[Primary intramedullary lymphoma in AIDS].
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
G Fischer
Publications and source records attributed to G Fischer.
Confirmation of the existence of a third family among peptidyl-prolyl cis/trans isomerases. Amino acid sequence and recombinant production of parvulin.
In addition to the major cyclophilin-like peptidyl-prolyl cis/trans isomerases (PPIases) of Escherichia coli an enzyme of very low relative molecular mass (10.1 kDa) was discovered in this organism which gave first indication of the existence of a novel family in this enzyme class [1994, FEBS Lett. 343, 65-69]. In the present report we describe the chemically determined amino acid sequence of four peptides derived from the 10.1 kDa protein by the treatment with either cyanogen bromide or endoproteinase Lys-C. Together with a continuous run of 75 amino acids starting N-terminally, the sequence of the mature enzyme, 92 residues in length, was elucidated. Cloning and determination of the primary structure of a DNA fragment encoding this enzyme were also performed. Overexpression of the enzyme by using multicopies of plasmid pSEP38 in E. coli and detecting an enhanced PPIase activity attributed to the 10.1 kDa enzyme provided additional proof that the 92 amino acid protein was a PPIase. The enzyme was called parvulin (lat.: parvulus, very small). Homology analyses indicated that several parvulin-like proteins could be found in the database screened. To further elucidate the functional role of PPIases it might be of some importance that homologous proteins like the PrtM protein of Lactococcus lactis and the PrsA lipoprotein of Bacillus subtilis are known to be involved in the protein export and maturation machinery of the bacteria.
Amino Acid Isomerases↗
A homodimer represents an active species of the peptidyl-prolyl cis/trans isomerase FKBP25mem from Legionella pneumophila.
The molecular mass of the native FK506-binding peptidyl-prolyl cis/trans isomerase (PPIase) FKBP25mem from Legionella pneumophila (Mip (macrophage infectivity potentiator) protein) was determined by two methods. By gel-permeation chromatography we found no indication of the presence of the monomeric enzyme. However, an oligomeric state with a molecular mass of about 62 kDa was detected. By cross-linking with dimethyl pimelimidate and subsequent SDS-PAGE of either the surface proteins of intact L. pneumophila cells or the purified recombinant FKBP25mem in solution, we observed an immunoreactive band indicative of a mass in the dimer range. In contrast to human recombinant FKBP12, the enzymatic activity of Legionella FKBP25men was strongly dependent on the protein concentration, pointing to a dimer as the most active species. However, the inhibition by FK506 yielded a nearly constant value of Ki of about 250 nM when measured in the same range of FKBP25mem concentration. These results may be explained by the fact that monomeric FKBP25mem has little, if any, influence on enzymatic activity when compared with the homodimer.
Amino Acid Isomerases↗
Four recombinant isoforms of Cor a 1, the major allergen of hazel pollen, show different reactivities with allergen-specific T-lymphocyte clones.
Purified preparations of allergenic proteins from plants, and in particular from pollens, consist of multiple closely related isoforms. These isoforms are highly similar in their amino acid sequences, yet they display different properties with respect to antibody binding. In this study we report of differential potencies of cross-reacting tree pollen allergens and cloned isoforms of these allergens to activate allergen-specific T-lymphocyte clones (T-cell clones; TCC). Six TCC with specificity for Bet v 1, a representative tree pollen major allergen, were established from peripheral blood of five birch-pollen-allergic donors. All TCC displayed the helper-cell phenotype. Five TCC reacted with distinct epitopes present on natural (n) and on recombinant (r) Bet v 1. One TCC could not be stimulated with r Bet v 1, in spite of strong reactivity with purified natural Bet v 1. The TCC were tested in proliferation assays using purified n Bet v 1, n Cor a 1 (the homologous major allergen of hazel pollen), r Bet v 1, four recombinant isoforms of Cor a 1 and peptides representing corresponding T-cell stimulating regions (isoepitopes) on these proteins. The clones showed different patterns of reactivity in response to stimulation with the five recombinant molecules and the corresponding peptides. Certain exchanges of amino acids within stimulating peptides correlated with a lack of proliferation of the TCC tested. These findings are important with respect to the use of broadly cross-reactive recombinant allergens or allergen-derived peptides for immunotherapy of type I allergy.
Allergens↗
Reassessment of the putative chaperone function of prolyl-cis/trans-isomerases.
