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R Grimm

Publications and source records attributed to R Grimm.

At least 73 records · Page 4Linked to original sources
PubMed1998

Four differently chromatin-associated maize HMG domain proteins modulate DNA structure and act as architectural elements in nucleoprotein complexes.

In contrast to other eukaryotes which usually express two closely related HMG1-like proteins, plant cells have multiple relatively variable proteins of this type. A systematic analysis of the DNA-binding properties of four chromosomal HMG domain proteins from maize revealed that they bind linear DNA with similar affinity. HMGa, HMGc1/2 and HMGd specifically recognise diverse DNA structures such as DNA mini-circles and supercoiled DNA. They induce DNA-bending, and constrain negative superhelical turns in DNA. In the presence of DNA, the HMG domain proteins can self-associate, whereas they are monomeric in solution. The maize HMG1-like proteins have the ability to facilitate the formation of nucleoprotein structures to different extents, since they can efficiently replace a bacterial chromatin-associated protein required for the site-specific beta-mediated recombination. A variable function of the HMG1-like proteins is indicated by their differential association with maize chromatin, as judged by their 'extractability' from chromatin with spermine and ethidium bromide. Collectively, these findings suggest that the various plant chromosomal HMG domain proteins could be adapted to act in different nucleoprotein structures in vivo.

Chromatin↗
PubMed1998

Presence of matrix metalloproteinase-9 activity in the cerebrospinal fluid of human immunodeficiency virus-infected patients.

To determine whether matrix metalloproteinase (MMP)-9 is a potential mediator involved in the frequently detected blood-brain barrier leakage in human immunodeficiency virus (HIV)-infected patients, zymography was used to detect MMP-9 activity in cerebrospinal fluid (CSF) samples of 80 HIV-infected patients and of 10 control patients. CSF MMP-9 activity was detected in 40% of HIV-infected patients (but not in controls) and was significantly more frequent in HIV-infected patients than in those without neurologic deficits (50% vs. 13.6%). The frequency of CSF MMP-9 activity did not significantly differ between neurologically symptomatic HIV-infected patients with or without opportunistic central nervous system disease (51.6% vs. 48.1%). Additionally, the presence of CSF MMP-9 activity in HIV-infected patients was associated with an increased CSF white blood cell count and an elevated CSF-to-serum albumin ratio, suggesting that it may play a role in blood-brain/CSF barrier leakage in HIV-infected patients.

CD4 Lymphocyte Count↗
PubMed1998

Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy.

Specific immunotherapy is an efficient treatment for patients suffering from type I allergy. The mechanisms underlying successful immunotherapy are assumed to operate at the level of T helper cells, leading to a modulation of the immune response to allergens. During immunotherapy, increasing doses of allergens are given on a regular basis, and the beneficial effects for the patient depend on the concentration of allergen used. On the other hand, the risk of IgE-mediated anaphylactic side effects also increase with the amount of allergen applied per injection. Therefore, we have proposed the use of hypoallergenic (low IgE binding activity) forms of allergens for immunotherapy. We evaluated by site-directed mutagenesis the contributions of individual amino acid residues/positions for IgE binding to Bet v 1, the major allergen of birch pollen. We found that IgE binding to Bet v 1 depended on at least six amino acid residues/positions. Immunoblot analyses and inhibition experiments showed that the multiple-point Bet v 1 mutant exhibited extremely low reactivity with serum IgE from birch pollen-allergic patients. In vivo (skin prick) tests showed that the potency of the multiple-point mutant to induce typical urticarial type I reactions in pollen-allergic patients was significantly lower than for wild-type Bet v 1. Proliferation assays of allergen-specific T cell clones demonstrated that these six amino acid exchanges in the Bet v 1 sequence did not influence T cell recognition. Thus, the Bet v 1 six-point mutant displayed significantly reduced IgE binding activity, but conserved T cell activating capacity, which is necessary for immunomodulation. The approach described here may be generally applied to produce allergen variants to be used in a safe therapy form of immediate-type allergies.

