Rotavirus 993/83, isolated from calf faeces, closely resembles an avian rotavirus

Polypeptides from purified virions of the calf rotavirus (RV) isolate 993/83 and those from the pigeon RV isolate PO-13 comigrated on SDS-polyacrylamide gels. Two polypeptides of 45K and 47K were detected at the position of VP6. Both proteins behaved like authentic VP6 protein with EDTA and heat treatment. RV 993/83 and PO-13 showed identical one-dimensional peptide maps for VP2, and the 45K and 47K proteins. More than 70% of sera from German cattle older than 1 year showed neutralizing serum antibodies to RV 993/83 and RV PO-1

Prevalence of antibodies to four different rotavirus strains in different age groups of cattle

Neutralizing antibody titers to four bovine rotavirus strains, representing three serotypes, were measured in 160 sera from cattle of different age groups. Age-specific seroprevalence analysis revealed serotype 6, represented by bovine rotavirus (BRV) NCDV, as the predominant rotavirus serotype infecting German cattle and serotype 10, represented by BRV V1005, as the least prominent. Infections with serotype 8, represented by BRV 678, occurred with intermediate frequency. Antibodies of young calves distinguished between NCDV and UK virus, two serotype 6 BRV strains differing in VP4 antige

Cattle develop neutralizing antibodies to rotavirus serotypes which could not be isolated from faeces of symptomatic calves

Neutralizing antibodies against 10 serotypes of rotavirus were measured in sera from different age groups of German cattle. Only five of 143 sera did not neutralize heterologous serotypes. Sera from 64 of 76 calves younger than 1 year neutralized bovine rotavirus NCDV (serotype 6). From these calves, sera 54, 26, 51, 24, 12, 10 and 37, in neutralized addition, the heterologous serotypes 1, 2, 3, 4, 5, 7 and 9, respectively. Thirty-eight of 46 rotavirus isolates from Bavarian calves with diarrhoea were serotyped by neutralization: 22, 2 and 14 isolates were typed as serotype 6, serotype 10 (B223) and a newly defined subtype of serotype 10 (V1005), respectively. All serotype 6 isolates and none of the serotype 10 or V1005-like viruses tested hybridized to a NCDV-specific cDNA probe. Eight isolates gave equivocal results by neutralization. We failed however to identify serotype 1, 2, 3, 4 or 8 bovine rotavirus isolates by neutralization with hyperimmune sera and dot blot hybridization with serotype-specific cDNA probes. Thus cross-reacting antibodies in cattle might not represent an anamnestic response, but the recognition of a cross-reacting neutralization epitope shared by many rotavirus serotype

Characterization of a Second Bovine Rotavirus Serotype

Bovine rotavirus (BRV) V 1005 was characterized by two-way cross-neutralization tests as a second serotype of BRV. Virions and inner shell particles of 65 nm and 55 nm diameter respectively, and empty capsids of 65 nm and 55 nm diameter were separated by density gradient centrifugation. Three polypeptides of molecular weight 60,000, 36,000 and 28,000 (minor protein) could be identified in the outer shell of virions and in the larger empty capsids. Inner shell particles contained three polypeptides of molecular weight 105,000, 83,000 and 43,000. Both sizes of empty capsids showed two polypeptides of molecular weight 75,000 and 55,000 not found in virions. Pulse-labelling of infected cells revealed eight major and three minor intracellular viral polypeptides. Viral polypeptide synthesis started at about 6 hours p.i. and correlated in time with double-stranded RNA synthesis. As soon as viral polypeptide synthesis was detectable, newly synthesized viral polypeptides were incorporated into intracellular viral particles. Radioactive viral polypeptides appeared without a longer lag period in extracellular viruses from 6 hours p.i. onward

ORFer – retrieval of protein sequences and open reading frames from GenBank and storage into relational databases or text files

BACKGROUND: Functional genomics involves the parallel experimentation with large sets of proteins. This requires management of large sets of open reading frames as a prerequisite of the cloning and recombinant expression of these proteins. RESULTS: A Java program was developed for retrieval of protein and nucleic acid sequences and annotations from NCBI GenBank, using the XML sequence format. Annotations retrieved by ORFer include sequence name, organism and also the completeness of the sequence. The program has a graphical user interface, although it can be used in a non-interactive mode. For protein sequences, the program also extracts the open reading frame sequence, if available, and checks its correct translation. ORFer accepts user input in the form of single or lists of GenBank GI identifiers or accession numbers. It can be used to extract complete sets of open reading frames and protein sequences from any kind of GenBank sequence entry, including complete genomes or chromosomes. Sequences are either stored with their features in a relational database or can be exported as text files in Fasta or tabulator delimited format. The ORFer program is freely available at http://www.proteinstrukturfabrik.de/orfer. CONCLUSION: The ORFer program allows for fast retrieval of DNA sequences, protein sequences and their open reading frames and sequence annotations from GenBank. Furthermore, storage of sequences and features in a relational database is supported. Such a database can supplement a laboratory information system (LIMS) with appropriate sequence information