The folding of proteins can be assisted by two unrelated groups of helper molecules. Chaperones suppress non-productive side reactions by stoichiometric binding to folding intermediates, and folding enzymes catalyze slow rate-limiting steps of folding. We reinvestigated, whether peptidyl-prolyl-cis/trans-isomerases of the cyclophilin type act simultaneously as chaperones and as folding catalysts in the reactivation of human carbonic anhydrase II, as reported recently [Freskgård, P.-O. et al. (1992) Science 258, 466-468; Rinfret, A. et al. (1994) Biochemistry 33, 1668-1673]. No increase in the yield of native carbonic anhydrase-II could be detected in the presence of three different prolyl isomerases, when reactivation was followed by a sensitive assay for an extended time of 4 h. We conclude that the role of prolyl isomerases in the refolding of carbonic anhydrase can be explained solely by their isomerase activity. There is no need to invoke simultaneous functions as chaperones for these folding catalysts.
Amino Acid Isomerases↗
Vulnerability to excitotoxic stimuli of cultured rat hippocampal neurons containing the calcium-binding proteins calretinin and calbindin D28K.
Rat embryonic hippocampal neurons cultured on astrocyte feeder-layers were sensitive to different excitotoxic stimuli after 10-12 DIV. Almost all neurons (approximately 95%) died within 20 h after a transient exposure for 10 min to 50 microM glutamate, a continuous exposure to either 25 microM NMDA or 250 microM kainate or after a 15-min deprivation of glucose and oxygen. Dizocilpine at 10 microM protected neurons against the glutamate- and NMDA-mediated toxicity as well as against 30 min glucose and oxygen deprivation. However, it failed to protect against kainate toxicity and prolonged glucose/oxygen deprivation (60 min). An additional treatment with CNQX (100 microM) protected neurons even under the latter two conditions. This indicates that the vast majority of neurons was sensitive to different excitotoxic stimuli acting through different types of glutamate receptors leading to calcium overload of the cells which might be the common denominator of triggering cell death under these conditions. Expression of calcium-binding proteins, such as calbindin D28K or calretinin, might increase the intracellular calcium buffer capacity of neurons, thus, rendering them more resistant to calcium overload. Therefore, we analysed whether neurons expressing these calcium-binding proteins would survive these toxic stimuli. Indeed, a small population of the neurons (3-5%) survived, including a subpopulation of calretinin-positive but not calbindin D28K-positive neurons. This implies that the expression of calcium-binding proteins per se does not render neurons more resistant towards these excitotoxic stimuli. Moreover, most of the surviving calretinin-positive neurons showed morphological damage as indicated by loss of neurites. When cytotoxicity due to calcium overload was induced by an exposure of the cells to the calcium ionophore 4-bromo-A23187 rather than by activation of glutamate receptors, calretinin-positive cells were found not to be significantly more resistant than the vast majority of neurons. This may indicate that the lower sensitivity of a subpopulation of calretinin-positive neurons to excitotoxic stimuli may be due to a lower expression of glutamate receptors.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Characterization of Mip proteins of Legionella pneumophila.
The Mip ('macrophage infectivity potentiator') protein of Legionella pneumophila has been shown to be an essential virulence factor, exhibiting peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. The cloning and sequencing of mip genes from three different L. pneumophila strains revealed a single amino acid substitution which did not affect the isomerase property of the enzyme. Mip proteins isolated from two wild-type L. pneumophila strains and from two corresponding Escherichia coli K-12 recombinant clones derived from these strains exhibited identical enzymatic properties and the precursor proteins are processed at identical cleavage sites. The mature Mip proteins exist in an oligomeric form. Site-directed mutagenesis demonstrated that a substitution of an Asp residue at position 142 by a Leu residue affects PPIase activity of Mip.
Amino Acid Isomerases↗
A novel peptidyl-prolyl cis/trans isomerase from Escherichia coli.
A novel peptidyl-prolyl cis/trans isomerase was isolated from Escherichia coli cell extract and characterized partially. Determination of the molecular mass by electrospray mass spectrometry indicated a protein of 10102 +/- 2 Da, smaller than cyclophilins or FK 506 binding proteins currently known. The specificity constant kcat/Km determined with Succinyl-Ala-Xaa-Pro-Phe-4-nitroanilide (Xaa = Leu) had a value comparable to those from cyclophilins from the same organism. However, the pattern of subsite specificity (Xaa = Gly, Ala, Val, Ile, Leu, Phe, Trp, His, Lys and Glu) was reminiscent of FK506 binding peptidyl-prolyl cis/trans isomerases. The enzyme activity was not inhibited by cyclosporin A or FK506 at inhibitor concentrations of < 5 microM, concentrations that affect most bacterial peptidyl-prolyl cis/trans isomerases. Computer-assisted analysis of 21 amino acid residues of the N-terminus determined by Edman degradation revealed no homology to known peptidyl-prolyl cis/trans isomerases.
