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R J Isfort

Publications and source records attributed to R J Isfort.

51 records · Page 3Linked to original sources
PubMed1992

Two-dimensional gel electrophoresis analysis of Syrian hamster embryo cells: morphological transformation is not cell type specific.

Studies were conducted to investigate the protein phenotype of normal and morphologically transformed Syrian hamster embryo (SHE) cells. Based upon two-dimensional gel protein phenotype analysis, we conclude that (i) SHE cells are a mixture of multiple cell types including mesenchymal and epithelial cells and (ii) several cell types present in the SHE cell population can be morphologically transformed by a variety of genotoxic and non-genotoxic carcinogens.

Animals↗
PubMed1992

Serum and growth factors stimulate ribosomal RNA processing in Syrian hamster embryo cells: divergence of this signalling pathway from immediate-early gene expression.

Growth factor stimulation of cells results in multiple intracellular biochemical changes including increases in calcium levels, phosphatidylinositol turnover, protein phosphorylation, immediate-early gene transcription and intracellular pH alterations. We report here that serum and growth factor stimulation of Syrian hamster embryo (SHE)-derived cell lines increases the ribosomal RNA (rRNA) processing rates. In addition, in several transformed SHE cell lines, the immediate-early response of fos/actin transcription could be separated from the immediate-early rRNA processing response. These results indicate that in these cell lines a mutation(s) which uncouples these responses has occurred. This is the first report describing (i) rRNA processing as an immediate-early effect following growth factor stimulation and (ii) the identification of a mutation(s) which can uncouple two immediate-early signalling responses.

Actins↗
PubMed1992

Enzymatic purification of plasmid DNA.

A novel method for plasmid DNA purification using enzymes that degrade all major types of contaminating nucleic acids present in crude plasmid DNA mixtures (but not plasmid DNA) has been devised. This method is quick (can be accomplished in two hours), requires no expensive laboratory equipment (an ultracentrifuge is not necessary) and is inexpensive. Plasmid DNA purified by this methodology can be used in a variety of molecular biological techniques including restriction enzyme digestions, subcloning, sequencing, nick translation and end labeling. This plasmid purification technique will be very useful for the molecular biologist performing cloning experiments.

DNA↗
PubMed1990

Characterization of the gene encoding herpesvirus of turkeys gp57-65: comparison to Marek's disease virus gp57-65 and herpes simplex virus glycoprotein C.

A gene encoding herpesvirus of turkeys (HVT) strain FC 126 gp57-65 has been mapped to the viral genome and sequenced. The HVT (FC 126) gp57-65 gene maps to BamHI fragments K1 and M, colinear with the gene from Marek's disease virus (MDV) strain GA. HVT gp57-65 gene sequences were compared to the MDV strain GA gp57-65 gene that we sequenced previously. Overall, the two sequences are 66% identical, with greater similarity in the 3' proximal two thirds of the genes. HVT gp57-65 gene sequences have a slightly higher overall guanosine plus cytosine (G + C) content than MDV gp57-65 gene sequences (46% vs. 41%, respectively). A single, long open reading frame capable of encoding 523 amino acids was identified within the HVT gp57-65 gene region. The predicted precursor polypeptide derived from this open reading frame would have a calculated molecular weight of 58,587. The predicted HVT gp57-65 amino acid sequences contain six potential N-linked glycosylation sites (asn-x-ser/thr). Five of these six potential N-linked glycosylation sites are conserved between the HVT and MDV predicted amino acid sequences. Hydropathic analysis of the predicted HVT gp57-65 amino acid sequences indicate the presence of an amino-terminal hydrophobic sequence, which may function as a signal peptide, and a hydrophobic carboxyl terminal sequence, which may function as a membrane anchor sequence. Overall, MDV gp57-65 and HVT gp57-65 precursor polypeptide sequences are 73% homologous and share many potential antigenic epitopes. Predicted MDV and HVT gp57-65 protein sequences are similar to those of herpes simplex virus glycoprotein C (gC) and gC-like proteins from other herpes-viruses. Similarities are scattered throughout the molecule, with a primary concentration near the carboxyl half of the molecule. One stretch of 60 amino acids (HVT amino acids 378-437 and MDV amino acids 350-410) are relatively well conserved among gC-like proteins from six herpesviruses. The possible implications of these homologies and the potential roles of gC-like proteins in virus infection, growth, and replication are discussed.

Amino Acid Sequence↗
PubMed1990

Frequency and mechanisms of factor independence in IL-3-dependent cell lines.

Interleukin-3 (IL-3) -dependent cell lines were tested for spontaneous mutation frequency with respect to two markers, growth factor-independent growth and 8-azaguanine-resistant growth. The mutation frequency for the growth factor-independent growth was approximately equal to that of 8-azaguanine-resistant growth. Investigation of the growth factor-independent phenotypes indicated that at least two different mechanisms for growth factor independence existed. One mechanisms was the activation of the IL-3 gene by mutation resulting in the constitutive expression of IL-3 and autocrine regulated growth. A second mechanisms results in IL-3-independent growth in a manner in which (1) constitutive tyrosine kinase activity was not seen and (2) c-myc transcription was constitutively activated. Interestingly, in these cells both the tyrosine phosphorylation and c-myc transcriptional pathways are still activatable following IL-3 stimulation. At present, the exact nature of the mutation that results in this phenotype is not known.

