Biomedical subjects

R Reichelt

Publications and source records attributed to R Reichelt.

At least 37 records · Page 2Linked to original sources

PubMed1999

Immunogold electron microscopic localization of the cross-reactive two-EF-hand calcium-binding birch pollen allergen Bet v 4 in dry and rehydrated birch pollen.

BACKGROUND: Recently, a novel family of low-molecular-weight (8-9 kD), two-EF-hand calcium-binding proteins has been described as allergens in plant pollens. Approximately 10% of pollen-allergic patients have IgE antibodies which cross-react with the two-EF-hand allergens in tree, grass and weed pollens. The aim of the present study was to localize Bet v 4, the two-EF-hand allergen from birch, in mature, dry pollen and to study the release of this allergen after hydration of the pollen by immunogold electron microscopy. METHODS: Using completely anhydrous fixation techniques in combination with immunogold electron microscopy, we localized Bet v 4 and, for control purposes, the major birch pollen allergen Bet v 1, in dry birch pollen as well as in pollen grains after different periods of hydration. Parallel with these morphological studies, we monitored the release of Bet v 4 and Bet v 1 into aqueous supernatants of hydrated birch pollen grains by immunoblotting. RESULTS: Bet v 4 was found in the electron-dense cytosol, in particular between the vesicles and cisternae of the endoplasmic reticulum, inside mitochondria and in the vegetative as well as in the generative nucleus. Bet v 1 was localized in similar cellular compartments except for the mitochondria. After 30 s to 1 min of hydration, Bet v 4 migrated into the pollen exine and into the aqueous supernatants. Bet v 1 also moved out of the pollen grain, though not as quickly as Bet v 4. CONCLUSION: Bet v 4 represents an intracellular pollen protein which, following hydration of pollen grains, rapidly migrates to the pollen surface (exine) and is washed out. This behavior explains how Bet v 4, being primarily an intracellular pollen protein, becomes available to sensitize patients.

Allergens↗

PubMed1998

In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum.

Purified recombinant poly(hydroxyalkanoic acid) (PHA) synthase from Chromatium vinosum (PhaECCv) was used to examine in vitro the specific synthase activity, turnover of R-(-)-3-hydroxybutyryl coenzyme A (3HB-CoA) and poly(3-hydroxybutyric acid) formation under various conditions. The 3HB-CoA consumption was terminated by a reaction-dependent inactivation of the PHA synthase. Salts (MgCl2, CaCl2, NaCl), proteins (bovine serum albumin, lysozyme, phasine) or detergent (Tween 20) increased the 3HB-CoA turnover to 2.5-fold. Specific PHA synthase activity was only partially affected by the added components. In general, a higher concentration of salt often inhibited the activity of PhaECCv without affecting the yield according to 3HB-CoA turnover. NAD+ and NADP+ (2 mM) inhibited PhaECCv completely, whereas NADH and NADPH did not. Macroscopic poly(3HB) granules were formed in vitro if PhaECCv was incubated in the presence of sufficient amounts of 3HB-CoA and if MgCl2 was present. The form and size of the granules synthesized in vitro were affected by the concentration of the PHA synthase protein as well as by bovine serum albumin and the GA24 protein, a poly(3HB)-granule-associated protein of Alcaligenes eutrophus. Scanning electron micrographs from the synthesized granules were obtained. The granules consisted of poly(3HB) that had a molar mass in the range (1-2) x 10(6) g/mol.

3-Hydroxybutyric Acid↗

PubMed1997

[Scanning electron microscopy studies of morphologic changes in chemically stabilized ultrahigh molecular weight polyethylene].

