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.     

‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immuno-histochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.     

Recognition of hepatitis B virus envelope proteins by liver-infiltrating T lymphocytes in chronic HBV infection

The Ag specificity and cytotoxic function of human T cell clones, generated from lymphocytes infiltrating the liver of a chronic hepatitis B patient, were studied. Both class I- and class II-restricted T clones specifically proliferated to hepatitis B virus envelope proteins, but not to hepatitis B core Ag. The fine specificity of T cells was studied by using rAg having different composition in relation to HBV-envelope proteins or synthetic peptides of preS regions. The antigenic determinant recognized by T cell clones mapped to the preS2 region based on the response to r(preS1+preS2+S) and to r(preS2+S) and the failure to respond to S or preS1 alone. More precise epitope mapping was based on synthetic preS2 peptides 120-150 or 120-134, which stimulated both class I- and class II-restricted T clones, whereas preS2 153-171 or preS1 1-110 peptides did not; thus, the preS2 120-134 appears to contain both the residues binding to class I molecules and the residues binding to class II molecules. Moreover, strong and specific cytotoxic responses of these clones were observed only when HLA-matched EBV-lines, used as target cells, were previously sensitized with r(preS1+preS2+S) or preS2 peptides, which were shown to stimulate the clones. Thus, a preS2 epitope can represent a target Ag for liver-infiltrating T cells, which could kill the hepatocytes expressing the Ag plus the appropriate MHC molecule.     

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.     

Kinetics of Vibrio cholerae sialidase action on gangliosidic substrates at different supramolecular-organizational levels

G_d1a, G_d1b and G_t1b gangliosides were dispersed in the following membrane-mimicking systems: (a) homogeneous micelles; (b) mixed micelles with G_m1 ganglioside (which is resistant to the enzyme action), Triton X-100 or bovine serum albumin; (c) small unilamellar vesicles of egg phosphatidylcholine. The effect of dispersion on sialic acid release by Vibrio cholerae sialidase was studied. As reference substrates freely interacting with the enzyme the lipid-free carbohydrates of G_d1a and 3′-sialosyl-lactose were employed. The apparent V_max. of the enzyme was, with all the gangliosides, dependent on the type of ganglioside dispersion. It was lowest for homogeneous micelles and mixed micelles with ganglioside G_m1, and increased about 6-fold for ganglioside/bovine serum albumin lipoprotein micelles, 15-fold for mixed-ganglioside/Triton X-100 micelles (optimal molar ratio 1:7.5) and 30-fold for phosphatidylcholine vesicles containing 2.5 mol% ganglioside (this proportion was optimal for enzyme activity on the vesicles). For ganglioside G_d1a, the activity on Triton X-100 mixed micelles and on mixed vesicles was even greater (3- and 6-fold respectively) than that displayed on G_d1a lipid-free carbohydrate. With each of the used gangliosides the apparent K_m values were very similar values for homogeneous micelles and vesicular dispersions, but showed marked increases for Triton X-100 mixed micelles, approaching the values exhibited by reference oligosaccharides. Triton X-100 micelles and phosphatidylcholine vesicles did not appreciably alter the kinetics of sialidase action on 3′-sialosyl-lactose and on G_d1a lipid-free carbohydrate, indicating that the above effects are dependent on the intrinsic characteristics of the membrane-like systems containing gangliosides.     

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．     

Production of an active anti‐CD20‐hIL‐2 immunocytokine in Nicotiana benthamiana

Anti‐CD20 murine or chimeric antibodies (Abs) have been used to treat non‐Hodgkin lymphomas (NHLs) and other diseases characterized by overactive or dysfunctional B cells. Anti‐CD20 Abs demonstrated to be effective in inducing regression of B‐cell lymphomas, although in many cases patients relapse following treatment. A promising approach to improve the outcome of mAb therapy is the use of anti‐CD20 antibodies to deliver cytokines to the tumour microenvironment. In particular, IL‐2‐based immunocytokines have shown enhanced antitumour activity in several preclinical studies. Here, we report on the engineering of an anti‐CD20‐human interleukin‐2 (hIL‐2) immunocytokine (2B8‐Fc‐hIL2) based on the C2B8 mAb (Rituximab) and the resulting ectopic expression in Nicotiana benthamiana. The scFv‐Fc‐engineered immunocytokine is fully assembled in plants with minor degradation products as assessed by SDS‐PAGE and gel filtration. Purification yields using protein‐A affinity chromatography were in the range of 15–20 mg/kg of fresh leaf weight (FW). Glycopeptide analysis confirmed the presence of a highly homogeneous plant‐type glycosylation. 2B8‐Fc‐hIL2 and the cognate 2B8‐Fc antibody, devoid of hIL‐2, were assayed by flow cytometry on Daudi cells revealing a CD20 binding activity comparable to that of Rituximab and were effective in eliciting antibody‐dependent cell‐mediated cytotoxicity of human PBMC versus Daudi cells, demonstrating their functional integrity. In 2B8‐Fc‐hIL2, IL‐2 accessibility and biological activity were verified by flow cytometry and cell proliferation assay. To our knowledge, this is the first example of a recombinant immunocytokine based on the therapeutic Rituximab antibody scaffold, whose expression in plants may be a valuable tool for NHLs treatment.