Allergens↗
PubMed1998

A high-conductance solute channel in the chloroplastic outer envelope from Pea.

The pea chloroplastic outer envelope protein OEP24 can function as a general solute channel. OEP24 is present in chloroplasts, etioplasts, and non-green root plastids. The heterologously expressed protein forms a voltage-dependent, high-conductance (Lambda = 1.3 nS in 1 M KCl), and slightly cation-selective ion channel in reconstituted proteoliposomes. The highest open probability (P open approximately 0. 8) is at 0 mV, which is consistent with the absence of a transmembrane potential across the chloroplastic outer envelope. The OEP24 channels allow the flux of triosephosphate, dicarboxylic acids, positively or negatively charged amino acids, sugars, ATP, and Pi. Structure prediction algorithms and circular dichroism spectra indicate that OEP24 contains seven amphiphilic beta strands. The primary structure of OEP24 shows no homologies to mitochondrial or bacterial porins on a primary sequence basis, and OEP24 is functionally not inhibited by cadaverine, which is a potent inhibitor of bacterial porins. We conclude that OEP24 represents a new type of solute channel in the plastidic outer envelope.

Amino Acid Sequence↗
PubMed1998

Allergens in pepper and paprika. Immunologic investigation of the celery-birch-mugwort-spice syndrome.

Mugwort and birch pollen allergy are frequently associated with IgE-mediated hypersensitivity to celery and spices. We analyzed 22 sera from patients with the mugwort-birch-celery-spice syndrome for IgE binding to the spices pepper and paprika by immunoblotting. Immunoblot results revealed two major allergens of 28 and 60 kDa in pepper and a 23-kDa allergen together with allergens of higher molecular weight in paprika. In immunoblot-inhibition studies, crude mugwort, birch pollen, and celery extracts significantly reduced the IgE binding to pepper and paprika allergens. However, no inhibition was achieved with rBet v 1 and rBet v 2, suggesting that no homologs of these birch proteins act as allergens in pepper or paprika extracts. N-terminal sequence analysis of the 14- and 28-kDa pepper and 23-kDa paprika allergens revealed no homology to known allergens. The 28-kDa pepper allergen showed homology to a wheat germin protein, and the 23-kDa paprika allergen was identified as a homolog of a osmotin-like or pathogenesis-related protein in tomato. Therefore, we conclude that the IgE cross-reactivity in the mugwort-birch-celery-spice syndrome to the spices pepper and paprika is not caused by homologs of Bet v 1 and profilin. N-terminal amino acid sequence analysis of the main allergens in pepper and paprika indicate a relation to frequently occurring plant proteins.

Adolescent↗
PubMed1998

Bell peppers (Capsicum annuum) express allergens (profilin, pathogenesis-related protein P23 and Bet v 1) depending on the horticultural strain.

BACKGROUND: Little is known about the role of bell peppers in food allergy. We collected sera from 11 patients with food allergy to bell peppers to analyze bell pepper extracts for allergen composition. METHODS: Proteins of mature fruits of eight horticultural strains of bell peppers were extracted and tested with patients' sera for IgE binding and with monoclonal and polyclonal antibodies in immunoblot. RESULTS: Profilin was detected in bell pepper extracts by an anti-celery profilin antibody. It showed high IgE binding activity in all extracts, which could be inhibited by recombinant birch pollen profilin. Anti-birch pollen monoclonal antibody BIP3, directed against birch pollen proteins between 30 and 69 kD, bound to bell pepper antigens of comparable molecular weights. A homologue of the major birch pollen allergen Bet v 1 was detected in four of eight horticultural strains of bell peppers, and was shown to bind IgE in 1 of the 11 patients. A 23-kD allergen of bell peppers was shown to correspond to the 23-kD major paprika allergen by IgE absorption experiments. Its N-terminal sequence showed 100% identity to P23 from tomatoes. CONCLUSION: The appearance of profilin in all and Bet v 1 in 50% of the tested horticultural strains indicates that bell peppers have to be considered potentially dangerous for Bet v 1- and profilin-sensitized patients. Moreover, in 4 of 8 horticultural strains of bell peppers a homologue of the osmotin-like protein P23 from tomatoes is responsible for substantial IgE binding. Contact with Bet v 1 and P23 homologues in bell peppers can therefore be minimized by avoidance of the respective horticultural strains.

