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Biomedical subjects

R Grimm

Publications and source records attributed to R Grimm.

At least 91 records · Page 5Linked to original sources
PubMed1997

Carrier-independent hapten recognition and promiscuous MHC restriction by CD4 T cells induced by trinitrophenylated peptides.

The elucidation of mechanisms underlying the recognition of haptens by class II MHC-restricted T cells is instrumental for the understanding of chemical- and drug-induced allergies. We have previously demonstrated that trinitrophenyl (TNP) peptides represent dominant antigenic epitopes for CD8+ and CD4+ mouse T cells triggered by chemically TNP-modified APC. Here, we report the characterization of TNP-specific, CD4+ mouse T cell lines and hybridomas that were induced in vivo and in vitro by defined hapten-conjugated peptides. These peptides, which we had previously shown to induce contact sensitivity to picryl chloride in vivo regardless of sequence homologies to mouse proteins, were found to activate carrier-independent TNP-specific T cells in vitro. We interpret these findings to support our view that carrier-independent T cells, reactive to particularly repetitive hapten epitopes, may play a crucial role in allergies to chemicals and drugs. In addition to carrier independence, one of our hybridomas (IT-H6/A11) exhibited a striking promiscuity of MHC restriction. Although absolutely dependent in its TNP reactivity on the presence of MHC class II molecules, the IT H6/A11 hybridoma completely ignored class II polymorphism and even reacted to TNP peptides presented on human DR molecules. Regarding hapten allergies in humans with a heterozygous situation for three types of class II molecules (DR, DP, and DQ), such promiscuous MHC restriction should lead to the presentation of even higher epitope densities to the respective T cell clones. Hybridoma IT-H6/A11, reacting to TNP independent of carrier peptide and of MHC haplotype, also allowed for an unusually systematic study of the minimal requirements for TNP recognition. Despite an almost complete ignorance of amino acid side chains on the carrier peptide, our data indicate a clearly position-specific interaction of hapten and TCR.

Animals↗
PubMed1997

Two-dimensional electrophoresis reveals a nuclear matrix-associated nucleolin complex of basic isoelectric point.

A monoclonal antibody was raised against a salt-extractable fraction of nuclear matrix / intermediate filament scaffolds of polarized MDCK cells. The antibody recognized an approximately 100 kDa protein in total cell lysates and nuclear matrices of various human cells and tissues and stained nucleolar structures in immunofluorescence microscopy. By partial sequencing of five peptides derived from immunoprecipitated protein, the targeted antigen was found to be homologous to human nucleolin. After two-dimensional electrophoresis of total HeLa cell lysates, immunoreactive bands were detected at isoelectric point (pI) 5.5--6.1, characteristic for nucleolin, and at pI 8.5--9. Whereas the protein focusing at acidic pI was found in Triton X-100-soluble cellular fractions, the antigen focusing at basic pI was exclusively contained in the residual nuclear fraction and was solubilized upon treatment of nuclear matrices with RNAse. The component solubilized by RNAse treatment was still detected at basic pI in two-dimensional electrophoresis. However, upon immunoprecipitation of the antigen from the RNAse-released fraction in the presence of sodium dodecyl sulfate (SDS), the nuclear matrix-derived antigen was positioned at pI 5--6. The present data indicate that the nuclear matrix-bound nucleolin is associated with ribonucleoproteins and a basic component resisting dissociation under conditions of isoelectric focusing.

Animals↗
PubMed1997

Perspectives of molecular and cellular electron tomography.

After a general introduction to three-dimensional electron microscopy and particularly to electron tomography (ET), the perspectives of applying ET to native (frozen-hydrated) cellular structures are discussed. In ET, a set of 2-D images of an object is recorded at different viewing directions and is then used for calculating a 3-D image. ET at a resolution of 2-5 nm would allow the 3-D organization of structural cellular components to be studied and would provide important information about spatial relationships and interactions. The question of whether it is a realistic long-term goal to visualize or--by sophisticated pattern recognition methods--identify macromolecules in cells frozen in toto or in frozen sections of cells is addressed. Because of the radiation sensitivity of biological specimens, a prerequisite of application of ET is the automation of the imaging process. Technical aspects of automated ET as realized in Martinsried and experiences are presented, and limitations of the technique are identified, both theoretically and experimentally. Possible improvements of instrumentation to overcome at least part of the limitations are discussed in some detail. Those means include increasing the accelerating voltage into the intermediate voltage range (300 to 500 kV), energy filtering, the use of a field emission gun, and a liquid-helium-cooled specimen stage. Two additional sections deal with ET of isolated macromolecules and of macromolecular structures in situ, and one section is devoted to possible methods for the detection of structures in volume data.

