Biomedical subjects

R Stohwasser

Publications and source records attributed to R Stohwasser.

18 recordsLinked to original sources

PubMed2003

Tumour necrosis factor alpha and nuclear factor kappaB inhibit transcription of human TFF3 encoding a gastrointestinal healing peptide.

BACKGROUND: and aims: Tumour necrosis factor alpha (TNF-alpha) induction of nuclear factor kappaB (NFkappaB) activation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). Trefoil factor family peptides TFF1, TFF2, and TFF3 exert protective, curative, and tumour suppressive functions in the gastrointestinal tract. In this study, we investigated effects of the TNF-alpha/NFkappaB regulatory pathway by TNF-alpha on expression of TFFs. METHODS: After TNF-alpha stimulation, expression of TFF genes was analysed by quantitative real time polymerase chain reaction and by reporter gene assays in the gastrointestinal tumour cell lines HT-29 and KATO III. Additionally, NFkappaB subunits and a constitutive repressive form of inhibitory factor kappaB (IkappaB) were transiently coexpressed. In vivo, morphological changes and expression of TFF3, mucins, and NFkappaB were monitored by immunohistochemistry in a rat model of 2,4,6-trinitrobenzene sulphonic acid induced colitis. RESULTS: TNF-alpha stimulation evoked up to 10-fold reduction of TFF3 expression in the colon tumour cell line HT-29. Downregulation of reporter gene transcription of TFF3 was observed with both TNF-alpha and NFkappaB, and was reversible by IkappaB. In vivo, the increase in epithelial expression of NFkappaB coincided with reduced TFF3 expression during the acute phase of experimental colitis. CONCLUSIONS: Downregulation of intestinal trefoil factor TFF3 is caused by repression of transcription through TNF-alpha and NFkappaB activation in vitro. In IBD, perpetual activation of NFkappaB activity may contribute to ulceration and decreased wound healing through reduced TFF3.

Animals↗

PubMed2000

Biochemical analysis of proteasomes from mouse microglia: induction of immunoproteasomes by interferon-gamma and lipopolysaccharide.

The 20S proteasome is a multicatalytic threonine protease and serves to process peptides that are subsequently presented as antigenic epitopes by MHC class I molecules. In the brain, microglial cells are the major antigen presenting cells and they respond sensitive to pathologic events. We used cultured mouse microglia and a microglial cell line, the BV-2 line, as a model to study the correlation between microglial activation parameters and structural plasticity of the 20S/26S proteasome. Lipopolysaccharide (LPS)- or interferon-gamma (IFN-gamma)-stimulated microglia or BV-2 cells exhibit properties of activated microglia such as high levels of TNFalpha and IL-6 release. In response to IFN-gamma or LPS, three constitutive beta subunits (beta1/Delta, beta2/MC14, beta5/MB1) were replaced by the immunoproteasome subunits ibeta1/LMP2, ibeta2/MECL-1, and ibeta5/LMP7, indicating that activated microglia adapts its proteasomal subunit composition to the requirements of an optimized MHC class I epitope processing. Induction of immunoproteasomes in BV-2 cells was solely provoked by IFN-gamma, but not by LPS. Moreover, LPS (but not IFN-gamma) triggered the expression of a novel protein of approximately 50 kD as part of the proteasome activator PA700, that is the substrate-recognizing and unfolding unit of the 26S proteasome. These results indicate that both the 20S core protease as well as the proteasome activator PA700 are targets of modulatory subunit replacements or transient association of regulatory components in the course of microglial activation.

Adenosine Triphosphatases↗

PubMed2000

Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28.

The activation kinetics of constitutive and IFNgamma-stimulated 20S proteasomes obtained with homomeric (recPA28alpha, recPA28beta) and heteromeric (recPA28alphabeta) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling. The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28alpha/RecP28beta/RecP28alpha + RecP28beta) exhibit biphasic characteristics which can be attributed to a low-level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28alphabeta complex is about two orders of magnitude higher than towards the homomeric PA28alpha and PA28beta complexes. Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides.

Animals↗

PubMed1999

The role of the proteasome system and the proteasome activator PA28 complex in the cellular immune response.

