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61.
The expression of p21ras proteins was investigated by immunocytochemistry in permanent cell lines and in fresh human leukaemic cells. While high and low levels of p21ras could be detected in most of the cell lines, no significant p21ras immunoreactivity was noted in cells of ten human acute and chronic leukaemias. Thus, notwithstanding its possible role in the initial transformation process in human leukaemias, p21ras expression appears not to be an irrevocable requirement for the maintenance of the transformed state. 相似文献
62.
Immunological detection of phytoene desaturase in algae and higher plants using an antiserum raised against a bacterial fusion-gene construct 总被引:3,自引:0,他引:3
A Schmidt G Sandmann G A Armstrong J E Hearst P B?ger 《European journal of biochemistry》1989,184(2):375-378
Immunological characterization of phytoene desaturase, a key enzyme of carotenoid biosynthesis, is reported. For this purpose, a phytoene-desaturase fusion protein has been employed. For its construction 921 base pairs of the crtI gene were fused to the N-terminal region of the Escherichia coli lacZ gene. Plasmid pGABX2 resulted from insertion of a BglI - XhoI fragment from the Rhodobacter capsulatus carotenoid biosynthesis gene cluster, carrying the crtI, crtA and crtB genes, into pBR322. A 968-base-pair SalI-fragment from pGABX2 was cloned into pUR288 at the 3' end of the lacZ gene. Isopropyl-beta-D-thio-galactopyranoside-dependent activation of the lacZ fusion gene resulted in expression of a stable 150-kDa protein. After electroelution from SDS/polyacrylamide slab gels, the protein was used for antibody production. The heterogenic antiserum obtained was tested by Western blotting against proteins from Rhodobacter capsulatus and several different photoautotrophic organisms including higher plants. Apparent molecular masses of immunoreactive proteins from Rhodobacter, Aphanocapsa, rape and spinach were around 64 kDa. In Bumilleriopsis a 55-kDa protein was found instead. The antibody also inhibited in vitro desaturation of phytoene when detergent-solubilized membranes were employed. 相似文献
63.
Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6-3H]glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H-NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N-terminal amino acid sequencing. The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5-8 mannose residues as well as small amounts of hybrid-type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N-acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(alpha 1-3) substituents and/or sulfate groups linked to position six of beta-galactosyl residues forming NeuAc(alpha 2-3)[HO3S-6]Gal(beta 1-4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (alpha 2-3)- or (alpha 2-6)-linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated. 相似文献
64.
Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis. 总被引:1,自引:0,他引:1 下载免费PDF全文
To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates. 相似文献
65.
B Mayer K Schmidt P Humbert E B?hme 《Biochemical and biophysical research communications》1989,164(2):678-685
In the presence of porcine aortic endothelial cytosol, soluble guanylyl cyclase purified from bovine lung was activated by L-arginine up to 2.5-fold, with an EC50 of about 6 microM. This activation was dependent on NADPH and Ca2+. The EC50 for Ca2+ was about 60 nM. No effect of L-arginine on guanylyl cyclase was observed when the cytosolic proteins were heat-denaturated. The effect of L-arginine was inhibited by NG-monomethyl-L-arginine and hemoglobin. These results indicate that endothelial cells contain a cytosolic enzyme which is directly or indirectly regulated by Ca2+ and converts L-arginine into a compound which in stimulating soluble guanylyl cyclase behaves similar to endothelium-derived relaxing factor. 相似文献
66.
J Schmidt M Aubele U Jütting K Rodenacker A Luz V Erfle G Burger 《Biochemical and biophysical research communications》1989,164(2):728-735
Established osteoblast-like (OB) cells infected with the bone tumor-inducing C-type retrovirus OA MuLV remained nontumorigenic over 104 cell culture passages. DNA histograms revealed a new cell population with a stem line peak at 5c. A second OA MuLV-infected OB cell line underwent neoplastic transformation with increasing passage level. These cells showed diffuse aneuploidy. Stepwise linear discriminant analysis of the chromatin structure of control, OA MuLV-infected, and FBR osteosarcoma virus-transformed cell lines resulted in various levels of discrimination ranging between 79.6% for control cells versus nontumorigenic OA MuLV-infected cells, and 96.6% for nontumorigenic OA MuLV-infected cells versus FBR osteosarcoma virus-transformed cells. OA MuLV-infected tumorigenic cells and FBR osteosarcoma virus-transformed cells were discriminated at a 93.6% level. 相似文献
67.
E D Schmidt H C van Beeren H Korfage J H Dussault W M Wiersinga W H Lamers 《Biochemical and biophysical research communications》1989,164(3):1053-1059
Monoclonal antibodies were raised against the nuclear thyroid hormone receptors encoded by c-ERB A genes and against a purified nuclear receptor fraction. These antibodies recognize the c-ERB A protein in nuclear extracts from rat liver and are able to compete with thyroid hormone in Scatchard analyses. In sections of rat liver they react with all the hepatocyte nuclei as well as with the cells of the hepatic bile ducts. Comparison with another putative T3 receptor antibody, described previously, showed that distinct 57 kD proteins with a different cellular distribution were recognized. 相似文献
68.
The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is the preferred substrate for the microsomal methyltransferase. Other nuclear double bond isomers support reduced rates of methenylation, while sterols which bear methyl groups at C-4 or C-14 are not substrates. Initial velocity and product inhibition studies are consistent with a rapid equilibrium ordered kinetic mechanism. A series of novel sterol analogues which contain heteroatoms substituted for C-24 or C-25 have been kinetically characterized as dead-end inhibitors of the methyltransferase, revealing three distinct mechanisms of interaction with the enzyme. Sterols which contain positively charged moieties in these positions are particularly potent inhibitors, supporting the proposed intermediacy of C-24 and C-25 carbocations. The methyltransferase is reversibly inhibited by low concentrations of 24-thiasterols, while behavior consistent with mechanism-based enzyme inactivation is apparent at higher concentrations. Possible mechanisms for this novel inactivation reaction are discussed. 相似文献
69.
G H Schmidt R Mead 《BioEssays : news and reviews in molecular, cellular and developmental biology》1990,12(1):37-40
Clonal studies of adult chimaeric mouse epithelium have demonstrated the monoclonal composition of crypts of Lieberkühn. In neonatal life, however, polyclonal crypts have been found, indicating that crypts are of polyclonal origin. We here relate these findings to studies of mosaic tissues which have addressed the question whether solid tumours are of monoclonal or polyclonal origin. The issues has so far remained unresolved because the expected frequencies of polyclonal tumours, given polyclonal origins, have not previously been estimated. A general approach for the calculation of such expected values is suggested. The consistent reports of tumours with polyclonal components suggest that autocrine or paracrine mechanisms play an important role during tumorigenesis. 相似文献
70.
Graham Pawelec Gerhard Ehninger Helmuth Schmidt Claudia Müller Hans-Jörg Bühring Markus Reutter Friedrich W. Busch 《Cancer immunology, immunotherapy : CII》1990,32(3):167-172
Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14+ and CD33+ cells but to a reduction in CD34+ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14+ and CD33+ as well as CD34+ cells but in a significant increase in CD3+ and CD56+ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120) 相似文献