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Biomedical subjects

K E Fry

Publications and source records attributed to K E Fry.

34 records · Page 2Linked to original sources
PubMed1990

Immunoglobulin V regions of a bactericidal anti-Neisseria meningitidis outer membrane protein monoclonal antibody.

C6 is a potentially therapeutic murine monoclonal antibody that recognizes the class 1 outer membrane protein of Neisseria meningitidis. C6 specifically immunoblots this antigen and augments in vitro killing of N. meningitidis bacteria. We describe a general method of obtaining the heavy and light chain variable-region sequence from immunoglobulin-secreting cells. The method uses mixed polymerase chain reaction (PCR) primers designed from the 5' end of the framework 1 (FR1) sequences of the heavy and light chains, and 3'-end primers for constant-region conserved sequences. The method has been applied to the cloning and sequencing of the variable region of C6 to construct a humanized monoclonal antibody. Rapid amplification and sequencing of variable regions by this general method have multiple applications in the study of the immune response to infectious diseases.

Amino Acid Sequence↗
PubMed1989

Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction.

A general method to directly obtain the DNA sequence of the variable regions of any immunoglobulin chain using a mixture of oligomer primers and the polymerase chain reaction (PCR) is described. Mixed oligonucleotide primers corresponding to the 5' signal peptide and a conserved 3' constant region primer were used for enzymatic amplification of each of the heavy and light chain variable regions of a human hybridoma producing a monoclonal antibody recognizing an epitope of gp120 of the human immunodeficiency virus 1. The amplified DNA segments were cloned and the sequence was determined for the heavy chain variable region. This method will greatly facilitate structural and functional studies of immunoglobulins by reducing the effort to clone and sequence the members of the immunoglobulin as well as other multigene families.

Amino Acid Sequence↗
PubMed1986

Altered regulation of c-myc expression in adenovirus-transformed cells.

Expression of the oncogenes c-myc, c-raski, and p53 is studied in normal primary mouse cultures and in two adenovirus-transformed mouse cell lines. In all cases oncogene expression is measured in cells arrested in G1 (or G0 for primary cells) by serum starvation and at different times after cell cycle traverse is stimulated by addition of high serum. For primary mouse cells, c-myc mRNA levels are found to increase four- to six-fold within 1 h of serum addition and then decline by 4 h to nearly the level observed in serum-starved cells. This level is maintained throughout the remainder of the cell cycle. The early induction of c-myc is dependent on serum concentration and is independent of cell density. These results confirm and extend previous observations for primary cells. By contrast, expression of c-raski does not vary at all through the cell cycle and p53 increases with time after mitogenic stimulation. In the adenovirus-transformed cell lines, the regulation of expression of c-myc with respect to the cell cycle is altered. There is an increase in c-myc in S phase cells which is dependent on cell density and the early induction in response to serum addition as seen in primary cells is absent. Expressions of c-raski and p53 are found to show similar profiles to those observed for primary cells.

Adenoviridae↗
PubMed1985

Patterns of thymocyte differentiation markers on virus and radiation induced lymphomas of C57BL/Ka mice.

To better understand the biology of tumorigenesis in virus and radiation lymphomas of C57Bl/Ka mice, we have examined the cell surface phenotypes of a large series of primary tumors induced by both agents. Data derived using flow cytometry and recently available monoclonal antibodies to thymocyte differentiation antigens supports three major conclusions. First, tumor cell populations are unimodal for staining with most antibodies and are probably of clonal origin. Second, many, but not all, tumor cells show surface phenotypes similar to those of previously defined subpopulations of normal thymocytes. Third, at the cell surface level, no major differences between virus- and radiation-induced lymphomas can be discerned. Our data thus further define the relationship between thymomas induced by these two agents.

Animals↗
PubMed1984

Nucleotide sequence of the large terminal repeat of two different strains of gibbon ape leukemia virus.

