Showing papers by "University of Basel published in 2002"

Functional profiling of the Saccharomyces cerevisiae genome.

Abstract: Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

Quantum computing in molecular magnets

Abstract: Shor and Grover demonstrated that a quantum computer can outperform any classical computer in factoring numbers1 and in searching a database2 by exploiting the parallelism of quantum mechanics. Whereas Shor's algorithm requires both superposition and entanglement of a many-particle system3, the superposition of single-particle quantum states is sufficient for Grover's algorithm4. Recently, the latter has been successfully implemented5 using Rydberg atoms. Here we propose an implementation of Grover's algorithm that uses molecular magnets6,7,8,9,10, which are solid-state systems with a large spin; their spin eigenstates make them natural candidates for single-particle systems. We show theoretically that molecular magnets can be used to build dense and efficient memory devices based on the Grover algorithm. In particular, one single crystal can serve as a storage unit of a dynamic random access memory device. Fast electron spin resonance pulses can be used to decode and read out stored numbers of up to 105, with access times as short as 10-10 seconds. We show that our proposal should be feasible using the molecular magnets Fe8 and Mn12.

Processing of gene expression data generated by quantitative real-time RT-PCR.

Abstract: Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.

Prospective Study of Polyomavirus Type BK Replication and Nephropathy in Renal-Transplant Recipients

Abstract: Background Nephropathy associated with the polyomavirus type BK (BKV) nephropathy has emerged as a cause of allograft failure linked to immunosuppressive regimens containing tacrolimus or mycophenolate mofetil. The presence of viral inclusions, known as “decoy cells,” in urine and the presence of BKV DNA in plasma have been proposed as markers for the replication of BKV and associated nephropathy, but data from prospective studies have been lacking. Methods In a prospective, single-center study, we followed 78 renal-transplant recipients who were receiving immunosuppressive therapy that included tacrolimus (37 patients) or mycophenolate mofetil (41 patients). Urine was tested for the presence of decoy cells at routine visits. BKV DNA was measured 3, 6, and 12 months after transplantation and whenever decoy cells were detected. The viral load in plasma was quantified with the use of a real-time polymerase-chain-reaction method. Renal biopsy was performed if allograft function deteriorated. Results Twenty-t...

Aquaporin water channels – from atomic structure to clinical medicine

Abstract: The water permeability of biological membranes has been a longstanding problem in physiology, but the proteins responsible for this remained unknown until discovery of the aquaporin 1 (AQP1) water channel protein. AQP1 is selectively permeated by water driven by osmotic gradients. The atomic structure of human AQP1 has recently been defined. Each subunit of the tetramer contains an individual aqueous pore that permits single-file passage of water molecules but interrupts the hydrogen bonding needed for passage of protons. At least 10 mammalian aquaporins have been identified, and these are selectively permeated by water (aquaporins) or water plus glycerol (aquaglyceroporins). The sites of expression coincide closely with the clinical phenotypes--ranging from congenital cataracts to nephrogenic diabetes insipidus. More than 200 members of the aquaporin family have been found in plants, microbials, invertebrates and vertebrates, and their importance to the physiology of these organisms is being uncovered.

Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex

Abstract: Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.

Nucleosynthesis in massive stars with improved nuclear and stellar physics

Abstract: We present the first calculations to follow the evolution of all stable nuclei and their radioactive progeni- tors in stellar models computed from the onset of central hydrogen burning through explosion as Type II supernovae. Calculations are performed for Population I stars of 15, 19, 20, 21, and 25 Musing the most recently available experimental and theoretical nuclear data, revised opacity tables, neutrino losses, and weak interaction rates and taking into account mass loss due to stellar winds. A novel '' adaptive '' reaction net- work is employed with a variable number of nuclei (adjusted each time step) ranging from � 700 on the main sequence to e2200 during the explosion. The network includes, at any given time, all relevant isotopes from hydrogen through polonium (Z ¼ 84). Even the limited grid of stellar masses studied suggests that overall good agreement can be achieved with the solar abundances of nuclei between 16 O and 90 Zr. Interesting dis- crepancies are seen in the 20 Mmodel and (so far, only in that model) are a consequence of the merging of the oxygen, neon, and carbon shells about a day prior to core collapse. We find that, in some stars, most of the '' p-process '' nuclei can be produced in the convective oxygen-burning shell moments prior to collapse; in others, they are made only in the explosion. Serious deficiencies still exist in all cases for the p-process isotopes of Ru and Mo. Subject headings: nuclear reactions, nucleosynthesis, abundances — stars: evolution — supernovae: general On-line material: machine-readable tables

Do Corrupt Governments Receive Less Foreign Aid

Abstract: Critics of foreign aid programs argue that these funds often support corrupt governments and inefficient bureaucracies. Supporters argue that foreign aid can be used to reward good governments. This paper documents that there is no evidence that less corrupt governments receive more foreign aid. On the contrary, according to some measures of corruption, more corrupt governments receive more aid. Also, we could not find any evidence that an increase in foreign aid reduces corruption. In summary, the answer to the question posed in the title is "no."

Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP

Abstract: Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.

Disruption of CREB function in brain leads to neurodegeneration

Abstract: Control of cellular survival and proliferation is dependent on extracellular signals and is a prerequisite for ordered tissue development and maintenance. Activation of the cAMP responsive element binding protein (CREB) by phosphorylation has been implicated in the survival of mammalian cells. To define its roles in the mouse central nervous system, we disrupted Creb1 in brain of developing and adult mice using the Cre/loxP system. Mice with a Crem(-/-) background and lacking Creb in the central nervous system during development show extensive apoptosis of postmitotic neurons. By contrast, mice in which both Creb1 and Crem are disrupted in the postnatal forebrain show progressive neurodegeneration in the hippocampus and in the dorsolateral striatum. The striatal phenotype is reminiscent of Huntington disease and is consistent with the postulated role of CREB-mediated signaling in polyglutamine-triggered diseases.

Photoremovable protecting groups: reaction mechanisms and applications

Abstract: Photolabile protecting groups enable biochemists to control the release of bioactive compounds in living tissue. ‘Caged compounds’ (photoactivatable bioagents) have become an important tool to study the events that follow chemical signalling in, e.g., cell biology and the neurosciences. The possibilities are by no means exhausted. Progress will depend on the development of photoremovable protecting groups that satisfy the diverse requirements of new applications—a challenging task for photochemists.

Electron spin decoherence in quantum dots due to interaction with nuclei.

Abstract: We study the decoherence of a single electron spin in an isolated quantum dot induced by hyperfine interaction with nuclei. The decay is caused by the spatial variation of the electron wave function within the dot, leading to a nonuniform hyperfine coupling A. We evaluate the spin correlation function and find that the decay is not exponential but rather power (inverse logarithm) lawlike. For polarized nuclei we find an exact solution and show that the precession amplitude and the decay behavior can be tuned by the magnetic field. The decay time is given by (planck)N/A, where N is the number of nuclei inside the dot, and the amplitude of precession decays to a finite value. We show that there is a striking difference between the decoherence time for a single dot and the dephasing time for an ensemble of dots.

Expression of Cytokeratins 17 and 5 Identifies a Group of Breast Carcinomas with Poor Clinical Outcome

Abstract: While several prognostic factors have been identified in breast carcinoma, the clinical outcome remains hard to predict for individual patients. Better predictive markers are needed to help guide difficult treatment decisions. In a previous study of 78 breast carcinoma specimens, we noted an association between poor clinical outcome and the expression of cytokeratin 17 and/or cytokeratin 5 mRNAs. Here we describe the results of immunohistochemistry studies using monoclonal antibodies against these markers to analyze more than 600 paraffin-embedded breast tumors in tissue microarrays. We found that expression of cytokeratin 17 and/or cytokeratin 5/6 in tumor cells was associated with a poor clinical outcome. Moreover, multivariate analysis showed that in node-negative breast carcinoma, expression of these cytokeratins was a prognostic factor independent of tumor size and tumor grade.

Cerebral Hemorrhage After Passive Anti-Aβ Immunotherapy

Abstract: Immunotherapy for Alzheimer's disease (AD) has been the subject of intense investigation. Both active and passive immunization against β-amyloid peptide (Aβ) in mouse models reduce levels of Aβ, prevent and clear amyloid plaques, and improve cognitive behavior ([1][1]). We studied passive

Arthroscopic Findings in Patients with Chronic Ankle Instability

Abstract: BackgroundThere are little objective data on structural changes of the chronically unstable ankle. Such knowledge could help with preoperative planning.HypothesisPreoperative ankle arthroscopy provides important insights into the causes and mechanisms of ankle instability and the resulting disability.Study DesignCase series.MethodsFrom 1993 to 1999, arthroscopic examination was performed in the ankles of 148 patients with symptomatic chronic ankle instability that had lasted 6 months or more. All structural changes were recorded and compared with the clinical diagnosis.ResultsA rupture or elongation of the anterior talofibular ligament was noted in 86% of ankles, of the calcaneofibular ligament in 64%, and of the deltoid ligament in 40%. Cartilage damage was noted in 66% of ankles with lateral ligament injuries, whereas 98% of the ankles with deltoid ligament injuries had cartilage damage. Although lateral instability could be verified arthroscopically in 127 patients, medial instability was presumed clin...

Oxidation-Resistant Gold-55 Clusters

Abstract: Gold nanoparticles ranging in diameter from 1 to 8 nanometers were prepared on top of silicon wafers in order to study the size dependence of their oxidation behavior when exposed to atomic oxygen. X-ray photoelectron spectroscopy showed a maximum oxidation resistance for “magic-number” clusters containing 55 gold atoms. This inertness is not related to electron confinement leading to a size-induced metal-to-insulator transition, but rather seems to be linked to the closed-shell structure of such magic clusters. The result additionally suggests that gold-55 clusters may act as especially effective oxidation catalysts, such as for oxidizing carbon monoxide.

Tumor response and clinical benefit in neuroendocrine tumors after 7.4 GBq (90)Y-DOTATOC.

Abstract: The aim of this prospective phase II study was to evaluate the tumor response of neuroendocrine tumors to high-dose targeted irradiation with 7.4 GBq/m2 of the radiolabeled somatostatin analog 90Y-1,4,7,10-tetra-azacyclododecan-4,7,10-tricarboxy-methyl-1-yl-acetyl-d-Phe-Tyr3-octreotide (DOTATOC). In addition, we investigated the clinical benefit of 90Y-DOTATOC regarding the malignant carcinoid syndrome and tumor-associated pain. Methods: Thirty-nine patients (mean age, 55 y) with progressive neuroendocrine gastroenteropancreatic and bronchial tumors were included. The treatment consisted of 4 equal intravenous injections of a total of 7.4 GBq/m290Y-DOTATOC, administered at intervals of 6 wk. After each treatment cycle, a standardized clinical benefit assessment using the National Cancer Institute grading criteria (NCI-CTC) was performed. Results: The objective response rate according to World Health Organization (WHO) criteria was 23%. For endocrine pancreatic tumors (13 patients), the objective response rate was 38%. Complete remissions were found in 5% (2/39), partial remissions in 18% (7/39), stable disease in 69% (27/39), and progressive disease in 8% (3/39). A significant reduction of clinical symptoms could be found in 83% of patients with diarrhea, in 46% of patients with flush, in 63% of patients with wheezing, and in 75% of patients with pellagra. The overall clinical benefit was 63%. All responses (both clinical benefit and WHO response) were ongoing for the duration of follow-up (median, 6 mo; range, 2–12 mo). Side effects were grade 3 or 4 (NCI-CTC) lymphocytopenia in 23%, grade 3 anemia in 3%, and grade 2 renal insufficiency in 3%. Conclusion: High-dose targeted radiotherapy with 7.4 GBq/m290Y-DOTATOC is a well-tolerated treatment for neuroendocrine tumors, with remarkable clinical benefit and objective response.

