Biomedical subjects
Publications and source records attributed to P Chandra.
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The present study describes the separation and purification of a reverse transcriptase and cellular DNA polymerases from the human spleen of a patient with myelofibrotic syndrome. The specific requirements with respect to bivalent cations and templateprimers for DNA polymerase-alpha, DNA polymerase-beta and DNA polymerase-gamma, as well as for the reverse transcriptase, are reported. Sedimentation velocity measurements of the purified enzymes gave values of 150 000, 40 000, 100 000 and 70 000 daltons for DNA polymerase-alpha, DNA polymerase-beta, and DNA polymerase-gamma and the reverse transcriptase respectively. The purified reverse transcriptase was specifically inhibited by antisera to the reverse transcriptases of the two primate viruses, SiSV and GaLV. Antisera raised against the myelofibrotic spleen reverse transcriptase inhibited the homologous enzyme and also the reverse transcriptase from SiSV and GaLV. DNA polymerases alpha, beta and gamma from the same spleen were not inhibited by the antisera. These results constitute the first indication of a possible retroviral etiology for myelofibrotic syndrome. Since SiSV and GaLV are exogenous to all primates the results indicate that this polymerase was acquired and the results are most simply interpreted as indicating that virus related to the SiSV-GaLV group is present in man.
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Identification of carriers of the Tay-Sachs gene during pregnancy is difficult because of the increase in serum of a heat stable hexosaminidase isozyme I (or P) as well as changes in the relative and absolute activities of the various molecular forms of the enzyme with advancing pregnancy. In contrast, isolation of blood platelets followed by ion exchange chromatographic separation and assay of the hexosaminidase isozymes in platelet extracts by an automated method provides a sensitive and reliable method for heterozygote identification during pregnancy. This method appears superior to procedures involving thermal inactivation of extracts of peripheral blood leukocytes because of significant differences in the content of the hexosaminidase isozymes in granulocytes, lymphocytes and other cell types, as well as variations in the proportion of these cell types in samples of peripheral blood. It also alleviates the problem inherent in any method involving thermal inactivation of hexosaminidase A by avoiding possible interconversion of the various molecular forms of the enzyme associated with heating.
Nucleated cells obtained from blood and/or bone marrow of patients with myelofibrosis with myeloid metaplasia (MMM) were cultured in diffusion chambers (DC) implanted into the peritoneal cavities of irradiated mice. A total of five blood studies and two bone marrow studies were performed using cells obtained from five patients. The DC were harvested at intervals from the host mice and the total and differential cellularity of DC contents were evaluated. The results obtained from MMM cultures were compared with those from similar cultures of blood cells and marrow cells of four and six normal individuals respectively. The proliferation and maturation of the granulocytic, erythrocytic, and megakaryocytic lines in MMM cultures occurred in an orderly fashion as they occur in vivo. The patterns of proliferation and maturation of the three cell lines in cultures after day 7 suggest that they primarily originate from progenitor cells. The numbers of granulocytes in the multiplicative pool, recognizable red cell precursors, and megakaryocytes recovered were significantly greater from the MMM cultures than those from the normal blood or marrow cultures. These results suggest that the blood and marrow cells of MMM patients have increased numbers of progenitors for granulocytes, erythrocytes, and megakaryocytes.
An RNA-direct DNA polymerase was purified from human melanoma tissue by successive column chromatography on DEAE-cellulose (DE-23 and DE-52) and phosphocellulose. The purified reverse transcriptase has a mol. wt. of 68,000, a pH optimum of 8.0, a Mn2+ optimum of 0.6 mM, and a KCl optimum of 60 mM. The purified enzyme transcribes (rA)n - (dT)12, (rC)n - (dG)18, (Ome-rC)n - (dG)18 and a 70s RNA from Rauscher leukemia virus (RLV), but failed to transcribe (dA)n - (dT)12. This enzyme has no terminal deoxynucleotidyl transferase activity. Serological studies have shown that the reverse transcriptase from human melanoma tissue is antigenically not related to DNA polymerases from Simian sarcoma virus (SiSV), Avian myeloblastosis virus (AMV), RLV, and human spleen of a patient with myelofibrosis. The purified enzyme showed a close antigenic resemblance to DNA polymerases from baboon endogenous virus (BEV) and rhabdomyosarcoma virus (RD-114), the endogenous virus of the cat.
