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991.
Yeast carboxypeptidase Y can be translocated and glycosylated without its amino-terminal signal sequence 总被引:15,自引:5,他引:10
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We have constructed a series of mutations in the signal sequence of the yeast vacuolar protein carboxypeptidase Y (CPY), and have used pulse-chase radiolabeling and immunoprecipitation to examine the in vivo effects of these mutations on the entry of the mutant CPY proteins into the secretory pathway. We find that introduction of a negatively charged residue, aspartate, into the hydrophobic core of the signal sequence has no apparent effect on signal sequence function. In contrast, internal in-frame deletions within the signal sequence cause CPY to be synthesized as unglycosylated precursors. These are slowly and inefficiently converted to glycosylated precursors that are indistinguishable from the glycosylated forms produced from the wild-type gene. These precursors are converted to active CPY in a PEP4-dependent manner, indicating that they are correctly localized to the vacuole. Surprisingly, a deletion mutation that removes the entire CPY signal sequence has a similar effect: unglycosylated precursor accumulates in cells carrying this mutant gene, and greater than 10% of it is posttranslationally glycosylated. Thus, the amino-terminal signal sequence of CPY, while important for translocation efficiency, is not absolutely required for the translocation of this protein. 相似文献
992.
Functional expression of plastid allophycocyanin genes in a cyanobacterium. 总被引:4,自引:2,他引:2
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In Cyanophora paradoxa, the allophycocyanin apoprotein subunits, alpha and beta, are encoded in the cyanelle (plastid) genome. These genes were transferred to the cyanobacterium Synechococcus sp. PCC 7002 on a plasmid replicon. Phycobilisomes isolated from transformed cyanobacteria were found to contain C. paradoxa allophycocyanin subunits. Thus, these plastid genes are expressed in the cyanobacterium as polypeptides which become linked to a chromophore and are incorporated into the light-harvesting apparatus. 相似文献
993.
R L Stevens K Otsu J H Weis R V Tantravahi K F Austen P A Henkart M C Galli C W Reynolds 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(3):863-868
Rat large granular lymphocyte (LGL) tumor cell lines were analyzed for the presence of proteoglycans and glycosaminoglycans in their cytolytic secretory granules. When isolated rat LGL tumor cells were incubated in vitro for 1 to 3 hr with [35S]sulfate, and the 35S-labeled macromolecules were purified by density-gradient centrifugation, they filtered on Sepharose CL-4B columns predominantly as approximately 500,000 m.w. macromolecules. After 19 hr of incubation with [35S]sulfate, however, an 85,000 m.w. species predominated. Pulse-chase experiments revealed that the larger macromolecules were proteoglycans that with time were processed to glycosaminoglycan-sized macromolecules. As assessed by their susceptibility to chemical and enzymatic degradation and by high pressure liquid chromatography of the chondroitinase ABC-generated unsaturated disaccharides, the cell-associated rat LGL tumor cell proteoglycans bore almost exclusively chondroitin sulfate A glycosaminoglycans. Northern blot analysis using a gene-specific probe revealed that both normal peripheral blood and transformed rat LGL expressed the same approximately 1.3-kb mRNA that encodes the peptide core of the proteoglycans in the secretory granules of rat and mouse mast cells. In vivo radiolabeling of rat LGL tumor cells and isolation of their intact granules after nitrogen cavitation and density sedimentation established that glycosaminoglycans compartmentalized with cytolytic activity. Thus these negatively charged macromolecules may play a role in the regulation of the packaging and delivery of the cytolysins and basically charged serine proteases that have been identified in the cytolytic secretory granules of LGL. 相似文献
994.
M E Rothenberg J P Caulfield K F Austen A Hein K Edmiston P E Newburger R L Stevens 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(8):2616-2625
Basophilic leukocytes from two patients with myelogenous leukemia were enriched to a purity of 10 to 45% by density gradient centrifugation. Ultrastructurally, these basophilic leukocytes contained segmented nuclei and granules with reticular patterns resembling those of normal basophils, and other granules with scroll and grating patterns resembling those of normal connective tissue mast cells. The 35S-labeled macromolecules isolated from these cells were approximately 140,000 m.w. Pronase-resistant proteoglycans bearing approximately 15,000 m.w. glycosaminoglycans. On incubation with chondroitinase ABC, nitrous acid, and heparinase, the 35S-labeled proteoglycans were degraded 50 to 84%, 16 to 43%, and 8 to 37%, respectively, indicating the presence of both chondroitin sulfate and heparin. As assessed by high performance liquid chromatography, the 35S-labeled chondroitin sulfate disaccharides liberated by chondroitinase ABC treatment were approximately 95% monosulfated chondroitin sulfate A and approximately 5% disulfated chondroitin sulfate E. The presence of heparin was confirmed by two-dimensional cellulose acetate electrophoresis of the 35S-labeled glycosaminoglycans. Cell preparations, enriched to 75% basophilic leukocytes by sorting for IgE+ cells, also synthesized 35S-labeled proteoglycans containing chondroitin sulfate and heparin. In one experiment, treatment of the cells with 1 microM calcium ionophore A23187 resulted in a 12% net release of both chondroitin sulfate and heparin containing 35S-labeled proteoglycans, a 57% net release of histamine, and the de novo generation of 8, 8, and 0.16 ng of immunoreactive equivalents of prostaglandin D2, leukotriene C4, and leukotriene B4, respectively, per 10(6) cells. Because only mast cells have been found to contain Pronase-resistant heparin proteoglycans, to generate PGD2 on cell activation, and to contain granules with scroll and grating patterns, these findings indicate that in some patients with myelogenous leukemia there are basophilic cells that possess properties of tissue mast cells. 相似文献
995.
