Bettina Schmidt

Search for a putative scrapie genome in purified prion fractions reveals a paucity of nucleic acids.

Scrapie can be transmitted by novel infectious pathogens termed prions. No evidence for a scrapie-specific nucleic acid has been detected to date. To investigate amounts, types and sizes of nucleic acid molecules associated with prions in purified preparations, aliquots were deproteinized, and the nucleic acids analysed by PAGE and silver staining. Digestion with nucleases and exposure to Zn2+ prior to analysis substantially diminished the content of nucleic acids, but did not alter the prion titre indicating that those nucleic acids which were removed are not essential for infectivity. Since a single species of scrapie-specific nucleic acid could not be identified, we explored the unprecedented possibility of scrapie-specific nucleic acids of variable length which are biologically active. If such molecules of variable length exist then they might be hidden within the background smear on silver-stained gels after PAGE. A new procedure designated return refocusing gel electrophoresis (RRGE) was developed to identify heterogeneous nucleic acids in purified prion fractions. The content of variable length nucleic acids was reduced by a factor of 10 by exhaustive Bal 31 exonuclease digestion after dispersion of purified prions into detergent-lipid-protein complexes. For example, a typical sample after Bal 31 digestion contained approximately 4 ng of nucleic acid of variable length and 10(8.7) ID50 units of scrapie prion infectivity. Consideration of different models for a hypothetical scrapie-specific nucleic acid suggests that such a molecule would have to be: (i) quite small (less than 100 nucleotides), (ii) possess a particle-to-infectivity ratio near unity or (iii) heterogeneous in size. Although our results do not eliminate the possibility that prions possess a scrapie-specific nucleic acid of variable length, they narrow considerably the spectrum of features specifying such a candidate molecule.

Cyclosporin synthetase is a 1.4 MDa multienzyme polypeptide Re‐evaluation of the molecular mass of various peptide synthetases

The earlier determined molecular mass of 0.8 MDa for the multifunctional polypeptide, cyclosporin synthetase, was re‐evaluated by SDS‐PAGE and CsCl density gradient centrifugation. In SDS‐PAGE, new molecular mass values as standards were available from sequencing data. In the CsCl density gradient extremly low protein concentrations, such as 10–50 nM could be analysed due to the fluorescence detection system of the analytical ultracentrifuge. Both methods yielded approximately the same value of about 1.4 MDa. Using this molecular mass of cyclosporin synthetase as a reference the molecular masses of various related enzymes could be re‐evaluated in SDS‐PAGE. The sedimentation coefficient of 26.3 S for cyclosporin synthetase indicates an oblate overall shape of the enzyme.

Over-expression and amplification of the c-myc gene in human urothelial carcinoma.

To understand the mechanisms underlying increased expression of Myc protein in human urinary bladder cancer, expression of c‐myc mRNA and the copy number of the c‐myc gene were determined. Expression of mRNA was measured by quantitative RT‐PCR in 40 urothelial carcinomas and in 18 histologically normal mucosae. Mean expression in tumors was significantly increased (3.23 ± 2.63 AU vs. 1.90 ± 0.95 AU, p < 0.023) and exceeded the highest level in normal mucosa in 15 (37.5%) tumors. The c‐myc gene copy number was higher than in leukocytes and normal bladder mucosa in 14 of 40 tumors, but only 3 among these showed a more than 4‐fold increase indicative of gene amplification. Most, but not all, tumors with elevated expression displayed an increased gene copy number (p < 0.0001). In line with other studies of the protein level, no significant association either of c‐myc mRNA over‐expression or of increased gene copy number with tumor stage or grade was observed. The data indicate that elevated mRNA expression as a consequence of increases in c‐myc gene copy number often underlies Myc protein over‐expression in bladder cancer. This increase may be a consequence of, most frequently, chromosome 8q gain and, occasionally, gene amplification, while in some tumors deregulation of mRNA expression occurs without evident changes in the c‐myc gene copy number. Int. J. Cancer (Pred. Oncol.) 84:169–173, 1999.

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 FKBP25mem 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 K i 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.