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Symptoms and signs of interstitial pregnancy cannot be differentiated from symptoms and signs of other ectopic pregnancies. Unruptured interstitial gestations are usually diagnosed incidental to the work-up of a suspected ectopic gestation. Unlike some teaching, a characteristic pelvic mass was palpable in 3 of 4 cases. Incomplete induced abortion helped in diagnosing 2 cases. Ultrasound and laparoscopy will aid in establishing the correct diagnosis.
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Proliferation of stimulated human lymphocytes was studied in cultures by determining cell counts and nuclear volume enlargement. DNA content and the nuclear volume increase before the cell divides. Volume-frequency curves were obtained of unstimulated and phytohemagglutinin (PHA) stimulated lymphocytes on 0, 3, 6 and 9 days of cultures with the Coulter Counter H4 system. As the number of lymphocytes in the cultures transformed and enlarged in volume, the area under the volume-frequency curve increased proportionately. The fraction of transforming cells was determined by comparing the area under the curve of the unstimulated and the stimulated lymphocytes. This method provides both the fraction of cells transformed and the increment of the number of cells in the culture and is therefore a better indicator of cell proliferation than the commonly used isotope-labeled thymidine uptake method which monitors the fraction of the cells in DNA synthesis phase only.
This article concerns the molecular mechanisms by which RNA tumor viruses, commonly called as oncornaviruses, transfer their genetic information from the genomic RNA (70 s RNA) of the virions to the cellular DNA, leading to neoplastic transformations. The article describes biochemical and serological properties of reverse transcriptase, its role in the life cycle of RNA tumor viruses and broader implications to molecular biology. In this connection, the authors report their own findings on the role of reverse transcriptase in a preleukemic disease, myelofibrosis. This enzyme, discovered in their laboratory, is antigenically closely related to reverse transcriptase of certain primate RNA tumor viruses, and of human leukemic cells. The article also describes the role of reverse transcriptase inhibitors in viral oncogenesis. Of particular interest, is the partially thiolated polycytidylic acid (MPC) which has been developed by the authors, and is known to have a very high binding affinity to the viral reverse transcriptase. The implication of these basic data on the clinical effectivity of MPC in human leukemia, documented in a few cases, has been discussed.
The interaction between 4'-6-Diamidino-2-Phenylindole-hydrochloride (DAPI) and a variety of DNAs and synthetic polydeoxynucleotides was investigated in order to delineate the nucleic acid structural features necessary for binding. The spectra of DAPI-DNA complexes, measured at various DAPI:DNA molar ratios (r), are hypochromic relative to DNA in the region of its maximum absorption. All the curves pass through an isosbestic point at 268 nm. A new maxima appears in the region of 380-392 nm for DAPI-DNA COMplexes. The magnitude of the peaks in the region are directly proportional to the amount of drug present in the complex. Studies with various DNA types and synthetic polydeoxynucleotides indicate that the drug preferentially binds to dAT-rich regions of DNA. This was also confirmed by enzymatic studies. The inhibition of template action by DAPI in a purified DNA-polymerase reaction was dependent on the dAT-content of the template. The implication of these data to explain a selective binding of DAPI to mitochondrial DNA have been discussed.
The present study describes the separation and purification of a reverse transcriptase and cellular DNA polymerases from the human spleen of a patient with myelofibrotic syndrome. The specific requirements with respect to bivalent cations and template-primers for DNA polymerase-alpha, DNA polymerase-beta and DNA polymerase-gamma, as well as for the reverse transcriptase, are reported. Sedimentation-velocity measurements of the purified enzymes gave values of 150000, 40000, 100000 and 70000 daltons for DNA polymerase-alpha DNA polymerase-beta, DNA polymerase-gamma and the reverse transcriptase respectively. Serological studies have shown that the reverse transcriptase from human spleen is not antigenically related to cellular DNA polymerase-alpha, -beta or -gamma, but is antigenically related to reverse transcriptase from simian sarcoma virus and gibbon-ape leukaemia virus.