Antigenic and biochemical characterization of bovine rotavirus V1005, a new member of rotavirus serotype 10

Bovine rotavirus (BRV) V1005 is serologically distinct from rotavirus serotypes 1, 2, 3, 4, 5, 6, 8 and 9. BRV V1005 showed cross-reactions with BRV B223, the American prototype of serotype 10 rotavirus, and with BRV E4049, a British serotype 10 isolate. BRV V1005 was, however, not neutralized by four monoclonal antibodies directed against VP7 of BRV B223. Two-way cross-reactions were observed between BRV V1005 and a reassortant rotavirus containing the VP4 from BRV UK. In addition the major tryptic cleavage product of VP4, VP5*, from BRV V1005 is indistinguishable by peptide mapping and its isoelectric point from the homologous protein of BRV UK, but is clearly different from VP5* of BRV NCDV. The peptide map of VP7 from BRV V1005 differed from that obtained for VP7 of BRV U

An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography

BACKGROUND: Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner. RESULTS: We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag (His-tag) or glutathione S-transferase (GST)-tag from bacterial expression systems. Proteins are expressed in 96-well microplates and are purified by a fully automated procedure on a pipetting robot. Up to 90 microgram purified protein can be obtained from 1 ml microplate cultures. The procedure is readily reproducible and 96 proteins can be purified in approximately three hours. It avoids clearing of crude cellular lysates and the use of magnetic affinity beads and is therefore less expensive than comparable commercial systems. We have used this method to compare purification of a set of human proteins via His-tag or GST-tag. Proteins were expressed as fusions to an N-terminal tandem His- and GST-tag and were purified by metal chelating or glutathione affinity chromatography. The purity of the obtained protein samples was similar, yet His-tag purification resulted in higher yields for some proteins. CONCLUSION: A fully automated, robust and cost effective method was developed for the purification of proteins that can be used to quickly characterise expression clones in high throughput and to produce large numbers of proteins for functional studies. His-tag affinity purification was found to be more efficient than purification via GST-tag for some proteins

