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Biomedical subjects

G Fischer

Publications and source records attributed to G Fischer.

At least 271 records · Page 15Linked to original sources
PubMed1995

Determination of the renin inhibitor Ro 42-5892 in human plasma by automated pre-column derivatization, reversed-phase high-performance liquid chromatographic separation and electrochemical detection after post-column irradiation.

The renin inhibitor Ro 42-5892 has been found to be very potent, thereby necessitating a sensitive assay method for the evaluation of its pharmacokinetics in man. We report here the development of a very sensitive and selective HPLC assay for the analysis of this compound in human plasma. Ro 42-5892 was extracted from plasma with dichloromethane, derivatized with 2,4-dinitrofluorobenzene and then chromatographed on a Novapak C18 column (150 x 3.9 mm I.D.) with acetic acid buffer (pH 7)-acetonitrile (100:85). Detection was performed by irradiation at 254 nm, followed by electrochemical oxidation at 550 mV. The extraction recovery of Ro 42-5892 from human plasma (mean 102%) was quantitative. With this method a limit of quantitation of 0.3 ng/ml was achieved. The assay was linear up to 5 ng/ml, had acceptable inter-assay precision (12.2%) and accuracy (9.3%) and was successfully tested for selectivity. This assay was successfully applied to over 250 samples from a pharmacokinetic study in hypertensive patients.

Autoanalysis↗
PubMed1995

A kinetic analysis of the folding of human carbonic anhydrase II and its catalysis by cyclophilin.

The kinetics of unfolding and refolding of human carbonic anhydrase II (HCAII) and its catalysis by the peptidyl-prolyl-cis/trans-isomerase cyclophilin were investigated. HCAII contains 15 trans- and 2 cis-prolyl peptide bonds, and, when long-term denatured, virtually all unfolded molecules contain non-native prolyl isomers. In unfolding these molecules (Us) are produced slowly in a biphasic process reflecting the isomerization of several trans-prolines and of one cis-proline. In refolding, the rapid formation of an intermediate of the molten globule type is followed by several slow prolyl isomerizations, which determine the rate of reactivation. By a short 10-s incubation in 5.0 M guanidinium chloride at 2 degrees C, unfolded HCAII species with all prolines still in the native conformation (Uf) could be produced. Surprisingly, only a fraction of Uf refolds rapidly, but the other molecules refold slowly. Evidently, some prolyl peptide bonds isomerize early in refolding, at the stage of the molten globule and as a consequence, molecules with incorrect prolyl isomers are formed in competition with the productive folding of Uf. This fraction of slow-folding molecules is strongly increased when cyclophilin is present, because it accelerates the formation of non-native prolyl isomers as long as the molecules remain in the molten globule state. Later cyclophilin catalyzes the isomerization of these prolyl peptide bonds toward the native state, which are stabilized in their conformation by further folding to the native state. This catalysis is very efficient, because only prolines that are accessible in the molten globule are involved in this sequence of isomerization and reisomerization.

Amino Acid Isomerases↗
PubMed1995

Time-dependent inhibition of peptidylprolyl cis-trans-isomerases by FK506 is probably due to cis-trans isomerization of the inhibitor's imide bond.

Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate constants of the slow phase were determined. We conclude that the cis to trans interconversion of the pipecolinyl bond of the three peptidomacrolides may be responsible for the slow process. There was no indication of a suicide catalysis of this process by FKBPs.

Amino Acid Isomerases↗
PubMed1995

Diversity of TCRAV and TCRBV sequences used by human T-cell clones specific for a minimal epitope of Bet v 1, the major birch pollen allergen.

T-cell clones (TCC) were raised from the peripheral blood of patients suffering from tree pollen allergy. All TCC were restricted by HLA-DR molecules. In order to investigate possible intervention targets in Type I allergic diseases, we examined T-cell receptor (TCR) alpha and beta chain nucleotide sequences of five allergen-reactive human CD4+ TCC specific for a C-terminal epitope (BV 144) of Bet v 1, the major birch pollen allergen. Proliferation assays using synthetic peptides revealed the 10-mer LRAVESYLLA as minimal epitope for three TCC; two TCC also displayed reactivity with the nonapeptide LRAVESYLL. Two TCC expressed TCRBV2S3, all other BV144-specific TCC used diverse TCRAV and TCRBV gene segments. Moreover, the junctional regions encoding the third complementary determining regions (CDR3) of the TCR showed a striking heterogeneity in length and amino acid composition. Nevertheless, all TCC showed an arginine residue in the N-terminal region of their TCRBV CDR3 loops. Therefore, therapeutical strategies aimed at the clonal deletion of allergen-specific T-cell clones, providing help for IgE synthesis, will not be feasible. Our results cast a doubt on the theory that the CDR3 exclusively provides the primary contact with the peptide bound in the major histocompatibility (MHC) groove, and suggest additional interaction with MHC class II.

