On the reaction of N-acetylchondrosine, N-acetylchondrosine 6-sulfate,chondroitin 6-sulfate,and heparin with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

N-Acetylchondrosine was activated at pH 4.75 with excess 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to give an O-acylisourea that consists of equimolar amounts of N-acetylchondrosine and 1-(3-dimethylaminopropyl)-3-ethylurea, with concomitant uptake of 0.94 mol of hydrogen ion per mol of N-acetylchondrosine. The product was treated with sodium borohydride to give a carboxyl-reduced disaccharide, but it did not react with a nucleophile reagent, such as glycine ethyl ester, over the pH range of 4.75–11.0. The O-acylisourea was hydrolyzed mostly into N-acetylchondrosine and 1-(3-dimethylaminopropyl)-3-ethylurea with 0.1m sodium carbonate overnight at room temperature, but a small proportion was transformed into the N-acylurea. N-Acetylchondrosine 6-sulfate, chondroitin 6-sulfate, and heparin were also activated at pH 4.75 with excess 1-(3-dimehtylaminopropyl)-3-ethylcarbodiimide hydrochloride to give the corresponding O-acylisoureas containing one mol of 1-(3-dimethylaminopropyl)-3-ethylurea moiety per mol of uronic acid residue, respectively.     

滇重楼地上部分的两个微量皂甙

从滇重楼地上部分中分离出两个新的微量的甾体皂甙ＰｏｌｙｐｈｙｌｌｏｓｉｄｅⅢ和Ⅳ,根据化学降解和光谱分析,它们的化学结构分别为２７－羟基－偏诺皂皂甙元－３－Ｏ［ａ－Ｌ－鼠李吡喃糖基（１→２）］［ａ－Ｌ－鼠李吡喃糖基（１→４－ａ－Ｌ鼠李吡喃糖基（１→４）］－β－Ｄ－葡萄吡喃糖甙,２３β,２７－二羟基－偏皂甙元－３－Ｏ－［ａ－鼠李吡喃糖（１→２）］     

Purification and Characterization of Two Epoxide Hydrolases from Corynebacterium sp. Strain N-1074

Enzymes II_a and II_b, which catalyze the conversion of epichlorohydrin (ECH) to 3-chloro-1,2-propanediol (MCP), were purified from Corynebacterium sp. strain N-1074, which catalyzes the formation of (R)-MCP from prochiral 1,3-dichloro-2-propanol via ECH. The specific activity of enzyme II_a for the formation of MCP from ECH was about 6.4-fold higher than that of enzyme II_b. Both enzymes catalyzed the conversion of 1,2-epoxides to the corresponding diol, although they differed in several enzymatic properties.     

Effect of Escherichia coli Lipopolysaccharide on Bacteroides fragilis Abscess Formation and Mortality in Mice

To study the mechanism of synergism between Bacteroides fragilis and Escherichia coli, the effect of sublethal dose of E. coli lipopolysaccharide (LPS) (25μg/mouse) was checked on B. fragilis abscess formation. LPS was administered prior or after inoculum injection. No significant difference in the abscess size was observed at necropsy on day 6. However, all the groups receiving LPS showed higher incidence of recovery of additional intestinal bacteria (23.5–45.5%) from the abscess pus. When LPS was given 4 hr prior to inoculum administration, 83–100% mortality was observed. Detailed investigation showed autoclaved cecal contents alone could also cause similar mortality. Studies with stimulation of endogenous cytokines by E. coli LPS demonstrated induction of all of them within 3 hr in the blood stream with TNF-α demonstrating peak at 1 hr, IL-1α and IL-6 at 4 hr and IFN-γ between 6–9 hr with moderately high levels at 4 hr. This E. coli LPS-triggered cytokine cascade possibly gets further stimulated by injection of autoclaved cecal contents containing high concentration of endotoxins (1.6 × 10⁵ EU/ml) contributed by dead bacteria and lead to the mortality of animals.     