Model checking combined Z and Statechart specifications

Eine der bedeutendsten Herausforderungen der Softwareentwicklung besteht darin, einen Entwicklungsprozess zu garantieren, der Fehlerfreiheit nicht nur gewährleistet sondern auch nachweisbar macht. Beides ist von besonderer Bedeutung, wenn sicherheitskritische Systeme entwickelt werden, etwa in den Bereichen der Medizin, der Produktionssteuerung oder der Verkehrstechnik. Softwarefehler können hier lebensbedrohlich sein. Aus diesem Grund ist es meist auch notwendig, die Fehlerfreiheit der Software einem Dritten nachzuweisen. Die Steuerung einer Verkehrsampel muss beispielsweise nicht nur fehlerfrei funktionieren, sondern auch vom TÜV abgenommen werden. Der Einsatz formaler Methoden stellt einen vielversprechenden Ansatz dar, diese Probleme zu lösen. Formale Sprachen haben gegenüber den üblichen, nicht-formalen Methoden (umgangssprachliche Spezifikationsdokumente oder Spezifikationssprachen ohne eindeutige Semantik) den Vorteil einer eindeutigen Semantik. Damit können Anforderungen an ein System eindeutig beschrieben und seine Eigenschaften mathematisch bewiesen werden. In der Praxis haben sich diese Methoden allerdings bisher noch nicht durchgesetzt. Zwei herausragende Ursachen hierfür sind: 1. Die formalen Spezifikationssprachen orientieren sich meist mehr an mathematischer Eleganz als an einfachen und intuitiven Sprachmitteln. Das stellt eine große Hürde für den praktischen Einsatz dar. Die Spezifikationssprache mSZ versucht dieses Problem zu lösen. Sie verbindet die von Harel entwickelte und in der Industrie akzeptierte grafische Sprache Statecharts mit der formalen Sprache Z. Damit liegt eine intuitive Sprache vor, die den Anforderungen einer formalen Sprache genügt. 2.Formale Spezifikationen haben zwar eine präzise Semantik, sie lassen aber dem Spezifikateur immer noch die Freiheit, inkonsistente oder fehlerhafte (nicht den tatsächlichen Anforderungen entsprechende) Spezifikationen zu erstellen. Andererseits ermöglichen sie es, Konsistenz und Eigenschaften formal zu beweisen und so zu einer fehlerfreien Spezifikation zu gelangen. Werden solche Beweise nicht geführt, ist gegenüber einer nicht-formalen Spezifikation wenig gewonnen. Um die aufwändige Beweisführung praktikabel zu machen, ist eine möglichst weitgehende Automatisierung unverzichtbar. Der Nachweis der Konsistenz sowie der Eigenschaften einer mSZ Spezifikation ist Ziel der vorliegenden Arbeit. Hierfür werden Model Checking Techniken eingesetzt. Um dies zu ermöglichen, wird die mSZ Spezifikation in drei Schritten übersetzt: 1. Übersetzung des Statechartanteils einer mSZ-Spezifikation nach Z. Damit werden zusätzlich die Semantik der Statecharts und die Semantik die Integration von Statecharts und Z definiert. Außerdem erlaubt diese Vorgehensweise andere, reine Z-Werkzeuge für die Analyse zu benutzen. 2. Vereinfachung der Z-Spezifikation in ein vereinfachtes Z (Simple Z), das vom Sprachumfang der Eingabesprache eines Model Checkers entspricht. Dieser Schritt erlaubt es, sowohl mSZ-Spezifikationen wie auch reine Z Spezifikationen für das Model Checking vorzubereiten. Das vereinfachte Z kann leicht in die Eingabesprache eines Model Checkers übersetzt werden. 3. Übersetzung von Simple Z in die Eingabesprache des SMV Model Checkers von McMillan. Der Model Checker kann dann Konsistenz und Eigenschaften der Spezifikation beweisen.One of today's major problems in software engineering is to achieve a high and comprehensive quality standard for the software development process, in order to maintain a reliable high quality for the resulting products. This holds particularly true for safety critical systems, where failure of the software may have life-threatening consequences. Here, not only the quality of the software itself is important, but also the ability to convince a third party of this very quality. The usage of formal methods is one promising approach to achieve these goals. Roughly speaking, formal methods introduce mathematical precision to the development process. They do so by using formalisms with well defined semantics, and so stipulate formal proofs to verify development steps. This approach is all too well feasible in theory. In practice, however, one will encounter various problems that impede a consequent usage of formal methods: 1. The formal character of the proposed languages and the need to use them for every aspect of the described system makes them too bulky. The reason for this is that they often times concentrate more on the mathematical elegance of their underlying semantics than on comfortable and intuitive usage. The specification language mSZ tackles this problem. It combines Harel s Statecharts with the formal specification language Z and offers very intuitive means to describe a system. 2. As adequate tool support is often missing, implementation of the formal proof obligations becomes practically impossible, because without any tools, these proofs are quite complicated, and their development takes a lot of time. Providing tool support for the verification of consistency and properties of an mSZ specification is the objective of this work. Model checking is used for the verification. An mSZ specification is translated in three steps into the input language of a model checker: 1. The Statecharts are translated to Z. With this, the Statechart semantics and the semantics of the Statechart integration with Z are defined. The result of this translation can also be used by Z tools that do not know Statecharts for analysis. 2. The Z specification is rewritten to a Z subset (Simple Z) that contains only language constructs, supported by the model checker. This step allows preparing mSZ as well as pure Z specifications for model checking. 3. Simple Z is translated to the input language of the SMV model checker

Neural networks as a prognostic tool in critically ill patients

Im Zeitraum 1. 11. 1993 bis 30. 3. 1997 wurden 1149 allgemeinchirurgische Intensivpatienten prospektiv erfaßt, von denen 114 die Kriterien des septischen Schocks erfüllten. Die Letalität der Patienten mit einem septischen Schock betrug 47,3%. Nach Training eines neuronalen Netzes mit 91 (von insgesamt n = 114) Patienten ergab die Testung bei den verbleibenden 23 Patienten bei der Berücksichtigung von Parameterveränderungen vom 1. auf den 2. Tag des septischen Schocks folgendes Ergebnis: Alle 10 verstorbenen Patienten wurden korrekt als nicht überlebend vorhergesagt, von den 13 Überlebenden wurden 12 korrekt als überlebend vorhergesagt (Sensitivität 100%; Spezifität 92,3%).Neural networks as a prognostic tool in critically ill patients Summary: From 1. 11. 93 to 30. 3. 97, 1149 patients were prospectively studied during their ICU stay. Of them,114 met the criteria of septic shock, with lethality of 47.3%. A neural network was trained with datasets from 91 of these 114 patients. Testing the trained neural network with the remaining 23 patients, the following result was obtained: all 10 patients dying from septic shock were correctly predicted; of 13 surviving patients, 12 were correctly identified (sensitivity 100%; specifity 92.3%)

Vectors for co-expression of an unrestricted number of proteins

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells