Four tetranortriterpenoids from the stem bark of Khaya anthotheca

Eight limonoids, anthothecanolide (1), 3-O-acetylanthothecanolide (2), 2,3-di-O-acetylanthothecanolide (3), 6R,8alpha-dihydroxycarapin (4), 3beta-acetoxy-3-deoxo-6R-hydroxycarapin (5), methyl angolensate, methyl 6-hydroxyangolensate and khayalactone together with sitosterol glucoside, have been isolated from the extracts of the stem bark of Khaya anthotheca. Compounds 1-4 are described for the first time. Their structures were established by analysis of the high-field NMR and MS data. The structure of compound 4 was confirmed by a single crystal X-ray structure analysis.     

Syntrophin gamma 2 regulates SCN5A gating by a PDZ domain-mediated interaction

SCN5A encodes the alpha subunit of the cardiac muscle and intestinal smooth muscle mechanosensitive Na(+) channel. Mechanosensitivity in the intestine requires an intact cytoskeleton. We report, using laser capture microdissection, single cell PCR, and immunohistochemistry, that syntrophins, scaffolding proteins, were expressed in human intestinal smooth muscle cells. The distribution of syntrophin gamma 2 was similar to that of SCN5A. Yeast two-hybrid and glutathione S-transferase pull-down experiments show that SCN5A and syntrophin gamma 2 co-express and that the PDZ domain of syntrophin gamma 2 directly interacts with the C terminus of SCN5A. In native cells, disruption of the C terminus-syntrophin gamma 2 PDZ domain interaction using peptides directed against either region result in loss of mechanosensitivity. Co-transfection of syntrophin gamma 2 with SCN5A in HEK293 cells markedly shifts the activation kinetics of SCN5A and reduces the availability of Na(+) current. We propose that syntrophin gamma 2 is an essential Na(+) channel-interacting protein required for the full expression of the Na(+) current and that the SCN5A-syntrophin gamma 2 interaction determines mechanosensitivity and current availability.     

Diterpenoids from Neoboutonia glabrescens (Euphorbiaceae)

Glabrescin, a daphnane diterpenoid, neoboutonin, a degraded diterpenoid with a novel skeleton, and neoglabrescins A and B, two rhamnofolane derivatives, have been isolated from the stem bark of Neoboutonia glabrescens Prain (Euphorbiaceae), together with the known tigliane derivative, baliospermin, and the known daphnane, montanin. Other constituents include squalene, 3-acetylaleuritolic acid, oleanolic acid and sitosterol, and the phenolic compounds 9-methoxy-1,7-dimethylphenanthrene and 2,3,8-tri-O-methylellagic acid. The structures were assigned on the basis of spectral studies and comparison with published literature data. The structures of neoglabrescins A and B were derived for their acetylated derivatives and, in the case of neoglabrescin A, confirmed by X-ray crystallographic analysis.     

Crystal structures of human antibodies: a detailed and unfinished tapestry of immunoglobulin gene products

Sequencing of all human immunoglobulin (Ig) germline gene segments has recently been completed. However, our first glimpses of the recombined products of this combinatorial gene system were in the 1970s, in landmark publications, reporting the crystal structures of two human myeloma proteins, the Mcg lambda light chain dimer and the New IgG1(lambda) Fab. Although numerous crystal structures of murine and human antibodies have now been determined, only a relatively small proportion of the human germline genes have had their corresponding protein three-dimensional structures resolved. Therefore, further structural investigations are required before the inherent diversity of the antibody repertoire can be fully appreciated. We discuss the detailed structural information available for human antibodies with regard to their immune functions. Also discussed, is how the structural information is finding application in the 'humanization' of murine antibodies as part of their development as 'biopharmaceuticals' for the treatment of human disease.     

Diversity among bacteria isolated from the deep subsurface

Culturable bacteria from the deep subsurface (179 m) at Cerro Negro, New Mexico were isolated and characterized. The average number of viable aerobic bacteria was estimated to be 5×10⁵g^–1 of sediment, but only about 0.1% of these could be recovered on agar medium when incubated under aerobic conditions. Of 158 strains isolated from this depth, 92 were characterized by cellular fatty acid profiles (FAME), 36 by analysis of partial 16S rDNA sequences, and 44 by rep-PCR genome fingerprint analysis using three different sets of oligonucleotide primers (REP, BOX, or ERIC). These analyses showed the majority of isolates (67%) were Gram-positive bacteria and primarily members of genera with a high %G+C DNA. The remaining isolates were -subdivisionProteobacteria (19%) and members of the flavobacteria group (14%). The diversity indices based on these different methods of characterization were very high suggesting this subsurface habitat harbors a highly diverse microbial community.     

