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171.
A wound-inducible proteinase Inhibitor I gene from tomato containing 725 bp of the 5 region and 2.5 kbp of the 3 region was stably incorporated into the genome of black nightshade plants (Solanum nigrum) using an Agrobacterium Ti plasmid-derived vector. Transgenic nightshade plants were selected that expressed the tomato Inhibitor I protein in leaf tissue. The leaves of the plants contained constitutive levels of the inhibitor protein of up to 60 g/g tissue. These levels increased by a factor of about two in response to severe wounding. Only leaves and petioles exhibited the presence of the inhibitor, indicating that the gene exhibited the same tissue specificity of expression found in situ in wounded tomato leaves. Inhibitor I was extracted from leaves of wounded transformed nightshade plants and was partially purified by affinity chromatography on a chymotrypsin-Sepharose column. The affinity-purified protein was identical to the native tomato Inhibitor I in its immunological reactivity and in its inhibitory activity against chymotrypsin. The protein exhibited the same M
r of 8 kDa as the native tomato Inhibitor I and its N-terminal amino acid sequence was identical to that of the native tomato inhibitor I, indicating that the protein was properly processed in nightshade plants. These expriments are the first report of the expression of a member of the wound-inducible tomato Inhibitor I gene family in transgenic plants. The results demonstrate that the gene contains elements that can be regulated in a wound-inducible, tissuespecific manner in nightshade plants. 相似文献
172.
173.
Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue. 总被引:1,自引:0,他引:1 下载免费PDF全文
The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase multienzyme complex (PDH complex). A thermoinducible expression system was developed to amplify a variety of genetically restructured PDH complexes, including those containing three, two, one and no lipoyl domains per E2p chain. Although large quantities of the corresponding complexes were produced, they had only 20-50% of the predicted specific activities. The activities of the E1p components were diminished to the same extent, and this could account for the shortfall in overall complex activity. Thermoinduction was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E2p component. This substitution abolished dihydrolipoamide acetyltransferase activity of the complex without affecting other E2p functions. The results support the view that His-602 is an active-site residue. The inactivation could mean that the histidine residue performs an essential role in the acetyltransferase reaction mechanism, or that the reaction is blocked by an irreversible modification of the cysteine substituent. Complementation was observed between the H602C PDH complex and a complex that is totally deficient in lipoyl domains, both in vitro, by the restoration of overall complex activity in mixed extracts, and in vivo, from the nutritional independence of strains that co-express the two complexes from different plasmids. 相似文献
174.
The use of 13C-n.m.r. spectroscopy to monitor alginate biosynthesis in mucoid Pseudomonas aeruginosa. 总被引:3,自引:0,他引:3 下载免费PDF全文
The biosynthesis of alginate by a mucoid strain of Pseudomonas aeruginosa, isolated from a cystic-fibrosis patient, was monitored by using 13C-n.m.r. spectroscopy of bacterial cultures incubated with 1-13C- or 2-13C-enriched fructose. When 1-13C- or 2-13C-enriched fructose was used as the precursor of alginate, enrichment with 13C in the constituent uronic acid monomers of the polysaccharide could only be detected in C-1 or C-2 respectively, indicating that alginate is synthesized in Ps. aeruginosa directly from fructose, with the hexose molecule being retained intact; this rules out the involvement of C3 intermediates, which occurs when glucose is the alginate precursor. The absence of detectable poly-L-gluluronate block sequences from the alginate of Ps. aeruginosa was confirmed, and it was shown that there is no modification of the arrangement of the constituent uronic acids between polymerization to form alginate and the appearance of the mature alginate in the extracellular medium. The 13C-n.m.r. data also provided independent evidence for acetylation on D-mannuronate residues and for the ratio of D-mannuronate to L-guluronate residues in newly synthesized alginate, which had previously been determined only for material secreted from bacteria into the extracellular medium. 相似文献
175.
Characterization of granulocyte chemotactic activity from human cytokine-stimulated chondrocytes as interleukin 8 总被引:2,自引:0,他引:2
Human articular chondrocytes, when stimulated with interleukin 1 beta (IL 1 beta), tumor necrosis factor-alpha (TNF-alpha), or with the double stranded RNA poly (rI).poly (rC), produce a chemotactic activity for granulocytes. The induction with IL 1 beta could be abolished by an antibody to IL 1 beta but not by an antibody to interleukin 6 (IL 6), indicating that the latter is not a mediator for the production of chemotactic activity. The inducers had no direct chemotactic effect on granulocytes. The granulocyte chemotactic factor from chondrocytes was characterized with a specific antibody against leukocyte-derived interleukin 8 (IL 8). The specificity of this antibody was demonstrated by immunochemical and biological criteria such that it could immunoprecipitate only the 6-7 kDa IL 8 protein from fibroblasts, and that it did not neutralize a structurally related monocyte chemotactic protein. This antibody against IL 8 completely neutralized the granulocyte chemotactic activity from stimulated chondrocytes. This demonstrates the identity of chondrocyte IL 8 with leukocyte- and fibroblast-derived IL 8. Our data show that leukocyte chemotaxis into the inflamed joint can be mediated by IL 8, induced in both synovial fibroblasts and chondrocytes by the inflammatory cytokines IL 1 and TNF-alpha. 相似文献
176.