Amino Acid Isomerases↗
Influence on proline-specific enzymes of a substrate containing the thioxoaminoacyl-prolyl peptide bond.
Dipeptidyl peptidase IV from porcine kidney and aminopeptidase P from Escherichia coli can utilize thioxoalanyl-proline 4-nitroanilide but with decreased kinetic constants compared to the normal substrates. Product analysis showed that exclusively thioxoalanyl-proline was liberated in the case of dipeptidyl peptidase IV catalysis and thioxo-alanine in the case of aminopeptidase-P-mediated thioxo peptide bond hydrolysis. For the proline-specific aminopeptidase P the kcat/Km value for the thioxo peptide is 1100-fold lower than for the corresponding oxo peptide. This difference is entirely due to kcat. Because the rotation about the thioxo amide bond is about 12.5 kJ mol-1 more difficult than rotation about an amide bond, these data support a mechanism involving rate-limiting rotation about the scissile peptide bond. It was found that the specificity rate constant for the reaction of thioxoalanyl-proline 4-nitroanilide and dipeptidyl peptidase IV is 100-1000-fold lower compared to the corresponding rate constant for alanyl-proline 4-nitroanilide. This remarkable effect is interpreted in terms of a distorted binding of the transition state for the thioxo substrate. The hydrolysis of the thioxo substrate by dipeptidyl peptidase IV is isomer-specific. The conformation about the nonscissile P2-P1 thioxo amide bond has to be in trans for successful cleavage of the scissile peptide bond. We can now directly compare the rotational energy barrier of the prolyl peptide bond for the oxo and the thioxo form.
Aminopeptidases↗
Lymphoma models for B cell activation and tolerance. X. Anti-mu-mediated growth arrest and apoptosis of murine B cell lymphomas is prevented by the stabilization of myc.
Treatment of the WEHI-2131 or CH31 B cell lymphomas with anti-mu or transforming growth factor (TGF)-beta leads to growth inhibition and subsequent cell death via apoptosis. Since anti-mu stimulates a transient increase in c-myc and c-fos transcription in these lymphomas, we examined the role of these proteins in growth regulation using antisense oligonucleotides. Herein, we demonstrate that antisense oligonucleotides for c-myc prevent both anti-mu- and TGF-beta-mediated growth inhibition in the CH31 and WEHI-231 B cell lymphomas, whereas antisense c-fos has no effect. Furthermore, antisense c-myc promotes the appearance of phosphorylated retinoblastoma protein in the presence of anti-mu and prevents the progression to apoptosis as measured by propidium iodide staining. Northern and Western analyses show that c-myc message and the levels of multiple myc proteins were maintained in the presence of antisense c-myc, results indicating that myc species are critical for the continuation of proliferation and the prevention of apoptosis. These data implicate c-myc in the negative signaling pathway of both TGF-beta and anti-mu.
Animals↗
Differentiation by preparative continuous free flow-isoelectric focusing of cyclosporin A inhibitable peptidyl-prolyl cis/trans isomerase of human erythrocytes.
Preparative continuous free flow-isoelectric focusing has been used to separate at least three different components of intrinsic peptidyl-prolyl cis/trans isomerase (PPIase) activity from erythrocytes lysate. By adding chemical spacer molecules like glycine and Bicine to commercial carrier ampholyte mixtures the resulting pH profile was predictably influenced. With an applied field strength of 125-170 V/cm a residence time of less than 15 min was sufficient for the separation of PPIases with isoelectric points of 5.4, 5.7 and 5.9 from the bulky hemoglobin. The recovery of the overall PPIase activities was about 100%. The purification factor has been determined as 20- to 100-fold. For each isoform of the enzyme the peptidyl-prolyl cis/trans isomerase activity of the separated proteins was inhibited by cyclosporin A but was resistant toward FK 506.
Amino Acid Isomerases↗
The immortalized astroglial cell line RC7 is a new model system for the study of nerve growth factor (NGF) regulation: stimulation by interleukin-1 beta and transforming growth factor-beta 1 is additive and affected differently by dibutyryl cyclic AMP.