The generation of antigenic peptides bound and presented to the immune system by MHC class I molecules predominantly depends on the function of the proteasome system. Stimulation of cells with interferon gamma induces the incorporation of three active site bearing beta-subunits into the 20S proteasome and the formation of the PA28 proteasome modulator complex. PA28 alters the cleavage properties of the proteasome and enhances MHC class I antigen presentation. Thus, by cytokine induced change of the proteasome system cells may alter the proteolytic properties of the 20S proteasome and may render an organism more flexible in its peptide generation capacity.

Cysteine Endopeptidases↗

PubMed1997

The subunits MECL-1 and LMP2 are mutually required for incorporation into the 20S proteasome.

Processing of antigens for presentation by major histocompatibility complex (MHC) class I molecules requires the activity of the proteasome. The 20S proteasome complex is composed of 14 different subunits, 2 of which can be substituted by the interferon gamma (IFN-gamma)-inducible and MHC-encoded subunits LMP2 and LMP7 (low molecular mass poylpeptides 2 and 7). A third subunit, MECL-1, is inducible by IFN-gamma but is encoded outside the MHC. Here we show by cotransfection experiments that the incorporation of MECL-1 into the 20S proteasome is directly dependent on the expression of LMP2 but independent of LMP7. Conversely, the uptake of LMP2 is strongly enhanced by MECL-1 expression. The expression of MECL-1 caused a replacement of the homologous subunit Z in the 20S proteasome complex. LMP2 is required for MECL-1 incorporation at the level of proteasome precursor formation that guarantees the concerted incorporation of two IFN-gamma-inducible proteasome subunits encoded inside and outside the MHC. The obligatory coincorporation of MECL-1 and LMP2 is an important parameter for the interpretation of results obtained with LMP2-deficient cell lines and mice as well as for the design of experiments addressing the function of MECL-1 in antigen presentation.

Animals↗

PubMed1997

Molecular cloning of the mouse proteasome subunits MC14 and MECL-1: reciprocally regulated tissue expression of interferon-gamma-modulated proteasome subunits.

The primary structures of the interferon-gamma-inducible mouse 20S proteasome subunit MECL-1 and its alternate homolog MC14 were determined. Northern analysis of mouse tissues revealed that MECL-1 mRNA predominantly occurred in thymus, lymph nodes, and spleen, whereas small amounts were detected in non-lymphoid tissues such as kidney, muscle, and testis. Unexpectedly, probing RNA blots with MC14 showed that tissues with high MECL-1 expression contained little MC14 and vice versa. A very similar reciprocal tissue expression was subsequently found for the homologous subunit pairs LMP2 and delta as well as LMP7 and MB1. The subunit protein composition of 20S proteasomes purified from liver, thymus, and lung reflected RNA expression. The impact of a regulated reciprocal tissue expression is discussed with respect to thymic selection and the induction of tolerance in potentially autoreactive T cells.

Amino Acid Sequence↗

PubMed1996

20S proteasome from LMP7 knock out mice reveals altered proteolytic activities and cleavage site preferences.

20S proteasomes of tissues from LMP7 knock out mice which show reduced MHC class I restricted antigen presentation were analyzed with regard to their subunit composition, peptide hydrolyzing activity and their ability to cleave a synthetic 25-mer polypeptide. LMP7 deficiency results in an enhanced incorporation of subunit MB1 and in a 2-3.8-fold increase in Vmax for the Suc-LLVY-MCA hydrolyzing activity. Since LMP7 deficiency also affects the cleavage site preference of 20S proteasomes the reduced MHC class I antigen presentation of LMP7 knock out mice is most likely due to an impairment in peptide generation.

Amino Acid Sequence↗

PubMed1996

A third interferon-gamma-induced subunit exchange in the 20S proteasome.

The 20S proteasome is a protease complex of functional importance for antigen processing. Two of the 14 proteasome subunits, delta and MB1, can be replaced by the major histocompatibility complex (MHC)-encoded and interferon-gamma (IFN-gamma)-inducible subunits LMP2 and LMP7, respectively. LMP2 and LMP7 alter the cleavage site specificity of the 20S proteasome and are required for the efficient generation of T cell epitopes from a number of viral proteins and for optimal MHC class I cell surface expression. We compared the 20S proteasome subunit pattern from IFN-gamma-induced and non-induced mouse fibroblasts on two-dimensional gels and identified a third subunit exchange by microsequencing: the non-MHC-encoded subunit MECL-1 is induced by IFN-gamma and replaces a sofar barely characterized beta subunit designated 'MC14'. In analogy to LMP2 and LMP7, MECL-1 may be functional in MHC class I-restricted antigen presentation.