A cyclophilin‐like peptidyl‐prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506‐binding protein, FKBP25mem), belongs to the enzyme family of peptidyl‐prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N‐terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164‐amino‐acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19‐amino‐acid polypeptide extension including a 6‐histi‐dine tag and an enterokinase cleavage site exhibits

A fluorescence detection system for the analytical ultracentrifuge and its application to proteins, nucleic acids, and viruses

A fluorescence detection system was developed for the analytical ultracentrifuge Spinco model E. Fluorescence is excited by a laser beam which is focussed into the cell and illuminates an area with a dimension of 60 μm in radial direction. For scanning the laser beam is moved in radial direction. After passing the cell, the laser beam is quenched by a carbon light trap and a set of optical filters. Fluorescence emission intensity is monitored by a photomultiplier located behind the light trap and the set of filters. The sensitivity of the detection system was tested by applying it to the sedimentation analysis of proteins and nucleic acids. Bovine serum albumin (BSA) was covalently labelled with the fluorescence-dye fluorescein-isothiocyanate (FITC), and its sedimentation coefficient could be determined even if BSA was analyzed in a concentration as low as 10−10 M. Nucleic acids were labelled non-covalently by the intercalating dye ethidium bromide. Only 8 ng RNA were needed for the determination of the sedimentation coefficient. The particular advantages of the fluorescence detection system were exploited for the establishment of a new method for quantitative virus detection. To tobacco mosaic virus (TMV) a monoclonal anti-TMV antibody from mouse was bound, and to this a second, anti-mouse antibody that carried the fluorescence-label FITC was attached. Either by UV-irradiation or by incubation with glutaraldehyde, the first antibody was covalently crosslinked to TMV, and the second antibody to the first. In CsCl density centrifugation with fluorescence detection as little as 3.2 ng virus/80 μl or 6×108 virus particles/ml were recorded in a well expressed band at the corresponding buoyant density. Tenfold lower concentration would result still in a significant band. The sensitivity compares well with those of the most advanced techniques from immunology. Due to the specific labelling of viruses by antibodies it will be possible to carry out quantitative physical characterization of virus containing samples without purifying the virus. Future applications of the fluorescence detection system and of the virus detection technique are discussed.

Expression of G1-->S transition regulatory molecules in human urothelial cancer.

Growth of cancer cells is characterized by accelerated passage through the cell cycle, which is often caused by deregulation of the G1→S transition. In this study the expression of G1→S transition regulatory molecules was analyzed in 32 transitional cell carcinoma specimens and fifteen normal tissues obtained by cystectomy or nephroureterectomy of mainly locally advanced tumors, as well as six bladder cancer cell lines. Expression of mRNAs for cyclins D1 and D2 and cyclin‐dependent kinases (CDK) 2 and 4 was investigated by quantitative reverse transcription‐poly‐merase chain reaction. Overexpression of cyclin D1 compared to normal mucosa was observed in 3 tumors (9.4%), but in neither of the cell lines. All tumors with overexpression were moderately differentiated (G2) pT1 or pT2 tumors, and thus among the less advanced specimens. Cyclin D2 was not expressed in normal bladder mucosa or in tumors. The expression of CDK4 mRNA varied within the same range in mucosa, tumors, and cell lines. CDK2 mRNA expression varied more strongly and was diminished in individual tumors and in four cell lines. It is concluded that cyclin D1 overexpression can play an important role in the early stage of urothelial tumorigenesis, driving cell proliferation. Ectopic expression of cyclin D2 or amplification of CDK4 does not occur at a significant frequency in urothelial carcinomas. Different expression patterns of cyclin D1 and CDK2 indicate heterogeneity in the mechanisms of G1→S transition deregulation in individual bladder tumors which may elicit differences in their biological and clinical behavior.

Regulation of CD95 (Apo-1/Fas) ligand and receptor expression in human embryonal carcinoma cells by interferon gamma and all-trans retinoic acid.

