Dammarane saponins of leaves of Panax pseudo-ginseng subsp. himalaicus

From dried leaves of Panax pseudo-ginseng subsp. himalaicus collected in Eastern Himalaya, new dammarane saponins, named pseudo-ginsenosides-F₁₁ and -F₈ were isolated along with the known Ginseng-root saponins, ginsenosides-Rb₃, Rd and -Re. Pseudo-ginsenoside-F₈ was proved to be a mono-acetyl-ginsenoside-Rb₃ and the location of its acetyl group was established mainly by ¹³C NMR spectroscopy. Pseudo-ginsenoside-F₁₁, was identified as the 6-O-α-rhamnopyransyl(1 → 2)-β-glucopyranoside of 3β,6α,12β,25-tetrahydoxy-(20S,24R)-epoxy-dammarane. The C-24 configuration of ocotillone and its related triterpenes was confirmed to be 24R excluding the recent comment by Lavie et al.     

Kinetics of biofilm nitrification

The reaction rates (r(NH(4) (+) ) and r(NO(2) (-) )) in the two-step nitrification reaction were measured in a fluidized-sand-bed biofilm reactor under a range of steady-state conditions with respect to bulk NH(4) (+), NO(2) (-), and O(2) concentrations. It was shown from theory and experiment that under low NH(4) (+) concentration conditions, if the O(2)/NH(4) (+) concentration ratio in the bulk liquid is less than the stoichiometric coefficient (3.4 mg/mg), then oxygen will be rate limiting. In all experiments r(NO(2) (-) ) decreased more than r(NH(4) (+) ) under low oxygen conditions. This resulted in high NO(2) (-) effluent concentrations under low residence time conditions. The influence of the oxygen penetration effects on the relative values of r(NH(4) (+) ) and r(NO(2) (-) ) was experimentally shown to be caused either by the Nitrobacter location in the inner biofilm regions or by a K(m) effect for oxygen. Theoretical support of these findings was provided by a differential diffusion-reaction model which was used to simulate the experimental results.     

Change in the intrinsic fluorescence of acetylcholine receptor purified from Narke japonica upon binding with cholinergic ligands

Effects of various cholinergic ligands on the intrinsic fluorescence of acetylcholine receptor purified from the electric organ of Narke japonica were investigated. Binding with acetylcholine decreased the fluorescence by 7–8%, and that with carbamylcholine by 4–5% at 20 °C. Decamethonium and d-tubocurarine did not affect significantly the fluorescence intensity, while hexamethonium enhanced it. These changes were completely inhibited by preincubation of the receptor with α-bungarotoxin, which indicated that the observed intrinsic fluorescence change was due to the specific binding of each ligand. Data of the quenching experiment using iodide ion as an extrinsic quencher suggested the occurrence of the conformational change in the receptor upon binding with various cholinergic ligands. Considering these results together with those on intrinsic fluorescence change, conformational change provoked by binding with acetylcholine or carbamylcholine seems to differ from that provoked by binding with other cholinergic ligands examined.     

The anti-inflammatory mechanism of MK-447 in rat carrageenin-induced pleurisy

Intrapleural injection of 2% λ-carrageenin caused the accumulation of exudate up to 19 hr. The rate of plasma exudation, measured by the exuded dye amounts for 20 min in the pleural cavity after intravenous injection of pontamine sky blue, showed a peak at 5 hr. Aspirin (100 mg/kg, i. p.) suppressed the dye exudation up to 5 hr, but did not at 7 hr. This inhibition coincided with the decrease of the PG and TXB₂ levels, which were measured by gas chromatography-mass spectrometry, in the pleural exudate. In _vitro experiments, MK-447, a phenolic compound, stimulates PG endoperoxide biosynthesis at lower doses and inhibits it at higher doses, acting as a tryptophan-like cofactor required by PG endoperoxide synthetase. This drug (0.3, 1.0 and 3.0 mg/kg, i. p.) suppressed the dye exudation dose-dependently up to 5 hr, but did not at 7 hr even at a higher dose, in combination with the dose-dependent decrease of the pleural level of PGE₂, which was reported to be a major PG among PGs and TXB₂ in the exudate in inducing the plasma exudation (Harada ; Prostaglandins, : 881, 1982). Thus, the anti-inflammatory action of MK-447 can be explained by inhibition of PGE₂ generation, giving no consideration to the role of oxygen-derived free radicals as a prime mediator in inflammation.     