UNLABELLED: Scanning electron microscopy (SEM) studies were performed on chemically stabilised ultra-high molecular weight polyethylene (UHMWPE) samples. The samples were sterilised by gamma radiation under a nitrogen atmosphere, packed under the exclusion of oxygen, and subjected to a special stabilising process. We investigated the surface as well as deeper surfaces in the samples using SEM: The samples showed minor surface irregularities resulting from the working of the material. Microtome sections of the specimens revealed no signs of cavitation. At high resolution, some typical lamination effects were to be seen. We were also able to demonstrate spherulites having diameters ranging from 10 to 40 microns. CLINICAL RELEVANCE: In contrast to other samples, the UHMWPE evaluated did not show any kind of cavitation or cracks, and this might be an indicator for a positive development.

Humans↗

PubMed1996

[Scanning electron microscopy studies of morphologic changes in ultra-high molecular weight polyethylene materials after various processing steps].

UNLABELLED: Scanning electron microscopic (SEM) studies were carried out on various orthopaedic implants made of ultra-high molecular weight polyethylene (UHMWPE). Both non-sterilized and gamma-sterilized machined (milled) implants were used. Milled implants showed significant surface irregularities, and cross-sections also revealed irregularities in deeper layers of the polyethylene. In gamma-sterilized implants, cracks measuring 10-30 microns in length were found at a depth of 1.5 to 2.5 mm. Crater-like defects were also occasionally seen. CLINICAL RELEVANCE: On the basis of these findings, direct compression moulding and a sterilization process in which oxygen is eliminated can be recommended as ways of improving the quality of UHMWPE used for orthopaedic implants.

Humans↗

PubMed1995

Mapping of nucleoporins to the center of the nuclear pore complex by post-embedding immunogold electron microscopy.

Ultrathin sections of Lowicryl K4M embedded cultured 3T3 cells, human keratinocytes and mouse/rat liver tissue were incubated with polyspecific primary antibodies against p62 and other nucleoporins followed by 10 nm gold labeled secondary antibodies. By quantitatively evaluating both cross sections and tangential sections of the NPC, we found that irrespective of the cell type antibodies predominantly bound within a radius of 25 nm around the central axis of the nuclear pore complex (NPC). Superposition of a current structural model of the NPC with the nucleoporin distribution observed by us showed that nucleoporins mapped predominatly to the controversely discussed 'central granule'. Our experimental approach was verified by mapping gp210, another nuclear pore protein, at or very close to the NPC in the perinuclear cisterna thus establishing a distribution pattern completely different from that of the nucleoporins.

3T3 Cells↗

PubMed1991

Biological structures imaged in a hybrid scanning transmission electron microscope and scanning tunneling microscope.

A hybrid scanning transmission electron microscope (STEM) and scanning tunneling microscope (STM) is described which allows simultaneous imaging of biological structures adsorbed to electron-transparent specimen supports in both modes of scanning microscopy, as demonstrated on uncoated phage T4 polyheads. We further discuss the reproducibility and validity of height data obtained from STM topographs of biomacromolecules and present raw data from topographs of freeze-dried, metal-coated nuclear envelopes from Xenopus laevis oocytes.

Animals↗

PubMed1991

Kindling does not induce persistent changes in fluorographic labeling patterns of benzodiazepine binding proteins in various rat brain regions.

The GABAA receptor has been implicated in the mechanisms underlying the phenomenon of kindling. Photoaffinity labeling with 3H-flunitrazepam followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allows the fluorographic visualization of GABAA receptor proteins with benzodiazepine binding sites which presumably correspond to different alpha-subtypes. This method offers an opportunity to investigate whether up- or down-regulation of single benzodiazepine binding proteins occurs. In the present study, labeling patterns of benzodiazepine binding proteins were determined in 12 brains regions of amygdala-kindled rats (2 weeks after the last fully kindled seizure) and sham-operated controls. For most brain regions, labeling patterns were separately determined for the ipsi- and contralateral side. A comparison of the labeling patterns thus obtained revealed no persistent changes between kindled animals and controls in any of the brain regions, including amygdala, substantia nigra and hippocampus. Thus, we conclude that kindling does not induce fluorographically detectable changes in the expression patterns of the benzodiazepine binding proteins. The results confirm the existence of regional heterogeneity of benzodiazepine binding proteins and extend the findings to brain regions which had previously not been investigated.

