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.

     

Bacterial cometabolic degradation of chlorinated paraffins

Summary Cometabolic dechlorination of chlorinated paraffins was demonstrated in the presence of n-hexadecane by bacterial strains (HK-3, HK-6, HK-8, and HK-10) isolated from soil samples.Eleven per cent of chlorine of chlorinated paraffin-150 (CP-150) was released by strain HK-3. The mixed culture of strain HK-3, catalyzing the dechlorination of terminal chlorine of chloroalkane, and strain H15-4, capable of releasing the chlorine from 2-chlorinated fatty acids, dechlorinated CP-150 up to 13%. The mixed culture of the four strains (HK-3, HK-6, HK-8, and HK-10) performed the dechlorination of CP-150 by cometabolism in a jar fermentor pH at 7.0. The amount of chloride released from the chlorinated paraffins tested was in the range of 15–57%.The activated sludge acclimatized to n-hexadecane for 60 days showed a little dechlorination activity to CP-150.     

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)     

Role of the essential thiol group in the thiol-activated cytolysin from Clostridium perfringens

A hemolysin, 0-toxin, produced by Clostridium perfringens has one cysteinyl residue in the free thiol form which is essential for its hemolytic activity. The cysteinyl residue was shown to be located at a position about 5 kDa from the C terminus of the molecule by the method of cysteine-specific chemical cleavage. Modification of the residue with a thiol-blocking agent, 5,5'-dithiobis(2-nitrobenzoic acid), reduced the binding affinity of the toxin to sheep erythrocytes to 1/100 that of intact toxin, resulting in a failure of binding at low cell concentrations (0.5%). Thus the failure of hemolysis at low cell concentrations is primarily ascribed to a decreased affinity of the toxin for erythrocytes. Effects of the modification on the lytic processes were examined using high cell concentrations where considerable amounts of modified toxin bound to the cells. The modified toxin hemolyzes erythrocytes once it binds to them; however, the efficiency of hemolysis is reduced by the modification. These, and additional results indicating that modification alters the sensitivity of toxin molecules to protease digestion, show that thiol-modification inactivates the toxin by affecting both binding and the subsequent lytic processes, probably through a conformational change introduced in the toxin molecules.     

Isolation and characterization of a novel 2-aminoethylphosphonyl-glycosphingolipid from the sea hare, Aplysia kurodai

A novel phosphonoglycosphingolipid named SGL-I' containing 1 mol of 2-aminoethylphosphonate residue was isolated from the skin of Aplysia kurodai using two silicic acid chromatography systems. Data obtained on methanolysis, permethylation, mild acid hydrolysis, and hydrogen fluoride treatment combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry showed that this glycolipid was 3-O-MeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 1----1Ceramide. Palmitic acid, octadeca-4-sphingenine and anteiso-nonadeca-4-sphingenine are its major aliphatic components. The new glycolipid has essentially the same structure as another major phosphonoglycosphingolipid in the skin of Aplysia, SGL-II, that contains 2 mol of 2-aminoethylphosphonate residue, suggesting a metabolic relationship between the two.     

Development of host-dependent high-grade tumor-specific immunity through a novel mechanism triggered by the Lyt-2+ tumor-specific T cell clone (K7L) that induces temporal growth of L1210 leukemia-K7L-variant

When a murine leukemia L1210-specific Lyt-2+ T cell clone, K7L, was injected i.p. into CD2F1 mice together with L1210, the normal growth of L1210 in the peritoneal cavity of the mice at the early stage (days 0 to 5) was strongly inhibited, but L1210 grew progressively at the middle-stage (days 5 to 10), and then was rejected at the late stage (days 10 to 20). The mice thus survived for long times (more than 60 days), whereas the normal control injected with L1210 alone died within 14 days. The L1210 that grew at the middle stage in mice initially inoculated with L1210 together with K7L was a K7L-insensitive (K7L-) variant. All of eight tumor clones established from L1210-K7L- by limiting dilution was insensitive to the antitumor activity of K7L, and this property of tumor clones was stable after repeated in vitro passage. The initial depression of the L1210 growth by K7L followed by growth and rejection of the variant L1210-K7L- by the host T cell activity was then found to prepare a strong, long-lasting (more than 3 mo) immunity to protect mice against the high-dose (10(7) cells per mouse) challenge of original L1210. Corresponding to this result, definite tumor (L1210)-specific cytotoxic T lymphocyte (CTL) activity against both variant and original L1210 targets was developed by antigen (L1210) restimulation in the culture of spleen cells from these mice, but was not increased to a detectable level before L1210-K7L- variant started to grow. It was suggested that the 1210-K7L- variant and the original L1210 should have the common tumor-specific antigen that was independent of the K7L-reactive antigen, and that original L1210, whose growth was retarded by K7L, primed the host with the common antigen to be enormously boosted by the subsequently growing L1210-K7L- variant.     

Effect of Exogenous Gangliosides on Amino Acid Uptake and Na+,K+-ATPase Activity in Superior Cervical and Nodose Ganglia of Rats

The effects of some gangliosides on active uptake of nonmetabolizable alpha-aminoisobutyric acid (AIB) and Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37 degrees C for 2 h. In NG, amino acid uptake was greatly accelerated with the addition of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylgluc osyl ceramide (GM1) (85%) and also with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide (GM2) or [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetyl- neuraminyl]-galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+, K+-ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca2+, Mg2+-ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (-27%) by addition of GM1, with a slight decrease in Na+, K+-ATPase but no change in Ca2+, Mg2+-ATPase activity in the tissue. Both asialo-GM1, in which N-acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+, K+-ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake.(ABSTRACT TRUNCATED AT 250 WORDS)