Biomedical subjects

G Fischer

Publications and source records attributed to G Fischer.

At least 163 records · Page 9Linked to original sources

PubMed1998

Ssp1, a site-specific parvulin homolog from Neurospora crassa active in protein folding.

Peptidyl-prolyl cis-trans-isomerases (PPIases) are enzymes capable of isomerizing a Xaa-Pro peptide bond. Three families of PPIases are known: cyclophilins, FKBPs, and parvulins. The physiological functions of the PPIases are only poorly understood. Eucaryotic members of the parvulin family have recently been shown to be essential for regulation of mitosis. Here we describe the purification and characterization of Ssp1, an abundant parvulin homolog from Neurospora crassa, which is unique among the known eucaryotic parvulins in containing a polyglutamine stretch between the N-terminal WW domain and the C-terminal PPIase domain. Ssp1 is a site-specific PPIase with respect to the amino acid N-terminal to the proline residue. Peptides with glutamate, phosphoserine, or phosphothreonine in the -1-position proved to be the best substrates. Ssp1 is not only able to isomerize small peptides but is also active in protein folding, as shown with mouse dihydrofolate reductase. Using the substrate specificity of Ssp1, we could identify Glu81-Pro82 as a PPIase-sensitive site in folding of dihydrofolate reductase. These results demonstrate that Ssp1 is a potent mediator of protein folding and that parvulins can serve as tools to elucidate rate-limiting steps in protein folding reactions.

Amino Acid Sequence↗

PubMed1998

Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of the linear chromosomal DNA of Streptomyces ambofaciens.

The chromosomal DNA of the bacteria Streptomyces ambofaciens DSM40697 is an 8-Mb linear molecule that ends in terminal inverted repeats (TIRs) of 210 kb. The sequences of the TIRs are highly variable between the different linear replicons of Streptomyces (plasmids or chromosomes). Two spontaneous mutant strains harboring TIRs of 480 and 850 kb were isolated. The TIR polymorphism seen is a result of the deletion of one chromosomal end and its replacement by 480 or 850 kb of sequence identical to the end of the undeleted chromosomal arm. Analysis of the wild-type sequences involved in these rearrangements revealed that a recombination event took place between the two copies of a duplicated DNA sequence. Each copy was mapped to one chromosomal arm, outside of the TIR, and encoded a putative alternative sigma factor. The two ORFs, designated hasR and hasL, were found to be 99% similar at the nucleotide level. The sequence of the chimeric regions generated by the recombination showed that the chromosomal structure of the mutant strains resulted from homologous recombination events between the two copies. We suggest that this mechanism of chromosomal arm replacement contributes to the rapid evolutionary diversification of the sequences of the TIR in Streptomyces.

Amino Acid Sequence↗

PubMed1998

[Conventional heart surgery with the fast-track-method: experiences from a pilot study].

Early release after cardiac surgery can be promoted by implementation of a standard protocol for accelerated perioperative and early postoperative care, with optimal education and support of the patient playing a key role. We report on our preliminary experience with 100 selected patients who underwent a "fast track" protocol following coronary artery bypass (n = 61), valve replacement or reconstruction (n = 34) or closure of an atrial septal defect (n = 5) between 1996 and 1998. Surgery was performed through a midline sternotomy using normothermic or mild hypothermic cardiopulmonary bypass. Patients undergoing cardiac surgery with less invasive techniques were excluded from this study. The following criteria had to be fulfilled for early hospital discharge: sinus rhythm, temperature below 37.5 degrees C, stable haematocrit around 0.30, uncomplicated wound healing and complete mobilisation including stair exercises. Mean duration of the operation was 137 +/- 24 minutes and mean intubation time was 4.5 +/- 3 hours. Mean duration of hospitalisation from the day of the operation was 4.9 +/- 2.1 days. There was no early or late mortality in this group of patients and only 2 patients had to be re-admitted on postoperative day 10 and 14 because of atrial fibrillation in one and a wound healing problem in the other. Accelerated recovery and early hospital discharge is highly attractive in selected patients; in helps to promote early cardiac rehabilitation and the costs of the procedure can be substantially reduced. According to our experience and the most recent literature, this approach does not expose patients to higher mortality or morbidity. In addition, fast-tracked patients have shown a higher level of satisfaction. Under optimal cooperation between surgery, anaesthesiology and intensive care unit, the fast-track protocol can be applied in approximately 30% of overall adult cardiac surgery patients.