Adult↗
PubMed1997

The chloroplastic protein import machinery contains a Rieske-type iron-sulfur cluster and a mononuclear iron-binding protein.

Transport of precursor proteins across the chloroplastic envelope membranes requires the interaction of protein translocons localized in both the outer and inner envelope membranes. Analysis by blue native gel electrophoresis revealed that the translocon of the inner envelope membranes consisted of at least six proteins with molecular weights of 36, 45, 52, 60, 100 and 110 kDa, respectively. Tic110 and ClpC, identified as components of the protein import apparatus of the inner envelope membrane, were prominent constituents of this complex. The amino acid sequence of the 52 kDa protein, deduced from the cDNA, contains a predicted Rieske-type iron-sulfur cluster and a mononuclear iron-binding site. Diethylpyrocarbonate, a Rieske-type protein-modifying reagent, inhibits the translocation of precursor protein across the inner envelope membrane, whereas binding of the precursor to the outer envelope membrane is still possible. In another independent experimental approach, the 52 kDa protein could be co-purified with a trapped precursor protein in association with the chloroplast protein translocon subunits Toc86, Toc75, Toc34 and Tic110. Together, these results strongly suggest that the 52 kDa protein, named Tic55 due to its calculated molecular weight, is a member of the chloroplastic inner envelope protein translocon.

Amino Acid Sequence↗
PubMed1997

Variability in Arabidopsis thaliana chromosomal high-mobility-group-1-like proteins.

The vertebrate high-mobility-group (HMG) protein HMG1 is an abundant non-histone protein which is considered as an architectural element in chromatin. In the monocotyledonous plant maize, four different HMG1-like proteins (HMGa, HMGc1/2, HMGd) have been identified, whereas other eukaryotes usually express only two different proteins of this type. We have examined here the HMG1-like proteins of the dicotyledonous plant Arabidopsis thaliana. The isolation and analysis of cDNAs encoding five different so far uncharacterised HMG1-like proteins (now termed HMG alpha, HMG beta1/2, HMG gamma, HMG delta) from Arabidopsis indicates that the expression of multiple HMG1-like proteins is a general feature of (higher) plants. The Arabidopsis HMG1-like proteins contain an HMG domain as a common feature, but outside this conserved DNA-binding motif the amino acid sequences are significantly different indicating that this protein family displays a greater structural variability in plants than in other eukaryotes. The five HMG1-like proteins were expressed in Escherichia coli and purified. They bind with somewhat different affinity to linear double-stranded DNA. The recognition of DNA structure is evident from their preferential interaction with DNA minicircles relative to linear DNA. Reverse-transcribed PCR suggested that the five HMG1-like genes are simultaneously expressed in Arabidopsis leaves and suspension culture cells.

Amino Acid Sequence↗
PubMed1997

The recombinant product of the Chryptomonas phi plastid gene hlpA is an architectural HU-like protein that promotes the assembly of complex nucleoprotein structures.

The HlpA protein which is encoded by the hlpA gene in the plastid genome of the cryptomonad alga Chryptomonas phi is structurally related to the non-sequence-specific DNA-binding and DNA-bending HU family of chromatin-associated proteins. The expression of the HlpA protein complements the mutant phenotype of Bacillus subtilis cells impaired in the Hbsu protein (B. subtilis HU), as measured by the resistance of the cells to methylmethane sulphonate. To analyse the interactions of HlpA with DNA, we expressed the protein in Escherichia coli and purified it to homogeneity. HlpA interacts preferentially with four-way junction DNA or DNA minicircles, when compared with linear DNA, recognising DNA structure. HlpA and E. coli HU display comparable affinities for all types of DNA tested; however, HlpA exhibits a stronger tendency to self-associate in the presence of DNA. Accordingly, HlpA oligomerises more readily than HU in protein crosslinking experiments. In the presence of topoisomerase I, HlpA constrains negative superhelical turns in closed circular plasmid DNA. The HlpA protein mediates the joining of distant recombination sites into a complex nucleoprotein structure, as judged by beta-mediated site-specific recombination. The results presented provide evidence that HlpA is a functional plastid equivalent of nuclear and mitochondrial HMG1-like proteins and bacterial HU proteins.

