High-performance anion-exchange chromatography of ultraviolet-absorbing constituents of human urine

A 100-μl urine sample was chromatographed on a column packed with a strongly basic macroreticular anion-exchange resin (Diaion CDR-10, 5– μm diameter with a nominal 35% cross linkage). The elution was performed with a linear acetate gradient from 0 to 6.0 M at an average flow-rate of 0.72 ml/min and at an average pressure of 104 kg/cm². The relative standard deviation of retention times and peak height was ± 4% or less. The properties of the macroreticular anion-exchange resin, the effect of the particle size, the pH of acetate buffers, and the effect of the flow-rate of the eluent on the separation were investigated. Thirty three components of urine were then resolved and named.     

Targeting Interleukin-6: All the Way to Treat Autoimmune and Inflammatory Diseases

Interleukin (IL)-6, a cytokine featuring redundancy and pleiotropic activity, contributes to host defense against acute environmental stress, while dysregulated persistent IL-6 production has been demonstrated to play a pathological role in various autoimmune and chronic inflammatory diseases. Targeting IL-6 is thus a rational approach to the treatment of these diseases. Indeed, clinical trials of tocilizumab, a humanized anti-IL-6 receptor antibody have verified its efficacy and tolerable safety for patients with rheumatoid arthritis, Castleman''s disease and systemic juvenile idiopathic arthritis, resulting in approval of this innovative biologic for treatment of these diseases. Moreover, a considerable number of case reports and pilot studies of off-label use of tocilizumab point to the beneficial effects of tocilizumab for a variety of other phenotypically different autoimmune and chronic inflammatory diseases. Elucidation of the source of IL-6 and of mechanisms through which IL-6 production is dysregulated can thus be expected to lead to clarification of the pathogenesis of various diseases.     

Mechanisms of modulation of neuronal nicotinic receptors by substance P and OAG

Substance P is known to modulate neuronal nicotinicacetylcholine receptors (nAChRs) in the sympathetic nervous system.There are two conflicting proposals for the mechanism of this effect, an indirect action mediated by protein kinase C (PKC) and a direct interaction with receptor subunits. We studied the mechanisms of thiseffect in PC-12 cells. Substance P enhanced the decay of thenicotine-induced whole cell current. This effect was fast in its onsetand was not antagonized by guanosine5'-O-(2-thiodiphosphate), a G protein blocker, orstaurosporine, a nonselective PKC blocker. Staurosporine failed toreverse the inhibition by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic diacylglycerol analog known to activate PKC. Theinhibitory effects of the peptide and OAG were preserved in excisedpatches, but substance P applied to the extra patch membrane wasineffective in the cell-attached patch configuration. We conclude thatsubstance P modulates neuronal nAChRs most likely by direct interactions with the receptors but independently from activation ofPKC or G proteins and that PKC does not participate in modulation by OAG.

     

Rate of DNA Synthesis during Meiotic Prophase in Lily Microsporocytes in the Presence of Deoxyadenosine and Nalidixic Acid

The rate and period of DNA synthesis during meiotic prophasewere examined using lily microsporocytes. Meiocytes at the earlyleptotene stage were cultured for discrete periods in the presenceof inhibitors of DNA synthesis, deoxyadenosine and nalidixicacid. Deoxyadenosine, which arrests meiotic development at theearly zygotene stage, markedly suppressed DNA synthesis to 35%of control at 2 mM. Nalidixic acid simply reduced the rate ofDNA synthesis, resulting in prolongation of the synthetic period.The relevance of DNA synthesis to meiotic development is discussed. (Received January 12, 1987; Accepted May 7, 1987)     

