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Biomedical subjects

L Philipson

Publications and source records attributed to L Philipson.

At least 163 records · Page 9Linked to original sources
PubMed1977

Purification and characterization of the phosphorylated DNA-binding protein from adenovirus-type-2-infected cells.

The virus-coded 72000-Mr DNA-binding protein from adenovirus-type-2-infected cells has been purified to homogeneity by DEAE-cellulose chromatography, selective precipitation and gel filtration. The 72000-Mr DNA-binding protein is phosphorylated and the phosphate is covalently linked predominantly to serine. Analysis of tryptic digests of the 32P-labeled 72000-Mr protein showed that the phosphate residue(s) is present in only one peptide. The DNA-binding fraction contains an additional non-phosphorylated protein with an approximate molecular weight of 45000. Tryptic peptide maps of [35S]methionine-labeled 72000-Mr and 45000-Mr polypeptides are indistinguishable. The amino acid compositions of the 72000-Mr and 45000-Mr polypeptides show closely related distributions. An antiserum produced against the purified 72000-Mr DNA-binding protein precipitates both the 72000-Mr and the 45000-Mr protein from extracts of adenovirus-infected cells. Immunofluorescence studies revealed DNA-binding protein to be accumulated in characteristic structures in nuclei of the infected cells.

Adenoviridae↗
PubMed1977

Purification of an endonuclease from adenovirus-infected KB cells.

This report describes the purification of an endonuclease from extracts of adenovirus-type-2-infected KB cells. Endonuclease activity can also be detected in extracts of uninfected KB cells and the enzyme activities from extracts of uninfected and adenovirus-infected cells are very similar, if not identical. The enzyme has its maximal activity at pH 4.0. The enzyme found in uninfected and adenovirus-infectedcells is, however, strikingly different from an endonuclease isolated from calf serum. Hence, the endonuclease described is probably not a contaminant derived from the medium in which the KB cells were propagated. The endonuclease in crude extracts from uninfected or adenovirus-infected KB cells can be activated or its activity enhanced by treatment of the extracts with proteolytic enzymes, like pronase or trypsin. Evidence has been presented suggesting that this activation is due to proteolytic cleavage of an inhibitor present in crude extracts of uninfected and adenovirus-type-2-infected KB cells. A second endonuclease has been found in extracts of infected and uninfected cells with optimal activity at pH 7.2 and this endonuclease can be separated from the one with a pH optimum at 4.0.

Adenoviruses, Human↗
PubMed1977

Distribution of cerebral blood flow in the dominant hemisphere during motor ideation and motor performance.

Distribution of activity in the dominant (left) hemisphere was studied with a multidetector instrument during diagnostic measurements of regional cerebral blood flow in 6 patients, 4 of them neurologically normal. Computercalculated charts, in color, of the flow/activity distribution--"cerebral ideograms"-- were obtained in three situations: at rest, during motor ideation (attempts to conceive of rhythmic clenching movements of the right hand), and during actual movements of the right hand. Motor ideation changed the normal "hyperfrontal" resting flow distribution, and an increase of the hemisphere mean flow was recorded. The increase was especially marked in frontal and temporal structures. This pattern differed from the one during actual hand movements, when a rolandic flow increase was seen. The result suggests that centers for motor ideation have a different cerebral location than those which control the actual hand movement.

Adult↗
PubMed1977

Inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate.

Ribavirin 5'-triphosphate (RTP), derived from the broad-spectrum antiviral compound ribavirin (Virazole), can selectively inhibit influenza virus ribonucleic acid polymerase in a cell-free assay. Ribavirin and its 5'-monophosphate have no effect on the polymerase. The inhibition is competitive with respect to adenosine 5'-triphosphate and guanosine 5'-triphosphate. RTP also inhibits ApG- and GpC-stimulated influenza virus ribonucleic acid polymerase. Since ribavirin is phosphorylated in the cell, the inhibition of influenza multiplication in the cell may also be caused by RTP.

DNA-Directed RNA Polymerases↗
PubMed1977

In vitro translation with adenovirus polyribosomes.

Polyribosomes isolated from adenovirus type 2 (Ad2)-infected HeLa cells late in productive infection can be used for translation in cell-free systems. At least eight viral polypeptides are synthesized, including the precursors to virion polypeptides VI and VII. Separation of polyribosomes by zonal rate centrifugation followed by translation in a cell-free system reveals a correlation between the sizes of the polyribosomes and the polypeptides synthesized. The cell-free extracts incorporate amino acid linearly for only 10 min and show little or no capacity to reinitiate protein synthesis. The elongation efficiency measured as the number of amino acids incorporated per ribosome in 20 min is low, ranging from 10 to 100. The maximum chain elongation rate is estimated to be 10 to 20 amino acids per min. The limited elongation has been used to assess the relative concentration of mRNA's engaged in translation.

Adenoviruses, Human↗
PubMed1977

Fidelity of adenovirus RNA transcription in isolated HeLa cell nuclei.

An in vitro nuclear system from adenovirus type 2-infected cells was developed to study transcription of viral RNA. Nuclei isolated from adenovirus-infected HeLa cells late in the infectious cycle synthesized in vitro only RNA from the r-strand of adenovirus DNA. Around 15% of the virus-specific RNA in isolated nuclei was polyadenylated. Short pulse labeling of nascent RNA followbd by hybrization of size-fractionated RNA to specific restriction endonuclease fragments of the genome suggested that the origin(s) for transcription is located on the r-strand in the left 30% of the adenovirus 2 genome at late times in the infectious cycle. Pulse-chase experiments were used to estimate the elongation rate of adenovirus high-molecular-weight RNA in isolated nuclei. An elongation of a least six nucleotides per second was observed in vitro. Viral RNA synthesis in the vitro nuclei showed several similarities to the in vivo system late in the infectious cycle.