The sporicidal efficacy of glutaraldehyde (2% w/v) was investigated under various conditions. Numerous factors influenced its activity: method of spore production, inherent spore resistance characteristics, alkalination, storage time and storage temperature. The sporicidal action of 2% alkaline glutaraldehyde at room temperature was compared with that of other aldehydes and commercially available formulations. Cidex (glutaraldehyde) and Sporicidin (glutaraldehyde + phenol full strength) were the most effective, followed by 8% (w/v) formaldehyde and 10% (v/v) Gigasept, a formaldehyde-containing product. Five per cent (v/v) Gigasept and 10% (w/v) glyoxal also had good sporicidal activity, though that of Sporicidin (1 : 16) was poor. No activity was observed with 10% (w/v) butyraldehyde. 相似文献
177.
Feeding and growth of juvenile sea bass: the effect of ration and temperature on growth rate and efficiency 总被引:7,自引:0,他引:7
The effect of ration on the growth of pairs of juvenile sea bass Dicentrarchus labrax fed squid mantle was recorded at four temperatures: 6, 10, 14 and 18) C, covering the range typical of Welsh coastal waters. Initial weight of the fish ranged from 2.8 to 15.9 g. A predictive model for the maximum meal size (Mmax) at temperatures between 10 and 18) C, accounted for 95% of the variance in lnMmax. Even when offered excess food, bass at 6) C had a low rate of food consumption [0.19% body weight (BW) day?1] and lost weight (G=?0.04% day?1). Predictive regression models for specific growth rate (G) accounted for 86% of the variance at reduced rations and 70% at maximum meals. The relationship between G (calculated for total biomass per tank) and ration was a decelerating curve. G at maximum meals increased with temperature, at lower rations G decreased with temperature. For a pair of bass with a combined weight of 15 g, predicted maintenance ration ranged between 0.7 and 2.3% BW day?1 and increased with temperature. Maximum meal size was more sensitive to temperature than maintenance ration. At 18) C optimum ration was 7.4% BW day?1. At lower temperatures, the optimum ration was the maximum meal. The maximum gross growth efficiency was 17.4% at 18) C. Mean absorption efficiency was 94.8%. Ration level had no significant effect on absorption efficiency, which was lowest at 6) C. Condition indices (Fulton condition factor, wet and dry liver—somatic indices and body depth index) increased with meal size at all temperatures except 6) C. An increase in temperature between 10 and 18) C generally resulted in a decrease in condition indices at a given ration. When comparisons were made at a given standard length, gut and carcass weight increased with ration. Visceral fat and gut weight decreased with increased temperature. 相似文献
178.
Human foamy virus (HFV) is a retrovirus of the spumavirus family. We have constructed vectors based on HFV that encode neomycin phosphotransferase and alkaline phosphatase. These vectors are able to transduce a wide variety of vertebrate cells by integration of the vector genome. Unlike vectors based on murine leukemia virus, HFV vectors are not inactivated by human serum, and they transduce stationary-phase cultures more efficiently than murine leukemia virus vectors. These properties, as well as their large packaging capacity, make HFV vectors promising gene transfer vehicles. 相似文献
179.
We have generated a number of chromosomal aberrations that disrupt the early-late ecdysone-induced 78C puff gene (Eip78C, ecdysone-induced protein, FlyBase name for the E78 gene of STONE and THUMMEL 1993), which encodes the two members of the nuclear hormone receptor superfamily Eip78C-A and Eip78C-B. The aberrations include deletions of the ligand-binding/dimerization domain of both, inversions that split Eip78C-A but retain residual Eip78C-B expression, and a small deletion specific for Eip78C-B. We find that wild-type Eip78C functions are completely dispensable for normal development under laboratory conditions. However, we show that Eip78C-B is required for the maximal puffing activity of a subset of late puffs (63E and 82F) since these puffs are reduced in size in Eip78C-B mutant backgrounds. Paradoxically the same late puffs are reduced, as well as at least one other, when the Eip78C-B cDNA is overexpressed from a heat shock promoter. These data indicate either that Eip78C function is redundant or that it plays a subtle modulating role in the regulation of chromosome puffing. 相似文献
180.
Elizabeth A. Kingsley Teresa E. Carter Kevin D. Barrow Pamela J. Russell 《Cancer immunology, immunotherapy : CII》1996,41(6):348-354
A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23. These components hadR
F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway. 相似文献