Spectroscopic studies on the interaction of phosphate with uteroferrin

The effect of phosphate on the binuclear iron center of pink (reduced) uteroferrin was examined by magnetic resonance and optical spectroscopy. The purple (oxidized) protein, which contains 1 mol of tightly bound phosphate per mol of enzyme at isolation, does not give rise to a 31P NMR signal. Phosphate binding to phosphate-stripped pink uteroferrin is indistinguishable from that in the native purple phosphoprotein. As measured by EPR and optical spectroscopy, the rate of reaction between phosphate and pink uteroferrin is pH-dependent, decreasing as the pH increases. Phosphate is capable of binding to the reduced protein between pH 3 and 7.8, resulting in formation of the purple uteroferrin-phosphate complex. Evans susceptibility measurements at pH 4.9 indicate that the EPR silent species with a maximum absorption at 535 nm, generated upon phosphate addition to pink uteroferrin, is diamagnetic. Moreover, phosphate causes disappearance of the hyperfine-shifted resonances in the 1H NMR spectra of the reduced protein. We therefore have not been able to identify the paramagnetic "purple reduced enzyme-phosphate complex" reported by Pyrz et al. (Pyrz, J. W., Sage, J. T., Debrunner, P. G., and Que, Jr., L. (1986) J. Biol Chem. 261, 11015-11020) using Mossbauer spectroscopy and dithionite-reduced 57Fe-reconstituted uteroferrin. Our present data with native unmodified enzyme are in accord with our earlier results (Antanaitis, B. C., and Aisen, P. (1985) J. Biol. Chem. 260, 751-756) and with the results of Burman et al. (Burman, S., Davis, J. C., Weber, M. J., and Averill, B. A. (1986) Biochem. Biophys. Res. Commun. 136, 490-497) on bovine spleen phosphatase, suggesting that phosphate binding to reduced protein rapidly induces oxidation of the binuclear iron center.     

Thymic stroma-derived T cell growth factor (TSTGF). I. Functional distinction of TSTGF from interleukins 2 and 4 and its preferential growth-promoting effect on helper T cell clones

A recently established thymic stroma-derived cell line (TSCL) supported the growth of the interleukin (IL) 2-dependent, antigen-specific helper T cell (Th) clone, 9-16, without requirement for IL-2 and antigen, and such growth was substituted by a factor produced into cultures by this established TSCL. This substance, thymic stroma-derived T cell-growth factor (TSTGF), was capable of inducing the proliferation of various Th clones including 9-16 Th clone, but not of cytotoxic T cell clones. TSTGF-induced growth promotion was obtained in a dose-dependent fashion and in maintaining antigen specificity of Th clones. The culture supernatant from the TSCL did not contain detectable level of IL-1, IL-2, IL-3, IL-4, or interferon activity. The proliferation of 9-16 Th clone was stimulated by recombinant IL-2 and IL-4 as well as TSTGF, but not by IL-1, IL-3, or interferons. However, the proliferation of this Th clone by IL-2 or IL-4 was almost completely inhibited by anti-IL-2 receptor or anti-IL-4 monoclonal antibody, respectively, whereas TSTGF-induced growth of 9-16 Th clones was not affected by either type of antibody, demonstrating that TSTGF is functionally distinct from IL-2 and IL-4. In addition, TSTGF activity was also obtained from the culture supernatant of the primary thymic explant, which was freshly prepared. These results indicate that the primary thymic explant as well as an established TSCL produce factors capable of promoting the growth of helper but not cytotoxic type of T cells in the absence of T cell growth factors thus far defined.     

The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs

We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: the stability of these eubacterial tRNA variants in the eukaryotic oocytes; the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-beta-D-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.     

Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes II. Chromosomal aberration tests with cultured mammalian cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on ptaquiloside and its related compounds, hypoloside B, hypoloside C, illudin M and illudin S. Ptaquiloside induced chromosomal aberrations at doses as low as 4.5 μg/ml (0.0113 mM). The clastogenic effect was ph-dependent. The same activity was observed at a 90-fold higher dose at pH 5.3 in the culture medium compared with the activity at pH 74. or pH 8.0. Both hypoloside B and hypoloside C were also clastogenic at almost the same dose levels as that of ptaquiloside. Illudin M and illudin S were also potet clastogens and induced aberrations at much lower doses than ptaquiloside. These results suggest that the clastogenic effect is involved in the mechanism of carcinogenic potency of ptaquiloside in animals.     

A restricted cytoplasmic region of IL-2 receptor beta chain is essential for growth signal transduction but not for ligand binding and internalization

The functional, high affinity form of interleukin-2 receptor (IL-2R) is composed of two receptor components, the IL-2R alpha (p55) and IL-2R beta (p70-75) chains. Unlike the IL-2R alpha chain, the IL-2R beta chain contains a large cytoplasmic domain that shows no obvious tyrosine kinase motif. In the present study, we report the establishment of a system in which the cDNA-directed human IL-2R beta allows growth signal transduction in a mouse pro-B cell line. This system enabled us to identify a unique region within the cytoplasmic domain of the human IL-2R beta chain essential for ligand-mediated signal transduction. We also demonstrate that certain cytoplasmic deletion mutants in the IL-2R beta chain, although deficient in signal transduction, can still form high affinity IL-2R in conjunction with endogenous mouse IL-2R alpha chain; the mutants are still able to internalize the ligand as well.