Production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile by the combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328

A new soil isolate, tentatively identified as Rhodococcus equi TG328, was found to be effective in the production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile. The conversion is catalysed by two enzymes. First, a nitrile hydratase converts the (R,S)-nitrile to (R,S)-2-phenylpropionamide. Second, a stereoselective amidase converts the S-(+)-amide to S-(+)-2-phenylpropionic acid. Conditions for optimal enzyme production and accumulation of S-(+)-2-phenylpropionic acid by resting cells were studied. The reaction of resting cells for 30 h at 10° C with (R,S)-2-phenylpropionitrile resulted in the production of 100 g of S-(+)-2-phenylpropionic acid per litre of reaction mixture. The enantiometric excess of the purified S-(+)-2-phenylpropionic acid was 99.4%. The amount of S-(+)-2-phenylpropionic acid accumulated was enhanced by lower reaction temperatures. In addition, unreacted R-(–)-2-phenylpropionamide with 99.0% enantiometric excess was isolated. Correspondence to: T. Nagasawa     

Relapses or reinfections: Analysis of a case of Clostridium difficile-associated colitis by two typing systems

Immunoblotting and pulsed-field gel electrophoresis of Clostridium difficile isolates were employed to differentiate reinfection by a newly acquired strain from relapse by an original strain in a 10-year-old patient with four episodes of C. difficile-associated colitis. Immunoblot typing demonstrated subserogroup K-1 of serogroup K for the first and second organisms, subserogroup A-1 of serogroup A for the third organism, and subserogroup G-4 of serogroup G for the fourth organism. PFGE analysis revealed consistent results with immunoblot analysis except that the strains from the fourth episode, whose DNA constantly degraded, were nontypable by this method. Five separate isolates of C. difficile from a specimen of each episode showed identical PFGE patterns, indicating that infections of multiple strains probably did not occur in this patient. These typing results suggested that the second episode after a 17-day course of vancomycin therapy represented a relapse by the strain causing the first episode, and that the third and fourth episodes after tapering vancomycin therapy were reinfections by other strains. Both immunoblot and PFGE typing systems are promising tools for analyzing recurrence of C. difficile infection. Received: 27 November 1995 / Revised: 1 January 1996 / Accepted: 22 March 1996     

Development of Functional Rat-Derived T Cells in SCID Mice Engrafted with the Fetal Thymus of LEC Rats Which Are Defective in CD4+ T Cells

We reported that LEC rats are genetically deficient in the development of thymic CD4⁺8^? cells and that this defect is caused by bone marrow (BM)-derived stem cells. To determine which BM-derived cells are responsible for the arrest of T-cell development in LEC rats, fetal thymuses of LEC rats, or LEA rats which bear the same major histocompatibility complex (MHC) as LEC rats but are immunologically normal, were engrafted under the kidney capsule of severe combined immunodeficiency (SCID) mice (LEC-TG and LEA-TG mice, respectively). We then examined the differentiation of T cells and their immunological functions in the SCID mice. A large number of rat-derived CD4⁺ T cells appeared in the peripheral blood, lymph nodes (LN) and spleens in LEC-TG mice. Furthermore, the peripheral LN cells in LEC-TG mice appeared to be functional. These cells produced IL-2 upon Con A stimulation, whereas LN cells from LEC rats produced no IL-2 in the same conditions. Thymopoiesis was observed at 3 weeks in LEC-TG as well as LEA-TG mice. The distribution of thymocyte subsets with respect to CD4 and CD8 expression in LEC-TG mice closely resembled that of LEA rat thymus and that in LEA-TG mice, suggesting that normal T-cell differentiation occurred in LEC-TG mice. The results indicated that BM-derived progenitor T cells of LEC rats could differentiate to functional CD4⁺ T cells.     

Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes

Muscarinic receptor-linked G protein, G _i , can directely activate the specific K⁺ channel (I _K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G _i to the K⁺ channel in the ventricle has not been well defined. G protein regulation of K⁺ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization, mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I _K(ACh) and/or the background inwardly-rectifying K⁺ channel (I _K1) in ventricular cell membranes. I _K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I _K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I _K(ACh), G protein-activated I _K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I _K(ACh) and 1.2 μm in I _K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic receptor-linked PTX-sensitive G protein, G _i , is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action potential by activating I _K(ACh) and I _K1 via muscarinic receptor-linked G _i proteins in human ventricular myocytes. Received: 23 September 1996/Revised: 18 December 1996