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R Hand

Publications and source records attributed to R Hand.

At least 37 records · Page 2Linked to original sources
PubMed1989

Medical Readiness Education and Training Exercises by United States Army medical personnel in Kenya.

Medical Readiness Education and Training Exercises (MEDRETEs) enable United States military medical personnel to sharpen their skills in diagnosis and treatment of common illnesses in rural third world populations. We report our experiences on MEDRETEs conducted during recent exercises in the Republic of Kenya involving United States and Kenyan forces. Respiratory, musculoskeletal, and eyes, ears, nose, and throat conditions comprised 55% of cases. In 73% of cases, the final diagnosis and the chief complaint were in the same diagnostic category. In asymptomatic patients, 17% had physical findings suggestive of significant disease. Anecdotal experiences demonstrated the importance of the local environment and social customs in the genesis of certain illnesses and symptoms.

Developing Countries↗
PubMed1988

Membrane D2 T antigen: characterization and comparison with its nuclear counterpart.

Up to 40% of membrane T antigen (D2 T antigen) produced by the adenovirus 2--simian virus 40 hybrid, Ad2+D2, remained tightly associated with membranes under alkaline conditions up to pH 11.5. The antigen could not be totally solubilized by treatment with ionic detergents, nonionic detergents, or both. These properties are characteristic of integral membrane proteins. Sephacryl S-300 chromatography in high salt in the presence of Brij-99 showed that the nuclear form of D2 T antigen was dissociated to low molecular weight species, while the membrane form eluted as a complex of high molecular weight. The membrane form, therefore, is able to bind more detergent than the nuclear form, indicating a difference in supramolecular structure.

Adenoviridae↗
PubMed1987

Isolation of a human sequence which complements a mammalian G1-specific temperature-sensitive mutant of the cell cycle.

We have isolated a human genomic sequence which corrects the temperature-sensitive phenotype of the G1-specific cell cycle mutant ts13. Transfection of a thymidine kinase negative (TK-) derivative of ts13 with high molecular weight human DNA from placenta of HeLa cells resulted in the isolation of thermocompetent transformants. These transformants were found to possess discrete Hind III fragments containing human Alu family sequences which were conserved in several independent secondary transformants. A transforming sequence was recovered from a HindIII recombinant bacteriophage library prepared from the DNA of one such secondary transformant. The transforming sequence was isolated as a 12.5 kb Hind III fragment.

Cell Cycle↗
PubMed1985

Defective DNA topoisomerase I activity in a DNAts mutant of Balb/3T3 cells.

Cell and polyomavirus DNA synthesis in ts20, a temperature-sensitive mutant derived from Balb/3T3 cells, is inhibited at an early step in chain elongation in vivo and in vitro. Virus DNA synthesized under restrictive conditions, when analyzed by gel electrophoresis and fluorography, contained a series of equally spaced bands migrating between form I and form II. If restrictive conditions were prolonged, the relative amount of these less-supercoiled topoisomers increased while the overall amount of virus DNA decreased. DNA topoisomerase I activity was lower and more heat-labile when prepared from mutant cells compared to wild-type and revertant cells. An assay in which extracts from wild-type cells corrected defective cell DNA synthesis in lysed mutant cells was applied to purification of the active factor from such extracts. Salt fractionation and three cycles of column chromatography resulted in the isolation of the activity in a fraction containing 10 major polypeptides. The specific activity in the final preparation was increased fivefold and was accompanied by the activity of DNA topoisomerase I. Our results provide evidence that DNA topoisomerase I functions at an early step in chain elongation of cell and polyomavirus DNA synthesis and that the enzyme activity may be decreased as a result of the mutation in ts20.

Animals↗
PubMed1985

Further characterization of the phenotype of ts20, a DNAts mutant of BALB/3T3 cells.

Ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells that is blocked at a step in DNA synthesis involving chain elongation. Following a shift from 33 degrees to 39 degrees C, mutant cells lost ability to grow or form colonies. When mutant cells were infected with polyomavirus, both cell and virus DNA synthesis were inhibited at the restrictive temperature of 39 degrees C. When cell extracts from wild-type cells were added in vitro to lysed infected mutant cells that had been incubated in vivo at 39 degrees C for expression of the mutation, cell DNA synthesis was increased 3-fold (similar to the effect in uninfected mutant cells), whereas virus DNA synthesis was increased only 60%. With harsher lysis conditions, the effect of added extract on virus DNA synthesis was greater, although baseline DNA synthesis (prior to addition of extracts) was much lower. Analysis by alkaline sucrose gradients showed that the addition of cell extract converted small cellular DNA molecules into larger ones, while it increased the synthesis of small virus DNA molecules rather than completed genomes. Analysis of cytosol extracts (in which the activity stimulating DNA synthesis resides) showed that DNA topo-isomerase I activity was more heat-labile when assayed in mutant extracts compared to wild-type extracts. In contrast, cytosol DNA polymerase activity was equally heat-labile in mutant and wild-type extract. This suggested the factor in extract was likely associated with the activity of DNA topo-isomerase I. Analysis of virus DNA synthesized in vitro in restricted mutant cells by gel electrophoresis and fluorography showed an accumulation of topo-isomers migrating between form I and II. These topo-isomers, thought to be a manifestation of the ts defect, did not disappear when extract from wild-type cells was added back in vitro or when mutant cells were shifted back to permissive temperature prior to lysis for in vitro synthesis. The results indicate that polyoma DNA synthesis and cell DNA synthesis differ in their response to the mutant gene product in ts20, although both are inhibited at a step early in DNA chain elongation that may involve DNA topo-isomerase I.