Animals↗
PubMed1990

Purification of genomic sized herpesvirus DNA using pulse-field electrophoresis.

A new method for the separation of genomic sized herpesvirus DNA was developed. This method utilizes the improved separation of extremely high molecular weight DNA molecules by pulse-field electrophoresis. This method has allowed us to separate and purify herpesvirus DNA (greater than 100 kbp) free of cellular DNA, directly from infected cells. In the case of cell associated herpesviruses (such as Marek's disease herpesvirus), this method is more efficient than present isolation procedures. The DNA obtained is able to be restriction enzyme digested and used in cloning experiments.

DNA, Viral↗
PubMed1990

Multiple hematopoietic growth factors signal through tyrosine phosphorylation.

The role of tyrosine phosphorylation in the signal transduction of four hematopoietic colony-stimulating factors (IL-3, IL-4, G-CSF, and GM-CSF) in myeloid cells was investigated. In DA-3 cells, both IL-3 and GM-CSF stimulated, within 10 min, the tyrosine phosphorylation of similar proteins with molecular masses of 70, 56, 51, 42, and 38 kD, as well as two different 140-kD proteins, one being the IL-3 receptor. Both of these growth factors were able to increase the transcription, within 1 hr, of two IL-3-inducible genes--c-myc and the gamma chain of the T-cell receptor. In NFS-60 cells, tyrosine phosphorylation on a common 56-kD protein occurred within 10 min in response to either G-CSF or IL-3. In addition, IL-3 stimulation increased the phosphorylation of the 140-kD IL-3 receptor. Both growth factors were able to increase transcription of the c-myc gene within 1 hr. Finally, in NFS-107 cells, IL-3 and IL-4 stimulated the tyrosine phosphorylation, within 10 min, of two different 140-kD proteins, one of which was the IL-3 receptor. Both growth factors were able, within 1 hr, to increase the transcription of the c-myc and gamma chain of the T-cell receptor genes.

Animals↗
PubMed1988

Interleukin 3 binds to a 140-kDa phosphotyrosine-containing cell surface protein.

Interleukin 3 (IL-3) is a hematopoietic growth factor that supports the proliferation and differentiation of early hematopoietic-lineage progenitors. By using growth factor-dependent cell lines, IL-3 has been shown to induce the appearance of several phosphotyrosine-containing proteins, including a 140-kDa cell surface phosphoprotein. Here we demonstrate that the 140-kDa phosphoprotein binds IL-3 and forms a stable complex with an apparent molecular mass of 170 kDa. The possible relationship of the 140-kDa phosphoprotein to the previously described 65-kDa IL-3-binding protein is discussed.

Affinity Labels↗
PubMed1988

The 4-6-8 method of sequence analysis.

A new method to obtain more sequence data from a single gel run is described. This method allows the reading of over 500 bases of sequence data from a single gel by taking advantage of the differential migration of specific sized dideoxy terminated chain lengths in sequencing gels containing variable percentages of acrylamide. The method is easy to use, requires no special equipment and requires no special technical abilities. We feel the methodology described will be useful for the average laboratory doing sequencing work.

Base Sequence↗
PubMed1987

Identification of the gene encoding Marek's disease herpesvirus A antigen.

The gene encoding the glycoprotein Marek's disease herpesvirus A antigen (MDHV-A) precursor polypeptide pr47 was delineated by using Northern blot (RNA blot) analysis and hybrid selection of its mRNA with cloned MDHV DNA, cell-free translation of the mRNA, and immunoprecipitation of the polypeptide. The resulting piece of DNA with strongly positive hybrid selection results was a 2.2-kilobase-pair (kbp) PvuII-EcoRI restriction fragment localized to the center of the 18.3-kbp MDHV BamHI B fragment of the total virus genome. The localization was specific since no other small restriction subfragment of the larger BamHI B fragment was able to hybrid select significant MDHV-A mRNA and the gene mapped only in the BamHI B fragment of the total virus genome. Northern blot analysis confirmed the localization of the MDHV-A gene on the 2.2-kbp fragment and detected its mRNA as a 1.8-kilobase species, a size consistent with encoding a 47-kilodalton polypeptide. This is the first report of an MDHV gene being mapped to the MDHV viral genome. This opens the way for the use of recombinant DNA technology to study the nature of the gene encoding a secreted virus-specific glycoprotein that could possibly be involved in immunoprevention, immunosuppression, or immunoevasion, immune phenomena known or speculated to be involved in this oncogenic herpesvirus system.

