PubMed Health⌕ Search

Biomedical subjects

D Blaas

Publications and source records attributed to D Blaas.

At least 73 records · Page 4Linked to original sources
PubMed1990

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site.

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP [( 125I]APTM), the monoanhydride of m7GDP with [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for [125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I]APTM. Like [32P]BPM, [125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP [( 32P]APGM), the monoanhydride of m7GDP with [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P]BPM and [125I]APTM, however, [32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.

Affinity Labels↗
PubMed1990

A raf/myc virus immortalized macrophage cell line which supports the growth of B-cell and B-cell hybridomas.

Using a combination of raf and myc oncogenes co-expressed by the recombinant retrovirus J-2 we have generated and characterized a cell line which very efficiently supports the growth of B-cells and B-cell hybridomas. Murine spleen cells were cultured under in vitro immunization conditions favoring the short term proliferation of splenic B lymphocytes and infected with J-2 virus. Screening of immortalized spleen cell pools for the capability to support long term B cell growth in vitro led to the selection of a clonal cell line termed alpha ChyJ2. The presence of macrophage specific features and surface markers suggest that alpha ChyJ2 belongs to the macrophage lineage. alpha ChyJ2 cells constitutively produce low levels of IL-1 like activity and high levels of IL-6. Expression of specific mRNAs as well as production of IL-1 alpha, IL-1 beta and IL-6 are inducible with LPS. Expression or production of other cytokines including IL-2, IL-3, IL-4, IL-5, TGF beta and GM-CSF could not be detected. As the biological effects of alpha ChyJ2 supernatant cannot be fully explained by the described pattern of cytokine production, participation of other, yet uncharacterized, factors is possible. Using alpha ChyJ2 as feeder cells for in vitro as well as in vivo immunizations increased the number of antibody secreting B-cell clones 2 to 15 fold, respectively.

B-Lymphocytes↗
PubMed1989

Low cost apparatus for primer-directed DNA amplification using Thermus aquaticus-DNA polymerase.

An apparatus is described which permits the incubation of samples at three different temperatures in a cyclic fashion. The parts for the incubator are either present in every biochemical laboratory (water baths) or can be easily obtained at a low price (timers and magnetic valves). Thus the new DNA amplification procedure employing Thermus aquaticus-DNA polymerase can be carried out automatically without major investments.

DNA-Directed DNA Polymerase↗
PubMed1989

Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis.

Evidence is presented that the protein 2A of human rhinovirus serotype 2 (HRV2) is a protease. On expression of the VP1-2A region of HRV2 in bacteria, protein 2A was capable of acting on its own N-terminus; derived extracts specifically cleaved a 16 amino acid oligopeptide corresponding to the sequence at the cleavage site. Cleavage of the oligopeptide substrate provides a convenient in vitro assay system. Deletion experiments showed that removal of 10 amino acids from the carboxy terminus inactivated the enzyme. Site-directed mutagenesis identified an essential arginine close to the C-terminus and showed that the enzyme was sensitive to changes in the putative active site. This analysis supports the hypothesis that 2A belongs to the group of sulfhydryl proteases, although sequence comparisons indicate that the putative active site of HRV2 2A is closely related to that of the serine proteases.

Amino Acid Sequence↗
PubMed1989

Human rhinovirus serotype 2: in vitro synthesis of an infectious RNA.

A complete cDNA copy of human rhinovirus serotype 2 RNA was placed under the control of a T7 RNA polymerase promoter. An in vitro transcribed RNA containing two extra G residues at the 5' end gave rise to plaques on transfection into HeLa cells. The efficiency was approximately half that obtained with viral RNA. On the contrary, an in vitro synthesized RNA containing 16 additional nucleotides at the 5' end was not infectious. This ability to make an infectious in vitro transcribed RNA will be useful in studying the characteristics of viruses using the human rhinoviral minor group receptor.

Cloning, Molecular↗
PubMed1989

Trypsin sensitivity of several human rhinovirus serotypes in their low pH-induced conformation.