Allergens↗
PubMed1995

Intermediate filament typing of the human embryonic and fetal notochord.

In order to characterize human notochordal tissue we investigated notochords from 32 human embryos and fetuses ranging between the 5th and 13th gestational week, using immunohistochemistry to detect intermediate filament proteins cytokeratin, vimentin and desmin, the cytokeratin subtypes 7, 8, 18, 19 and 20, epithelial membrane antigen (EMA), and adhesion molecules pan-cadherin and E-cadherin. Strong immunoreactions could be demonstrated for pan-cytokeratin, but not for desmin or EMA. Staining for pan-cadherin and weak staining for E-cadherin was found on cell membranes of notochordal cells. Also it was demonstrated that notochordal cells of all developmental stages contain the cytokeratins 8, 18 and 19, but not 7 or 20. Some cells in the embryonic notochord also contained some vimentin. Vimentin reactivity increased between the 8th and 13th gestational week parallel to morphological changes leading from an epithelial phenotype to the chorda reticulum which represents a mesenchymal tissue within the intervertebral disc anlagen. This coexpression reflects the epithelial-mesenchymal transformation of the notochord, which also loses E-cadherin expression during later stages. Our findings cannot elucidate a histogenetic germ layer origin of the human notochord but demonstrate its epithelial character. Thus, morphogenetic inductive processes between the human notochord and its surrounding vertebral column anlagen can be classified as epithelial-mesenchymal interactions.

Antibodies, Monoclonal↗
PubMed1995

Virtual reality arthroscopy training simulator.

This paper describes the result of the interdisciplinary cooperation of traumatologists of the Berufsgenossenschaftliche Unfallklinik (BGU) in Frankfurt am Main and a team of computer graphics scientists of the Franunhofer Institute for Computer Graphics in Darmstadt. We have developed a highly interactive training simulator system by means of computer graphics and virtual reality techniques.

Arthroscopy↗
PubMed1995

Quantitative measurement of neuronal degeneration in organotypic hippocampal cultures after combined oxygen/glucose deprivation.

Organotypic hippocampal cultures were used to study cell degeneration during the recovery period after defined periods (30 and 60 min) of combined oxygen/glucose deprivation mimicking transient ischemic conditions. Staining with the fluorescent dye propidium iodide allowed detection of damaged cells. Fluorescence intensity was measured by an image analysis system and used to quantify cell damage at different time points during the recovery period (up to 22 h). At 30 min of oxygen/glucose deprivation cells in the CA1 area were relatively more sensitive compared to CA3 and dentate gyrus cells, with respect to the time course of degeneration and the percentage of affected cells. Expanding the oxygen/glucose deprivation period from 30 to 60 min drastically increased the percentage of cells dying in all hippocampal areas. Still, however, cells in CA1 degenerated faster compared to those in the CA3 area and dentate gyrus. A histological analysis of toluidine blue as well as MAP2-immunostained sections revealed that almost all neurons degenerated in all hippocampal areas following the 60-min deprivation period, whereas GFAP-stained astrocytes appeared to be unaffected. Therefore, neuronal degeneration could be quantified by taking the fluorescence intensity values 22 h after 60 min of oxygen/glucose deprivation as 100% neuronal damage. The possibility to quantify neuronal damage in organotypic cultures offers a useful tool for detailed studies on mechanisms of neuronal cell death in a cell culture system which is closer to in situ conditions than monolayer cell cultures.

Animals↗
PubMed1995

T-cell epitopes of Phl p 1, major pollen allergen of timothy grass (Phleum pratense): evidence for crossreacting and non-crossreacting T-cell epitopes within grass group I allergens.

BACKGROUND: The use of peptides representing T-cell epitopes of allergens is a modern concept for improvement of specific immunotherapy. A prerequisite for this approach is the identification of T-cell epitopes of atopic allergens. METHODS: T-cell lines and 40 T-cell clones (TCC) specific for Phl p 1, the group I allergen of timothy grass (Phleum pratense), were established from the peripheral blood of nine patients allergic to grass pollen and mapped for epitope specificity by using overlapping dodecapeptides. Phenotype and cytokine production profile of TCC were investigated. Representative TCC were analyzed for HLA-restriction, T-cell receptor V beta gene usage, and crossreactivity with grass pollen extracts from Dactylis glomerata, Poa pratensis, Lolium perenne, Secale cereale, and selected amino acid sequence-derived peptides. RESULTS: Patients displayed IgE binding to all grass species investigated. Forty TCC were established. Fifteen T-cell epitopes could be identified on Phl p 1. Of 40 TCC, 39 displayed the helper cell (Th) phenotype; one clone was CD8+. Specific stimulation induced a Th2-like type of cytokine production in 20 of 39 TCC. Crossreactivity studies revealed crossreacting and non-crossreacting T-cell epitopes. CONCLUSION: Phl p 1, a major grass pollen allergen, harbors multiple T-cell epitopes. Species-specific and crossreacting T-cell epitopes exist among group I allergens of grasses. Epitope recognition patterns could not be correlated with particular HLA haplotypes. A restricted T-cell receptor V beta gene usage was not observed.