Amino Acid Sequence↗

PubMed1996

Cytokine induced changes in proteasome subunit composition are concentration dependent.

In eukaryotes, 20S proteasome subunit composition is controlled by the cytokine interferon-gamma (IFN-gamma). IFN-gamma induces the synthesis of the beta-subunits LMP2, LMP7 and MECL-1, which in consequence replace their constitutive subunit homologs delta, MB1 and MC14/Z in the 20S complex. By pulse labeling mouse RMA cells and immunoprecipitation of proteasome complexes with the antibody MP3, we have analysed the effect of different IFN-gamma concentrations on proteasomal subunit composition. Our experiments show that IFN-gamma concentrations as low as 5 U/ml induce subunit substitutions and that overall proteasomal subunit composition is dependent on the cytokine concentration used. An IFN-gamma concentration of 50 U/ml is sufficient for complete replacement of subunit delta by LMP2. In contrast, IFN-gamma treatment never induces a complete replacement of subunit MC14 by MECL-1. These subunits are present at an approximate 1:1 molar ratio, suggesting that both subunits coexist in the same 20S proteasome complex. Furthermore, different regulatory mechanisms have to be postulated for the synthesis and incorporation of the three IFN-gamma inducible proteasome subunits. Both IFN-gamma as well as IL-2 also seem to influence the modification state of the alpha subunit C8. Since the subunit composition is dependent on the cytokine concentration used and strongly influences the proteolytic properties of the 20S proteasome complex, our experiments represent a caveat for experiments in which IFN-gamma dependent proteasomal enzyme characteristics have been analysed without monitoring the subunit composition.

Animals↗

PubMed1995

Coding strategy of the S and M genomic segments of a hantavirus representing a new subtype of the Puumala serotype.

The hantavirus strain Vranica was previously reported to have been isolated from a bank vole in Bosnia-Hercegovina and associated with the occurrence of hemorrhagic fever with renal syndrome (HRFS) in humans. The complete cDNA nucleotide sequences of the small (S) and medium (M) genomic RNA segments of this virus were determined. Major open reading frames were found in the S and M segment between nucleotide positions 43 and 1341 coding for a polypeptide of 433 amino acid residues and between nucleotide positions 41 and 3,484 coding for 1,148 amino acid residues, respectively. The analysis and the alignment of the nucleotide and the derived amino acid sequences with known sequences of other hantavirus strains demonstrate that Vranica resembles Swedish strains and represents a new virus subtype of the Puumala serotype distinct from the subtypes represented by virus strains CG18-20 and Sotkamo.

Amino Acid Sequence↗

PubMed1993

RNA binding of recombinant nucleocapsid proteins of hantaviruses.

Genes encoding the nucleocapsid (N) proteins of two hantaviruses, Hantaan virus strain 76-118 (HTN) and Puumala virus strain CG 18-20 (PUU), were expressed in Escherichia coli as histidine-tagged proteins. They were purified by metal-chelate affinity chromatography under native or denaturing conditions to near homogeneity. The soluble form of HTN N protein was associated with RNA of E. coli. Renatured N proteins were shown to bind in vitro transcribed RNA representing the hantaviral small genomic (S) RNA segment. RNA binding was shown by affinity to filter-immobilized N proteins and by gel mobility shift assays. Competition experiments using tRNA, poly(U) and poly(A)+ U indicated that binding of RNA by the N protein is nonspecific. However, direct binding of ds-RNA resulted in efficient formation of large complexes suggesting that double-stranded nucleic acids are bound preferentially. Carboxyterminal fragments of HTN and PUU N proteins containing about 100 amino acids of the carboxy termini retained full binding capacity indicating that RNA binding occurs via a carboxyterminal domain.

Capsid↗

PubMed1993

Identification of the gene encoding the major capsid protein of insect iridescent virus type 6 by polymerase chain reaction.