Adult↗

PubMed1998

Cerebral CT findings in male opioid-dependent patients: stereological, planimetric and linear measurements.

Cerebrospinal fluid (CSF) space enlargement has been demonstrated in substance-related disorders like alcohol and cocaine dependence. Experimental animal studies showed a reduction in shape and size of mesolimbic dopaminergic neurons after chronic morphine administration. Other studies indicated a change of neurofilament and glial fibrillary acid proteins after chronic opiate administration. Furthermore, frequent overdosing and toxicological effects of 'street'-heroin may lead to CSF space enlargement in opioid dependence. In our study the pericortical and ventricular CSF space of 21 male opioid-dependent patients was compared with an age- and sex-matched normal control group. Considering serious problems with ratio and proportion measures, we used a battery of linear (cella media index, Huckman number, frontal horn index), planimetric (cortical atrophy score) and stereological volumetric measures in order to detect differences in cranial computerized tomography scans. We found a significant ventricular and cortical volume loss of the brain in opioid-dependent patients. A higher degree of frontal lobe volume loss seemed to be associated with a shorter period of abstinence before relapse. However, the etiology of volume loss of the brain in opioid-dependent patients is still unclear, but experimental animal studies provide some evidence that long-term, chronic opiate exposure is associated with visible changes of specific structures in the brain.

Adult↗

PubMed1998

Mapping the stereospecificity of peptidyl prolyl cis/trans isomerases.

The stereospecificity of peptidyl prolyl cis/trans isomerases (PPIases) was studied using tetrapeptide substrate analogs in which one amino acid residue was replaced by the cognate D-amino acid in various positions of the peptide chain. Reversed stereocenters around proline markedly increased the rate of the spontaneous trans to cis isomerization of the prolyl bond whereas cis to trans isomerizations were less sensitive. PPIases like human cyclophilin18, human FKBP12, Escherichia coli parvulin10 and the PPIase domain of E. coli trigger factor exhibited stereoselectivity demanding at the P1 to P2' position of the substrate chain. The discriminating factor for stereoselectivity was the lack of formation of the Michaelis complexes of the diastereomeric substrates. However, D-alanine at the P1 position preserved considerable affinity to the active site, and largely prevented activation of the catalytic machinery for all PPIases investigated.

Alanine↗

PubMed1998

Side-chain effects on peptidyl-prolyl cis/trans isomerisation.

Peptidyl-prolyl cis/trans isomerisation has been frequently found as a rate limiting step in the folding of proteins. In order to determine whether the nature of the amino acid preceding proline controls the probability of cis prolyl bonds in native proteins, systematic studies on the thermodynamics and kinetics of the prolyl isomerisation in the pentapeptide series Ac-Ala-Xaa-Pro-Ala-Lys-NH2 were performed. All proteinogenic amino acids were substituted in the position preceding proline. When measured by 1H-NMR and CD spectroscopy both isomers proved to be devoid of ordered structure in the whole series of the oligopeptides in aqueous solution. Thus, isomerization rates and cis/trans ratios calculated from solvent jump and 1H-NMR magnetisation transfer experiments exclusively reflect the side-chain effects of the Xaa position in the peptide series. There is a rough correlation between the cis content in the oligopeptides and the propensity of Xaa-Pro cis prolyl bonds in proteins. This correlation suggests that the prolyl bond conformation is mainly determined by local effects in proteins. The rate constants kc-->t of pentapeptides containing unionised amino acids preceding proline range from 3.2 x 10(-3) s-1 (Xaa = Ala) to 0.5 x 10(-3) s-1 (Xaa = Trp) at 4 degrees C. Proline clustering led to an isomerisation cycle indicating considerable influence on the isomerisation rates of the peptide bond conformations flanking the rotating bond. Both tyrosine and histidine specifically reduce isomerisation rates severalfold by deprotonation of their respective side-chains.

Amino Acid Sequence↗

PubMed1998

Spinal cord ependymomas.

Ependymomas are the most frequent spinal cord tumors in adult patients. Although magnetic resonance imaging can be a highly accurate diagnostic tool, it does not always provide accurate differentiation between ependymomas and astrocytomas. This is why the authors recommend surgical resection and histological evaluation in all intraspinal cord tumors. It cannot be said that a tumor is unresectable without first attempting to remove it. Complete removal should be accomplished whenever possible, and patients should undergo operation before they become neurologically impaired. Quality of life depends on preoperative neurological status. Postoperative radiotherapy should be avoided in all low-grade ependymomas even after partial removal. Radiotherapy may be used to treat anaplastic ependymomas, which are quite rare in the spinal cord. Patients with low-grade ependymomas must be followed for years and undergo reoperation if necessary. In our experience treating 93 spinal cord ependymomas, complete removal was achieved in 86 patients and only one patient underwent reoperation 18 years later for tumor recurrence. The gold standard in treatment protocol is gross-total resection without adjunctive radiation therapy. Good long-term outcomes have been achieved by using this strategy.

Journal Article↗

PubMed1998

Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone.

In contrast to FK506 binding proteins and cyclophilins, the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases; E.C. 5.2.1.8) cannot be inhibited by either FK506 or cyclosporin A. We have found that juglone, 5-hydroxy-1,4-naphthoquinone, irreversibly inhibits the enzymatic activity of several parvulins, like the E. coli parvulin, the yeast Ess1/Ptf1, and human Pin1, in a specific manner, thus allowing selective inactivation of these enzymes in the presence of other PPIases. The mode of action was studied by analyzing the inactivation kinetics and the nature of products of the reaction of E. coli parvulin and its Cys69Ala variant with juglone. For all parvulins investigated, complete inactivation was obtained by a slow process that is characterized by pseudo-first-order rate constants in the range of 5.3 x 10(-)4 to 4. 5 x 10(-)3 s-1. The inactivated parvulin contains two juglone molecules that are covalently bound to the side chains of Cys41 and Cys69 because of a Michael addition of the thiol groups to juglone. Redox reactions did not contribute to the inactivation process. Because thiol group modification was shown to proceed 5-fold faster than the rate of enzyme inactivation, it was considered as a necessary but not sufficient condition for inactivation. When measured by far-UV circular dichroism (CD), the rate of structural alterations following thiol group modification parallels exactly the rate of inactivation. Thus, partial unfolding of the active site of the parvulins was thought to be the cause of the deterioration of PPIase activity.

Amino Acid Isomerases↗

PubMed1998

Room-temperature vibrational difference spectrum for S2QB-/S1QB of photosystem II determined by time-resolved Fourier transform infrared spectroscopy.

Time-resolved FTIR spectroscopy has been used to kinetically characterize the vibrational properties of intact photosystem II-enriched membrane samples undergoing the S1QB-to-S2QB- transition at room temperature. To optimize the experimental conditions for the FTIR measurements, oxygen polarographic and variable chlorophyll a fluorescence measurements were used to define the decay of S2 and QA-, respectively. The flash-induced S2QB-/S1QB difference spectra were measured at a temporal resolution of 4.44 s and a spectral resolution of 4 cm-1. An intense positive band is observed at 1480 cm-1 in the difference spectrum and shows a slow decay with a half time of approximately 13 s. Based on its decay kinetics and analogy to the infrared absorption of QA- of photosystem II and QB- in bacterial reaction centers, we conclude that the 1480 cm-1 band arises from QB- of PSII and tentatively assign it to the upsilon(CO) mode of the semiquinone anion QB-. The infrared spectral features attributed to the S1-to-S2 transition of the Mn cluster at room temperature show striking similarity to the S2/S1 difference spectrum measured at cryogenic temperatures (Noguchi, T., Ono, T.-A., and Inoue, Y. (1995) Biochim. Biophys. Acta 1228, 189-200).