Affinity Labels↗

PubMed1991

Mass analysis of bacteriophage T4 proheads and mature heads by scanning transmission electron microscopy and hydrodynamic measurements.

Quantitative mass analysis of bacteriophage T4 proheads by scanning transmission electron microscopy (STEM) revealed a mass of 79.5 +/- 0.6 MDa, while hydrodynamic measurements yielded a prohead mass of about 80 MDa. This is 25% less than the prohead mass deduced from its polypeptide composition, and this finding implies that the bacteriophage T4 prohead is built of fewer polypeptide copies than previously reported. In contrast, the mass of mature heads measured by STEM, 194 +/- 2 MDa, is in agreement with previous mass measurements of DNA and protein content, and it is consistent with the previously determined stoichiometry. This good agreement of average STEM values for proheads and mature heads with corresponding hydrodynamic measurements suggests that STEM allows faithful evaluation of the masses of large supramolecular assemblies (i.e., greater than or equal to 200 MDa) such as whole viruses or cellular organelles.

Formaldehyde↗

PubMed1991

Ontogeny of the benzodiazepine receptor in human brain: fluorographic, immunochemical, and reversible binding studies.

The prenatal and postnatal human ontogeny of the central benzodiazepine receptor was investigated in six different brain regions between week 24 postconception and age 14 years. Binding studies, which were performed with [3H]flunitrazepam [( 3H]FNZ), revealed a steep increase in receptor density postnatally in frontal cortex and cerebellum. Bmax values were higher in medulla oblongata, pons, and thalamus than in cortex and cerebellum up to week 26. After that, receptor densities declined significantly in medulla and olive. The same tendency was apparent in pons, whereas receptor density remained unchanged in thalamus. The early ontogeny of the benzodiazepine receptor was also evaluated in fluorographs [( 3H]FNZ) and immunoblots using the alpha 1-subunit-specific monoclonal antibody (mAb) bd-24. Specific radiolabeled proteins with molecular weights of 53K and 59K were visible in cortical membranes from gestational week 8, the earliest time investigated. During further development, the intensity of the 53K band increased without changes in the 59K band. As in other species, postmortem proteolysis in human brain led to a specifically labeled peptide of 47K. The mAb bd-24 immunolabeled only the 53K protein and the 47K peptide.

Adolescent↗

PubMed1990

Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.

Nuclear pore complexes (NPCs) prepared from Xenopus laevis oocyte nuclear envelopes were studied in "intact" form (i.e., unexposed to detergent) and after detergent treatment by a combination of conventional transmission electron microscopy (CTEM) and quantitative scanning transmission electron microscopy (STEM). In correlation-averaged CTEM pictures of negatively stained intact NPCs and of distinct NPC components (i.e., "rings," "spoke" complexes, and "plug-spoke" complexes), several fine structural features arranged with octagonal symmetry about a central axis could reproducibly be identified. STEM micrographs of unstained/freeze-dried intact NPCs as well as of their components yielded comparable but less distinct features. Mass determination by STEM revealed the following molecular masses: intact NPC with plug, 124 +/- 11 MD; intact NPC without plug, 112 +/- 11 MD; heavy ring, 32 +/- 5 MD; light ring, 21 +/- 4 MD; plug-spoke complex, 66 +/- 8 MD; and spoke complex, 52 +/- 3 MD. Based on these combined CTEM and STEM data, a three-dimensional model of the NPC exhibiting eightfold centrosymmetry about an axis perpendicular to the plane of the nuclear envelope but asymmetric along this axis is proposed. This structural polarity of the NPC across the nuclear envelope is in accord with its well-documented functional polarity facilitating mediated nucleocytoplasmic exchange of molecules and particles.

Animals↗

PubMed1990

Calpain inhibitor I prevents rapid postmortem degradation of benzodiazepine binding proteins: fluorographic and immunological evidence.