Gibbon ape leukemia virus, SEATO strain (GaLV-SEATO), a virus that induces myeloid leukemia in gibbon apes, and GaLV, San Francisco strain (GaLV-SF), a virus associated etiologically with lymphocytic leukemia in gibbon apes, have been molecularly cloned. The complete nucleotide sequence of the large terminal repeats (LTRs) of both viruses are reported and compared to the previously published nucleotide sequence of the LTR of another member of the same virus group, the simian sarcoma virus (SSV). Substantial homology is evident among all three LTR sequences. The most striking feature of the GaLV-SEATO LTR is the presence of a 45-bp tandem direct repeat in the U3 region, an area likely to contain transcriptional enhancers. Both GaLV-SEATO and GaLV-SF contain a deletion in U3 when compared to SSV. Each of the three LTRs differ from the other two by short deletions in R-U5 and short additions in U3, as well as by numerous point mutations. The possibility that the structural changes observed in the LTR contribute to the differences in the pathogenic effects of these viruses is discussed.

Animals↗
PubMed1984

Establishment and characterization of a cloned giant cell line from a patient with Hodgkin's disease.

The phenotypic characteristics of a cloned giant cell line, SU/RH-HD-1, established from the spleen of a patient with Hodgkin's disease were studied. The cells grew slowly, adhered to the culture vessel surface, and had an elongated, irregular shape. After trypsinization, they became spherical and measured 30-100 micron in diameter. Although most cells were mononuclear, binucleated and multinucleated cells could be identified in expanded cultures. The cells phagocytized latex and ink particles and were nonspecific esterase-positive, but they did not secrete lysozyme. They were Epstein-Barr nuclear antigen-negative, and their culture fluid supernatants were devoid of reverse transcriptase activity. Electron microscopy revealed cells with a pronounced smooth endoplasmic reticulum, free ribosomes, some filaments, and mitochondria. Many 0.5- to 1.0-micron invaginations (pits) were seen along the cell membrane. Nucleoli were enlarged and prominent in the very heterochromatic nuclei. The SU/RH-HD-1 cells had 10- to 100-micron-long pseudopodia that were sometimes forked or branching, as well as multiple stress fibers. Electron microscopic appearance was suggestive of that of macrophages. This interpretation of the results was substantiated by monoclonal antibody studies, which revealed that the cells express antigenic determinants distinctive for cells of the monocyte-macrophage lineage and by functional studies demonstrating that the cells are capable of specific antigen presentation to immune T-cells. The SU/RH-HD-1 cells were aneuploid and could be cloned, first in liquid culture by limiting dilution and later in semisolid medium. It was likely that the SU/RH-HD-1 cells were derived from the neoplastic giant cell population in Hodgkin's disease and that they originated from cells of the mononuclear phagocyte-reticulum cell lineage.

Animals↗
PubMed1982

Characterization of an infective molecular clone of the B-tropic, ecotropic BL/Ka(B) murine retrovirus genome.

Using molecular cloning techniques, we amplified the unintegrated, linear proviral DNA of the BL/Ka(B) virus, a non-leukemogenic retrovirus of mouse strain C57BL/Ka. Two independent clones in lambda phage vector 607 and one subclone in pBR322 were infective when transfected into mouse fibroblasts. Analysis of the progeny virus revealed biological properties and a restriction map identical to those of the parental viral shock. Comparison of the restriction map with the maps of other ecotropic murine viruses reveals many similarities. Particularly interesting is the comparison of the N-tropic Akv virus and the B-tropic BL/Ka(B) virus. The long terminal repeats of the two viruses are virtually identical, as are 22 of 23 restriction sites located outside of the region which spans from 1.8 to 3.8 kilobases from the left end of the genome. Within this region, however, only three of nine sites examined are shared. This suggests that the BL/Ka(B) virus was derived from an endogenous N-tropic virus closely related to Akv by recombinational events which altered the sequence in the last half of the gag gene and the first third of the pol gene. This change is probably responsible for the observed difference in the Fv-1 tropism of the two viruses.

Animals↗
PubMed1981

Molecular cloning and partial characterization of unintegrated linear DNA from gibbon ape leukemia virus.

