with one proton transition in the rate determining step and resembles the rate limiting deacylation of Ala-Pro-DP IV. The hypothetical reaction model now locates splitting in both reactions prior to formation of a covalent intermediate during the catalytic cycle.

Dipeptidyl Peptidase 4↗
PubMed1989

[The occurrence of paramyxoviruses in game birds: epizootiological aspects, characteristics in vivo and in vitro. A review of the literature].

The recent review is based on recent literature on the epizootiology and on properties of avian paramyxoviruses in vivo and in vitro. Paramyxoviruses are classified according to their pathogenicity (especially serotype 1), in addition to serotyping by means of hemagglutination-inhibition tests and by monoclonal antibody binding assays. Also, neuraminidase-inhibition tests, double-immunodiffusion tests can be performed. Growth properties of the different paramyxovirus-serotypes are accessory techniques to classify paramyxoviruses parallel to the determination of the pathogenicity. Structural polypeptides can be demonstrated in polyacrylamide-electrophoresis, thus leading to a distinction of the different serotypes.

Animals↗