Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.     

Incorporation of fluorotryptophan into triostin antibiotics by Streptomyces triostinicus

The quinoxaline chromophores of the antibiotics produced by Streptomyces triostinicus are derived from tryptophan. Protoplasts of this organism made novel products when they were incubated with DL-5-fluorotryptophan or DL-6-fluorotryptophan. When added to batch cultures of the organism, DL-5-fluorotryptophan, at concentrations as low as 10 microM, inhibited both mycelial growth and triostin production, but gave rise to novel products. These have been characterized, using fast atom bombardment mass spectrometry, as novel triostins in which one or both of the quinoxaline rings contain an atom of fluorine. The chromatographic properties of the triostins arising from the incorporation of DL-5-fluorotryptophan are very similar to those of triostins containing chlorine or bromine at position 6 of the quinoxaline ring; they are clearly different from those having a chlorine atom at position 7. Accordingly, it is suggested that the carbon atom at position 5 of the indole ring of tryptophan ends up at position 6 of the quinoxaline ring system in triostins A and C.     

Characterization of a membrane surface glycoprotein associated with T-cell activation

A cell surface antigen (gp140) was previously shown to exist on T cell subsets as well as on monocytes and macrophages in normal peripheral blood. Elevated expression of this antigen was associated with immune system disorders, acute lymphocytic leukemias, and in vitro activation of T cells. The antigen could be identified with monoclonal antibody (MAb) T305. Gp140 was a biosynthetic product of T cells because it could be labeled with [3H]leucine or [3H] glucosamine. Biochemical studies of gp140 used high performance liquid chromatography with nitrocellulose blotting to isolate aliquots suitable for 125I radiolabeling and immunoprecipitation to demonstrate: a) a reduction in m.w. of gp140 KD to 90 KD after deglycosylation by trifluoromethanesulfonic acid, b) alteration of isoelectric point from 4.1 to 5.7 after neuraminidase treatments, c) absence of N-linked sugars based on resistance to endoglycosidase F, d) resistance to trypsin and chymotrypsin digestion but susceptibility to pronase, and e) presence of sialic acid and lactosaminoglycan as O-linked sugars. Gp140 could be labeled with the periodate/NaB[3H]4 technique, indicating its similarity to a class of sialoglycoproteins previously described on activated T-cells in mouse and man. The antigenic epitope recognized by MAb T305 contains sialic acid linked (2----3) to galactose; however, periodate oxidation of the exocyclic ring of sialic acid did not affect binding by MAb T305. In an attempt to determine the functional role of gp140, we tested the ability of MAb T305 to block: a) proliferation of peripheral blood lymphocytes to mitogens, b) response to interleukin 2 (IL 2) of an IL 2 dependent T cell line, and c) growth of a T-ALL derived cell line. No inhibition of proliferation or growth was noted. Although the function of gp140 remains unknown, its association with lymphocyte activation and certain disease states suggests that it may provide a target for modulation of the immune response. These studies characterize the structural features of gp140 and further define the epitope recognized by MAb T305.     

Activation of human thymocytes via the 50KD T11 sheep erythrocyte binding protein induces the expression of interleukin 2 receptors on both T3+ and T3- populations

Recent studies have demonstrated that the 50KD T11 molecule is a surface component of a macrophage-independent alternative pathway of human T cell activation that is unrelated to the T3/Ti antigen-MHC receptor complex. Given the expression of T11 on all human thymocytes, it was of interest to determine whether they could be activated via this pathway. The triggering of T11 by monoclonal antibodies anti-T112 and anti-T113, directed at two unique epitopes on the molecule, induced IL 2 receptor expression on both T3+ and T3- thymocytes but did not induce IL 2 production. Consequently, in contrast to peripheral blood T cells, thymocytes did not proliferate in response to anti-T112 and anti-T113 in the absence of exogenous IL 2. These studies suggest that IL 2 receptor gene activation precedes IL 2 gene activation in T cell development. The ability of the alternative pathway of T cell activation to induce IL 2 receptor expression on T3- thymocytes implies that the T11 molecule may have an important role in early thymocyte ontogeny.     