Actins↗
PubMed1997

Are the characteristics of betanidin glucosyltransferases from cell-suspension cultures of Dorotheanthus bellidiformis indicative of their phylogenetic relationship with flavonoid glucosyltransferases?

Uridine 5'-diphosphoglucose:betanidin 5-O- and 6-O-glucosyltransferases (5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific formation of betanin (betanidin 5-O-beta-glucoside) and gomphrenin I (betanidin 6-O-beta-glucoside), respectively. Both enzymes were purified to near homogeneity from cell-suspension cultures of Dorotheanthus bellidiformis, the 5-GT by classical chromatographic techniques and the 6-GT by affinity dye-ligand chromatography using UDP-glucose as eluent. Data obtained with highly purified enzymes indicate that 5-GT and 6-GT catalyze the indiscriminate transfer of glucose from UDP-glucose to hydroxyl groups of betanidin, flavonols, anthocyanidins and flavones, but discriminate between individual hydroxyl groups of the respective acceptor compounds. The 5-GT catalyzes the transfer of glucose to the C-4' hydroxyl group of quercetin as its best substrate, and the 6-GT to the C-3 hydroxyl group of cyanidin as its best substrate. Both enzymes also catalyze the formation of the respective 7-O-glucosides, but to a minor extent. Although the enzymes were not isolated to homogeneity, chromatographic, electrophoretic and kinetic properties proved that the respective enzyme activities were based on the presence of single enzymes, i.e. 5-GT and 6-GT. The N terminus of the 6-GT revealed high sequence identity to a proposed UDP-glucose: flavonol 3-O-glucosyltransferase (UF3GT) of Manihot esculenta. In addition to the 5-GT and 6-GT, we isolated a UF3GT from D. bellidiformis cell cultures that preferentially accepted myricetin and quercetin, but was inactive with betanidin. The same result was obtained with a UF3GT from Antirrhinum majus and a flavonol 4'-O-glucosyltransferase from Allium cepa. Based on these results, the main question to be addressed reads: Are the characteristics of the 5-GT and 6-GT indicative of their phylogenetic relationship with flavonoid glucosyltransferases?

Amino Acid Sequence↗
PubMed1997

Energy filtered electron tomography of ice-embedded actin and vesicles.

Semiautomatic single-axis tilt electron tomography has been used to visualize the three-dimensional organization of actin filaments in "phantom cells," i.e. lipid vesicles. The instrumentation consisted of a 120-kV electron microscope equipped with a postcolumn energy filter, which was used in the zero-loss imaging mode. Apart from changing the tilt angle, all steps required for automated tomography, such as recentering the image area, refocusing, and centering the energy-selecting slit, were performed by external computer control. This setup permitted imaging of ice-embedded samples up to a thickness of 800 nm with improved image contrast compared with that produced by tomography with a conventional electron microscope. In spite of the missing-wedge effect that is especially obvious in the study of membrane-filament interaction, single-axis tilt tomography was found to be an appropriate (in fact the only available) method for this kind of investigation. In contrast to random actin networks found in actin gels, actin filaments in and on vesicles with a bending radius of less than approximately 2 microns tend to be arranged in single layers of parallel filaments and often induce an elongated shape of the vesicles. Actin filaments located on the outside usually associate with the vesicle membrane.

Actins↗
PubMed1997

Complex patterns of immediate early gene induction in rat brain following brightness discrimination training and pseudotraining.