Protein kinase C in rat brain synaptosomes. Beta II-subspecies as a major isoform associated with membrane-skeleton elements.

A small fraction (approximately 5%) of protein kinase C (PKC) in the adult rat brain synaptosomes is tightly associated with Triton X-100-insoluble components (most likely membrane-skeleton elements), and is solubilized only after denaturation with sodium dodecyl sulfate. The kinase domain of this PKC can be released as a soluble form after limited proteolysis with calpain, whereas the regulatory domain which binds phorbol ester remains insoluble. The PKC in this fraction was identified as the beta II-subspecies or its related molecule. Presumably, this enzyme subspecies is responsible for the phosphorylation of a major PKC substrate protein, growth-associated protein-43, which is located in nerve endings as well as in growth cones in association with the membrane-skeleton elements.     

Cloning and characterization of the CYS3 (CYI1) gene of Saccharomyces cerevisiae.

A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYI1) gene was cloned. The clone had a single open reading frame of 1,182 bp (394 amino acid residues). By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine gamma-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme. In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine gamma-lyase (41%), Escherichia coli cystathionine gamma-synthase (36%), and cystathionine beta-lyase (25%). The N-terminal half of it was homologous (39%) with the N-terminal half of S. cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase. The cloned CYS3 (CYI1) gene marginally complemented the E. coli metB mutation (cystathionine gamma-synthase deficiency) and conferred cystathionine gamma-synthase activity as well as cystathionine gamma-lyase activity to E. coli; cystathionine gamma-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate. We therefore conclude that S. cerevisiae cystathionine gamma-lyase and E. coli cystathionine gamma-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S. cerevisiae and the scarceness of cystathionine in E. coli.     

Further studies on aspartate aminotransferase of thermophilic methanogens by analysis of general properties, bound cofactors, and subunit structures.

Aspartate aminotransferase (AspAT) [EC 2.6.1.1] of thermophilic methanogen was further characterized with the enzyme from Methanobacterium thermoautotrophicum strain FTF-INRA as well as M. thermoformicicum strain SF-4. AspAT of strain FTF-INRA was similar in the amino donor specificity to the enzyme of M. thermoformicicum strain SF-4, in that it was active on L-cysteine and L-cysteine sulfinate in addition to L-glutamate and L-aspartate. The enzymes gave similar absorption spectra having maxima at around 326 and 415 nm with no pH-dependent shift but were found to contain 1 mol of tightly bound pyridoxal 5'-phosphate (PLP) per subunit. Reconstitution of each apoenzyme with added PLP resulted in partial recovery of the original enzymatic activity, suggesting a significant conformational change of the active site region upon removal of the cofactor. Polyacrylamide gel electrophoresis (PAGE) and gel filtration analyses revealed a tetrameric structure (180 kDa) of identical subunits with a molecular mass of 43 kDa for each of these enzymes. Electric current was found to affect the interaction or affinity of each subunit, promoting dissociation of the native enzyme into the monomeric form. Alkaline treatment was effective only for dissociation of the enzyme from strain SF-4. They were distinguishable by the more rapid reassociation of the monomer to the native aggregated form in the enzyme of strain FTF-INRA.     

Inhibition of actin-activated myosin Mg(2+)-ATPase in smooth muscle by ruthenium red.

Ruthenium red was found to inhibit actin-activated myosin Mg(2+)-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg(2+)-ATPase was of the competitive type with respect to actin (Ki 4.4 microM) and of the non-competitive type with respect to ATP (Ki 6.6 microM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg(2+)-ATPase.