Antigens, Viral↗
PubMed1986

Synthesis, processing, and secretion of the Marek's disease herpesvirus A antigen glycoprotein.

The 57,000- to 65,000-dalton (Da) Marek's disease herpesvirus A (MDHV-A) antigen glycoprotein (gp57-65) has a 47,000-Da unglycosylated precursor polypeptide (pr47), as determined by immunological detection after cell-free translation of infected-cell mRNA. Cleavage of its signal peptide yielded a 44,000-Da precursor polypeptide molecule (pr44), detected both in vivo after tunicamycin inhibition of glycosylation and in vitro after dog pancreas microsome processing of pr47. High-resolution pulse-chase studies showed that pr44 was quickly glycosylated (within 1 min) to nearly full size, a rapid processing time consistent with a cotranslational mode of glycosylation. This major glycosylation intermediate was further modified 6 to 30 min postsynthesis (including the addition of sialic acid), and mature MDHV-A was secreted 30 to 120 min postsynthesis. Limited apparent secretion of pr44 occurred only in the first minute postsynthesis, in contrast to the later secretion of most of the MDHV-A polypeptide as the fully glycosylated form described above. In addition, in the presence of tunicamycin a small fraction of the newly synthesized MDHV-A protein appeared as a secreted, partially glycosylated, heterogeneously sized precursor larger than pr44. pr44 constituted the major fraction of the new MDHV-A made in the presence of the inhibitor but the precursor was smaller than mature MDHV-A. These data indicate that there is a minor glycosylation pathway not sensitive to tunicamycin and that "normal" glycosylation is not necessary for secretion. Collectively, the data demonstrate that the rapid release of most of the fully glycosylated form of MHDV-A from the cell shortly after synthesis is true secretion in a well-regulated and precisely programmed way and not the result of cell death and disruption.

Animals↗
PubMed1986

Molecular characterization of Marek's disease herpesvirus B antigen.

The Marek's disease herpesvirus (MDHV) B antigen (MDHV-B) was identified and molecularly characterized as a set of three glycoproteins of 100,000, 60,000, and 49,000 apparent molecular weight (gp100, gp60, and gp49, respectively) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immunoprecipitation from [35S]methionine-labeled infected cells by specific rabbit antiserum directed against MDHV-B (R alpha B), as previously determined by immunodiffusion. Further identification was accomplished by blocking this immunoprecipitation with highly purified MDHV-B. The same set of three polypeptides was also immunoprecipitated from [35S]methionine- and 14C-labeled infected cells with two other sera shown to have anti-B activity, i.e., rabbit anti-MDHV-infected-cell plasma membrane (R alpha PM) and immune chicken serum from birds naturally infected with MDHV. The three herpesvirus of turkeys (HVT) B-antigen (HVT-B) glycoproteins immunoprecipitated with all three sera containing anti-B activity were also shown to be identical in size to those of MDHV-B by immunoprecipitation and SDS-PAGE. These data serve to clarify the molecular identification of the polypeptides found in common between MDHV and HVT by linking them to MDHV-B, previously identified only by immunodiffusion, and to a similarly sized set of immunologically related common glycoproteins called gp100, gp60, and gp49, detected with monoclonal antibody by other workers. Tunicamycin inhibition of N-linked glycosylation resulted in either nonglycosylated or O-linked glycosylated putative precursors of MDHV-B and HVT-B with apparent molecular weights of 88,000, called pr88, and 44,000, tentatively called pr44, both immunoprecipitable with all three sera. However, the relationships of these two polypeptides to each other and to the overall precursor-processing relationship of the MDHV-B complex remains to be elucidated. The three fully glycosylated B-antigen polypeptides were not connected by disulfide linkage. Collectively, the data presented here and by others support the conclusion that all three glycoproteins now identified as gp100, gp60, and gp49 have MDHV-B determinants. Finally, detection of the same three polypeptides with well-absorbed R alpha PM, which was directed against purified infected-cell plasma membranes, suggests that at least one component of the B-antigen complex has a plasma membrane location in the infected cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Viral↗
PubMedDate not supplied

Phenotypic differences between Syrian hamster embryo cells cultured at pH 6.7 or 7.3.

We have investigated the molecular phenotypic differences between Syrian hamster embryo (SHE) cells cultured at pH 6.7 or 7.3. Multiple pH-sensitive phenotypic differences were noted including changes in cellular morphology, a unique charge differential in a major cellular protein, nine uniquely expressed proteins, two unique phosphoserine/threonine phosphoproteins, one unique phosphotyrosine phosphoprotein, and the pH dependent mRNA level of a gap junctional gene (connexin 43). These differences, combined with previously described pH-specific differences (differential transformation rates and gap junctional communication), illustrate that culturing SHE cells in media that differ by 0.6 pH units (0.3 units on either side of pH 7.0) can have a profound influence on the cellular phenotype.

Animals↗
PubMedDate not supplied

Establishment of immortalized alveolar type II epithelial cell lines from adult rats.

We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37 degrees C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham's F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