Heterologous expression and initial characterization of recombinant RbcX protein from Thermosynechococcus elongatus BP-1 and the role of RbcX in RuBisCO assembly.

2008 ◽  
Vol 55 (4) ◽  
pp. 777-785 ◽  
Author(s):  
Miroslaw Tarnawski ◽  
Beata Gubernator ◽  
Piotr Kolesinski ◽  
Andrzej Szczepaniak
Keyword(s):  
Soluble Fraction ◽  
Core Complex ◽  
Functional Studies ◽  
Initial Characterization ◽  
Thermophilic Organism ◽  
Dimeric Form ◽  
Rbcs Genes ◽  
Rubisco Subunits

In the cyanobacterial RuBisCO operon from Thermosynechococcus elongatus the rbcX gene is juxtaposed and cotranscribed with the rbcL and rbcS genes which encode large and small RuBisCO subunits, respectively. It has been suggested that the rbcX position is not random and that the RbcX protein could be a chaperone for RuBisCO. In this study, the RbcX protein from T. elongatus was overexpressed, purified and preliminary functional studies were conducted. The recombinant protein purified from Escherichia coli extracts was predominantly present in a soluble fraction in a dimeric form. Coexpression experiments have demonstrated that RbcX can mediate RbcL dimer (L(2)) formation, and that it is essential for the L(8) core complex assembly. This is the first characterization of the RbcX protein from a thermophilic organism.

The role of Golgi bodies in polysaccharide sulphation in Fucuszygotes

1978 ◽  
Vol 32 (1) ◽  
pp. 337-356
Author(s):  
M.E. Callow ◽  
S.J. Coughlan ◽  
L.V. Evans
Keyword(s):  
Cell Wall ◽  
Alginic Acid ◽  
Soluble Fraction ◽  
Amorphous Layer ◽  
Acceptor Molecule ◽  
Golgi Bodies ◽  
Isolation And Characterization ◽  
Fucus Serratus

The cell wall of 24-h zygotes of Fucus serratus is composed of 3 layers—an inner fibrillar layer (sulphated fucan), an outer fibrillar layer (alginic aicd/cellulose) and an exterior amorphous layer (sulphated fucan, alginic acid). The 2 layers containing sulphated fucan are preferentially thickened at the rhizoid pole. Light- and electron-microscope autoradiographic pulse-chase experiments on 22-h zygotes using 35SO2-(4) show the Golgi bodies to be the sites of fucan sulphation. The isolation and characterization of isolated Golgi-rich fractions from 22-h zygotes shows that the first detectable labelled macromolecule is associated with these fractions 2 min after addition of 35SO2-(4). The sulphate acceptor molecule has been partially characterized. 35S-APS and 35S-paps are detectable in the soluble fraction 0.5 min after addition of 35SO2-(4). The results are discussed in relation to other published work on the differentiation of Fucus embryos and on polysaccharide sulphation.

Identification and Partial Characterization of Fanconi Anemia Associated Polypeptides (FAAPs) Using a Versatile Multiprotein-Complex Purification Method.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 989-989 ◽  
Author(s):  
Abdullah M. Ali ◽  
Thiyam R. Singh ◽  
Ruhikanta A. Meetei
Keyword(s):  
Mass Spectrometry ◽  
Cell Lines ◽  
Fanconi Anemia ◽  
Hela Cells ◽  
Affinity Purification ◽  
Gene Products ◽  
Core Complex ◽  
Purification Method