Endogenous proteolysis of the major central benzodiazepine (BZ) binding protein of 53K occurs rapidly postmortem and leads to a fragment of 47K. To determine indirectly the protease responsible for this proteolysis, membranes of porcine cortex were prepared from homogenates, which were either frozen immediately or left at room temperature for 12 h in the presence or absence of various representative protease inhibitors. Membranes were subsequently photolabeled with [3H]flunitrazepam, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography or immunoblotted using an alpha-subunit-specific monoclonal antibody bd-24. Both fluorographs and immunoblots revealed that calpain inhibitor I, Ep-459 (E-64 analogue), and EDTA (greater than or equal to 1 mM) prevent endogenous proteolysis. In future studies one of these inhibitors should be added to receptor preparations. The results indicate that calpain is the responsible protease.

Affinity Labels↗

PubMed1989

Photolabeled tryptic degradation products of benzodiazepine-binding proteins are glycopeptides. Implications for localization of cleavage sites.

Crude synaptic membranes of avian and mammalian brain tissue were photolabeled with the benzodiazepine-receptor ligand [3H]flunitrazepam and subsequently treated extensively with trypsin followed by incubation with endoglycosidase F. SDS-polyacrylamide gel electrophoresis and fluorography revealed that the final tryptic degradation product of 25 kDa in both pigeon and calf brain is deglycosylated in two steps. These results were confirmed by immunoblots of similarly pretreated membranes of pig brain using the alpha-subunit-specific monoclonal antibody bd-24. Benzodiazepine-receptor binding and its enhancement by GABA are largely retained after trypsinization. Based on the proposed transmembrane topology for the alpha-subunits of the GABA/benzodiazepine receptor, we suggest that the large N-terminal domain of benzodiazepine-binding proteins is protected against tryptic cleavage.

Animals↗

PubMed1989

Characterization of the promastigote surface protease of Leishmania as a membrane-bound zinc endopeptidase.

The effects of a variety of inhibitors suggested that the promastigote surface protease (PSP) of Leishmania might be a zinc metalloprotease. To investigate this possibility, we conducted atomic emission and absorption spectroscopic analyses, which show that PSP contains 1 atom of zinc per 63-kDa monomer. Further studies showed that the enzyme can be biosynthetically labeled with 65ZnCl2. The comparison of the amino acid sequence of Leishmania major PSP with nine other zinc metalloproteinases revealed significant similarity in the area of their zinc-binding sites. These data show clearly that the promastigote surface protease of Leishmania is a zinc metalloproteinase. Secondary structure analysis by circular dichroism spectroscopy indicates that PSP contains over 40% beta-strand and less than 20% alpha-helical structure. The molecular masses of amphiphilic PSP (152 kDa) and of hydrophilic PSP (142 kDa), determined by quantitative electron scattering, suggest that the purified enzyme occurs in solution, and presumably at the cell surface, as a non-covalent homodimer.

Amino Acid Sequence↗

PubMed1989

Postmortem degradation alters fluorographic labeling patterns and affinities of benzodiazepine binding proteins.

To investigate the effect of endogenous proteolysis on the molecular weights of the benzodiazepine binding proteins, brains of trout, chicken, and rat were removed immediately after death and stored at room temperature for various periods of time before they were frozen. Photoaffinity labeling of membranes with [3H]flunitrazepam, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, revealed proteolytic fragments of 47K in trout, chicken, and rat. The proteolysis set in rapidly after death. Seemingly in parallel with the degradation observed fluorographically, the affinity for [3H]flunitrazepam increased without systematic changes in receptor density. The degradation pattern was not identical to that of the photolabeled trypsinized benzodiazepine binding proteins. The endogenous proteolytic fragments were deglycosylated in two steps. In conclusion, proteolytic effects must be taken into account when interpreting labeling patterns and binding parameters.

Animals↗

PubMed1989

Determination of protein and nucleic acid concentration and localization of specific elements by scanning transmission electron microscopy.

