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W J Neubert

Publications and source records attributed to W J Neubert.

35 records · Page 2Linked to original sources
PubMed1992

Rapid sequencing of the Sendai virus 6.8 kb large (L) gene through primer walking with an automated DNA sequencer.

The determination of the complete DNA sequence of the large (L) polymerase gene of Sendai virus strain Fushimi was used to explore the potential and feasibility of primer walking with fluorescent dye-labelled dideoxynucleotide terminators on an automated ABI DNA sequencer. The rapid identification of the complete sequence demonstrated that this approach is a time- and cost-saving alternative to classical sequencing techniques. Analysis of the data revealed that the L gene of Sendai virus strain Fushimi consists of exactly 6800 nucleotides and that the deduced amino acid sequence identifies a single open reading frame encoding a protein of 252.876 kDa. In contrast to Sendai virus strain Enders, the L mRNA of strain Fushimi is monocistronic. The comparison of the deduced amino acid sequences of the L genes of three different Sendai virus strains confirmed the existence of conserved as well as variable regions in the L protein and revealed a high grade of conservation in the carboxyterminal third. Furthermore, functional amino acid sequence motifs, like elements of RNA-dependent RNA polymerases and ATP-binding sites as postulated previously, were identified.

Base Sequence↗
PubMed1992

Comparison of modulation transfer function and through focus response with monofocal and bifocal IOLs.

We analyzed modulation transfer function (MTF) and through focus response (TFR) of the TRUE VISTA bifocal IOL and monofocal IOLs and compared the experimental and clinical data. MTF and TFR were measured at different pupil sizes (2, 3, 4 mm), base powers (14, 21, 27 D), add powers (2, 3, 4 D), and distance zone diameters (1.25, 1.5, 1.75 mm). Standards were established to correlate MTF and contrast sensitivity, as well as TFR and the defocus curve measured clinically. MTF of TRUE VISTA decreased with increasing pupil size and base power. A near add of 4 D showed the best MTF at distant and near focus. MTF of TRUE VISTA at near focus was lower than at distant focus and MTF at distant focus was lower than MTF of monofocal IOLs. TFR and the defocus curve correlated closely. MTF and contrast sensitivity of TRUE VISTA at far and near focus also correlated closely. MTF and contrast sensitivity of monofocal IOLs and TRUE VISTA at distant focus did not correlate. MTF was considerably better with monofocal IOLs while contrast sensitivity was slightly better only at low contrast. However, MTF of monofocal IOLs was much higher than MTF of a normal eye while MTF of TRUE VISTA at distant focus was only slightly lower than the MTF of a normal eye. Using MTF of a normal eye as a threshold, our results demonstrated a close correlation between experimental and clinical findings. MTF and TFR may therefore reliably predict the performance of bifocal IOLs.

Contrast Sensitivity↗
PubMed1991

Sendai virus NP gene codes for a 524 amino acid NP protein.

The complete nucleoprotein (NP) gene sequences of the Sendai virus Fushimi and 6/94 strains were determined. For both viruses an open reading frame of 524 amino acids can be predicted for the NP proteins. By comparing the sequences with others reported in the literature, the 5' noncoding region and the middle third of the coding region were found to be highly conserved. The carboxyl terminal part carries nine amino acid changes and a completely different sequence of the carboxyl terminus with a seven amino acid extension. This carboxyl terminus of the Sendai virus NP protein was confirmed using tryptic peptide sequence analysis.

Amino Acid Sequence↗
PubMed1991

Sendai virus protein-protein interactions studied by a protein-blotting protein-overlay technique: mapping of domains on NP protein required for binding to P protein.

Proteins from Sendai virus particles and from infected cells were analyzed in a protein-blotting protein-overlay assay for their interaction with in vitro-synthesized, [35S]methionine-labeled viral proteins NP, P, and M. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer onto polyvinylidene difluoride membranes, and renaturation, the immobilized proteins were found to interact specifically with radiolabeled proteins. NP proteins from virus particles and from infected cells retained 35S-P protein equally well. Conversely, P protein from virus particles and from infected cells retained 35S-NP protein. 35S-M protein was retained mainly by NP protein but also by several cellular proteins. To determine the domains on NP protein required for binding to immobilized P protein, a series of truncated and internally deleted 35S-NP proteins was constructed. The only deletion that did not affect binding resides between residues 426 and 497. The carboxyl-terminal 27 residues (positions 498 to 524) contribute significantly to the binding affinity. Removal of 20 residues (positions 225 to 244) in the hydrophobic middle part of NP protein completely abolished its binding to P protein.

Animals↗
PubMed1990

Sendai virus gene expression in lytically and persistently infected cells.

Sendai virus RNA species were quantitated in lytically and persistently infected cultured cells by Northern blot hybridization to region- and strand-specific cloned cDNA probes. Levels of NP, P and M mRNA in lytically infected cells were equally high, but F and HN mRNA were present in about 3-fold, and L mRNA in 30-fold, lower amounts, reflecting transcriptional attenuation especially at the M-F and HN-L gene junction. Two persistently infected cell lines, which release only 1% of the virus particles of lytically infected cells, were shown to contain only 4- to 8-fold-less amounts of each viral mRNA and 2- to 3-fold-less genomic RNA than lytically infected cells. Additionally, transcription was neither defective nor more attenuated as compared to the lytical infection. Taken together the results suggest the existence of an additional regulatory mechanism for the virus release. A cell-associated state of infection therefore seems to be achievable by a relatively weak general reduction of the copy numbers of viral mRNA and genomic RNA.

Animals↗
PubMed1990

Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro.