The use of Cephalosporium eichhorniae 152 (ATCC 38255) (reclassified as Acremonium alabamense; see Addendum in Proof), a thermophilic, acidophilic, amylolytic fungus, for the conversion of potato processing wastes into microbial protein for use as animal feed was studied. The fungus was not inhibited by alpha-solanine or beta-2-chaconine, antimicrobial compounds in potatoes, or by morpholine or cyclohexylamine (additives to steam used in the peeling process) at levels likely to be encountered in this substrate. Mixed effluent from holding tanks at a potato-processing plant contained about 10 bacteria per ml and inhibited fungal growth. The fungus grew well on fresh potato wastes containing up to 5% total carbohydrate and utilized both starch and protein at 45 degrees C and pH 3.75. On potato homogenate medium containing 2% carbohydrate (about 14% fresh potato) supplemented with monoammonium phosphate (0.506 g/liter) and ferric iron (0.1 g/liter), with pH control (at 3.75) and additional nitrogen supplied by the automatic addition of ammonium hydroxide, typical yields were 0.61 g (dry weight) of product and 0.3 g of crude protein per g of carbohydrate supplied. An aerobic, spore-forming bacterium, related to Bacillus brevis, commonly contaminated nonsterilized batch cultures but was destroyed by heating for 15 min at 100 degrees C. 相似文献
996.
Intestinal mucosal mast cells from rats infected with Nippostrongylus brasiliensis contain protease-resistant chondroitin sulfate di-B proteoglycans 总被引:15,自引:0,他引:15
R L Stevens T D Lee D C Seldin K F Austen A D Befus J Bienenstock 《Journal of immunology (Baltimore, Md. : 1950)》1986,137(1):291-295
Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [35S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The cell-associated 35S-labeled proteoglycans were extracted from the MMC-enriched cell preparation by the addition of detergent and 4 M guanidine HCl and were partially purified by density gradient centrifugation. The isolated proteoglycans were of approximately 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. Analysis by high-performance liquid chromatography of chondroitinase ABC-treated 35S-labeled proteoglycans from these rat MMC revealed that the chondroitin sulfate chains consisted predominantly of disaccharides with the disulfated di-B structure (IdUA-2SO4----GalNAc-4SO4) and disaccharides with the monosulfated A structure (G1cUA----GalNAc-4SO4). The ratio of disaccharides of the di-B to A structure ranged from 0.4 to 1.6 in three experiments. Small amounts of chondroitin sulfate E disaccharides (GlcUA----GalNAc-4,6-diSO4) were also detected in the chondroitinase ABC digests of the purified rat MMC proteoglycans, but no nitrous acid-susceptible heparin/heparan sulfate glycosaminoglycans were detected. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain such a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched population of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leukemia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans as well as rat serosal mast cell heparin proteoglycans are all highly sulfated, protease-resistant proteoglycans. 相似文献
997.
Location and cloning of the ultraviolet-sensitizing function from the chromosomally associated IncJ group plasmid, R391 总被引:1,自引:0,他引:1
The IncJ plasmid R391, which specifies a uv-sensitizing function, has been shown to be associated with chromosomal DNA. Deletions originating from Tn10 insertion into the kanamycin-resistance determinant of plasmid R391 gave rise to uv-resistant derivatives. This apparent linkage between the kanamycin-resistance determinant and the uv-sensitizing gene(s) was used to clone the uv-sensitizing function from plasmid R391 into pUR222. A recombinant plasmid containing both functions (KanR and Uvs+) was obtained. The uv-sensitizing function was mapped to a 4-kb EcoRI fragment. 相似文献
998.
Certain combinations of alleles at the apterous locus generate wings with extra copies of wing margin structures, some of which are located far from the normal margin. We have examined wing imaginal discs from these mutants, using position-specific antibodies as probes for two-dimensional patterning in the discs. Our results indicate that the adult phenotypes arise from unprecedented disruptions in the two-dimensional pattern of the disc epithelia. Examination of other apterous mutants suggest that pattern alterations may be a general consequence of lesions at this locus. 相似文献
999.
The unicellular cyanobacterium Agmenellum quadruplicatum PR-6 grows in the presence of light on agar containing 10 microM 3-(3,4 dichlorophenyl)-1,1-dimethylurea and 1 to 30 mM glycerol. A derivative strain, PR-6G2, was tolerant of 100 mM glycerol. Photoheterotrophic growth conditions had little effect on transformation competence but did decrease the viability of single cells plated onto agar, particularly cells of the parent strain. 相似文献
1000.
The activity of proteinases in mycelial extracts of Aspergillus nidulans increased during storage. The rate of activation increased with temperature. Three separate proteinase activities, differing in their electrophoretic mobilities on polyacrylamide gels, were readily detected at pH 6.5. Inhibitory activity, effective against all three proteinase activities, was also detected in fractions prepared from fresh mycelial extracts. The inhibitory factor(s) were heat-stable and non-dialysable. The inhibitory activity was lost during storage of mycelial extracts. It is proposed that the inhibitory factor(s) are digested by the proteinases during storage. 相似文献