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Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme (1993)

Avila, Concepción ; Márquez, Antonio J. ; Pajuelo, Purificación ; [et al.]

Springer

Planta 189 (1993), S. 475-483

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ISSN: 1432-2048

Keywords: Amidotransferase ; Amino-terminal sequences ; Chromosomal assignment ; Glutamate synthase ; Hordeum (mutants) ; Photorespiration mutants (barley) ; Polymerase chain reaction

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract The NH2-terminal sequences of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) purified from barley (Hordeum vulgare L.) and Chlamydomonas reinhardtii (Dangeard), and of a barley peptide, were determined and the barley sequences were used to design oligonucleotide primers for the polymerase chain reaction. A specific 1.3-kilobase (kb) cDNA fragment specifying the NH2-terminal one-third of the mature barley polypeptide, was amplified, cloned and sequenced. The NH2-terminus of plant Fd-GOGAT is highly conserved and homologous to the NH2-terminus of the heavy subunit of Escherichia coli NADPH-GOGAT. Based on sequence homologies, we tentatively identified the NH2-terminal region of Fd-GOGAT as the glutamine-amidotransferase domain, which is related to the corresponding domain of the purF-type amidotransferases. The Fd-GOGAT cDNA clone, and polyclonal antibodies raised against the barley enzyme, were used to analyse four Fd-GOGAT-deficient photorespiratory mutants. Three mutants (RPr 82/1, RPr 82/9 and RPr 84/82) had no detectable Fd-GOGAT protein in leaves, while the fourth (RPr 84/42) had a small amount of cross-reacting material. Hybridization to Northern blots of total leaf RNA revealed that both RPr 82/9 and RPr 84/82 were indistinguishable from the parental line (Maris Mink), having normal amounts of a 5.7-kb mRNA species. On the other hand, RPr 82/2 and RPr 84/42 each contained two distinct hybridizing RNA species, one of which was larger than 5.7 kb, the other smaller. Using a set of wheat-barley telosomic addition lines we have assigned the Fd-GOGAT structural locus to the short arm of chromosome 2.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00198209

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Human prostate cancer model: Roles of growth factors and extracellular matrices (1992)

Chung, Leland W.K. ; Li, Wei ; Gleave, Martin E. ; [et al.]

New York, N.Y. : Wiley-Blackwell

Journal of Cellular Biochemistry 50 (1992), S. 99-105

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ISSN: 0730-2312

Keywords: extracellular matrices (ECMs) ; bFGF ; NGF ; HGF and KGF ; growth factors (GFs) ; human prostate cancer model ; prostate cancer-bone interaction ; stromal-epithelial interaction ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: A human prostate cancer model was established by inoculating a prostate specific antigen (PSA)-producing LNCaP cell line with either prostate or bone fibroblasts. Alternatively, this human prostate cancer model can also be established by inoculating LNCaP cell with growth factor(s) (GFs) and extracellular matrix (ECM) immobilized on Gelfoam®. The resulting LNCaP tumors were used to evaluate PSA production and excretion athymic hosts. This model was also employed to examine the biochemical nature of mesenchymal cell-derived growth-promoting protein(s) and to assess the efficacy of potential chemotherapeutic agents. Because of the propensity of human prostate cancer to metastasize to the bone, this study defined a 1.0 M NaCI-eluted fraction, MS1, from the conditioned medium of a bone stromal cell line (MS) by heparin-affinity column chromatography. The growth-promoting activity was assayed both in vivo (e.g., tumor formation) and in vitro (e.g., soft agar colony formation). We found that the growth-promoting activity was trypsin-and heat-sensitive, and partially degraded by acid and dithiothreitol. Immunochemical studies indicated that the polyclonal antibody raised against MS1 blocked the growth-promoting effect elicited by the bone-conditioned media. This growth-promoting factor was found to be immunochemically dissimilar to KGF, HGF, and bFGF. However, addition of bFGF, HGF and NGF, but not a FGF, TGFβ, IGF1, IGF2, PDGF, EGF, TGFα and KGF, stimulated anchorage-independent growth of prostate cells, a condition closely parallel to tumor formation in vivo. We found that the MS1 fraction also contained fibronectin and tenascin but not laminin or collagen IV. None of the ECM proteins induced soft agar colony formation by normal prostate epithelial cells. Therefore, it is possible that the ECM protein(s) may potentiate the tumor-inducing activity of locally produced GFs. © 1992 Wiley-Liss, Inc.

Additional Material: 5 Ill.