Bacillus subtilis↗
PubMed1997

Effect of different surfactants on the separation by micellar electrokinetic chromatography of a complex mixture of dipeptides in urine of prolidase-deficient patients.

Prolidase deficiency is a severe disorder characterized by massive excretion of metabolites with closely related structures. At present, micellar electrokinetic chromatography is the separation method which provides the highest selectivity of structurally similar solutes. However, the structure of a surfactant can greatly affect the selectivity of separation depending on factors such as the length of hydrophobic alkyl chain or the nature of the hydrophilic group. Here we investigated the effect of three non-ionic and four anionic detergents for obtaining the best separation conditions for resolving imidodipeptide mixtures. The effect on resolution of variables such as temperature, surfactant concentrations and organic solvents was also examined. The greatest resolution was obtained at the lowest temperature studied (10 degrees C) using 50 mM sodium borate, pH 9.3 containing 50 mM pentanesulfonate and 10% (v/v) methanol. Under these experimental conditions almost all excreted components were baseline separated and identified.

Dipeptidases↗
PubMed1997

Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system.

To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon gamma induction in vivo or addition of recombinant proteasome activator 28alpha in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

Acetylcysteine↗
PubMed1997

Direct monitoring of prolidase activity in cultured skin fibroblasts using capillary electrophoresis.

Capillary electrophoresis (CE) was used as an alternative to current analysis schemes for detecting prolidase activity in erythrocytes and skin fibroblast cultures because of its unique selectivity and high resolving power. Kinetic measurement of peptide bond hydrolysis was performed using porcine kidney prolidase on different substrates (Gly-Pro, Leu-Pro and Ala-Pro) and by following the disappearance of the peptide-substrate's peak. The K(m) values obtained were in agreement with those previously reported. Interestingly, in the case of Phe-Pro as the substrate, simultaneous analysis of the product and parent peptide was possible, thus showing the superiority of the capillary electrophoresis (CE) assay with respect to the standard spectrophotometric method. The application of the CE technique to the characterization of prolidase activity in control and prolidase-deficient skin cultured fibroblasts was successful. Enzyme activity was easily calculated in all controls tested and the K(m) values determined were slightly lower than those obtained with the colorimetric reaction, thus confirming our assumption that the CE assay shows higher specificity than the ninhydrin technique. Our results demonstrate the feasibility of using CE as a simple and reliable technique for determining prolidase activity.

Adolescent↗
PubMed1997

Postimport methylation of the small subunit of ribulose-1,5-bisphosphate carboxylase in chloroplasts.

Electron impact mass spectronomy analysis of the amino-terminal amino acid of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) showed that the amino-terminal methionine residue is post-translationally modified to N-methyl-methionine. Modification of the amino-terminal methionine residue was found in mature SSU proteins from the dicotyledonous plants pea and spinach as well as the monocotyledonous plants barley and corn. SSU methyltransferase is a soluble protein in the chloroplast stroma and accepts heterologously expressed non-methylated SSU as a substrate using S-adenosylmethionine as methyl-group donor. We show that this modification occurs after post-translational uptake of the precursor form of SSU into chloroplasts and processing to its mature size. This reaction represents a new step in the import and assembly pathway of Rubisco holoenzyme.

Chloroplasts↗
PubMed1997

Altered hapten ligands antagonize trinitrophenyl-specific cytotoxic T cells and block internalization of hapten-specific receptors.

Low molecular chemicals (haptens) frequently cause T cell-mediated adverse immune reactions. Our previous work provided evidence that hapten-specific T cells, in analogy to those specific for nominal peptide antigens, direct their TCR towards hapten-modified, MHC-associated peptides. We now demonstrate that trinitrophenyl (TNP)-specific, class I MHC-restricted CTL from mice may exhibit exquisite specificity for subtle structural details of these hapten determinants, surpassing even the specificity of immunoglobulins. More importantly, these CTL could be antagonized by ligands altered either in their peptide sequence or in their hapten structure. The system was employed to examine the molecular basis of T cell antagonism. Whereas agonists resulted in a dose-dependent downregulation of TCR in different mouse T cell clones, antagonistic peptides totally failed to do so despite engaging the specific TCR. Moreover, simultaneous presentation of antagonist and agonist on the same antigen presenting cell prevented TCR internalization. No signs of anergy or functional receptor inactivation were observed in CTL treated with antagonist-loaded target cells. Based on a serial triggering model of T cell activation, our data favor a model in which antagonists block T cell functions by competitively engaging the specific TCR in unproductive interactions.

Amino Acid Sequence↗
PubMed1997

Purification and cDNA cloning of maize HMGd reveal a novel plant chromosomal HMG-box protein with sequence similarity to HMGa.

We have purified the chromosomal high mobility group (HMG) protein HMGd from maize suspension culture cells, determined the N-terminal amino acid (aa) sequence, and isolated the corresponding cDNA. Sequence analysis showed that the cDNA encoded a protein of 126 aa residues with a theoretical mass of 14,104 Da. The protein contains an HMG-box DNA-binding domain and a short acidic C-terminal tail. HMGd is in approx. 65% of its residues identical to maize HMGa, whereas it is only approx. 46% identical to maize HMGcl/2. The differences to the previously reported HMG proteins in aa sequence, in overall charge and in protein size indicate that we have identified a third type of plant chromosomal HMG-box protein belonging to the HMG1 protein family. Immunoblot analysis with a HMGd antiserum reveals that HMGd is expressed in all tissues tested.

Amino Acid Sequence↗
PubMed1997

Complete resolution of imidodipeptide mixtures in urine of prolidase-deficient patients using micellar electrokinetic chromatography.

The use of capillary zone electrophoresis as an efficient method for the identification of urinary imidodipeptides of prolidase-deficient patients has already been reported. However, owing to the complexity of the components excreted, the resolution of electrophoretic patterns obtained was poor. Here we examine the use of micellar electrokinetic chromatography to enhance peak resolution in order to obtain better insight into the electropherograms of patients' urine. The usefulness of sodium dodecyl sulphate as surfactant is reported: refined electropherograms were achieved using 35 mM sodium borate, pH 8.3 containing 65 mM sodium dodecyl sulphate. Almost all peaks were baseline separated, collected and sequenced. This allowed us to define the exact imidodipeptide composition of patients' urine. The possibility of identifying and thus quantifying each single peak means that comparison of urinary imidodipeptide excretion patterns from different patients can be made and the hypothesis that peptide patterns can be correlated with differing clinical severity can be investigated.

Adolescent↗
PubMed1997

Identification of human common nuclear-matrix proteins as heterogeneous nuclear ribonucleoproteins H and H' by sequencing and mass spectrometry.

Nuclear-matrix proteins were prepared from different rat and human cells and separated by two-dimensional gel electrophoresis. By computer-assisted analysis of the images, two of the proteins were identified as ubiquitously occurring (common) nuclear-matrix proteins, which appeared in tissue-dependent concentrations. The two proteins that originated from human blood mononuclear cells were analyzed further. Tryptic digests of the blotted proteins were analyzed by partial peptide sequencing and matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The two human common nuclear-matrix proteins were identified as heterogeneous nuclear ribonucleoproteins (hnRNP) H and H' or their variants. Furthermore, mass analysis revealed details on the N terminus of hnRNP H.

Amino Acid Sequence↗