scholarly journals Biochemical Characterization of the Drosophila Wingless Signaling Pathway Based on RNA Interference

2004 ◽  
Vol 24 (5) ◽  
pp. 2012-2024 ◽  
Author(s):  
Hiroko Matsubayashi ◽  
Sonoka Sese ◽  
Jong-Seo Lee ◽  
Tadaoki Shirakawa ◽  
Takeshi Iwatsubo ◽  
...  
Keyword(s):  
Rna Interference ◽  
Biochemical Characterization ◽  
The Other ◽  
Protein Levels ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Mediated Priming ◽  
Kinase A

ABSTRACT Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase Iα (CKIα) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKIα and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKIα- or Zw3-mediated Arm phosphorylaytion in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R+ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKIα and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKIα-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKIα-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKIα-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphoylation is due to CKIα and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.

GENETIC LOCALIZATION AND BIOCHEMICAL CHARACTERIZATION OF A TRANS-ACTING REGULATORY EFFECT IN DROSOPHILA

Genetics ◽  
1983 ◽  
Vol 105 (1) ◽  
pp. 55-69
Author(s):  
Joseph J King ◽  
John F McDonald
Keyword(s):  
Drosophila Melanogaster ◽  
Biochemical Characterization ◽  
Regulatory Gene ◽  
Regulatory Effect ◽  
Protein Levels ◽  
The Third ◽  
Genetic Localization ◽  
Glycerophosphate Dehydrogenase

ABSTRACT A region-specific, trans-acting regulatory gene that alters in vivo protein levels of α-glycerophosphate dehydrogenase (α-GPDH) has been mapped to position 55.4 on the third chromosome of Drosophila melanogaster. The gene has been found to affect the in vivo stability of α-GPDH in adult thoracic tissue but has no effect on α-GPDH levels in the abdomen. Although no other thoracic proteins were found to be influenced by the locus, it appears to modify the level of one additional abdominal protein. The action of the gene over development and its possible mode of control are discussed.

Further characterization of the antigen defined by the monoclonal antibody M27

1989 ◽  
Vol 94 (4) ◽  
pp. 725-731
Author(s):  
M.E. Bramwell ◽  
S.M. Humm
Keyword(s):  
Monoclonal Antibody ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Reducing Conditions ◽  
Rat Lymphocytes ◽  
Foetal Liver ◽  
Cultivation In Vitro

Using immunoblotting techniques, the antigen that binds the monoclonal antibody M27 has been clearly defined in terms of apparent molecular mass and distribution. In reducing conditions it has an apparent mass of 178K (K = 10(3) Mr) and is present in the cytoplasm and membranes of all mammalian tissue culture cells so far examined. It is absent from lines derived from avian, piscine and amphibian sources. It is also absent from foetal liver of both rat and mouse, but subsequently appears after cultivation in vitro. Similarly, it can be detected on rat lymphocytes only after mitogenic stimulation. However, it is found on both hepatoma and lymphoma cells in vitro, and on in vivo tumours from murine sources. It thus appears to be associated with cell proliferation.

scholarly journals The cbiS Gene of the Archaeon Methanopyrus kandleri AV19 Encodes a Bifunctional Enzyme with Adenosylcobinamide Amidohydrolase and α-Ribazole-Phosphate Phosphatase Activities

2006 ◽  
Vol 188 (12) ◽  
pp. 4227-4235 ◽  
Author(s):  
Jesse D. Woodson ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
The Other ◽  
Bifunctional Enzyme ◽  
Visible Spectroscopy ◽  
Uv Visible ◽  
Cobyric Acid ◽  
Methanopyrus Kandleri

ABSTRACT Here we report the initial biochemical characterization of the bifunctional α-ribazole-P (α-RP) phosphatase, adenosylcobinamide (AdoCbi) amidohydrolase CbiS enzyme from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri AV19. The cbiS gene encodes a 39-kDa protein with two distinct segments, one of which is homologous to the AdoCbi amidohydrolase (CbiZ, EC 3.5.1.90) enzyme and the other of which is homologous to the recently discovered archaeal α-RP phosphatase (CobZ, EC 3.1.3.73) enzyme. CbiS function restored AdoCbi salvaging and α-RP phosphatase activity in strains of the bacterium Salmonella enterica where either step was blocked. The two halves of the cbiS genes retained their function in vivo when they were cloned separately. The CbiS enzyme was overproduced in Escherichia coli and was isolated to >95% homogeneity. High-performance liquid chromatography, UV-visible spectroscopy, and mass spectroscopy established α-ribazole and cobyric acid as the products of the phosphatase and amidohydrolase reactions, respectively. Reasons why the CbiZ and CobZ enzymes are fused in some archaea are discussed.

