scholarly journals Construction and Homologous Expression of a Maize Adh1 Based NcoI Cassette Vector

1987 ◽  
Vol 85 (2) ◽  
pp. 327-330 ◽  
Author(s):  
Lisa Lee ◽  
Carmen Fenoll ◽  
Jeffrey L. Bennetzen
Keyword(s):  
Homologous Expression ◽  
Cassette Vector

scholarly journals Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei

2001 ◽  
Vol 268 (24) ◽  
pp. 6498-6507 ◽  
Author(s):  
Johan Karlsson ◽  
Markku Saloheimo ◽  
Matti Siika-aho ◽  
Maija Tenkanen ◽  
Merja Penttilä ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Homologous Expression

Simple strategy for bone regeneration with a BMP-2/7 gene expression cassette vector

2009 ◽  
Vol 390 (3) ◽  
pp. 1012-1017 ◽  
Author(s):  
Mariko Kawai ◽  
Hiroki Maruyama ◽  
Kazuhisa Bessho ◽  
Hiromitsu Yamamoto ◽  
Jun-Ichi Miyazaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Regeneration ◽  
Expression Cassette ◽  
Simple Strategy ◽  
Gene Expression Cassette ◽  
Cassette Vector

Abstract 066: The Undescribed Protein Caskin2 Is a Novel Regulator of eNOS Phosphorylation and Systemic Blood Pressure

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Sarah B Mueller ◽  
Susan B Gurley ◽  
Christopher D Kontos
Keyword(s):  
Blood Pressure ◽  
Molecular Mechanisms ◽  
Systemic Blood Pressure ◽  
Regulatory Subunit ◽  
Systemic Blood ◽  
Homologous Expression ◽  
Ongoing Work ◽  
Inducing Factors

Disruptions in the function of the quiescent endothelial cells (ECs) that line mature vessels can both result in and contribute to the progression of numerous cardiovascular diseases including hypertension, atherosclerosis, and disorders of vascular permeability. Despite recent attention, the signaling pathways that are active in quiescent ECs remain poorly characterized relative to those that regulate EC activation. In an effort to provide mechanistic insight into these pathways, we have characterized the previously undescribed protein Caskin2, which we hypothesize is a novel regulator of EC quiescence. Caskin2 is expressed in ECs throughout the vasculature, including the aorta, coronary arteries, and renal glomeruli. In vitro, Caskin2 promotes a quiescent EC phenotype characterized by decreased proliferation and increased resistance to apoptosis-inducing factors. Caskin2 knockout mice are viable and fertile. However, preliminary radiotelemetry measurements indicate that Caskin2 knockout (KO) mice have mildly elevated systemic blood pressure (BP). Compared to wild type (WT) littermates (n=8), Caskin2 KO mice (n=7) had increased mean arterial pressure (119+/-1 vs. 113+/-1, p=0.012), systolic BP (138+/-2 vs. 132+/-2, p=0.023), and diastolic BP (99+/-1 vs. 93+/-1, p=0.014) at baseline. To explore the molecular mechanisms of Caskin2’s effects, we used mass spectrometry to identify interacting proteins. Among the 67 proteins identified were the Ser/Thr phosphatase protein phosphatase 1 (PP1) and eNOS. Using standard in vitro biochemical techniques, we demonstrated that Caskin2 acts as a PP1 regulatory subunit. Interestingly, homologous expression of Caskin2 in vitro resulted in a marked increase in phosphorylation of eNOS on S1177, which is known to promote eNOS activity, and a decrease in phosphorylation on T495, which is associated with eNOS inhibition. Finally, PP1 has been shown to dephosphorylate eNOS T495 in vitro, suggesting a molecular mechanism for our in vivo findings. Ongoing work aims to determine if the interaction of Caskin2 and PP1 is required for the Caskin2-induced increase in activating phosphorylation of eNOS and to characterize the physiological mechanisms responsible for Caskin2’s effects on BP in more detail.

scholarly journals Engineering and Production of the Light-Driven Proton Pump Bacteriorhodopsin in 2D Crystals for Basic Research and Applied Technologies

2020 ◽  
Vol 3 (3) ◽  
pp. 51
Author(s):  
Mirko Stauffer ◽  
Stephan Hirschi ◽  
Zöhre Ucurum ◽  
Daniel Harder ◽  
Ramona Schlesinger ◽  
...  
Keyword(s):  
Applied Sciences ◽  
Proton Pump ◽  
Genetically Modified ◽  
Functional Characterization ◽  
Basic Research ◽  
Halobacterium Salinarum ◽  
Homologous Expression ◽  
Scientific Disciplines ◽  
2D Crystals ◽  
Purple Membranes

The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.

scholarly journals Biotin Synthesis in Ralstonia eutropha H16 Utilizes Pimeloyl Coenzyme A and Can Be Regulated by the Amount of Acceptor Protein

2020 ◽  
Vol 86 (18) ◽  
Author(s):  
Jessica Eggers ◽  
Carl Simon Strittmatter ◽  
Kira Küsters ◽  
Emre Biller ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Acyl Carrier Protein ◽  
Value Added ◽  
Auxotrophic Mutant ◽  
Bioinformatic Analysis ◽  
Biotechnological Production ◽  
Content Type ◽  
Homologous Expression ◽  
Regulator Protein ◽  
Eukaryotic Microbes

ABSTRACT The biotin metabolism of the Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha (syn. Cupriavidus necator), which is used for biopolymer production in industry, was investigated. A biotin auxotroph mutant lacking bioF was generated, and biotin depletion in the cells and the minimal biotin demand of a biotin-auxotrophic R. eutropha strain were determined. Three consecutive cultivations in biotin-free medium were necessary to prevent growth of the auxotrophic mutant, and 40 ng/ml biotin was sufficient to promote cell growth. Nevertheless, 200 ng/ml biotin was necessary to ensure growth comparable to that of the wild type, which is similar to the demand of biotin-auxotrophic mutants among other prokaryotic and eukaryotic microbes. A phenotypic complementation of the R. eutropha ΔbioF mutant was only achieved by homologous expression of bioF of R. eutropha or heterologous expression of bioF of Bacillus subtilis but not by bioF of Escherichia coli. Together with the results from bioinformatic analysis of BioFs, this leads to the assumption that the intermediate of biotin synthesis in R. eutropha is pimeloyl-CoA instead of pimeloyl-acyl carrier protein (ACP) like in the Gram-positive B. subtilis. Internal biotin content was enhanced by homologous expression of accB, whereas homologous expression of accB and accC2 in combination led to decreased biotin concentrations in the cells. Although a DNA-binding domain of the regulator protein BirA is missing, biotin synthesis seemed to be influenced by the amount of acceptor protein present. IMPORTANCE Ralstonia eutropha is applied in industry for the production of biopolymers and serves as a research platform for the production of diverse fine chemicals. Due to its ability to grow on hydrogen and carbon dioxide as the sole carbon and energy source, R. eutropha is often utilized for metabolic engineering to convert inexpensive resources into value-added products. The understanding of the metabolic pathways in this bacterium is mandatory for further bioengineering of the strain and for the development of new strategies for biotechnological production.

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