The gene encoding the major capsid protein of Chilo iridescent virus (CIV) has been identified by PCR using oligonucleotide primers corresponding to different regions of the major capsid proteins of Tipula iridescent virus (TIV) and iridescent virus 22 (IV22). A DNA fragment of 0.5 kbp was amplified using two oligonucleotide primers corresponding to the amino acid positions 146 to 153 and 304 to 313 of the major capsid protein of TIV, respectively. The radioactively labelled DNA fragment derived from PCR was hybridized to a CIV gene library. This analysis revealed that only the EcoRI CIV DNA fragment X [2.85 kbp; 0.589 to 0.603 viral map units (m.u.)] hybridized to the amplified DNA fragment. An RNA transcript of about 1.5 kb was identified when the PCR product was used as a hybridization probe. The same RNA transcript was detected when the EcoRI fragments X and Q (5.9 kbp; 0.603 to 0.631 viral m.u.) were used as probes. This indicates that the expected gene is located within map coordinates 0.589 to 0.631 and harbours part of the DNA sequences of fragments Q and X. The analysis of the DNA sequences of this particular region of the CIV genome revealed the presence of one open reding frame of 1401 bp. The DNA sequences of this region encode a protein of 467 amino acid residues with an M(r) of 51.4K. A high degree (64.7%) of amino acid sequence identity was detected between the major capsid protein of TIV and/or IV22 and the amino acid composition of the identified CIV protein.

Amino Acid Sequence↗

PubMed1991

Primary structure of the large (L) RNA segment of nephropathia epidemica virus strain Hällnäs B1 coding for the viral RNA polymerase.

The L RNA segment of the nephropathia epidemica virus (NEV) strain Hällnäs B1 was characterized by molecular cloning of the corresponding cDNA and subsequent determination of the DNA nucleotide sequence. The L RNA segment is 6550 nucleotides long with complementarity of 20 bases at the 3' and 5' termini. The viral messenger sense RNA contains one major open reading frame (ORF) with a coding capacity of 2156 amino acid residues encoding a protein with a calculated molecular weight of 246 kDa and an IEP of pH 7.4. Comparison of the deduced amino acid sequences from NEV hantavirus and Bunyamwera virus (BWV) L segment messenger sense RNAs, revealed a high degree of diversity (overall amino acid identity, 17%). However, three clusters of 30-40% amino acid identity were detected. One of these domains, containing an Asp-Asp motif found in many RNA polymerases, also shares amino acid sequence homology with the PB1 polymerase component of influenza type A. These results indicate that the L RNA segment of the NEV codes for the viral RNA-dependent RNA polymerase. The data presented here complete our previous studies on the characterization of the NEV genome by cDNA sequencing of the viral M and S RNA segments.

Amino Acid Sequence↗

PubMed1991

Antigenicity of hantavirus nucleocapsid proteins expressed in E. coli.

DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hällnäs B1 (NEV) and Hantaan virus strain 76-118 (HTV) encoding their nucleocapsid proteins were inserted into the E. coli vector pIN-III-ompA for secretion of proteins into the periplasmic space. The complete HTV and NEV nucleocapsid proteins and two truncated versions of the NEV nucleocapsid proteins were expressed as fusion proteins. Unexpectedly, all products accumulated as insoluble aggregates. Most of the ompA signal peptide remained uncleaved. However, nucleocapsid fusion proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution. Rabbits were immunized with the eluted proteins and the resulting antibodies reacted specifically with authentic viral nucleocapsid proteins of HTV and NEV. The recombinant nucleocapsid proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens. This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques.

Antigens, Viral↗

PubMed1990

Molecular characterization of the RNA S segment of nephropathia epidemica virus strain Hällnäs B1.

The S segment RNA of nephropathia epidemica virus (NEV) strain Hällnäs B1 was isolated by molecular cloning of the corresponding cDNA. The RNA is 1785 nucleotides long with the 3' and 5' termini being complementary for 23 bases. The viral messenger-sense RNA contains one major open reading frame (ORF) with a coding capacity of 433 amino acids encoding a 49-kDa polypeptide. Compared to the Hantaan S segment cDNA sequence there is a nucleotide homology of 60 and 61% at the amino acid level. Many of the amino acid differences are conservative exchanges. The C-termini of the NEV and Hantaan nucleocapsid proteins are nearly identical and the hydrophilicity profiles are very similar. In contrast, the following differences are significant: The calculated isoelectric points of the NEV and Hantaan nucleocapsid proteins are 5.6 and 6.7, respectively. The most prominent antigenic determinants predicted by the hydrophilicity profiles are located close to the C-terminus of NEV and close to the N-terminus of Hantaan virus nucleocapsid polypeptides.