Allergens↗
PubMed1995

Maternal alloimmunization against fetal platelet antigens: a prospective study.

Neonatal alloimmune thrombocytopenia (NAIT) is induced by maternal alloantibodies to fetal platelet antigens. This prospective study was carried out to evaluate the incidence of anti-platelet antibodies in 933 mother-child pairs where the mother and child were typed for the human platelet antigens (HPA)-1, -2, -3, -5. Sera from mismatched mother-child pairs were screened for anti-platelet antibodies, anti-HLA class I and blood group ABO IgG antibodies. Platelet-specific antibodies were anti-HPA-3a in one and anti-HPA-5b in 17 neonates, respectively. All these neonates had normal platelet counts. One woman had autoreactive antibodies. Anti-HLA class I and anti-blood group A IgG antibodies were detected in five and four neonates, respectively, born with a platelet count < 150 x 10(9)/l. None of the 11 homozygous HPA-1b mothers became immunized against their heterozygous offspring. The maternal HLA-allotypes HLA-DR52 and -DR6, typically found in individuals immunized against HPA-1a and -5b, respectively, were found in three of 11 HPA-b/b nonresponders and eight of the anti-HPA-5b responders. The results indicate that a risk for NAIT due to HPA-2 and -3 alloimmunization is low. The HLA allotypes do not predict the risk for NAIT due to HPA-1 or -5 alloimmunization. Maternal anti-HPA-5b antibodies do not correlate with the platelet count in the neonate.

ABO Blood-Group System↗
PubMed1995

The chronically symptomatic vulva: aetiology and management.

OBJECTIVE: To determine the causes and management of chronic vulval symptoms and to compare the findings in patients first presenting to a gynaecologist with those in patients first presenting to a dermatologist. DESIGN: A prospective study of 144 patients, approximately half each being referred to a gynaecologist and a dermatologist. Diagnosis was based on clinical history, vulvoscopy, vulval biopsy and bacteriology. Biopsies were examined by a histopathologist experienced in dermatopathology and gynaecological pathology. RESULTS: The two patient groups were similar in both range and frequency of conditions. The commonest cause of chronic vulval symptoms was dermatitis, which was found in 64% of our patients. Dermatitis occurred alone in 55% and was found in association with histological evidence of human papilloma virus (HPV) in a further 9%. These patients responded to simple dermatological methods, mainly topical corticosteroids. Histopathological evidence of HPV was encountered in only 23% of our patients, and of these 36% also demonstrated dermatitis on biopsy. Most responded to topical corticosteroids. Another 7% had lichen sclerosus, and all responded to potent topical corticosteroid. The remaining 15% demonstrated a range of diagnoses, including psoriasis, dysaesthetic vulvodynia, vulval intraepithelial neoplasia (VIN) and chronic candidiasis. The majority of patients had a corticosteroid responsive dermatosis rather than a gynaecological condition. CONCLUSIONS: The majority of patients with a chronically symptomatic vulva who present to either a gynaecologist or a dermatologist have a dermatological condition that responds to simple dermatological treatments. We believe that the presence or absence of the human papilloma virus is not relevant to most patients with a chronically symptomatic vulva and treatments should not be aimed at eradicating this virus. Histopathologists and gynaecologists who have focused on gynaecological disorders have often missed simple dermatological conditions that are easily treatable.

Adolescent↗
PubMed1995

Neutrophil elastase in crevicular fluid: comparison of a middle-aged general population with healthy and periodontitis groups.

Neutrophil elastase (NE) was measured in crevicular fluid (GCF) collected from 3 subject groups. GCF was harvested at a single visit of subjects with periodontal health (n = 21) and with periodontitis (n = 28). Samples were obtained from 132 middle-aged, middle-class health conscious patients of a health maintenance organization (HMO) at baseline and 1 year later. GCF NE was higher in periodontitis than in health. Mean GCF NE of HMO subjects was much closer to health than to periodontitis. Few members of the HMO population had enzyme levels typical of periodontitis. Subjects and sites of the HMO population were segregated into 3 categories based on enzyme levels of the healthy and periodontitis subjects. Most HMO subjects and sites were in the activity category corresponding to healthy subjects. Only a small portion were in the activity category common in periodontitis. Enzyme levels in the highest activity category at both samplings were infrequent. High enzyme levels in the HMO population were not associated with attachment loss. Thus, assay of GCF NE provided little evidence of disease in a middle-aged, middle-class health conscious general population. This finding confirms an analysis of epidemiological surveys which concluded that a population such as studied here would not benefit from periodontal diagnostic testing.