Dynamic recrystallization of quartz: correlation between natural and experimental conditions

Abstract: Quartz veins in the Eastern Tonale mylonite zone (Italian Alps) were deformed in strike-slip shear. Due to the synkinematic emplacement of the Adamello Pluton, a temperature gradient between 280°C and 700°C was effected across this fault zone. The resulting dynamic recrystallization microstructures are characteristic of bulging recrystallization, subgrain rotation recrystallization and grain boundary migration recrystallization. The transitions in recrystallization mechanisms are marked by discrete changes of grain size dependence on temperature. Differential stresses are calculated from the recrystallized grain size data using paleopiezometric relationships. Deformation temperatures are obtained from metamorphic reactions in the deformed host rock. Flow stresses and deformation temperatures are used to determine the strain rate of the Tonale mylonites through integration with several published flow laws yielding an average rate of approximately 10−14s−1 to 10−12s−1. The deformation conditions of the natural fault rocks are compared and correlated with three experimental dislocation creep regimes of quartz of Hirth & Tullis. Linking the microstructures of the naturally and experimentally deformed quartz rocks, a recrystallization mechanism map is presented. This map permits the derivation of temperature and strain rate for mylonitic fault rocks once the recrystallization mechanism is known.

The Yersinia Ysc-Yop 'type III' weaponry.

Abstract: 'Type III secretion'--the mechanism by which some pathogenic bacteria inject proteins straight into the cytosol of eukaryotic cells to 'anaesthetize' or 'enslave' them--was discovered in 1994. Important progress has been made in this area during the past few years: the bacterial organelles responsible for this secretion--called 'injectisomes'--have been visualized, the structures of some of the bacterial protein 'effectors' have been determined, and considerable progress has been made in understanding the intracellular action of the effectors. Type III secretion is key to the pathogenesis of bacteria from the Yersinia genus.

RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles

Abstract: The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5′-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.

Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity

Abstract: Expression of oncogenic Ras in primary human cells activates p53, thereby protecting cells from transformation. We show that in Ras-expressing IMR-90 cells, p53 is phosphorylated at Ser33 and Ser46 by the p38 mitogen-activated protein kinase (MAPK). Activity of p38 MAPK is regulated by the p53-inducible phosphatase PPM1D, creating a potential feedback loop. Expression of oncogenic Ras suppresses PPM1D mRNA induction, leaving p53 phosphorylated at Ser33 and Ser46 and in an active state. Retrovirus-mediated overexpression of PPM1D reduced p53 phosphorylation at these sites, abrogated Ras-induced apoptosis and partially rescued cells from cell-cycle arrest. Inactivation of p38 MAPK (the product of Mapk14) in vivo by gene targeting or by PPM1D overexpression expedited tumor formation after injection of mouse embryo fibroblasts (MEFs) expressing E1A+Ras into nude mice. The gene encoding PPM1D (PPM1D, at 17q22/q23) is amplified in human breast-tumor cell lines and in approximately 11% of primary breast tumors, most of which harbor wildtype p53. These findings suggest that inactivation of the p38 MAPK through PPM1D overexpression resulting from PPM1D amplification contributes to the development of human cancers by suppressing p53 activation.

Yersinia type III secretion send in the effectors

Abstract: Pathogenic Yersinia spp (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) have evolved an exquisite method for delivering powerful effectors into cells of the host immune system where they inhibit signaling cascades and block the cells' response to infection Understanding the molecular mechanisms of this system has provided insight into the processes of phagocytosis and inflammation