Abstract Fanconi Anemia (FA) is an autosomal recessive and X-linked disorder characterized by congenital abnormalities, progressive bone marrow failure, and a high incidence of hematological (acute leukemia) and non-hematological malignancies (squamous cell carcinomas of the head and neck or gynecologic system). FA is genetically heterogeneous disease and to date 12 complementation groups are known of which 11 gene products have been identified (FANC- A, B, C, D1, D2, E, F, G, J, L, M). Eight of the FA gene products, FANCA, FANCB, FANC, FANCE, FANCF, FANCG, FANCL and FANCM form a multiprotein FA core complex. This complex is required for the monoubiquitination of FANCD2 upon DNA damage by various genotoxic agents. The other two FA proteins; FANCD1/BRCA2 and FANCJ are believed to act “downstream” of FANCD2. In order to understand the role of FA proteins in DNA repair pathway it is necessary to find all the FA genes and their interacting partners. We have established a two-step purification method using 6XHis and FLAG tags for the biochemical and functional characterization of the FA core complex proteins. In an attempt to isolate interacting partners of FANCM and FANCL proteins; we have established two different HeLa cell lines; HeLa-HF-FANCM and HeLa-HF-FANCL, stably expressing HF-FANCM and HF-FANCL recombinant proteins respectively. Two step affinity purification was carried out to isolate the complexes from the extracts prepared from stable cell lines. Two polypeptides, namely, FAAP16 and FAAP100 were identified by mass-spectrometry as major interacting partners of FANCM and FANCL respectively. The interaction of FAAP16 and FAAP100 with other FA core complex proteins was confirmed by reciprocal affinity purification coupled mass-spectrometry using HeLa cells stably expressing HF-FAAP16 and HF-FAAP100 proteins. Furthermore, suppression of FAAP16 and FAAP100 in HeLa cells using siRNA resulted in a reduced MMC-induced FANCD2 monoubiquitination. Studies are being carried out to understand the precise role of these proteins in the FA core complex. These data suggest additional proteins interact with FA core complex members and demonstrate the utility of the purification method in delineating interacting proteins involved in FA.

scholarly journals Phospholipase C in mouse oocytes: characterization of β and γ isoforms and their possible involvement in sperm-induced Ca2+ spiking

1996 ◽  
Vol 316 (2) ◽  
pp. 583-591 ◽  
Author(s):  
Genevieve DUPONT ◽  
Orla M. McGUINNESS ◽  
Martin H. JOHNSON ◽  
Michael J. BERRIDGE ◽  
Franck BORGESE
Keyword(s):  
Monoclonal Antibodies ◽  
Phospholipase C ◽  
Kinase Inhibitors ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Functional Studies ◽  
Mouse Oocytes ◽  
Mature Mouse

This study involved an investigation of the role of phospholipase C (PLC) in generating repetitive Ca2+ spikes at fertilization. Using a PCR-based strategy we have demonstrated that mouse oocytes have mRNA coding for PLCβ1, PLCβ3 and PLCγ isoenzymes. Furthermore, immunodetection of PLCγ1 using monoclonal antibodies reveals that PLCγ1 protein is present in mature mouse oocytes, ruling out the possibility that mRNA was being transcribed but not expressed. We were unsuccessful at detecting the presence of PLCβ protein, but the presence of this isoform can be inferred from functional studies. The PLC inhibitor, U73122, exerted an inhibitory effect on oocytes activated by spermatozoa or acetylcholine at concentrations of 10 and 30 μM respectively, while its inactive analogue had no effect. The soluble tyrosine kinase inhibitors, genistein (100 μM), herbimycin (10 μM) and geldanamycin (0.6 μM) which could affect signalling through PLCγ hindered but never completely inhibited Ca2+ spiking in response to fertilization. We conclude that the activation of PLC to generate InsP3 may play a critical role in fertilization.

scholarly journals Renal nitric oxide production in rat pregnancy: role of constitutive nitric oxide synthases

2010 ◽  
Vol 299 (4) ◽  
pp. F830-F836 ◽  
Author(s):  
Cheryl A. Smith ◽  
Beth Santymire ◽  
Aaron Erdely ◽  
Vasuki Venkat ◽  
György Losonczy ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Renal Cortex ◽  
Soluble Fraction ◽  
Splice Variants ◽  
Normal Pregnancy ◽  
No Production ◽  
Protein Abundance ◽  
Functional Studies ◽  
Enos Protein