Adult↗
PubMed1995

Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells.

Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive intracellularly in eukaryotic cells such as monocytes, macrophages, and protozoan organisms. The Mip (macrophage infectivity potentiator) protein represents a factor of L. pneumophila necessary for optimal intracellular survival. Interestingly, Mip belongs to the substance class of FK 506-binding proteins and exhibits peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. In order to identify amino acids most likely to be involved in the enzymatic activity of Mip, site-directed mutagenized Mip proteins were constructed and characterized. It was shown that an Asp-142 to Leu-142 mutation and a Tyr-185 to Ala-185 substitution resulted in strongly reduced PPIase activity of the recombinant Mip proteins (5.3 and 0.6% of the activity of the wild-type Mip, respectively). Genes coding for the wild-type and for site-directed-mutagenized Mip proteins were used to complement three different Mip-negative mutants of the L. pneumophila Corby, Philadelphia I, and Wadsworth. While Mip protein expression could be restored in the corresponding complementants, significant Mip-specific PPIase activity could be detected only in Mip mutants complemented with wild-type mip genes. To investigate the influence of the PPIase activity of Mip on intracellular survival of L. pneumophila, invasion assays were performed using the macrophage-like cell line U937, human blood monocytes, and Acanthamoeba castellanii. The Mip-negative mutants were approximately 50- to 100-fold less infective for A. castellanii and for human mononuclear phagocytes in vitro compared with their isogenic Mip-positive parental strains. The wild-type invasion rate could be restored by introducing an intact copy of the mip gene into Mip-negative strains. In addition, no differences in intracellular survival were observed between the wild-type isolates and the Legionella strains exhibiting strongly reduced PPIase activity. These data indicated that the enzymatic activity of Mip does not contribute to intracellular survival of L. pneumophila.

Amino Acid Isomerases↗
PubMed1995

Patterns of free calcium in multicellular stages of Dictyostelium expressing jellyfish apoaequorin.

To examine the patterns of high free cytosolic calcium or [Ca2+]i during Dictyostelium's development, we expressed apoaequorin in D. discoideum, reconstituted aequorin and observed the resultant patterns of calcium-dependent luminescence. Specific, high calcium zones are seen throughout normal multicellular development and are roughly coincident with those regions that later differentiate into stalk or stalk-like cells. A slug, for example, shows a primary high calcium zone within its front quarter and a secondary one around its tail; while a mound shows such a zone around the periphery of its base. Combined with previous evidence, our findings support the hypothesis that high [Ca2+]i feeds back to favor the stalk pathway. We also discovered several high calcium zones within the mound's base that do not coincide with any known prepatterns in D. discoideum. These include two, relatively persistent, antipodal strips along the mound's periphery. These various persistent zones of high calcium are largely made up of frequent, 10 to 30 second long, semiperiodic calcium spikes. Each of these spikes generates a correspondingly short-lived, 200 to 500 microns long, high calcium band which extends along the nearby surface. Similar, but relatively large and infrequent, spikes generate cross bands which extend across migrating slugs and just behind their advancing tips as well as across the peripheries of rotating mounds and midway between their antipodal strips. Moreover, calcium has a doubling time of about a second as various spikes rise. This last observation suggests that the calcium bands seen in Dictyostelium may be generated by so-called fast calcium waves.

Aequorin↗
PubMed1995

Isolated tuberculosis of the pancreas masquerading as a pancreatic mass.

A 65-yr-old woman presented for evaluation of a pancreatic mass. She had been suffering from severe constitutional symptoms for 18 months; those symptoms included weight loss, increasing fatigue, night sweats, and recurrent fever attacks up to 40 degrees C. Later, bluish subcutaneous nodules developed on her lower limbs. Laboratory tests yielded signs of chronic inflammation and impaired glucose tolerance with elevated serum insulin and glucagon concentrations. Skin biopsy revealed lobular panniculitis. Ultrasonography and a CT scan demonstrated enlargement of the pancreas, and endoscopic retrograde pancreaticography disclosed displacement and stenosis of the main pancreatic duct. The patient was referred for explorative laparotomy, which was highly suggestive of a malignant pancreatic tumor. However, histological examination of the resected pancreatic and peripancreatic mass revealed tuberculous pancreatitis. This form of isolated tuberculous pancreatitis, associated with lobular panniculitis and laboratory features consistent with a tumor of the endocrine pancreas, has not been reported previously. Active tuberculosis should be a leading differential diagnosis in a patient with an enlarged pancreas when the usual diagnostic reasoning does not yield conclusive results.

Aged↗