Animals↗
PubMed1985

Plasma membrane orientation of simian virus 40 T antigen in three transformed cell lines mapped with monoclonal antibodies.

Simian virus 40 large T antigen transforms cells from several species. Recent studies show that it is present on the cell surface. As in other tumor virus systems, this may be important for transformation. We have used a radioimmunoassay to map antigenic determinants on living and formaldehyde-fixed transformed cells with six different monoclonal antibodies to T antigen. Nonrelevant monoclonal antibodies of the same subclasses served as controls. With the transformed mouse line SVT2, antibody PAb 101, which reacts with the C-terminal region of T antigen, and PAb 1700, which is directed against an internal region of T, reacted with both formaldehyde-fixed and living cells. Antibodies PAb 402 (C terminus) and 419 (N terminus) reacted only with living cells, their determinants being destroyed upon formaldehyde fixation. Antibodies PAb 405 (C terminus) and 100 (internal) fail to react on either fixed or living cells. Similar results were obtained on the simian virus 40-transformed human line SV80 and the fixed hamster line CHLwt23, although all antibodies failed to react with living CHLwt23 cells. The data suggest that T antigen is inserted into the plasma membrane of transformed cells in a specific, nonrandom manner, with the C and N termini exposed on the cell surface and the midportion either buried in the lipid bilayer, hidden by the tertiary structure of T antigen, or masked by a post-translational modification such as fatty acid acylation.

Animals↗
PubMed1985

Simian virus 40 large T antigen oligomers: analysis of electrophoresis in the absence of detergent.

Large T antigen of simian virus 40 is found as monomeric and oligomeric species in transformed cells. These can be demonstrated in cell extracts by velocity centrifugation in sucrose gradients. We analyzed them further in a transformed human line cell (SV80) and a transformed mouse line cell (SVT2). Individual fractions from sucrose gradients were subjected to polyacrylamide gel electrophoresis in the absence of detergent. T-antigen species were then detected by protein blotting and antibody overlay with polyclonal anti-D2 T antibody or monoclonal Pab419, Pab101, or Pb1700 antibody. The rapidly sedimenting species (14S and larger) of large T antigen from both cell lines reproducibly showed two major bands with estimated molecular weights of 670,000 and 850,000. A third band of 1,200,000 was more prominent in SVT2 cells than in SV80 cells. In SV80 cells the slowly sedimenting species of large T antigen (5S to 11S) contained two reproducible bands. A band with a molecular weight of 95,000 was the predominant one in all fractions between 5S and 11S. A relatively minor band with a molecular weight of 230,000 was found in fractions between 9S and 11S. The low-molecular-weight forms were seen in SVT2 cells only when a prominent peak at 5S to 7S was present, that is, when extracts were stored before analysis. In fresh extracts, the low-molecular-weight bands and slowly sedimenting forms were absent.

Animals↗
PubMed1985

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells.

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

Animals↗
PubMed1984

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.

Animals↗
PubMed1984

The distribution of large T antigen in simian virus 40 nucleoprotein complexes.

Simian virus 40 large T antigen binds to two types of nucleoprotein complexes from lytically infected cells: those containing replicating virus DNA (100S complexes) and those containing nonreplicating virus DNA (70S complexes). Analysis by agarose gel electrophoresis showed that replicating DNA was found exclusively in 100S complexes, although these complexes also contained large amounts of form I and form II DNA. In contrast, no replicating DNA was found in 70S complexes, and pulse-labeled DNA in these complexes migrated as form I and form II DNA that presumably had recently completed replication. Immunoprecipitation and gel electrophoresis showed that large T antigen was associated with both types of complexes. From 21 to 62% of replicating DNA in 100S complexes was bound to T antigen. Our estimates indicated, however, that more than three-fourths of the DNA molecules in 100S complexes were nonreplicating and unassociated with T antigen. In 70S complexes, 12 to 31% of pulse-labeled DNA was bound to T antigen, but because there were more DNA molecules in the 70S complexes, they contained a greater absolute amount of T antigen.

Animals↗
PubMed1982

Phosphorylation and dephosphorylation alter the structure of D2 hybrid T antigen.

D2 hybrid T antigen is a protein closely related to simian virus 40 large T antigen and is synthesized in large quantities in cells infected with Ad2+D2, an adenovirus-simian virus 40 hybrid. We have analyzed the effects of phosphorylation on the structure and DNA binding of this protein. On nondenaturing pore-gradient gels, the purified protein migrated with an apparent molecular weight of 135,000, with a minor band at 330,000 molecular weight. In vitro phosphorylation catalyzed by the protein kinase activity associated with the protein resulted in a structural change so that most of it migrated with an apparent molecular weight of 740,000. Treatment of the phosphorylated form of the protein with alkaline phosphatase (which removed 95% of the phosphate) caused the disappearance of the 740,000-molecular-weight form and reappearance of the smaller forms. Partial tryptic digestion showed that D2 T antigen has two major regions of phosphorylation, only one of which was phosphorylated in vitro. The region phosphorylated in vitro was responsible for the aggregation of D2 T antigen and was tentatively assigned to the N-terminal part of the protein. As shown by protein blotting onto nitrocellulose filters, it was mainly the form of 740,000 molecular weight that bound to simian virus 40 DNA. However, sucrose gradient analyses showed that only a fraction of the in vitro-phosphorylated protein bound to DNA, suggesting that aggregation alone is not sufficient for binding.

Adenoviruses, Human↗
PubMed1982

Case report: management of methotrexate toxicity in an anephric patient.

We describe the case of an anephric woman on chronic hemodialysis who developed breast cancer with positive axillary nodes. After mastectomy, we attempted to treat her with adjuvant chemotherapy. She suffered severe toxicity involving the skin, gastrointestinal tract and bone marrow after a single dose of 15 mg (10 mg m-2) of methotrexate. She received high doses of leukovorin and hemodialysis was continued. Although hemodialysis caused little acute change in the serum methotrexate levels, over a period of 3 weeks the levels did decrease, her symptoms of toxicity subsided, and she recovered. The case demonstrates the limited effectiveness of hemodialysis for treating methotrexate toxicity and suggests that leukovorin is useful for reversing it.

Adult↗
PubMed1981

Immunological cross-reactivity between simian virus 40 large T antigen and D2 hybrid T antigen.

A specific antiserum was raised in rabbits against D2 hybrid T antigen that had been purified from HeLa cells infected with the adenovirus/simian virus 40 hybrid, Ad2(+)D2. The specificity of this serum was compared with that of a conventional hamster antiserum against simian virus 40-induced tumors by immunoprecipitation and by a new radioimmune assay that can detect nanogram quantities of D2 hybrid T antigen.

Adenoviruses, Human↗
PubMed1981

Cell surface T antigen in cells infected with simian virus 40 or an adenovirus-simian virus 40 hybrid, Ad2+D2.

Cell surface T antigen, detected by a radioimmune assay that uses 125I-labeled Staphylococcus aureus protein A and antibodies against either authentic T antigen or D2 hybrid T antigen, was found in simian virus 40-transformed and -infected cells and in cells infected with an adenovirus-simian virus 40 hybrid, Ad2+D2. In simian virus 40 lytic infection, the surface T antigen appeared at the same time as the nuclear T antigen.

Adenoviruses, Human↗
PubMed1981

DNA binding of a nonstructural reovirus protein.

The specific early inhibition of DNA synthesis in reovirus-infected cells suggests that the cell nucleus is a target for virus-induced damage. We have now examined the affinity of reovirus proteins for DNA, postulating that such affinity could provide a mechanism for the inhibition. Cytoplasmic and nuclear extracts of cells labeled with [35S] methionine from 6 to 8.5 h after infection at high multiplicity was subjected to chromatography on denatured DNA - cellulose columns. Fractions from both cytoplasm and nucleus eluted with 0.6 N NaCl contained a protein with the same electrophoretic mobility of polyacrylamide slab gels as the nonstructural (NS) reovirus protein of the sigma size class. The protein also exhibited affinity for native DNA - cellulose and denatured DNA - agarose. Electrophoretic analysis is tube gels of cell extracts labeled for 48 h before infection with [14C] leucine and from 6 to 8.5 h after infection with [3H] leucine showed increased 3H label in this protein indicating it is reovirus specific. Small amounts of mu proteins also had DNA affinity. Purified virus did not bind strongly to DNA, suggesting that the binding protein is not a structural protein of the sigma size class on the outer surface of the virus. Our results provide evidence that the sigma NS protein binds to DNA. This affinity could interfere with chromosome function in the infected cell.

Animals↗