Functional studies show that increased renal nitric oxide (NO) mediates the renal vasodilation and increased glomerular filtration rate that occur during normal pregnancy. We investigated whether changes in the constitutive NO synthases (NOS), endothelial (eNOS) and neuronal (nNOS), were associated with the increased renal NO production in normal midterm pregnancy in the rat. In kidneys from midterm pregnant (MP: 11–13 days gestation), late-term pregnant (LP: 18–20 days gestation), and similarly aged virgin (V) rats, transcript and protein abundance for eNOS and the nNOSα and nNOSβ splice variants, as well as the rate of l-arginine-to-l-citrulline conversion, were determined as a measure of NOS activity. At MP, renal cortical abundance of the total eNOS protein and phosphorylated (Ser1177) eNOS was reduced, and l-arginine-to-l-citrulline conversion in the cortical membrane fraction was decreased; these declines were also seen in LP. There were no changes in the eNOS transcript. In contrast, l-arginine-to-l-citrulline conversion in the soluble fraction of renal cortex increased at MP and then declined at LP. This MP increase was ablated by S-methylthiocitrulline, a nNOS inhibitor. Using Western blotting, we did not detect a change in the protein abundance or transcript of the 160-kDa nNOSα, but protein abundance and transcript of the nNOSβ were increased at MP in cortex. Collectively, these studies suggest that the soluble nNOSβ is responsible for the increased renal cortical NO production during pregnancy.

scholarly journals Short Communication: Identification of Mildew Locus O (MLO) genes in Durio zibethinus genome corresponding with the Powdery Mildew disease

2018 ◽  
Vol 19 (6) ◽  
pp. 2204-2212
Author(s):  
RAHMAT AZHARI KEMAL ◽  
ERIC BERNARDUS L. SANDJAJA ◽  
AUDI PUTRA SANTOSA ◽  
JEREMIAS IVAN
Keyword(s):  
Powdery Mildew ◽  
Short Communication ◽  
Functional Characterization ◽  
Functional Studies ◽  
Durio Zibethinus ◽  
Common Motif ◽  
Powdery Mildew Disease ◽  
Intracellular Domains

Kemal RA, Sandjaja EBL, Santosa AP, Ivan J. 2018. Short Communication: Identification of Mildew Locus O (MLO) genes in Durio zibethinus genome corresponding with the Powdery Mildew disease. Biodiversitas 19: 2204-2212. Mildew Locus O (MLO) is a protein consisting of seven transmembrane domains and appears in the various type of plants. MLO proteins are classified into seven clades. It is known that specific clades have different roles in a plant. MLOs from Clades IV and V have been linked to plant's susceptibility to Powdery Mildew (PM) disease. This study aimed to provide an overview of MLO genes present in durian (Durio zibethinus) genome. Bioinformatic analyses were conducted to analyze the phylogeny and structure of MLO genes and proteins in durian. The result showed that there were 20 putative DzMLO genes in durian, encoding 39 putative DzMLO proteins. Durian MLOs belong to Clade I-VI with one protein belongs to Clade IV and five proteins belong to Clade V. Those six MLO proteins shared a common motif in C-terminal and second intracellular domains. Putative alternative splicing and differential expressions were observed among Clade V DzMLO genes. These findings will facilitate the functional characterization of MLO genes and proteins in durian. Functional studies, especially on C-terminal and second intracellular domains, need to be conducted to elucidate the role of MLO in PM susceptibility in durian.

Variations in the Photoluminescence Intensity of Chemically and Anodically Etched Silicon Films

1993 ◽  
Vol 298 ◽  
Author(s):  
P.S Williams ◽  
J.N. Kidder ◽  
H. Yun ◽  
D. Crain ◽  
T.P. Pearsall
Keyword(s):  
Luminescent Properties ◽  
Silicon Films ◽  
Photoluminescence Intensity ◽  
Ambient Conditions ◽  
X Ray ◽  
Initial Characterization ◽  
Etched Silicon ◽  
Free Films