Elastic and inelastic dark-field micrographs simultaneously recorded by the scanning transmission electron microscope (STEM) provide structural and quantitative chemical information at a spatial resolution of some nanometers. Simultaneous acquisition is essential as it warrants: (i) the geometrical identity of picture elements (pixels) in these micrographs, and (ii) the identity of recording conditions (focus, electron dose) for the elastic and inelastic images. Suitable off-line processing of such multichannel images allows the concentration of protein or nucleic acid within embedding material to be evaluated. In addition, location and number of chemical elements such as phosphorus can be determined. Theoretical sensitivity is a few phosphorus atoms in a volume of approximately 1000 nm3 (not demonstrated in this paper). Quantitative electron microscopy of this kind, however, requires the influence of mass loss as well as plural scattering to be correctly accounted for. Experimental conditions that provide a sound basis for concentration determination as well as element mapping are discussed.

Animals↗

PubMed1988

A comparative phylogenetic study of the distribution of cerebellar GABAA/benzodiazepine receptors using radioligands and monoclonal antibodies.

In mammalian cerebella the distribution of high affinity GABAA and benzodiazepine binding sites does not parallel each other. Differences in the molecular structure of the receptor complex or conformational changes have been proposed to explain the lack of a co-localization of these binding sites. Using radio- and immunohistochemistry we were able to obtain similar results for cerebella of non-mammalian species, indicating that the respective distribution of the binding sites is well conserved throughout vertebrate evolution. The gamma-aminobutyric acid agonist [3H]muscimol and the beta-subunit specific antibody bd-17 strongly labeled the granular layer. The radioligands for the benzodiazepine binding site [3H]flunitrazepam and [3H]Ro 15-1788 revealed species variation in the ratio of benzodiazepine receptor density in the granular and molecular layers. In human and pig cerebellum, the localization of antigenic sites recognized by the alpha-subunit specific antibody bd-24 deviates from the distribution of the benzodiazepine binding sites.

Animals↗

PubMed1988

Persistence of species variation and regional heterogeneity of the apparent molecular masses of benzodiazepine-binding proteins after deglycosylation.

Brain membrane preparations of different vertebrates were photoaffinity labeled with [3H]flunitrazepam and subsequently deglycosylated with endoglycosidase F and peptide N-glycopeptidase. SDS-polyacrylamide gel electrophoresis followed by fluorography revealed that each benzodiazepine-binding protein is deglycosylated in two steps, indicating that each protein has two glycosylation sites. Species variation of the apparent molecular masses of the benzodiazepine-binding proteins and regional heterogeneity in avians persist after deglycosylation. These results indicate that the alpha-subunit(s) of the GABA/benzodiazepine receptor has undergone electrophoretically detectable changes in its amino acid composition during vertebrate evolution. The existence of at least two different alpha-subunits in avians is further substantiated.

Animals↗

PubMed1988

The shark GABA-benzodiazepine receptor: further evidence for a not so late phylogenetic appearance of the benzodiazepine receptor.

Whilst the brain-specific benzodiazepine receptor has been assumed to show a late evolutionary appearance, we present evidence for the presence of a central benzodiazepine binding site in sharks, which shows a high affinity for [3H]Ro 15-1788. However, the receptor density and the affinities of several benzodiazepine receptor ligands are lower than in mammals, thus presumably explaining why the benzodiazepine binding sites had previously escaped detection in elasmobranchs. Additionally, radio- and immunohistochemistry were performed to localize the radioligand binding sites and the antigenic sites of the shark gamma-aminobutyric acid (GABA)-benzodiazepine receptor. In cerebellum, the granular layer reveals a high density of [3H]muscimol binding sites. The immunoreaction obtained with the beta-subunit-specific monoclonal antibody bd-17 seemingly parallels the distribution of high-affinity GABA binding sites. In contrast, [3H]Ro 15-1788 binding sites are evenly distributed in the molecular and granular layers, thus the results are similar to those previously described for rat cerebellum. Apparently, the respective distributions in this brain region are well conserved throughout vertebrate evolution.

Animals↗