Adenoviruses, Human↗
PubMed1977

Reassociation of complementary strand-specific adenovirus type 2 DNA with viral DNA sequences of transformed cells.

Complementary strand-specific adenovirus DNA, either full length or from restriction enzyme cleavage fragments, was used to estimate the fractional representation and abundance of viral sequences in two adenovirus type 2 (Ad2)-transformed rat cell lines, A2F19 and A2T2C4. The reassociation method introduced is based on the linear relationship, after exhaustive hybridization, between the inverted fraction of hybrid DNA and the molar ratio of probe to cellular DNA in the reaction mixture. The amount of viral DNA in A2F19 cells represents 12 to 14% of the viral genome at a level of around seven copies per diploid cell equivalent. For the cell line A2T2C4, the pattern of integrated viral DNA sequences is more complex. With full-length Ad2 DNA strands as a probe, about 56% of the probe was represented in cellular DNA. When each of the four BamHI fragment strands of Ad2 DNA was used as a probe, the fraction of the viral DNA present also amounted to around 56% with one to five copies from different regions of the viral genome. The results demonstrate the advantage of using strand-specific viral DNA as a probe in reassociation analysis with denatured cell DNA. The method should be useful in any system in which complementary strand separation of viral DNA sequences can be achieved.

Adenoviruses, Human↗
PubMed1976

Purification of adenovirus messenger ribonucleic acid by an aqueous polymer two-phase system.

An aqueous polymer phase system containing 6.3% (w/w) dextran and 3.5% (w/w) poly(ethylene glycol) in 10 mM phosphate buffer (pH 8.0) was developed to select RNA-DNA hybrids from unhybridized RNA. The top phase of this phase system, which contains DNA and the RNA-DNA hybrids, can be used to purify adenovirus messenger RNA both early and late in the infectious cycle. The hybrids can be melted by heat in the top phase and the messenger RNA selected by oligo(dT)cellulose chromatography whereupon the polymers and the DNA percolate and the polyadenylated messenger RNA absorb to the column. The isolated messenger RNA appears to be almost quantitatively recovered at a purity from 70 to 90% depending on the concentration of the specific messenger RNA in the starting material. Early and late viral messenger RNA were selected on the complementary strands of adenovirus DNA according to this procedure.

Adenoviruses, Human↗
PubMed1976

A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2.

A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA. Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to alpha-amanitin. In isolated nuclei, both RNA species are labeled with beta-32P-labeled GTP, which suggests that they are initiated at separate promotor sites.

Adenoviridae↗
PubMed1976

Propagation in E. coli of bacteriophage lambda with integrated fragments of adenovirus 2 DNA.

Hybrid genomes of bacteriophage lambda with integrated fragments of adenovirus type 2 (Ad2) DNA have been constructed in vitro and propagated in E. coli. DNA from a derivative of bacteriophage lambdaplac5 (Rambach and Tiollais, 1974) was used as a vector. The two fragments of the vector DNA contain all the essential genes for the replication of the lambda DNA but are too short to be encapsidated. Insertion of DNA is therefore essential for plaque formation which constitutes a selection method for phages containing hybrid genomes. Fragments EcoRI-B and EcoRI-F of Ad2 DNA were purified, and separately ligated with the vector fragments. Clones of hybrid phage could readily be isolated. Two clones of hybrid phage containing fragment EcoRI-B inserted in opposite directions were used to study the transcription of adenovirus-specific sequences. Hybridization experiments showed that transcripts from both strands of fragment Ad2-Eco RI-B could be detected and that transcription probably was controlled by the "early" leftward and the "late" rightward promoters on the lambda genome. No polypeptides specified by the adenovirus fragment have so far been identified.

Adenoviridae↗
PubMed1976

Kinetic studies on the cleavage of adenovirus DNA by restriction endonuclease Eco RI.

The kinetics of cleavage of DNA from Adenovirus Type 1 (Ad1), Type 5 (Ad5) and Type 6 (Ad6) by restriction endonuclease EcoRI was investigated by quantitative evaluation of the fluorescence from ethidium stained DNA fragments separated on agarose gels. The apparent rate constants of cleavage at different cleavage sites have been determined and large differences in the cleavage rates of the individual sites within one type of DNA were found. From the kinetics of cleavage information on the sequence of the DNA fragments can be obtained. The order of the fragment A, B, C, D of Ad6 DNA obtained after complete cleavage by restriction endonuclease Eco RI was found to be A-D-C-B; the order of the corresponding fragments A, B, C of Ad1 and Ad5 DNA was found to be A-C-B.

Adenoviruses, Human↗
PubMed1976

Intermediates in adenovirus assembly.

Three intermediates in adenovirus assembly have been defined; nuclear intermediates, young virions, and mature virions. The nuclear intermediates are fragile and heterogenous in size (550S-670S) and withstand separation on ficoll gradients but fall apart upon CsCl gradient centrifugation unless prefixed with glutaraldehyde. They contain both capsid and core structures, and the core structures are preferentially released during purification in CsCl. The precursor polypeptides pVI and pVII are present in the intermediates without any corresponding mature polypeptide. The young virions (Ishibashi and Maizel, 1974) are stable and preferentially confined to the nuclei after cell fractionation. They contain both uncleaved precursor polypeptides and their cleavage products. The mature virions accumulate in the cytoplasm during cell fractionation and contain the final mature polypeptides. Pulse-chase labeling kinetics, focusing on the precursor polypeptides, suggest that these three classes participate in assembly of adenovirus. Tryptic peptide maps establish that polypeptide pVI is the precursor of polypeptide VI, but only a small fraction of polypeptide 26K can in vivo account for polypeptide VIII.

Adenoviridae↗