Five serotypes of human rhinovirus (HRV) were examined for sensitivity to trypsin at physiological pH, HRV1A, HRV2, and HRV14 were found to be resistant whereas in serotypes HRV49 and HRV89 degradation of VP2 was observed. However, exposure to low pH followed by neutralization, a treatment which causes irreversible conformational changes in the capsid, led to rapid cleavage by trypsin of VP1 in HRV1A, HRV2, and HRV49 at defined sites followed by degradation of VP2. In the case of HRV2, the cleavage site in VP1 was determined by direct protein sequencing and was shown to occur between Arg260 and Thr261, close to the C-terminus. HRV49 behaves similarly to HRV2 as expected from extensive sequence similarity in this region, whereas VP1 in HRV1A is most probably cleaved at a site closer to the C-terminus than that in HRV2. Although HRV14 contains the same amino acid pair present in HRV2 and HRV49, it was not cleaved under these conditions. HRV89, which lacks a basic residue at the corresponding position, was also insensitive. Examination of the cleavage site on the three-dimensional structural map of native HRV2 reveals that it is most probably buried inside the capsid and thus not accessible. Structural rearrangements of the viral capsid are thus necessary to account for the cleavage observed after low pH treatment.

Capsid↗
PubMed1989

Typing of human rhinoviruses based on sequence variations in the 5' non-coding region.

Unambiguous assignment of restriction enzyme patterns to six individual serotypes of human rhinovirus was accomplished after amplification of a 380 bp DNA fragment derived from the 5' non-coding region. This was possible even though serotypes 1A and 1B and serotypes 2 and 49 differed only at 10 and 15 positions respectively. The method utilizes the conserved and variable components of this part of the genome and provides the basis for a simple and rapid method for typing of human rhinoviruses.

Base Sequence↗
PubMed1988

Characteristics of the minor group receptor of human rhinoviruses.

The receptor for the minor group of human rhinoviruses was solubilized from HeLa cell membranes with various detergents. Virus binding activity was determined in a filter binding assay using 35S-labeled human rhinovirus 2 (HRV2) as a probe. The receptor protein was enriched on Lens culinaris lectin columns and the active fractions were further purified by gel permeation and anion exchange chromatography. The receptor has an apparent molecular weight of 450 kDa in the presence of detergent. The binding activity is sensitive to trypsin, sulfhydryl modifying agents, but insensitive to neuraminidase. Divalent cations are essential for virus binding.

Acetylglucosaminidase↗
PubMed1987

Assembly of pre-mRNA splicing complex is cap dependent.

To study the influence of the ubiquitous cap structure of nuclear pre-mRNAs on the assembly of a functional splicing complex, the in vitro splicing of a truncated human metallothionein pre-mRNA was examined in the presence of the cap analogue m7GTP. Significant inhibition of splicing was observed at a concentration as low as 5 microM m7GTP. Analysis of the splicing reaction on glycerol density gradients showed two complexes sedimenting at 45S and 22S. When the reaction was carried out in presence of m7GTP a marked decrease of the material sedimenting at 45S, representing the active splicing complex, was observed. When capped pre-mRNA was replaced by uncapped pre-mRNA, complex formation was significantly reduced. These data indicate that the cap structure plays an important yet unknown role in the assembly of spliceosomes.

Exons↗
PubMed1987

Comparison of the three-dimensional structure of two human rhinoviruses (HRV2 and HRV14).

An attempt has been made to build a model of human rhinovirus 2 (HRV2) based on the known human rhinovirus 14 (HRV14) structure. HRV2 was selected because its amino acid sequence is known and because it belongs to the minor rhinovirus receptor class as compared to HRV14, which belongs to the major class. Initial alignment of HRV2 with HRV14 based on the primary sequence and the knowledge of the three-dimensional structure of HRV14 showed that the most probable position of the majority of insertions and deletions occurred in the vicinity of the neutralizing immunogenic sites (NIm). Out of a total of 855 amino acids present in one copy of each of the capsid proteins VP1 through VP4 of HRV14, 411 are different between the two viruses. There are also 6 amino acid residues inserted and 14 residues deleted in HRV2 relative to HRV14. Examination of amino acid interactions showed several cases of conservation of function, e.g., salt bridges or the filling of restricted space. The largest variation amongst the residues lining the canyon, the putative receptor binding site, was in the carboxy-terminal residues of VP1.

Amino Acid Sequence↗
PubMed1987

Mechanism of entry of human rhinovirus 2 into HeLa cells.