Sodium dodecyl sulfate-solubilized Sendai virus large (L) protein was highly purified by a one-step procedure, using hydroxylapatite column chromatography. Monoclonal antibodies addressed to the carboxyl-terminal amino acid sequence of the L protein were used for monitoring L protein during purification. By removing sodium dodecyl sulfate from purified L protein, a protein kinase activity was successfully renatured. P and NP proteins served as its substrates. After immunoprecipitation with anti-L antibodies, the immunocomplex already showed protein kinase activity. In the presence of P protein, the NP protein was more highly phosphorylated. The results show that Sendai virus L protein possesses a protein kinase activity phosphorylating the other proteins of the viral nucleocapsid in vitro.

Amino Acid Sequence↗
PubMed1988

Simple method for rapid and highly sensitive detection of antiviral-antibodies in serum and cerebrospinal fluid of small laboratory animals.

A technique is described for the small-scale determination of antibodies in blood and liquor. Small sample volumes of about 3 microliter blood and 15 microliter liquor are sufficient to detect even low antibody titers against different proteins. Thus, for studying the timecourse of antibody titers, repeated examinations of small laboratory animals are possible. The method includes the highly reproducible separation of the antigens in a 45 X 35 X 0.5 mm polyacrylamide gel (6.5%) using the Phast-system (Pharmacia), followed by electrotransfer and immunostaining. The whole procedure takes less than five hours.

Animals↗
PubMed1985

Selective protection of in vitro synthesized cDNA against nucleases by incorporation of phosphorothioate-analogues.

The conditions for the stepwise synthesis of single- (ss) and double-stranded (ds) cDNA using thio-analogues instead of dNTPs are described in this paper. RNA of paramyxovirus Sendai (strain 6/94) serves as template in these experiments. The increased resistance of this alpha S-modified cDNA against several nucleases, like S1-Nuclease, DNase I, Exonuclease III, snake venom Phosphodiesterase (PDE) and the combination of DNase I and PDE is demonstrated.

DNA↗
PubMed1984

Lifelong persistence of paramyxovirus Sendai-6/94 in C129 mice: detection of a latent viral RNA by hybridization with a cloned genomic cDNA probe.

C129 mice infected intracerebrally with Sendai-6/94 virus were examined periodically for the presence of viral proteins and viral RNA over a span of 423 days. On postinfection Day 15 (PID 15) an acute infection was demonstrated by increased anti-6/94 antibody titers and expression of viral proteins. More than a year later, on PIDs 373 and 423, no viral antigens were detected. Rescue of infectious virus from the mouse brains was only observable for 74 days following cocultivation of the cells on PID 15. Later, no signs of viral persistence were found at the protein level. In a further experiment, the murine tissues and cell cultures were examined for the presence of viral RNA. Virus-specific cDNA was cloned in the bacteriophage lambda system and used as a highly specific hybridization probe. Surprisingly, 6/94 viral RNA was detectable lifelong in the brain tissue of infected mice although no proteins were expressed. Murine brain cells cocultivated on PID 15 still contained viral RNA after 245 days in culture, although the expression of viral proteins was measureable for only 80 days. The results indicate a stable latency of the 6/94 virus in cell cultures and in animals at the RNA level without detectable protein expression.

Animals↗
PubMed1983

Transient rescue of Sendai-6/94 cl virus from the persistently infected cell line Cl-E-8 by cocultivation.

The cell line Cl-E-8 showing expression of Sendai-6/94 viral antigens after the original isolation was reexamined after approximately 160 subcultures. In the virus fraction of cell supernatants 6/94 virus particles (termed 6/94 cl) could be demonstrated; no infectivity, however, was monitored. We were also unable to activate the viral infectivity of 6/94 cl virus by trypsin treatment. The analysis of viral RNA revealed that the virus contains a high-molecular-weight (50 S) single-stranded RNA. After cocultivation of Cl-E-8 cells with several standard cell lines the production of an infectious 6/94 virus, termed 6/94 co, was detected. The infectivity titer in the supernatants was very low, about three orders of magnitude lower than in cultures infected with the egg-grown 6/94 virus (6/94 ST). Surprisingly, the production of infectious 6/94 cl virus invariably ceased several subcultures after cocultivation even in the presence of foreign cells. However, the infectivity could be repeatedly reinduced by adding fresh foreign cells.

Antigens, Viral↗
PubMed1983

Search for Sendai 6/94 viral RNA in the antigen-free cell line Cl-C-2 isolated from human multiple sclerosis brain tissue.

The viral antigen-free cell line Cl-C-2, obtained from multiple sclerosis brain tissue by cell fusion with CV-1 cells, was examined for the presence of intracellular virus-specific RNA sequences of the persistent Sendai 6/94 virus by nucleic acid hybridization. As a specific probe for this assay, an in vitro synthesized cDNA was used. Oligodeoxyguanidylic acid served as a primer for the initiation of cDNA synthesis. The 6/94 RNA was detectable as expected in the viral antigen-expressing cell lines Cl-E-8 and Cl-F-2, which were used as a reference of the same source. In the viral antigen-free cell line Cl-C-2, however, no viral RNA sequences have been found by hybridization experiments. Corresponding superinfection studies confirmed the conclusion that in cell line Cl-C-2 no viral components are present. The lack of expression of viral proteins and of protection against superinfection seems to be correlated with the lack of viral RNA in Cl-C-2 cells, which may eliminate the persistent virus by a cellular defense mechanism.

Animals↗
PubMedDate not supplied

Evidence of measles virus RNA in otosclerotic tissue.

By use of the very sensitive polymerase chain reaction technique, evidence is given that otosclerosis is a measles-virus-associated disease of the otic capsule. These results support recent observations which demonstrated the expression of measles antigen within active otosclerotic foci by immunohistochemical methods. Thus it is hypothesized that the viral infection acts as at least one pathogenetic factor during the development of this obscure, locally restricted inflammatory bone disease.

Blotting, Southern↗