Nerve growth factor (NGF) synthesis was studied with an astroglial cell line derived from rat cerebellar astrocytes by transfection with a simian virus 40 T containing retroviral vector. As in primary astrocytes, NGF synthesis/secretion could be stimulated dose-dependently with interleukin-1 beta (IL-1 beta) and transforming growth factor-beta 1 (TGF-beta 1). We therefore have used this cell line as a model system to analyze putative intracellular signalling pathways underlying the effects of these factors. Protein kinase C inhibitors (calphostin and Ro 31-8830) as well as a lipoxygenase inhibitor (nordihydroguaiaretic acid) did not affect stimulation of NGF synthesis/secretion by IL-1 beta or TGF-beta 1. However, dibutyryl cyclic AMP partly inhibited the stimulation by TGF-beta 1 but did not affect that evoked by IL-1 beta. This finding, together with the fact that IL-1 beta and TGF-beta 1 stimulate NGF production/secretion in an additive manner, indicates that different intracellular signalling pathways are involved in the mediation of IL-1 beta and TGF-beta 1 induced NGF production/secretion.
Animals↗
L- and M2-pyruvate kinase expression in renal cell carcinomas and their metastases.
Using immunohistochemical and enzyme biochemical methods we investigated the expression of L- and M2-pyruvate kinase (PK) in normal renal tissue, renal cell carcinomas (RCCs; of clear cell, chromophilic cell and mixed cell type) and RCC metastases. L-PK was expressed in the proximal tubules of normal renal tissue and, to a variable extent, in 23/25 primary RCCs, in 1 RCC recurrence and in 10 RCC metastases. Staining intensity and percentage of stained tissue did not correlate with tumour grade. One renal oncocytoma and all extrarenal malignancies examined lacked L-PK immunoreactivity. M2-PK was mainly expressed in the distal tubules of the normal kidney and was found in all renal tumours as well as extrarenal malignancies. Quantitative biochemical investigations yielded a two- to seventeen-fold increase in PK activity in RCCs compared to the normal renal cortex taken from the same patient, whereas fructose-1,6-bisphosphatase and cytosolic glycerol-3-phosphate dehydrogenase activity was dramatically lower in RCCs. Otherwise, the activity of all other enzymes investigated (glucose-6-phosphate dehydrogenase, enolase and lactate dehydrogenase) was not significantly changed in the RCCs. The immunocytochemical results suggest that L-PK is a useful marker for RCC and its metastases, if acetone-fixed tissue is available. The quantitative changes of the concentration of PK and other enzymes in RCCs when compared with normal renal tissue probably reflect metabolic alterations related to tumour growth.
Carcinoma, Renal Cell↗
Non-concordance by gender for schizophrenia and related disorders in sibships.
We analysed gender-concordance rates among 29 prospectively sampled schizophrenic probands and their 39 affected and 71 unaffected siblings. We did not find any unusual concordance rates. We found no same-gender concordance particularly in siblings affected by schizophrenia and related disorders. We considered unaffected siblings in an additional attempt to make valid and unbiased comparisons between genders, but this reduced the number of informative sibships to 20. We stratified the siblings of probands by sibship and by the proband's gender in order to check gender distribution within families. The data do not support hypotheses that schizophrenia is pseudo-autosomal or male-female chromosomally transmitted.
Adult↗
[Fulminating E. coli sepsis in Fournier's gangrene].
Fournier's gangrene is a necrotising soft-tissue infection of the scrotum and perineal region caused by gram-negative and gram-positive Enterobacteriaceae. The disease is characterised by its unique appearance, its speed of onset, and its high mortality. CASE REPORT. A 26-year-old male presented to the emergency room complaining of a painful, tremendously swollen scrotum and penis (Fig. 1) that had developed within the past 24 h. Later, slurred speech, pallor, and hypotension were recognised, leading to the patient's admission to the intensive care unit. Suspecting a severe internal haemorrhage, vigorous volume therapy was started using crystalloids and colloids until blood and fresh frozen plasma were available. One hour later, septic shock was presumed and therapy augmented by IV antibiotics, tracheal intubation, and mechanical ventilation. Despite all efforts, the patients condition deteriorated rapidly and he died a few hours later due to multiple organ failure in septic shock. Postmortem, a perforated external hemorrhoidal node was found to be the primary focus of sepsis. Microbiologic cultures revealed Escherichia coli in blood and tissue samples. DISCUSSION. Fournier's gangrene is a rare disease; nevertheless, its clinical picture has to be recognised immediately in order to provide appropriate treatment in time. It occurs predominantly in males after minor trauma, colorectal or urological disease, and perineal or abdominal surgery. Fournier's gangrene usually begins with itching and pain in the scrotal region followed by swelling and dark-blueish discolouration of the scrotum and penis, occasionally including the lower abdominal wall. Fever and chills are usually present. The illness progresses to severe prostation and septic shock with a mortality of 20%-50%. Tissue cultures mostly reveal E. coli, gram-positive enterococci, Pseudomonas, Proteus, and various anaerobes. The treatment should include immediate radical surgical debridement, i.v. administration of broad-spectrum antibiotics, and cardiopulmonary support. CONCLUSION. The dramatic course of Fournier's gangrene requires early recognition, extensive surgical debridement, as well as intensive care treatment in order to prevent irreversible septic shock.
Adult↗
[Special aspects of histopathologic diagnosis in laser microsurgery of cancers of the upper aerodigestive tract].
Histological examination of CO2 laser-resected specimens reveals a thin layer of carbonisation, beneath which the cellular architecture is preserved, permitting adequate assessment. With early staged laryngeal, oral and pharyngeal carcinomas, it is usually possible to remove the tumour as a single block, and the margins can be studied by parallel histologic sections. Advanced tumours, regardless of their localisation, demand resection in several blocks. Each tissue block is carefully orientated and the deep surface of every specimen is stained. Accurate mapping of every block is essential to enable the surgeon to identify the precise location of each specimen in the patient. The specimens are vertically sectioned after paraffin wax embedding. Grading, depth of invasion, lateral and basal margins are examined. Like in conventional surgery, it is technically impossible to examine all margins of a large specimen. With vertical sections only limited information can be obtained about lateral and basal margins. If deep tumour infiltration reaches cartilage or bony structures, clear microscopic margins are difficult to prove after laser resection. Finally it must be possible for the surgeon to recreate a complete, 3-dimensional mosaic of the entire lesion to achieve accurate pT-staging.
Carcinoma, Squamous Cell↗
A preliminary report on endoscopic transanal resection of the rectum.
Since 1986 we have performed 62 endoscopic transanal resections (TAR) on 43 patients with malignant or benign tumours. Initially a transurethral resectoscope was used, and later the recently developed rectal resectoscope was introduced, enabling diathermy resection of a rectal lesion growing into the lumen of the rectum and making it possible to take the resection level up to or below the level of the muscularis propria. The wound is left to epithelialise spontaneously. 32 patients with malignant lesions of the rectum underwent TAR. The aim was to palliate symptoms and avoid colostomy. 11 patients with benign tumours (8 adenomas, 2 polyps, 1 stricture) also underwent TAR. No patient died within 25 days of operation. TAR is a minimally invasive procedure. It is quick, safe and effective if performed caudal to the peritoneal reflection fold.
Aged↗
[Intramedullary spinal cord tumors. Report. French Society of Neurosurgery. 45th annual congress. Angers, June 12-15 1994].
Since Guidetti and Slooff's masterbooks (1964), numerous papers have been devoted to intramedullary spinal cord tumors (IMT) and their treatment, most of them were focused on ependymomas and astrocytomas. Informed opinion was that these tumors were difficult to cure and that biopsy plus radiotherapy was the treatment of choice. Thanks to microsurgery and bipolar coagulation surgeons grew bolder and more efficient as illustrated by the contributions from Hurth or Resche on hemangioblastomas, Fischer on ependymomas, Epstein on childhood astrocytomas and Guidetti, Malis, Stein on IMT in general. Meanwhile, in the eighties, Magnetic Resonance Imaging (MRI) and Cavitron Ultrasonic Surgical Aspirator (CUSA) drastically modified diagnostic and therapeutic strategies of IMTs, as may be judged by the significant increase in the number of publications on this topic in current medical literature. However, no updated work of synthesis is currently available. This prompted us to publish our common experience based on 171 patients and 200 surgical procedures. Thanks to the collaboration of many colleagues of the "Société de Neurochirurgie de Langue Française" (SNCLF) who completed a questionnaire sent to them, we are now in the position to give an epidemiological estimation based on 1117 cases. This work is divided in three main parts. The first part deals with considerations common to all IMTs, the second with particular aspects specific of each tumoral type, the third with the results and concluding recommendations.
Humans↗