Biochemical characterization of Yin Yang 1 – RNA complexes

2008 ◽  
Vol 86 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Zachery R. Belak ◽  
Andrew Ficzycz ◽  
Nick Ovsenek
Keyword(s):  
Rna Binding ◽  
Biochemical Characterization ◽  
Ribonucleoprotein Complexes ◽  
Yin Yang 1 ◽  
Significant Difference ◽  
Yin Yang ◽  
Rna Interaction ◽  
Rna Complexes

YY1 (Yin Yang 1) is present in the Xenopus oocyte cytoplasm as a constituent of messenger ribonucleoprotein complexes (mRNPs). Association of YY1 with mRNPs requires direct RNA-binding activity. Previously, we have shown YY1 has a high affinity for U-rich RNA; however, potential interactions with plausible in vivo targets have not been investigated. Here we report a biochemical characterization of the YY1–RNA interaction including an investigation of the stability, potential 5′-methylguanosine affinity, and specificity for target RNAs. The formation of YY1–RNA complexes in vitro was highly resistant to thermal, ionic, and detergent disruption. The endogenous oocyte YY1–mRNA interactions were also found to be highly stable. Specific YY1–RNA interactions were observed with selected mRNA and 5S RNA probes. The affinity of YY1 for these substrates was within an order of magnitude of that for its cognate DNA element. Experiments aimed at determining the potential role of the 7-methylguanosine cap on RNA-binding reveal no significant difference in the affinity of YY1 for capped or uncapped mRNA. Taken together, the results show that the YY1–RNA interaction is highly stable, and that YY1 possesses the ability to interact with structurally divergent RNA substrates. These data are the first to specifically document the interaction between YY1 and potential in vivo targets.

scholarly journals Morphological and Biochemical Characterization of Macrophages Activated by Carrageenan and Lipopolysaccharide In Vivo

2004 ◽  
Vol 29 (2) ◽  
pp. 27-34 ◽  
Author(s):  
Valéria Pereira Nacife ◽  
Maria de Nazaré Correia Soeiro ◽  
Rachel Novaes Gomes ◽  
Heloísa D’Avila ◽  
Hugo Caire Castro-Faria Neto ◽  
...  
Keyword(s):  
Biochemical Characterization

Biochemical characterization of bona fide polycystin-1 in vitro and in vivo

2001 ◽  
Vol 38 (6) ◽  
pp. 1421-1429 ◽  
Author(s):  
Alessandra Boletta ◽  
Feng Qian ◽  
Luiz F. Onuchic ◽  
Alessandra Bragonzi ◽  
Marina Cortese ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Bona Fide

scholarly journals Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis

1997 ◽  
Vol 328 (3) ◽  
pp. 847-854 ◽  
Author(s):  
E. Margaret TOME ◽  
M. Steven FISER ◽  
M. Claire PAYNE ◽  
W. Eugene GERNER
Keyword(s):  
Ornithine Decarboxylase ◽  
Competitive Inhibitor ◽  
Initiation Factor ◽  
Protective Mechanism ◽  
Eukaryotic Initiation Factor ◽  
Post Translational Modification ◽  
Resistant Variant ◽  
Culture Cells ◽  
Tissue Culture Cells

DH23A cells, an α-difluoromethylornithine-resistant variant of the parental hepatoma tissue culture cells, express high levels of stable ornithine decarboxylase. Aberrantly high expression of ornithine decarboxylase results in a large accumulation of endogenous putrescine and increased apoptosis in DH23A cells when α-difluoromethylornithine is removed from the culture. Treatment of DH23A cells with exogenous putrescine in the presence of α-difluoromethylornithine mimics the effect of drug removal, suggesting that putrescine is a causative agent or trigger of apoptosis. Accumulation of excess intracellular putrescine inhibits the formation of hypusine in vivo, a reaction that proceeds by the transfer of the butylamine moiety of spermidine to a lysine residue in eukaryotic initiation factor 5A (eIF-5A). Treatment of DH23A cells with diaminoheptane, a competitive inhibitor of the post-translational modification of eIF-5A, causes both the suppression of eIF-5A modification in vivo and induction of apoptosis. These data support the hypothesis that rapid degradation of ornithine decarboxylase is a protective mechanism to avoid cell toxicity from putrescine accumulation. Further, these data suggest that suppression of modified eIF-5A formation is one mechanism by which cells may be induced to undergo apoptosis.

scholarly journals Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea

2012 ◽  
Vol 194 (19) ◽  
pp. 5434-5443 ◽  
Author(s):  
H. Tomita ◽  
Y. Yokooji ◽  
T. Ishibashi ◽  
T. Imanaka ◽  
H. Atomi
Keyword(s):  
Coenzyme A ◽  
Biochemical Characterization ◽  
Coenzyme A Biosynthesis ◽  
Kinase A
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