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Biomedical subjects

F R Masiarz

Publications and source records attributed to F R Masiarz.

51 records · Page 3Linked to original sources
PubMed1985

Characterization of the polypeptide composition of human factor VIII:C and the nucleotide sequence and expression of the human kidney cDNA.

Human coagulation factor VIII:C has been purified approximately 5000-fold from commercial preparations with an average activity yield of 35%. Proteins of 92 kD and 77-80 kD enriched during purification are precipitated by a human serum polyclonal antibody which inhibits factor VIII:C activity. Evidence suggests that these polypeptides are linked by a calcium ion bridge. Partial amino acid sequence information from these proteins has been obtained from the intact polypeptides and from products of digestion with thrombin, endoproteinase lysC, or trypsin after citraconylation. An oligonucleotide probe designed from one of the amino acid sequences was used to isolate a partial genomic clone from a human 4X chromosome library in bacteriophage lambda. The genomic segment was used to isolate two cDNA molecules encompassing the entire human kidney factor VIII:C mRNA. Biologically active factor VIII:C has been produced in a mammalian cell line utilizing a complete cDNA construction.

Amino Acid Sequence↗
PubMed1984

Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone alpha-factor. These cells accurately process the hybrid protein and efficiently secrete authentic biologically active human epidermal growth factor into the medium.

Amino Acid Sequence↗
PubMed1981

Reversible chemical modification of the scrapie agent.

The scrapie agent causes a degenerative nervous system disease in sheep and goats. Studies with extensively purified preparations demonstrated that the agent contains a protein that is required for infectivity. Chemical modification of the scrapie agent by diethyl pyrocarbonate reduced the titer 1000-fold. Exposure of the inactivated agent to hydroxylamine, a strong nucleophile, resulted in complete restoration of infectivity. Presumably, nucleophilic residues within a scrapie agent protein undergo carbethoxylation on reaction with diethyl pyrocarbonate, and subsequent addition of hydroxylamine displaces these carbethoxy groups.

Animals↗
PubMed1981

Scrapie agent contains a hydrophobic protein.

The scrapie agent causes a degenerative nervous system disorder of sheep and goats. Considerable evidence indicates that the scrapie agent contains a protein that is necessary for infectivity [Prusiner, S. B., Groth, D. F., Cochran, S. P., Masiarz, F. R., McKinley, M. P. & Martinez, H. M. (1980) Biochemistry 19, 4883-4891], but direct demonstration of a protein moiety has been hampered by lack of sufficiently purified preparations. Employing preparations of the scrapie agent enriched 100- to 1000-fold with respect to protein, we found that digestion by proteinase K destroyed more than 99.9% of the infectivity. Diethylpyrocarbonate, which chemically modifies amino acid residues in proteins with high efficiency, also inactivated the scrapie agent in these purified preparations. Reductions of infectivity by proteinase K and diethylpyrocarbonate were not observed with less purified preparations. The agent bound to phenyl-Sepharose could not be eluted with 8.5 M ethylene glycol; however, a combination of ethylene glycol and detergents did release the agent. These observations provide good evidence for a protein and for hydrophobic domains within the scrapie agent. Whether the protein required for infectivity is the same protein responsible for the hydrophobic properties of the scrapie agent remains to be established.

Animals↗
PubMed1980

Molecular properties, partial purification, and assay by incubation period measurements of the hamster scrapie agent.