Production of interleukin 2 (IL 2) by salivary gland lymphocytes in Sj?gren's syndrome. Detection of reactive cells by using antibody directed to synthetic peptides of IL 2

Because abnormalities in interleukin 2 (IL 2) production have been reported in the blood of patients with certain autoimmune diseases, we have examined the lymphocytes from patients with Sj?gren's syndrome (SS) in which it is possible to obtain simultaneous samples of inflammatory site (i.e., salivary gland) lymphocytes and blood lymphocytes. We found that IL 2 production by peripheral blood lymphocytes (PBL) after mitogen stimulation was markedly diminished (4 +/- 2 U/ml) in 8/32 SS patients. However, salivary gland lymphocytes (SGL) from six out of six SS patients (including three patients with low IL 2 production by their PBL) had a high level of IL 2 production (97 +/- 32 U/ml), suggesting that IL 2 production by inflammatory site lymphocytes may differ from blood lymphocytes in the same patients. Low IL 2 production by a patient's PBL was not correlated with the patient's age, duration of disease, immunoglobulin level, or presence of antinuclear antibodies. Low IL 2 production was associated with a decreased ratio of Leu-3a/Leu-2a positive cells (p less than 0.05) and with an increased proportion of "activated" T cells expressing HLA-DR and gp140 (p less than 0.05). To determine the proportion of PBL and SGL containing cytoplasmic IL 2-like material, we used affinity-purified rabbit antibodies prepared against chemically synthesized peptides of human IL 2. Before mitogen stimulation, PBL were not stained by these antibodies (less than 1% reactive cells), whereas SGL T cells eluted from the salivary gland of SS patients contained a small (3.4% +/- 1.8) proportion of reactive cells. A similar proportion (2.4% +/- 1.2) of reactive cells was noted when frozen tissue sections of salivary gland biopsies were examined with these antibodies. After mitogen stimulation, 35% +/- 17 of PBL and 56% +/- 18 of SS SGL were specifically stained with anti-IL 2 peptide antibodies. In summary, these studies demonstrate a significant difference in IL 2 production between PBL and SGL of the same patients. Furthermore, antibodies against IL 2 peptides provide a powerful tool for detection of T cells producing IL 2 in vitro and in situ, and for understanding the role of this lymphokine in pathogenesis.     

The cell cycle dependence of thermotolerance. III. HeLa cells heated at 45.0 degrees C

We have extended our studies on the cell cycle dependence of thermotolerance to include HeLa cells heated at 45.0 degrees C to compare the results to Chinese hamster ovary (CHO) cells. We found that asynchronous HeLa cells were more resistant to heat than CHO cells but showed a similar development and decay of thermotolerance. Flow cytometry (FCM) was used to study redistributions in the cell cycle after an initial heat dose. Cells heated for 35 min at 45.0 degrees C were delayed in G1 by about 7 h compared to controls, with delays in late S and G2/M phase also. The heat sensitivity varied through the cell cycle; G1 cells were the most resistant to heat, while S-phase cells were uniformly sensitive throughout S phase, and G2 cells were resistant. Thermotolerance could be induced and expressed in early or late S-phase cells, but to a lesser extent than for G1 cells. The results were similar in many respects to CHO cells, but there were significant differences.     

The localization and rate of disappearance of a synaptic vesicle antigen following denervation

Summary A proteoglycan-specific antiserum has been used to monitor the effects of denervation in the electric organ of Torpedo marmorata. The antiserum was produced by injecting a highly purified synaptic vesicle fraction prepared from the electric organs of Torpedo marmorata. Following absorption the serum appears to be specific towards synaptic vesicles. The ultrastructural localization of the antigen determined by immuno-electron microscopy confirmed the specificity of the antiserum and showed that it did not crossreact with the proteoglycans of the basal lamina. The rate of disappearance of the vesicle proteoglycans following denervation was evaluated by means of the antiserum and was compared to the rate of disappearance of other vesicular and nerve terminal-associated markers. The results suggest that degeneration affects the vesicular constituents at varying rates resulting in a progressive disappearance of the entire functional capacity of the synaptic vesicles.     

Investigations into the sequence-selective binding of mithramycin and related ligands to DNA.

The preferred binding sites for mithramycin on four different DNA fragments have been investigated by DNAase I footprinting. Sites containing at least two contiguous GC base pairs are protected by the antibiotic, the preferred binding site consisting of the dinucleotide step GpG (or CpC). Related antibiotics chromomycin and olivomycin produce similar, but not identical footprinting patterns suggesting that they can recognize other sequences as well. All three antibiotics induce enhanced rates of enzyme cleavage at regions flanking some of their binding sites. These effects are generally observed in runs of A and T and are attributed to DNA structural variations induced in the vicinity of the ligand binding site. The reaction of dimethylsulphate with N7 of guanine was modified by the presence of mithramycin so that we cannot exclude the possibility that these antibiotics bind to DNA via the major groove.