Robert E. Handschumacher

A rapid quantitative determination of deoxyribonucleoside triphosphates based on the enzymatic synthesis of DNA

Abstract An assay has been developed for deoxyribonucleoside triphosphates; it is based on the observation that all four nucleotides are necessary for the continued synthesis of DNA. A linear standard curve is obtained with one of the nucleotides as a limiting factor and measurement of the amount of incorporation into the polymer of a radioactive deoxyribonucleoside triphosphate present in excess. The method has been utilized to determine the concentration of dTTP in HClO 4 extracts of mouse leukemia cells and Streptococcus faecalis . Other materials present in the extracts did not influence the assay. The method offers the advantages of simplicity, speed, and sensitivity.

Potentiation of the T lymphocyte response to mitogens: III. Properties of the mediator(s) from adherent cells

Abstract Thymus lymphocyte activating factor was obtained in supernatants from cultures of human leukocytes or mouse peritoneal cells and exhibited the following properties: A. Lymphocyte activating factor activity was stable for 2 hr at pH 5.5 or 10.5. A reduction in activity was noted at pH 3.0. B. Up to two thirds of the lymphocyte activity factor activity could be precipitated by ammonium sulfate and recovered in a pattern different from that of total serum proteins. Maximum activity was precipitated by 60% saturation. C. Lymphocyte activating factor is stable at temperatures up to 37 °C, but is partially destroyed by heating at 60 or 80 °C. In the presence of normal serum, heating at 80 °C completely suppresses lymphocyte activating factor activity, due to the formation of an inhibitory factor from the serum. D. Lymphocyte activating factor is not dialyzable. E. Chromatography of supernatants on columns of Sephadex G-50 or G-100 showed that lymphocyte activating factor activity localized in fractions containing molecules estimated at 5000 to 25,000 daltons with a peak at about 15,000 daltons. Similar fractionation patterns were obtained with lymphocyte activating factor activity from human or mouse cells, stimulated with various agents, E. Short incubation (1 hr) with lymphocyte activating factor was insufficient to stimulate thymocytes or to potentiate their response to phytohemagglutinin.

Preparation of lymphocyte-activating factor from continuous murine macrophage cell lines☆

Abstract Lymphocyte-activating factor (LAF), a mitogen for thymocytes and T lymphocytes, is released into the culture medium by human mononuclear cells and mouse peritoneal exudate cells following treatment with various macrophage stimulants. Experiments were performed to determine if recently described mouse macrophage cell lines released LAF in response to stimulation with bacterial lipopolysaccharide. Four continuous cell lines (P388-D 1 , J774, WEHI 3, and PU5-1.8) were found to release LAF in serum-free medium following endotoxin stimulation. The results of partial purification indicated that LAF obtained from cell lines had a higher molecular weight and lower isoelectric point than LAF from human mononuclear cells.

Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update

Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and bio-membranes, is a crucial element in the regulation of normal physiological processes as well as pathological states. The biological effects of uridine have been associated with the regulation of the cardio-circulatory system, at the reproduction level, with both peripheral and central nervous system modulation and with the functionality of the respiratory system. Furthermore, uridine plays a role at the clinical level in modulating the cytotoxic effects of fluoropyrimidines in both normal and neoplastic tissues. The concentration of uridine in plasma and tissues is tightly regulated by cellular transport mechanisms and by the activity of uridine phosphorylase (UPase), responsible for the reversible phosphorolysis of uridine to uracil. We have recently completed several studies designed to define the mechanisms regulating UPase expression and better characterize the multiple biological effects of uridine. Immunohistochemical analysis and co-purification studies have revealed the association of UPase with the cytoskeleton and the cellular membrane. The characterization of the promoter region of UPase has indicated a direct regulation of its expression by the tumor suppressor gene p53. The evaluation of human surgical specimens has shown elevated UPase activity in tumor tissue compared to paired normal tissue.

5-Diazo-4-Oxo-L-Norvaline: Reactive Asparagine Analog with Biological Specificity

The L-asparagine analog, 5-diazo-4-oxo-L-norvaline, specifically inactivates L-asparaginase and inhibits the growth of L-asparagine-dependent or L-asparaginase-sensitive tumor cells in culture. With 5-14C-labeled compound, a biphasic incorporation into sensitive cells occurs, but the inhibition of cell multiplication is manifest much later than the rapid phase of incorporation of the analog.