Chloroplasts↗

PubMed1998

Role of phosphorylation in determining the backbone dynamics of the serine/threonine-proline motif and Pin1 substrate recognition.

Proline residues provide a backbone switch in a polypeptide chain, which is controlled by the cis/trans isomerization about the peptidyl-prolyl bond. Phosphorylation of serine- and threonine-proline motifs has been shown to be a critical regulatory event for many proteins. The biological significance of these motifs has been further highlighted by the discovery of a novel and essential peptidyl-prolyl cis/trans isomerase Pin1. Pin1 is required for progression through mitosis via catalyzing the isomerization of phosphorylated Ser/Thr-Pro motifs specifically present in mitosis-specific phosphoproteins. However, little is known whether the phosphorylation regulates the conformational switch of the Ser/Thr-Pro bonds. Here, we report the synthesis and conformational characterization of a series of peptides that contain the phosphorylated or nonphosphorylated Ser/Thr-Pro motifs. Phosphorylation affected the rate of the cis to trans isomerization of the Thr/Ser-Pro bonds. As determined by a protease-coupled assay, the isomerization rate of phosphorylated Thr-Pro bond was found to be 8-fold slower than that of the nonphosphorylated analogue. Furthermore, studies of the pH dependence of the isomerization of the phosphopeptides reveal that both cis content and the rate constant of prolyl cis to trans isomerization are lower for the dianionic state of the phosphothreonine-containing peptides. These effects of phosphorylation are specific for phosphorylated Ser/Thr since neither phosphorylated Tyr nor glutamic acid was able to affect the prolyl isomerization. Finally, our experiments provide evidence that effective catalysis of cis/trans isomerization of phosphorylated Ser/Thr-Pro bonds by Pin1 is specific to the dianionic form of the substrate. Thus, our results demonstrate that protein phosphorylation specifically regulates the backbone dynamics of the Ser/Thr-Pro motifs and that Pin1 specifically isomerizes the certain conformation of the phosphorylated Ser/Thr-Pro motifs.

Animals↗

PubMed1998

Effects of FK506-binding protein 12 and FK506 on autophosphorylation of epidermal growth factor receptor.

FK506-binding proteins and cyclophilins are intracellular proteins that express peptidylproline cis-trans-isomerase (PPIase) activity. The effects of FK506-binding protein 12 (FKBP12) and the cyclophilins 18 and 23 on autophosphorylation of the epidermal growth factor (EGF) receptor prepared from plasma membranes of the human epidermoid cell line A431 have been investigated. Whereas FKBP12 inhibited EGF receptor tyrosine kinase activity in a concentration-dependent manner, the cyclophilins did not affect autophosphorylation. In contrast to the wild-type enzyme, several variants of FKBP12 with greatly reduced PPIase activity were unable to suppress EGF receptor tyrosine kinase significantly. Pervanadate an inhibitor of protein tyrosine phosphatases, abolished the effect of FKBP12 on EGF receptor autophosphorylation. Finally, FK506 and rapamycin, which are known to block the PPIase activity of FKBP12, induced a significant stimulation of EGF receptor autophosphorylation in intact A431 cells suggesting suppression of EGF receptor autophosphorylation by intracellular FKBP12 in vivo. Taken together the data point to an inhibitory function of FKBP12 in EGF receptor signaling, possibly induced by stimulation of a protein tyrosine phosphatase coupled to the EGF receptor. Both PPIase activity and substrate specificity of FKBP12 seem to be indispensable for this effect.

Adenosine Triphosphate↗

PubMed1998

The mode of action of peptidyl prolyl cis/trans isomerases in vivo: binding vs. catalysis.