Expression of CD95 (Apo-1/Fas) ligand and its two receptor isoforms, in response to all-trans retinoic acid and interferon gamma (IFNgamma), was analyzed atthe mRNA and protein levels in human Tera-2 embryonal carcinoma cells. Exposure of Tera-2 cells to all-trans retinoic acid for up to 16 days led to a decrease of CD95 ligand expression when compared to the control conditions, whereas expression of both CD95 isoforms increased. These changes were functionally significant since Tera-2 cells treated with all-trans retinoic acid for six to 16 days were more susceptible to CD95-mediated apoptosis. On the other hand, Tera-2 cells lost their capacity to induce apoptosis in CD95 receptor bearing Jurkat T lymphocytes after six days of incubation with all-trans retinoic acid. When Tera-2 cells were treated with IFNgamma, expression of CD95 ligand and both CD95 receptor isoforms increased within 24 hours. Tera-2 cells were then more susceptible to CD95 mediated apoptosis but also killed more CD95 receptor bearing Jurkat T lymphocytes via CD95 ligation compared to the control conditions. The results are indicative of differential regulation of CD95-mediated apoptosis by all-trans retinoic acid and IFNgamma in Tera-2 embryonal carcinoma cells, with likely impact on antitumor immunity.

Analysis of the DCC tumor suppressor gene in testicular germ cell tumors: mutations and loss of expression.

Inactivation of the DCC (Deleted in Colon Carcinoma) tumor suppressor gene by allelic loss and/or reduced expression is associated with the development of colon cancer, gliomas, gastric and prostatic malignancies. In a total cohort of 51 testicular germ cell tumors (GCT) of different histologies we analyzed restriction fragment length polymorphism (RFLP) for DCC at two specific DNA sites in 37 GCT and DCC mRNA expression compared to that of the adjacent normal testicular tissue in 41 GCT, one Leydig cell tumor and one testicular metastasis of a non-small cell lung cancer (NSCLC). Two of 17 tumors (11.7%) informative for the Msp I polymorphic site of the DCC gene and 6/25 tumors (24.0%) informative for variable number of tandem repeat (VNTR) showed loss of heterozygosity (LOH). DCC expression was analyzed by semi-quantitative polymerase chain reaction after initial reverse transcription (RT-PCR). Thirty of 41 GCT (73.1%) and both, the Leydig cell tumor and the testicular metastasis of NSCLC, had a nearly complete or total loss of DCC mRNA expression. Six of 11 (54.5%) seminomas and 24/30 (80.0%) nonseminomas had this loss of expression. Twelve of 17 (70.5%) localized tumors, 9/13 (69.2%) tumors with lymph node involvement and 9/11 (82.2%) tumors with distant metastases showed decreased or absent DCC expression. This data suggests that inactivation of the DCC gene, especially the loss of DCC expression, is associated with the development and progression of human GCT.

Enzymatic biosynthesis of cyclosporin A and analogues

The final assembly of the undecapeptide chain of cyclosporin A and its cyclization is accomplished in Beauveria nivea by cyclosporin synthetase. This multienzyme is the largest integrated enzyme structure so far reported. Its size has been estimated at approximately 1,400 kDa by two different methods: 1), by 3% SDS-PAGE using the related multienzymes ACV synthetase and gramicidin S synthetase 2 as references (420 and 556 kDa, respectively); and 2), by CsCl density gradient centrifugation experiments using fluorescence-labeled cyclosporin synthetase. Besides cyclosporin A and a number of cyclosporins known from fermentation studies cyclosporin synthetase is capable of synthesizing some new cyclosporins which are so far unobtainable by fermentation. So, for example the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid1]CyA, dihydro-CyA, [L-norvaline2,5, N-methyl-L-norvaline11]CyA, [L-allo-isoleucine5, N-methyl-L-allo-isoleucine11]CyA, [D-2-aminobutyric acid8]CyA, [beta-chloro-D-alanine8]CyA and some related compounds could be established. By using a related but different enzyme from Cylindrotrichum Bonorden, the peptolide [L-threonine2, L-leucine5,10, D-2-hydroxyisovaleric acid8]CyA could be synthesized in vitro. We were able to synthesize these cyclosporins in sufficient quantities to examine their structure by FAB mass spectroscopy and explore their immunosuppressivity. It was found that all new cyclosporins so far synthesized in the in vitro system are immunosuppressive.