We have cloned the complete genome of an oncogenic primate retrovirus, the San Francisco isolate of gibbon ape leukemia virus, in a lambda phage vector. DNA sequence analysis and restriction endonuclease mapping of the inserted linear provirus demonstrated 9-base pair inverted repeats at its ends, flanking direct terminal repeats 470 base pairs in length. The (-) strong stop region of this DNA showed surprisingly low sequence homology to that of another gibbon ape leukemia virus isolate from an animal with similar disease. Analysis of the clone also revealed the terminal phosphate configuration of the linear provirus. The recombinant phage is suitable for direct use as a hybridization probe to detect homologous retroviral sequences in human cell lines.

Animals↗
PubMed1977

Electron microscopic studies of purified eukaryote messenger RNA.

Rat liver mRNA, hen and rabbit globin messenger RNA (mRNA) were investigated by electron microscopy. No secondary structures were visible in the molecules of these mRNAs under conditions where short secondary structures of other types of RNA, i.e. MS2 phage RNA and 28 S rRNA, were clearly demonstrated. The contour lengths of these mRNAs were also determined by electron microscopy and compared with the sizes estimated by other techniques. The contour lengths of rabbit short and long globin mRNA are 0.1498 +/- 0.0019 and 0.1908 +/- 0.0021 micrometer, respectively. The former is assumed to be globin mRNA for alpha chain and the latter for beta chain. Similarly, hen "alpha" and "beta" globin mRNA have mean lengths of 0.1449 +/- 0.0011 and 0.1891 +/- 0.0017 micrometer, respectively. Hen and rabbit reticulocytes contain 1.8-2.0 times as much mRNA for alpha globin chains as for beta.

Animals↗
PubMed1970

Some factors affecting the Hill reaction activity in cotton chloroplasts.

A method of plant culture was developed for growing large leaves of glandless cotton on single stems. Chloroplasts isolated from these leaves actively reduced ferricyanide when assayed for the Hill reaction. Hill reaction activity increased 133% when the 0.5 m sucrose isolation medium was replaced with 10% (w/v) polyethylene glycol, both buffered at pH 7.6. The presence of 2 or 5% (w/v) bovine serum albumin in the sucrose buffer did not increase Hill activity. Ferricyanide reduction in the dark occurred in all assays, and the possibility of gossypol as the reductant is discussed. Half-life of the chloroplasts stored in 10% glycerol at -23 C was 23 days. The ammonium ion at 0.01 m enhanced Hill reaction activity up to 171%. Leaves containing chloroplasts with the highest Hill reaction activity were found near the 8th node below the apex. Leaf water potentials less than -28 bars reduced the activity about 50%. Daylight conditions during the winter months in the greenhouse reduced the activity about 30%.

Chloroplasts↗
PubMed1969

Variations in genetic recombination due to amber mutations in T4D bacteriophage.

Recombination experiments were performed to assess the affect of amber mutations in 12 genes of T4D bacteriophage on genetic recombination. Crosses were performed in various suppressor-containing bacterial hosts to permit the production of progeny phage. Amber mutations in genes 32, 46, and 47 caused decreased recombination, amber mutations in genes 30, 41, 42, 43, 56, 61, and 62 caused increased recombination, whereas mutations in genes 63 and 37 showed no demonstrable effect on recombination.

Coliphages↗
PubMedDate not supplied

Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNAs from small numbers of cells.

A rapid and highly sensitive technique (MAPPing: message amplification phenotyping) has been developed to simultaneously analyze the array of messenger RNAs made by small numbers of cells. The technique incorporates a micro-procedure for isolating RNA, reverse transcription of total cellular RNA to produce cDNA, and enzymatic amplification of cytokine-specific DNA fragments using the polymerase chain reaction. In this study, the technique has been applied to the analysis of cytokines produced by lymphoid cells ranging in number from a single cell to 10(6) cells. The technique should be applicable to virtually any tissue or cell type.

Base Sequence↗