Polypeptides often display proline-mediated conformational substates that are prone to isomer-specific recognition and function. Both possibilities can be of biological significance. Distinct families of peptidyl prolyl cis/trans isomerases (PPIases) evolved proved to be highly specific for proline moieties arranged in a special context of subsites. Structural and chemical features of molecules specifically bound to the active site of PPIases served to improve catalysis of prolyl isomerization rather than ground state binding. For example, results inferred from receptor Ser/Thr or Tyr phosphorylation in the presence of site-directed FKBP12 mutant proteins provided evidence for the crucial role of the enzymatic activity in downregulating function of FKBP12.

Binding Sites↗

PubMed1998

Evidence for the absolute conformational specificity of the intestinal H+/peptide symporter, PEPT1.

This study was initiated to determine whether the intestinal H+/peptide symporter PEPT1 differentiates between the peptide bond conformers of substrates. We synthesized a modified dipeptide where the peptide bond is replaced by the isosteric thioxo peptide bond. The Ala-Pro derivative Ala-psi[CS-N]-Pro exists as a mixture of cis and trans conformation in aqueous solution and is characterized by a low cis/trans isomerization rate. The compound was recognized by PEPT1 with high affinity. The Ki value of Ala-psi[CS-N]-Pro for the inhibition of the uptake of radiolabeled glycylsarcosine in Caco-2 cells was 0.30 +/- 0.02 mM, determined in solution with 96% trans conformation. In contrast, the Ki value was 0.51 +/- 0.02 mM when uptake media with 62% trans conformer were used. We conclude that only the trans conformer interacts with the transport system. From our data, a significant affinity of the cis conformer at PEPT1 cannot be derived. In a second approach, conformer-specific uptake of Ala-psi[CS-N]-Pro was studied by analyzing the intracellular content of Caco-2 cells following transport as well as the composition of the extracellular medium using capillary electrophoresis. The percentage of trans conformer that was 62% in the uptake medium increased to 92% inside the cells. This is the first direct evidence that an H+/peptide cotransport system selectively binds and transports the trans conformer of a peptide derivative.

Biological Transport↗

PubMed1998

Consanguinity in question in two patients with congenital adrenal hyperplasia who bear an identical first and last name--second look.

Two patients with identical first and last name, both suffering from Congenital Adrenal Hyperplasia due to 21-hydroxylase deficiency, were investigated in our endocrine outpatient unit. The families of the two girls did not know of each other's existence. Although paternal ancestors were identified up to the 11th and 9th previous generations, respectively, and a common area of origin, roughly 50 km apart, in the northwestern part of the Czech Republic (formerly Austrian Silesia) was established, no common ancestor was found. The patients' respective paternal CYP 21B allele was identical. However, by means of analysis of class I-and class II-HLA regions in both patients and their respective parents no common paternal haplotype was established. In order to still postulate consanguinity by the same paternal haplotype it is necessary to assume that two recombinations in the HLA region must have occurred. The probability that these two events have taken place within the past 11 generations is 0.11%.

Adolescent↗

PubMed1998

Separation of cis/trans conformers of human and salmon calcitonin by low temperature capillary electrophoresis.

Conformer-specific recognition of peptides and proteins has often been proved with the aid of indirect methods. Here we provide an analytical approach for a direct investigation of separated isomers. Cis/trans conformers of the peptide hormones human (hCT) and salmon (sCT) calcitonin exhibit different migration properties in capillary zone electrophoresis at subambient temperatures. Calcitonin consists of 32 amino acids with two proline residues incorporated. It is the longest unstructured peptide for which a conformer separation by capillary electrophoresis has yet been achieved. Lowering the temperature yielded a splitting into two and three peaks for sCT and hCT, respectively, in acidic buffer. The peak ratios of 66:34 for sCT and 71:23 for hCT are in good agreement with the conformer distribution previously reported from nuclear magnetic resonance (NMR) studies. The addition of different organic modifiers (5-20% v/v) to the running buffer does not improve the separation. The observed merging of conformer peaks in buffer containing 20% v/v 2,2,2-trifluoroethanol (TFE) is attributed to structure formation.