Internalized human rhinovirus 2 (HRV2) undergoes a rapid conformational change leading to recognition by the C-determinant-specific monoclonal antibody 2G2. In the presence of the ionophore monensin, the virus accumulates in the cells in its native conformation and infection is strongly inhibited. At 20 degrees but not at 34 degrees the inhibitory effect of monensin can be overcome by a short incubation of the infected cells at low pH as late as 2 hr after inoculation. Incubation of infected cells at 20 degrees prior to addition of monensin permits virus synthesis to occur, depending on the time of preincubation.

Capsid↗
PubMed1987

Evolutionary relationships within the human rhinovirus genus: comparison of serotypes 89, 2, and 14.

The complete nucleotide sequence of the genome of human rhinovirus type 89 was determined from the cDNA that had been cloned into Escherichia coli. The genome is 7152 nucleotides long and contains a single large open reading frame of 2164 codons. Translation commences at position 619 and ends 42 nucleotides before the poly(A) tract. The positions of three proteolytic cleavage sites in the polyprotein were determined by N-terminal amino acid sequencing of the capsid proteins; the remainder were predicted from comparisons with other picornaviruses. Extensive similarity between the derived amino acid sequences of human rhinovirus types 89 and 2 was found, whereas the similarity between human rhinovirus types 89 and 14 was considerably less. It is apparent that human rhinoviruses may be more closely related than has been previously thought.

Amino Acid Sequence↗
PubMed1987

Cleavage site between VP1 and P2A of human rhinovirus is different in serotypes 2 and 14.

The viral capsid protein VP1 of human rhinovirus serotype 2 (HRV2) was cleaved with cyanogen bromide. The peptides thus obtained were separated on an HPLC butyl reversed phase column. Their positions on VP1 were determined by amino-terminal sequencing using the known nucleotide sequence of the genomic RNA of HRV2. The putative carboxy-terminal peptide was further cleaved with trypsin and the resulting fragments were separated on a C18 reversed phase column. Amino-terminus of sequencing of the C-terminal peptide revealed alanine as being the carboxy terminus of VP1 in HRV2. This indicates that the processing of the polyprotein is different in HRV2 from the processing previously reported for HRV14 and poliovirus.

Amino Acid Sequence↗
PubMed1987

A neutralizing epitope on human rhinovirus type 2 includes amino acid residues between 153 and 164 of virus capsid protein VP2.

Use has been made of a monoclonal antibody (designated 8F5) to map a neutralizing epitope on the viral capsid protein VP2 of human rhinovirus 2 (HRV2). This antibody which was raised against the native virus, neutralizes HRV2 and is also capable of recognizing denatured VP2 on Western blots. To examine the binding site of 8F5, VP2 of HRV2 was expressed in Escherichia coli. Deletions starting at the 3' end were then introduced into the gene for VP2 using Bal-31 nuclease. Polypeptides shortened at the carboxy terminus of VP2 were obtained from the deletions and were blotted onto nitrocellulose. The samples were then probed with monoclonal antibody 8F5. Recognition by 8F5 was maintained as long as the expressed polypeptide contained the VP2 sequence up to amino acid 164 or beyond. However, when the VP2 sequence was truncated to amino acid 153 or less 8F5 was no longer able to bind. The neutralization epitope (or part of it) recognized by 8F5 on VP2 is therefore located between amino acids 153 and 164.

Amino Acid Sequence↗
PubMed1987

The molecular biology of human rhinoviruses.

A brief review of recent advances in the understanding of human rhinovirus molecular biology is presented. The importance of recent findings on the elucidation of serotypic diversity and their implications for the viral host-cell receptor site are emphasized. An introduction to the genome structure and to the pathway of gene expression in rhinoviruses leads on to a discussion of the crystal structure of human rhinovirus 14 (HRV14) and the antibody-inducing regions on the surface of the capsid. Evidence from these experiments indicates that four sites on HRV14 are responsible for inducing neutralizing antibodies. Amino-acid sequence comparisons reveal that these sites are different in other serotypes, strengthening the view that the sequences of these regions are fundamental in determining and defining rhinovirus serotypes. The crystal structure of HRV14 points to a depression in the viral capsid as being the site of binding to the host-cell receptor; however, residues involved in binding cannot yet be identified.

Amino Acid Sequence↗