Following training of rats on a footshock-motivated brightness discrimination task in a Y-maze, different sets of transcription factor encoding immediate early genes (IEGs) were induced in anatomically distinct brain regions. As revealed by Northern analysis, mRNA levels of c-fos, jun-B and zif/268 increased in the hippocampus, while the expression of c-jun remained unchanged over a period of 7 h. In the cerebral cortex, c-jun was induced in addition to the other genes examined. In contrast, only c-fos, but not c-jun or zif/268 mRNAs were increased in the cerebellum. The induction of IEGs was rapid and transient, reaching maximal levels immediately after training and returning to basal levels within 2 h. Similar spatiotemporal expression patterns were observed in rats that received identical, but unpaired, stimuli in a pseudotraining procedure. Our results suggest that the initial prerequisites of learning, such as stimulus novelty, lead to an increased expression of IEG mRNAs after training and pseudotraining as an early necessary but not sufficient precondition for memory consolidation. Additional converging inputs might control at the transcriptional, translational or post-translational level the synthesis and biological effectiveness of proteins necessary to complete the formation of the memory trace in trained animals.

Animals↗
PubMed1997

Tricorn protease exists as an icosahedral supermolecule in vivo.

Tricorn protease is the core enzyme of a recently discovered modular proteolytic system. We present evidence that tricorn protease exists in vivo in the form of a higher-order assembly, namely as an icosahedral capsid. Its size exceeds that of many virus particles and represents by far the largest known homooligomeric enzyme complex. Each capsid is built from 20 copies of the tricorn hexameric toroid and thus has a molecular weight of 14.6 MDa. Three-dimensional reconstructions of ice-embedded capsids from electron micrographs show that it is hollow and has large void volumes in its wall. We suggest that the tricorn capsid, in addition to its intrinsic proteolytic activity, serves as the organizing center of a multienzyme complex.

Archaeal Proteins↗
PubMed1996

Maize chromosomal HMGc. Two closely related structure-specific DNA-binding proteins specify a second type of plant high mobility group box protein.

The chromosomal high mobility group (HMG) proteins are small and abundant non-histone proteins common to eukaryotes. We have purified the maize HMGc protein from immature kernels and characterized it by mass spectrometry and amino acid sequence analysis. HMGc could be resolved into two similar proteins by reversed phase chromatography. Cloning and characterization of the corresponding cDNAs revealed that they encode two closely related maize HMGc proteins, now termed HMGc1 and HMGc2. Their theoretical masses of 15,316 and 15,007 Da are >300 Da lower than the masses determined for the proteins purified from maize, indicating post-translational modifications of the proteins. Despite sequence similarity to maize HMGa (and previously described homologous proteins of other species) amino acid sequence alignments reveal that HMGc is in several conserved regions distinct from these proteins. Consequently, we have identified a novel type of plant protein containing an HMG box DNA binding domain and belonging to the HMG1 protein family. HMGc1 and HMGc2 were expressed in Escherichia coli, purified to homogeneity, and analyzed for their DNA binding properties. They proved to bind to DNA structure-specifically since they formed complexes with DNA minicircles at concentrations approximately 100-fold lower than the concentrations required to form complexes with linear fragments of identical sequence. Furthermore, HMGc1 and HMGc2 can constrain negative superhelical turns in plasmid DNA.

Amino Acid Sequence↗
PubMed1996

Use of capillary zone electrophoresis for analysis of imidodipeptides in urine of prolidase-deficient patients.

Prolidase deficiency (PD) is characterized by massive urinary excretion of imidodipeptides X-Pro and X-Hyp. We report the applicability of capillary zone electrophoresis to urinary imidodipeptide determination. The protocol is fast, simple, reliable, only small amounts of sample are required and there is minimal sample preparation. Electropherograms of urine samples from control subjects and four patients with prolidase deficiency were compared. The presence of imidodipeptides normally absent in urine was evident in patients' urine. Further analysis of urine samples enabled identification of excreted imidodipeptides and the pattern of excretion appeared to be heterogeneous for different patients. This method appears to be useful for identification of imidodipeptides in biological samples, as an efficient aid in diagnosis of PD, and as a method for providing more information about this disease.

Dipeptidases↗
PubMed1996

The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides.

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.

Amino Acid Sequence↗
PubMed1996

An essential lysine in the substrate-binding site of ornithine carbamoyltransferase.

Treatment of ornithine carbamoyltransferase from dolphin Stenella with pyridoxal phosphate, followed by reduction with NaBH4 resulted in complete loss of enzyme activity. The phosphate alone or the substrate analogue 2-aminovaleric acid moderately decreased the extent of inactivation, while carbamoyl phosphate plus 2-aminovaleric acid provided complete protection from inactivation. The partially inactivated enzyme showed K(m) values for substrates equivalent to those of native enzyme and lowered Kcat values. Two lysyl residues were substantially modified in the absence of ligands but only one of them was responsible for the inactivation of catalytic activity. Modification of a single subunit was sufficient to completely abolish the catalytic activity of the trimeric enzyme. The lysine involved has been identified as lysine 56 on the known primary structure of homologous human liver enzyme.

Amino Acid Sequence↗
PubMed1996

Non-native capillary isoelectric focusing for the analysis of the microheterogeneity of glycoproteins.

A simple capillary isoelectric focusing method in bare fused-silica capillaries under denaturing conditions was developed for the separation of the glyco-isoforms of recombinant tissue plasminogen activator (rTPA) which have pI values ranging from pH 6 to 8. The glycoprotein was denatured in 8 M urea and separated in the presence of 0.75% carrier ampholytes, 0.4% hydroxypropylmethylcellulose and 4 M urea. Excellent separation of the glyco-isoforms of rTPA was achieved. Good reproducibilities for peak area and migration times were obtained using pressure mobilization of the focused protein zones. The method optimized for rTPA was also successfully applied to other glycoproteins (recombinant human erythropoietin and a monoclonal antibody) with different carbohydrate contents (3-55%).

Electrophoresis, Capillary↗
PubMed1996

Change in depression as a precursor of cardiovascular events. SHEP Cooperative Research Group (Systoloc Hypertension in the elderly).

OBJECTIVE: To determine the relationship between increasing depressive symptoms and cardiovascular events or mortality. DESIGN: Cohort analytic study of data from randomized placebo-controlled double-blind clinical trial of antihypertensive therapy. Depressive symptoms were assessed semi-annually with the Center for Epidemiological Studies-Depression (CES-D) scale during an average follow-up of 4.5 years. SETTING: Ambulatory patients in 16 clinical centers of the Systolic Hypertension in the Elderly Program. PATIENTS: Generally healthy men and women aged 60 years or older randomized to active antihypertensive drug therapy or placebo who were 70% white and 53% women and had follow-up CES-D scores and no outcome events during the first 6 months (N=4367). MAIN OUTCOME MEASURES: All-cause mortality, fatal or nonfatal stroke, or myocardial infarction. RESULTS: Baseline depressive symptoms were not related to subsequent events; however, an increase in depression was prognostic. Cox proportional hazards regression analyses with the CES-D scale as a time-dependent variable, controlling for multiple covariates, indicated a 25% increased risk of death per 5-unit increase in the CES-D score (relative risk [RR], 1.25;95% confidence interval [CI], 1.15 to 1.36). The RR for stroke or myocardial infarction was 1.18(95%CI,1.08 to 1.30). Increase in CES-D score was an independent predictor in both placebo and active drug groups, and it was strongest as a risk factor for stroke among women (RR,1.29;95%CI,1.07 to 1.34). CONCLUSIONS: Among elderly persons, a significant and substantial excess risk of death and stroke or myocardial infarction was associated with an increase in depressive symptoms over time, which may be a marker for subsequent major disease events and warrants the attention of physicians to such mood changes. However, further studies of casual pathways are needed before wide-spread screening for depression in clinical practice is to be recommended.

Aged↗
PubMed1996

Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy.

We dissected the T cell activation potency and the immunoglobulin (Ig) E-binding properties (allergenicity) of nine isoforms of Bet v 1 (Bet v 1a-Bet v 1l), the major birch pollen allergen. Immunoblot experiments showed that Bet v 1 isoforms differ in their ability to bind IgE from birch pollen-allergic patients. All patients tested displayed similar IgE-binding patterns toward each particular isoform. Based on these experiments, we grouped Bet v 1 isoforms in three classes: molecules with high IgE-binding activity (isoforms a, e, and j), intermediate IgE-binding (isoforms b, c, and f), and low/no IgE-binding activity (isoforms d, g, and 1). Bet v 1a, a recombinant isoform selected from a cDNA expression library using IgE immunoscreening exhibited the highest IgE-binding activity. Isoforms a, b, d, e, and 1 were chosen as representatives from the three classes for experimentation. The potency of each isoallergen to activate T lymphocytes from birch pollen-allergic patients was assayed using peripheral blood mononuclear cells, allergen-specific T cell lines, and peptide-mapped allergen-specific T cell clones. Among the patients, some displayed a broad range of T cell-recognition patterns for Bet v 1 isoforms whereas others seemed to be restricted to particular isoforms. In spite of this variability, the highest scores for T cell proliferative responses were observed with isoform d (low IgE binder), followed by b, 1, e, and a. In vivo (skin prick) tests showed that the potency of isoforms d and 1 to induce typical urticarial type 1 reactions in Bet v 1-allergic individuals was significantly lower than for isoforms a, b, and e. Taken together, our results indicate that hypoallergenic Bet v 1 isoforms are potent activators of allergen-specific T lymphocytes, and Bet v 1 isoforms with high in vitro IgE-binding activity and in vivo allergenicity can display low T cell antigenicity. Based on these findings, we propose a novel approach for immunotherapy of type I allergies: a treatment with high doses of hypoallergenic isoforms or recombinant variants of atopic allergens. We proceed on the assumption that this measure would modulate the quality of the T helper cell response to allergens in vivo. The therapy form would additionally implicate a reduced risk of anaphylactic side effects.

Allergens↗
PubMed1996

Enhanced in situ gel digestion of electrophoretically separated proteins with automated peptide elution onto mini reversed-phase columns.

An improved method for the generation and automated isolation of internal peptides by in situ gel digestion of electrophoretically separated proteins is described. To enhance the sensitivity of the method, and to reduce the amount of sample handling steps, we have automated the extraction procedure of peptides after protein cleavage in a sodium dodecyl sulfate (SDS) gel matrix. The excised protein-containing polyacrylamide bands or spots are first minced to defined particles of about 30 microns. After in situ gel digestion, the gel slurry is transferred into a mini reversed-phase column-funnel assembly in the sample loading station of the Hewlett-Packard protein sequencer. Applying nitrogen pressure elutes peptides from the gel slurry onto the reversed-phase material. The mini reversed-phase column is then placed in an in-line column adapter and connected to a micropreparative high performance liquid chromatography (HPLC) column, where separation of the peptides under standard conditions is achieved. In the work described here complete digestions and excellent peptide recoveries allowed the generation of extensive internal sequence information from low picomole amounts of proteins. The method has been routinely applied in both laboratories for two years.

Acrylic Resins↗
PubMed1996

Determination of the inelastic mean free path in ice by examination of tilted vesicles and automated most probable loss imaging.

Using electron microscopy, the thickness of ice-embedded vesicles is estimated examining tilted and untilted views and assuming an ellipsoidal shape of the vesicles that appear to be circular in the untilted view. Another thickness measure is obtained from the ratio of the unfiltered and zero-loss-filtered image intensities of the vesicle. From these two measurements, the mean free path A for inelastic scattering of electrons in ice is calculated as 203 +/- 33 nm for 120 kV acceleration voltage. It is found that vesicles in thin ice films (< or = 1.5 lambda) significantly protrude out of the ice film. Due to surface tension the shape becomes an oblate ellipsoid. In holes covered with a thick ice film (> or = 3 lambda) and strong thickness gradients, vesicles are predominantly found in regions where the ice thickness is appropriate for their size. Also, a way of imaging the most probable loss under low-dose conditions involving thickness measurement is proposed. Even at large ice thicknesses zero-loss filtering always gives better image contrast. Most probable loss imaging can only help where there is no intensity in the zero-loss image, at very large thicknesses (lambda > 8).

Dimyristoylphosphatidylcholine↗