Amino Acid Sequence↗

PubMed1998

Graft-versus-host disease following second syngeneic stem cell transplantation for relapsed chronic myeloid leukemia.

Allogeneic bone marrow transplantation is the only curative treatment for patients with Philadelphia chromosome-positive chronic myeloid leukemia (CML); however, recurrence of disease remains a major cause of treatment failure. A 26-year-old man with chronic myeloid leukemia who had a cytogenetic relapse 49 months after his first syngeneic bone marrow transplant (BMT) and hematologic relapse 23 months thereafter progressed to blast crisis despite treatment with IFN-alpha for 15 months. He underwent a second transplantation in early second blast crisis, 92 months after the first BMT with PBPC from his previous donor. Successful hematological reconstitution occurred. On day 50 after the second transplantation the patient developed a generalized rash, hepatomegaly, and cholestatic signs. Skin and liver biopsy revealed changes compatible with acute graft-versus-host disease (GVHD). Treatment with cyclosporin A (CSA) and prednisone was started, and the GVHD resolved. Fifteen months after PBPC transplantation he had a molecular relapse. Despite discontinuation of CSA, the patient progressed into blast crisis 7 months later. The occurrence of GVHD and disappearance of the BCR-ABL-positive clone suggest that a graft-versus-leukemia (GVL) effect may have been operative for 15 months in a patient given a second syngeneic BMT in blast crisis.

Adult↗

PubMed1998

Analytical validation of the BEM--application of the BEM to the electrocardiographic forward and inverse problem.

The objective of this study is to analytically validate a boundary element (BE) formulation for the relationship between the transmembrane potential on the heart's surface and the potential on the body surface applying a concentric spherical test geometry. The relative difference (reldif) between the potential on the outer sphere of the test geometry computed analytically and numerically is determined by 3.59% for the coarse discretization (48 BEs) and by 0.46% in the case of the finer subdivision (192 BEs). In the inverse problem, the transmembrane potential on the inner sphere is estimated numerically from the electric potential on the outer sphere by using a minimum-norm least-square approach. The relative differences found are 20.2% when no measurement noise is added and 26.4% in the presence of 2% additional Gaussian noise. The BE formulation is also applied to real world data for solving the electrocardiographic inverse problem. A normal volunteer's inhomogeneous thorax (outer thorax surface, surfaces of the lungs, epicardial heart surface) is modelled by 424 BEs. The same inverse method is then applied in order to reconstruct the transmembrane potential on the epicardium from the measured body surface potential (BSP) data during normal ventricular depolarisation.

Electrocardiography↗

PubMed1998

Improved investigation of portal-hepatic veins by echo-enhanced Doppler sonography.

Microbubbles of air released from a galactose vehicle (Levovist) amplify the intensity of Doppler signals. They survive both pulmonary and systemic capillary passage, leading to echo enhancement in the entire vascular system. The aim of this study was to investigate this agent in patients with liver disease and insufficient Doppler signals. A total of 275 Doppler examinations were performed in 176 patients; 20 of these patients could not be studied conventionally due to bowel gas, obesity or noncompliance. They received Levovist to examine portal or hepatic veins or TIPS patency. Angiography, computed tomography (CT) scan or magnetic resonance tomographic angiography (MRTA) was performed subsequently as a control. After administration of Levovist, portal or hepatic veins and TIPS patency could be unequivocally assessed in 18 of the 20 patients. In two patients, suspected occlusion of the portal vein was disproved because the diagnosis was not confirmed later. Only minor adverse effects were encountered. Echo-enhanced Doppler sonography with Levovist is well tolerated. Further study of the value of Levovist for the assessment of portal-hepatic vessels not amenable to conventional Doppler sonography is justified.

Contrast Media↗