Stimulation of Growth and Nucleic Acid Biosynthesis at Low Concentration of Abscisic Acid in Tissue Culture of Spinacia oleracea

The effect of abscisic acid on growth, ultrastructure and nucleic acid biosynthesis was studied in tissue culture of spinach (Spinacia oleracea L.). Low concentration (0.01 mg l^?1) of abscisic acid increased fresh and dry weight of calluses, whereas 1.0 mg l^?1 was inhibitory. The stimulating effect was observed only in the presence of a relatively high concentration of kinetin (1 mg l^?1). The inhibitory effect was partly overcome by the same kinetin concentration. The low concentration of abscisic acid probably accelerated the induction of callus growth after subculture and stimulated cell division in the exponential phase of growth. Electron microscopy showed the presence of numerous polysomes and rough endoplasmic reticulum in callus cells grown at the stimulating abscisic acid concentration. Control cells and cells at the inhibitory concentration had slightly hyaline cytoplasm and were more vacuolated. Incubation of callus tissue with ³²P in the presence of stimulating concentration of abscisic acid showed a significant increase in the rate of biosynthesis of all nucleic acid classes after 8 h, whereas inhibitory concentration produced a decrease in ³²P incorporation. However, when the tissue was grown in the presence of abscisic acid for 20 days, both concentrations decreased the rate of nucleic acid biosynthesis, as compared to the controls.     

Intracellular calcium buffering capacity in isolated squid axons

Changes in ionized calcium were studied in axons isolated from living squid by measuring absorbance of the Ca binding dye Arsenazo III using multiwavelength differential absorption spectroscopy. Absorption changes measured in situ were calibrated in vitro with media of ionic composition similar to axoplasm containing CaEGTA buffers. Calcium loads of 50-2,500 μmol/kg axoplasm were induced by microinjection, by stimulation in 112 mM Ca seawater, or by soaking in choline saline with 1-10 mM Ca. Over this range of calcium loading of intact axoplasm, the ionized calcium in the axoplasm rose about 0.6 nM/μM load. Similar loading in axons preteated with carbonyl cyanide 4- trifluoromethoxyphenylhydrazone (FCCP) to inhibit the mitochondrial proton gradient increased ionized calcium by 5-7 percent of the imposed load, i.e. 93-95 percent of the calcium load was buffered by a process insensitive to FCCP. This FCCP- insensitive buffer system was not saturated by the largest calcium loads imposed, indicating a capacity of at least several millimolar. Treatment of previously loaded axons with FCCP or apyrase plus cyanide produced rises in ionized calcium which could be correlated with the extent of the load. Analysis of results indicated that, whereas only 6 percent of the endogenous calcium in fresh axons is stored in the FCCP-sensitive (presumably mitochondrial) buffer system, about 30 percent of an imposed exogenous load in the range of 50-2,500 μM is taken up by this system.     

安徽庐江鸭乙型肝炎病毒LJ-76转染细胞系的建立

为建立鸭乙型肝炎病毒ＬＪ－７６的转染细胞系,将ＬＪ－７６病毒ＤＮＡ插入到ｐＵＣ１９的ＥｃｏＲⅠ位点上,分离得到含有双拷贝ＬＪ－７６ＤＮＡ的重组质粒．通过磷酸钙沉淀方法,将经ＣｓＣｌ等密度离心纯化的ＬＪ－７６ＤＮＡ双体导入到人肝癌细胞ＢＥＬ７４０２中．收集转染细胞的培养液进行蔗糖密度梯度离心,所得沉淀经检测发现含有ＬＪ－７６ＤＮＡ并具有特异性ＤＨＢＶ内源性ＤＮＡ多聚酶活性;对上述样品通过ＤｏｔＥＩＡ检测ＤＨＢＶ核心抗原及表面抗原结果为阳性．Ｓｏｕｔｈｅｒｎｂｌｏｔ分析表明转染细胞内存在病毒ＤＮＡ复制中间体ｃｃｃＤＮＡ、ｓｓＤＮＡ和ｒｃＤＮＡ,而ｃｃｃＤＮＡ被认为是复制活动较为活跃的标志．电镜观察转染细胞的上清发现有病毒颗粒的存在．     

G-Protein Regulation of an L-Type Calcium Channel Current in Canine Jejunal Circular Smooth Muscle

Calcium entry into smooth muscle cells is essential to maintain contractility. In canine jejunal circular smooth muscle cells the predominant calcium entry pathway is through L-type calcium channels. The aim of this study was to determine the G-protein regulation of L-type calcium channel current (I _CaL) in isolated canine jejunal circular smooth muscle cells. Barium (80 mm) was used as the charge carrier. GTP-γS and GTP increased maximal inward current from 118.7 ± 12 pA to 227.5 ± 21.5 pA (n= 8) and 174.6 ± 10.1 pA (n= 6) respectively. The increase in inward current was blocked by nifedipine suggesting it was through L-type calcium channels. Pertussis toxin did not alter baseline I _CaL while cholera toxin increased I _CaL from 125 ± 19 pA in controls (n= 6) to 347 ± 30 pA (n= 4). Staurosporine inhibited the increase in current evoked by GTP-γS and calyculin further increased I _CaL over the increase evoked by GTP-γS. The results suggest that cholera toxin sensitive G-proteins activate L-type calcium channels in isolated canine jejunal circular smooth muscle cells through protein phosphorylation. Received: 27 March 1997/Revised: 3 July 1997