cyclophilin, A Primary Molecular Target For Cyclosporine

An understanding of the mechanism of action of cyclosporine requires the identification and functional characterization of its molecular target or targets in the cell. Our laboratory has presented evidence that cyclophilin (CYP), a low‐molecular‐weight (Mr 17,737) basic protein, is the primary cytosolic receptor for CsA. The high affinity of CYP for CsA (Kd 30 nM) and specificity for immunosuppressive cyclosporine analogs implicate CYP as a pivotal regulator of T cell and B cell activation. CYP exists in at least two isoforms, is abundant (0.05% to 0.4% of total protein) in the cytosol, and is ubiquitous in cells and tissues of eukaryotic organisms. Its amino acid sequence is highly conserved and there is strong evidence that CYP is a member of a new multigene family. These features suggest that one or more CYP isoforms must play a crucial role in lymphocyte activation and perhaps a multifunctional role in cellular physiology. The ability of CsA to suppress expression of several lymphokine and proto‐oncogene products via a transcriptional control mechanism suggests that CYP may function at some level in a signaling pathway linking membrane receptor stimulation to gene regulatory elements in lymphocytes, and possibly nonlymphoid cell types as well.

Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter

Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [3H]adenosine 1.8-fold within 30 s, approx. half of the [3H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [14C]Inosine, [14C]hypoxanthine and [3H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC50 approx. 0.7 microM), but not nitrobenzylthioinosine (15 microM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 microM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [3H]adenosine and [14C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purines may also be present.

Fluoroolefin peptide isosteres - tools for controlling peptide conformations

Abstract Fluoroolefin dipeptide isosteres were synthesized applying the Peterson reaction as a novel method for fluoroolefination. The dipeptide isosteres were elaborated to provide the conformationally constrained analogs ( 1 -( R ), 1 -( S ) and 2 -( R ), 2 -( S )) of the Suc-Ala-Gly-Pro-Phe-pNA tetrapeptide, a synthetic substate of cyclophilin.

Identification of two calcineurin B-binding proteins: Tubulin and heat shock protein 60

Calcineurin (CaN) is a Ca++/calmodulin-dependent protein phosphatase with two subunits: a catalytic subunit (CaNA) and a regulatory subunit (CaNB). With four Ca(++)-binding sites and a sequence homology to calmodulin, CaNB has been defined as the regulatory subunit for CaNA. However, we have shown that mitochondrial expression of CaNB far exceeds that of CaNA. To investigate the role of this excess CaNB, we have generated glutathione-S-transferase-CaNB (GST-CaNB) fusion protein and demonstrated that the fusion protein predominantly bound to alpha-tubulin, a 57 kDa protein in bovine brain extracts, and heat shock protein 60 (Hsp60) in bovine kidney extracts. Their Ca(++)-dependent interactions with CaNB were verified by immunoprecipitation. The binding of CaNB could be demonstrated with purified alpha/beta tubulins and Hsp60, but not GroEL, a bacterial Hsp60 analog. The interaction of CaNB and Hsp60 was not disrupted by the incubation with Hsp10, ATP and Mg++, suggesting that CaNB was not associated with Hsp60 as a misfolded substrate, and may serve as a regulatory protein. Thus, CaNB may play other regulatory roles in Ca(++)-dependent events in addition to its interaction with CaNA, and may be important for Ca(++)-dependent processes in mitochondria.

Aberrant cell cycle inhibition pattern in human colon carcinoma cell lines after exposure to 5-fluorouracil

In this report, we describe the use of two human colon carcinoma cell lines, HCT-8 and HT-29, as potential models to study DNA- and RNA-directed cytotoxicity due to 5-fluorouracil (FUra) exposure by flow microfluorimetric analysis of DNA cell content. The sensitivity of the HT-29 line (EC50 = 0.9 microM) to FUra was somewhat greater than that of the HCT-8 line (EC50 = 4 microM), but each presented a dramatically different DNA histogram after exposure to FUra. In HCT-8, an unexpected and nearly complete disappearance of cells in S-phase occurred, whereas in HT-29 the expected accumulation of cells at the G1-S border was observed. The absence of HCT-8 cells in S-phase also occurred as a result of two RNA polymerase inhibitors: actinomycin D and dichloro-D-ribofuranosylbenzimidazole. However, an accumulation of cells in S-phase was observed in the presence of 5-fluorodeoxyuridine. These results suggest that in the HCT-8 cell line, FUra predominantly causes an RNA-related toxicity. By comparison, the rate of formation of 5-fluorodeoxyuridine monophosphate, the increased dUMP pool size, and low thymidylate synthase activity in the HT-29 line are consistent with its greater susceptibility to DNA-directed toxicity. Further evidence was seen in the prevention of FUra cytotoxicity by thymidine in HT-29, but not in HCT-8 cells. Similarly, Leucovorin synergized the action of FUra in HT-29 but not in HCT-8. Enzymatic correlates supporting these observations are seen in the greater activity of uridine kinase than thymidine kinase (20:1) in HCT-8 cells compared with that in HT-29 cells (4:1).