The scrapie agent causes a progressive degeneration of the central nervous system of animals after a prolonged incubation period. Measurements of incubation period length, defined as the time from inoculation to the onset of clinical signs of neurological dysfunction, were related to the titer of the agent and the dilution of the inoculated sample. Equations defining the relationship provide a new assay for the agent requiring fewer animals than end point titrations. By use of this incubation period assay, the scrapie agent from hamster brain was found to have an s20,w of < 300 S but > 30 S assuming rho p = 1.2 g/cm3. A partially purified fraction P3 was obtained by differential centrifugation and sodium deoxycholate extraction. When P3 was extracted with phenol, virtually no infectivity was found in the aqueous phase even after examining such variables as pH, salt concentration, and predigestion of samples with proteinase K. Nonionic and nondenaturing, anionic detergents did not inactivate the scrapie agent; in contrast, denaturing detergents inactivated the agent. Sodium dodecyl sulfate (NaDodSO4) inactivated greater than 90% of the agent at a NaDodSO4 to protein ratio of 1.8 g/g. Inactivation by NaDodSO4 appears to be a cooperative process. Addition of a nonionic detergent to form mixed micelles with NaDodSO4 prevented inactivation of the agent by NaDodSO4. Weak chaotropic ions do not inactivate the scrapie agent while strong chaotropic ions like SCN- and Cl3CCOO- destroy infectivity at concentrations of 0.2 M. These data provide evidence in support of a protein component within the scrapie agent which is essential for maintenance of infectivity. Thus, it is unlikely that the scrapie agent is composed only of a "naked" nucleic acid as is the case for the plant viroids.

Animals↗
PubMed1980

Electrophoretic properties of the scrapie agent in agarose gels.

The molecular properties of the scrapie agent were investigated by subjecting partially purified preparations to electrophoresis on agarose gels. When electrophoresis was performed at room temperature in the presence of sodium dodecyl sulfate (NaDodSO4), most of the recoverable agent was found at the top of the gel, consistent with previous studies indicating aggregation of the agent upon exposure to elevated temperatures. In addition, less than 5% of the agent applied to the gel was found after electrophoresis, even though the study was performed with a low concentration of NaDodSO4 (0.1%). Further studies on the inactivation of the agent by NaDodSO4 suggest that this may be, in part, a function of the NaDodSO4: protein ratio in the sample. In contrast, sodium N-lauroyl sarcosinate (Sarkosyl) did not inactivate the agent in concentrations as high as 5% (wt/vol). Virtually all of the infectivity could be recovered after electrophoresis of the agent into 0.6% agarose gels at 4 degrees C in the presence of 0.2% Sarkosyl. Digestion of the preparations with micrococcal nuclease and proteinase K prior to Sarkosyl electrophoresis caused a substantial portion of the agent to migrate ahead of DNA fragments of 1 x 10(6) daltons. The behavior of the scrapie agent in electrophoretic gels is consistent with earlier studies showing that the monomeric form of the agent has a sedimentation coefficient of less than or equal to 40 S. Thus, the smallest or monomeric form of the agent is smaller than any known animal virus.

Detergents↗
PubMed1977

Ribosomal RNA genes of Saccharomyces cerevisiae. II. Physical map and nucleotide sequence of the 5 S ribosomal RNA gene and adjacent intergenic regions.

A DNA fragment containing the structural gene for the 5 S ribosomal RNA and intergenic regions before and after the 35 S ribosomal RNA precursor gene of Saccharomyces cerevisiae has been amplified in a bacterial plasmid and physically mapped by restriction endonuclease cleavage and hybridization to purified yeast 5 S ribosomal RNA. The nucleotide sequence of the DNA fragments carrying the 5 S ribosomal RNA gene and adjacent regions has been determined. The sequence unambiguously identifies the 5 S ribosomal RNA gene, determines its polarity within the ribosomal DNA repeating unit, and reveals the structure of its promoter and termination regions. Partial DNA sequence of the regions near the beginning and end of the 35 S ribosomal RNA gene has also been determined as a preliminary step in establishing the structure of promoter and termination regions for the 35 S ribosomal RNA gene.

Base Sequence↗
PubMed1970

Incubation effects in behavior induction in rats.

Incubation (rest) periods interposed during donor training regimens significantly enhance the "memory transfer" effect reported by some investigators. When extracts from ihe brains of donor rats given interpolated rest during acquisition training were injected into recipient animals, statistically reliable and experimentally reproducible "memory transfer" effects were found.

Animals↗