AbstractWe are studying the effects of etch conditions on the surface morphology, chemistry, and luminescent properties of porous silicon (PS) films. Luminescent silicon films are produced by chemical etching using solutions of HNO3 in HF and by anodic etching using aqueous HF electrolytes. Films produced by both methods are analyzed and compared using photoluminescence (PL), vibrational, and X-ray photoelectron (XPS) spectroscopies. The initial characterization of PS is performed immediately following the etching process, resulting in oxide-free films (as confirmed by XPS). In chemically etched PS films, the luminescent intensity decreases as the vol. % HNO3 in etch solution increases. Spectral features evolve in the PL spectrum of chemically etched films as the result of aging under ambient conditions and when the films are cooled under illumination. Moreover, we have also found that increased electrolyte convection results in a decrease in photoluminescence intensity of PS films formed anodically. The role of electrolyte flow in modifying the luminescent properties of PS is being evaluated in an etch cell with well-characterized hydrodynamics.

scholarly journals Identification of the maize Mediator CDK8 module, and transposon-mediated mutagenesis of ZmMed12a

2020 ◽  
Vol 52 ◽  
Author(s):  
Ana Laura Alonso-Nieves ◽  
Tania Núñez-Ríos ◽  
Julio A. Massange-Sánchez ◽  
Kevin R. Ahern ◽  
Daniel Lepe-Soltero ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Insertional Mutagenesis ◽  
Single Copy ◽  
Functional Studies ◽  
Cyclin C ◽  
Transposon Insertions ◽  
Nutrient Homeostasis

Background: Mediator is a conserved transcriptional co-activator that links transcription factors bound at enhancer elements to RNA Polymerase II. Mediator-RNA Polymerase II interactions can be sterically hindered by the Cyclin Dependent Kinase 8 (CDK8) module, a submodule of Mediator that acts to repress transcription in response to discrete cellular and environmental cues. The CDK8 module is conserved in all eukaryotes and consists of 4 proteins: CDK8, CYCLIN C (CYCC), MED12, and MED13. Methods: In this study, we have characterized the CDK8 module of Mediator in maize using genomic, molecular and functional resources. Results: The maize genome contains single copy genes for Cdk8, CycC, and Med13, and two genes for Med12. Analysis of expression data for the CDK8 module demonstrated that all five genes are broadly expressed in maize tissues, and change their expression in response to phosphate and nitrogen limitation. We performed Dissociation (Ds) insertional mutagenesis, recovering two independent insertions in the ZmMed12a gene, one of which produces a truncated transcript. Conclusions: Our molecular identification of the maize CDK8 module, assays of CDK8 module expression under nutrient limitation, and characterization of transposon insertions in ZmMed12a establish the basis for molecular and functional studies of the role of these important transcriptional regulators in development and nutrient homeostasis in Zea mays.

Digital x-ray mapping of nanometer-size supported bimetallic catalysts

1988 ◽  
Vol 46 ◽  
pp. 530-531
Author(s):  
L. T. Germinario
Keyword(s):  
Background Radiation ◽  
Metal Cluster ◽  
Single Element ◽  
Beam Diameter ◽  
X Rays ◽  
X Ray ◽  
Major Interest ◽  
Induced Changes

Understanding the role of metal cluster composition in determining catalytic selectivity and activity is of major interest in heterogeneous catalysis. The electron microscope is well established as a powerful tool for ultrastructural and compositional characterization of support and catalyst. Because the spatial resolution of x-ray microanalysis is defined by the smallest beam diameter into which the required number of electrons can be focused, the dedicated STEM with FEG is the instrument of choice. The main sources of errors in energy dispersive x-ray analysis (EDS) are: (1) beam-induced changes in specimen composition, (2) specimen drift, (3) instrumental factors which produce background radiation, and (4) basic statistical limitations which result in the detection of a finite number of x-ray photons. Digital beam techniques have been described for supported single-element metal clusters with spatial resolutions of about 10 nm. However, the detection of spurious characteristic x-rays away from catalyst particles produced images requiring several image processing steps.

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