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.     

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.

     

Purification and characterization of spermidine N1-acetyltransferase from chick duodenum

We have reported that spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in putrescine synthesis in chick duodenum induced by 1 alpha,25-dihydroxycholecalciferol (calcitriol) [Shinki, T., Kadofuku, T., Sato, T. and Suda, T. (1986) J. Biol. Chem. 261, 11712-11716]. In the present study, spermidine N1-acetyltransferase was purified from the duodenal cytosol of calcitriol-treated chicks to homogeneity judged by SDS/polyacrylamide gel electrophoresis. The purified enzyme converted spermidine only to N1-acetyl-spermidine. The apparent molecular mass of the purified spermidine N1-acetyltransferase was found to be 36 kDa by gel filtration on Sephacryl S-200 and 18 kDa by SDS/polyacrylamide gel electrophoresis. When duodenal crude 105,000 x g extracts were directly applied to a Sephacryl S-200 column without prior purification, three peaks with spermidine N1-acetyltransferase activity appeared. The first peak was in the void volume, the second peak was in the fraction corresponding to an apparent molecular mass of 70 kDa, and the third peak was in the fraction corresponding to 36 kDa. These results suggest that spermidine N1-acetyltransferase exists as a dimer of the 18 kDa subunits and is stabilized in (a) form(s) bound to other components or proteins in intact cells.     

Effects of a newly synthesized leukotriene antagonist, NZ-107, on immediate-type hypersensitivity reaction in rats and guinea-pigs.

The effects of 4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone (NZ-107) on immediate type hypersensitivity reactions in rats and guinea-pigs were studied. 1. When NZ-107, at a dose of 50 mg/kg (i.p.) or 100 mg/kg (orally), was administered to rats, 48-h homologous passive cutaneous anaphylaxis (PCA) reaction and histamine-, leukotriene C4 (LTC4)- and leukotriene D4 (LTD4)-induced skin reactions were suppressed by the agent. 2. NZ-107 (10(-6) g/ml) inhibited both LTC4- and LTD4-induced contractions of isolated rat stomach smooth muscle. 3. NZ-107 inhibited antigen-induced histamine release from rat peritoneal mast cells by 26% at a concentration of 10(-4) g/ml. 4. NZ-107, at doses of 25 and 50 mg/kg (orally), significantly inhibited guinea-pig 3-h heterologous PCA reaction. 5. NZ-107 inhibited antigen-induced histamine release from guinea-pig lung tissue by 17% and 48% at concentrations of 5 x 10(-5) and 10(-4) g/ml, respectively. 6. NZ-107, at doses of 25 and 50 mg/kg (i.p.), inhibited antigen-induced bronchoconstriction and eosinophil accumulation in the bronchoalveolar lavage fluid (BALF) of guinea-pigs. These results suggest that NZ-107 has anti-allergic action including inhibition of eosinophil accumulation in an antigen-challenged airway lesion in rats and guinea-pigs. The anti-allergic action of this agent is thought to be due to its action as a histamine and LT antagonist and its consequent inhibition of antigen-induced histamine release.     

Presence of immunoreactive endothelin in human saliva and rat parotid gland.

The presence of immunoreactive endothelin (IR-ET) in human saliva and rat parotid gland was investigated by radioimmunoassay. The IR-ET concentration (mean +/- SEM) in saliva taken from normal volunteers was 2.0 +/- 0.2 pmol/l (n = 15). The IR-ET concentration in rat parotid gland was 19.2 +/- 2.2 fmol/g wet weight (n = 10). Fast protein liquid chromatography (FPLC) of human saliva extract revealed 6 peaks; one peak eluting in the void volume, one in a position between ET-1 and -3, and the other four in the positions of synthetic ET-1, -2, -3 and big ET(1-38), respectively. A similar pattern of rat parotid gland extract was noted with FPLC, except that there was no peak after the void volume. Presence of endothelin, a potent growth factor, in saliva and salivary gland points to a role in maintaining the integrity of the oral and gastrointestinal tract mucosa.     

Establishment of a new perchloric acid treatment method to allow determination of the total endotoxin content in human plasma by the limulus test and clinical application.

We established a new method of plasma treatment for the removal of interfering factors in the plasma to allow detection of endotoxin by limulus test. The limulus test used was an endotoxin-specific chromogenic test, the Endospecy test. Perchloric acid (PCA) treatment and centrifugation (PCA method) is usually used to remove interfering factors from plasma, with the precipitate being discarded and the supernatant used to detect endotoxin. As the solubilized precipitates of endotoxin-spiked plasma and some patient plasma were found to contain the Endospecy activity, we have devised a new method assaying endotoxin in both the supernatant and precipitate. This study confirmed that the solubilized precipitate of endotoxin-spiked plasma had Endospecy activity and found that the precipitate had other endotoxin activities, such as lethality in galactosamine-sensitized mice and pyrogenicity in rabbits. We also confirmed that interfering factors were completely removed from plasma samples by this new method. The endotoxin level after the new PCA method was found to be about 8 times higher than that determined after PCA treatment and the new PCA method surpasses the conventional PCA method with regard to the positive rate of endotoxin contents in clinical samples. These results indicate that the new PCA method is superior to the PCA method as a plasma pretreatment method for limulus test.     

O3 Tolerance and the Ascorbate-Dependent H2O2 Decomposing System in Chloroplasts

The relationship between O₃ tolerance and the chloroplast H₂O₂scavenging system (PS I     

Presence of immunoreactive dynorphin in human cerebrospinal fluid

Immunoreactive dynorphin (I-dynorphin) was measured by radioimmunoassay in human cerebrospinal fluid (CSF). I-dynorphin concentration in CSF was 30 +/- 2 pg/ml. Sephadex G-25 column chromatography showed the main peak eluted at the position of dynorphin-(1-17). HPLC elution profile of this major peak from gel filtration showed a large peak corresponding to the position of dynorphin-(1-17) and small peaks corresponding to the positions of dynorphin-(1-13), dynorphin-(1-12) and other unknown peptides.