Amino Acid Sequence↗

PubMed1989

Immunoblot analysis of the serological response in Hantavirus infections.

Sera from patients with nephropathia epidemica (NE) or Korean hemorrhagic fever (KHF) were tested for specific antibody response to antigens of Hällnäs virus and Hantaan virus strain 76-118. A Vero E6 derived cell line persistently infected with Hällnäs virus strain B1, and Vero E6 cells freshly infected with Hantaan virus type strain 76-118 were used as antigens in the immunofluorescence assay (IFA) and the immunoblot. Blots were prepared from whole cell lysates. The convalescent-phase sera of NE patients tested in this study regularly revealed a marked reaction with a 52 kilodalton (Kd) protein of Hällnäs virus and a 50 Kd protein of Hantaan virus. A convalescent serum from a patient with Korean hemorrhagic fever and a rat antiserum against Hantaan virus could recognize the 50 Kd band of Hantaan virus but showed no apparent reactivity with the 52 Kd component of Hällnäs virus in the standard dilutions. Some sera could additionally identify minor bands in the 55 Kd and/or 67 Kd region of the blots. A one-way cross reactivity between Hantaan and Hällnäs viruses was also evident from the results of the immunofluorescence assays in that NE convalescent sera reacted with both viruses, whereas KHF convalescent or anti-Hantaan sera gave strongly positive results with Hantaan virus but only faint reaction with Hällnäs virus.

Antibodies, Viral↗

PubMed1989

Determination of the coding capacity of the M genome segment of nephropathia epidemica virus strain Hällnäs B1 by molecular cloning and nucleotide sequence analysis.

The M genome RNA segment of nephropathia epidemica virus (NEV) strain Hällnäs B1 was characterized by molecular cloning and DNA nucleotide sequencing of the corresponding cDNA clones. The size of the M RNA segment is 3682 nucleotides. The 3' and 5' terminal sequences are complementary for 21 bases and their predicted secondary structure is very stable. The viral complementary messenger RNA possesses a single long open reading frame with a coding capacity of 1148 amino acids (polypeptide of 126 kDa). A comparison of the NEV M segment to that of Hantaan virus strain 76-118 reveals 61% sequence homology at the nucleotide level and 53% at the deduced amino acid level. Four out of five potential asparagine-linked glycosylation sites of the encoded glycoproteins have been conserved between NEV and Hantaan M. The isoelectric points (IEP) are nearly identical. Furthermore it was found that 90% of all cysteine residues have been conserved. Putative NEV G1 and G2 are preceded by a short hydrophobic sequence as shown for G1 and G2 of Hantaan virus. Hydrophilicity profiles of the two segments are of striking similarity. These data indicate that NEV- and Hantaan virus M-encoded polypeptides seem to be very similar in structure and function despite the relatively low amino acid sequence homology.

Amino Acid Sequence↗

PubMedDate not supplied

PA28alphabeta double and PA28beta single transfectant mouse B8 cell lines reveal enhanced presentation of a mouse cytomegalovirus (MCMV) pp89 MHC class I epitope.

PA28 is an interferon-gamma inducible modulator of proteasome function composed of two subunits, i.e. PA28alpha and PA28beta. Previously we showed that stabile overexpression of the PA28alpha subunit alone supported MHC class I antigen presentation of two viral epitopes. However, no information was obtained on the consequences when PA28alpha and PA28beta function in concert or when PA28beta is overexpressed on its own. Here we demonstrate that overexpression of PA28alpha and beta together is similarly efficient in supporting MHC class I antigen presentation of the MCMV pp89 9mer epitope as PA28alpha alone, excluding a potentially potentiating role of PA28beta. Surprisingly, and despite the fact that PA28beta alone was thought to be inactive and to only stabilize PA28 activity, overexpression of PA28beta also resulted in improved antigen presentation. However, by northernblot and immunoprecipitation experiments we show that while PA28alpha is able to act alone the observed effect in the PA28beta and PA28alphabeta transfectant cell lines is due to increased levels of PA28alphabeta complexes.

Animals↗