The non-catalytic domain of the Xenopus laevis auroraA kinase localises the protein to the centrosome

2001 ◽  
Vol 114 (11) ◽  
pp. 2095-2104
Author(s):  
Régis Giet ◽  
Claude Prigent
Keyword(s):  
Xenopus Laevis ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Terminal Domain ◽  
Egg Extracts ◽  
Green Fluorescent ◽  
Xenopus Egg Extracts

Aurora kinases are involved in mitotic events that control chromosome segregation. All members of this kinase subfamily possess two distinct domains, a highly conserved catalytic domain and an N-terminal non-catalytic extension that varies in size and sequence. To investigate the role of this variable non-catalytic region we overexpressed and purified Xenopus laevis auroraA (pEg2) histidine-tagged N-terminal peptide from bacterial cells. The peptide has no effect on the in vitro auroraA kinase activity, but it inhibits both bipolar spindle assembly and stability in Xenopus egg extracts. Unlike the full-length protein, the N-terminal domain shows only low affinity for paclitaxel-stabilised microtubules in vitro, but localises to the centrosomes in a microtubule-dependent manner. When expressed in Xenopus XL2 cells, it is able to target the green fluorescent protein to centrosomes. Surprisingly, this is also true of the pEg2 catalytic domain, although to a lesser extent. The centrosome localisation of the N-terminal peptide was disrupted by nocodazole whereas localisation of the catalytic domain was not, suggesting that in order to efficiently localise to the centrosome, pEg2 kinase required the non-catalytic N-terminal domain and the presence of microtubules.

scholarly journals Modulation of Virulence by Two Acidified Nitrite-Responsive Loci of Salmonella enterica Serovar Typhimurium

2003 ◽  
Vol 71 (6) ◽  
pp. 3196-3205 ◽  
Author(s):  
Charles C. Kim ◽  
Denise Monack ◽  
Stanley Falkow
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner ◽  
Inducible Promoters ◽  
Serovar Typhimurium ◽  
Bacterial Genes ◽  
Green Fluorescent

ABSTRACT Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. The nipAB promoter was regulated by oxygen in an Fnr-dependent manner. The nipC promoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.

scholarly journals Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate

2000 ◽  
Vol 182 (11) ◽  
pp. 3254-3258 ◽  
Author(s):  
D. K. Stafslien ◽  
P. P. Cleary
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
High Sensitivity ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Group B Streptococci ◽  
Mutant Proteins ◽  
Green Fluorescent

ABSTRACT A glutathione-S-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca2+, Mg2+, and Mn2+ but was inhibited by the same concentrations of Zn2+. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp130-Ala, His193-Ala, Asn295-Ala, and Ser512-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), and recombinant SCPB, fromStreptococcus agalactiae (group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP−1liter−1 min−1.

Activity of Cardiorespiratory Networks Revealed by Transsynaptic Virus Expressing GFP

2001 ◽  
Vol 85 (1) ◽  
pp. 435-438 ◽  
Author(s):  
Mustapha Irnaten ◽  
Robert A. Neff ◽  
Jijiang Wang ◽  
Arthur D. Loewy ◽  
Thomas C. Mettenleiter ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Nucleus Ambiguus ◽  
Dependent Manner ◽  
Neural Basis ◽  
Electrophysiological Properties ◽  
Functional Studies ◽  
Neuronal Systems ◽  
Green Fluorescent

A fluorescent transneuronal marker capable of labeling individual neurons in a central network while maintaining their normal physiology would permit functional studies of neurons within entire networks responsible for complex behaviors such as cardiorespiratory reflexes. The Bartha strain of pseudorabies virus (PRV), an attenuated swine alphaherpesvirus, can be used as a transsynaptic marker of neural circuits. Bartha PRV invades neuronal networks in the CNS through peripherally projecting axons, replicates in these parent neurons, and then travels transsynaptically to continue labeling the second- and higher-order neurons in a time-dependent manner. A Bartha PRV mutant that expresses green fluorescent protein (GFP) was used to visualize and record from neurons that determine the vagal motor outflow to the heart. Here we show that Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties and that the labeled baroreflex pathways that control heart rate are unaltered by the virus. This novel transynaptic virus permits in vitro studies of identified neurons within functionally defined neuronal systems including networks that mediate cardiovascular and respiratory function and interactions. We also demonstrate superior laryngeal motorneurons fire spontaneously and synapse on cardiac vagal neurons in the nucleus ambiguus. This cardiorespiratory pathway provides a neural basis of respiratory sinus arrhythmias.

scholarly journals Natural Competence Rates Are Variable Among Xylella fastidiosa Strains and Homologous Recombination Occurs In Vitro Between Subspecies fastidiosa and multiplex

2017 ◽  
Vol 30 (7) ◽  
pp. 589-600 ◽  
Author(s):  
Prem P. Kandel ◽  
Rodrigo P. P. Almeida ◽  
Paul A. Cobine ◽  
Leonardo De La Fuente
Keyword(s):  
Homologous Recombination ◽  
Fluorescent Protein ◽  
Xylella Fastidiosa ◽  
Dna Recombination ◽  
Bacterial Cells ◽  
Disease Emergence ◽  
Natural Competence ◽  
In Silico Studies ◽  
Green Fluorescent

Xylella fastidiosa, an etiological agent of emerging crop diseases around the world, is naturally competent for the uptake of DNA from the environment that is incorporated into its genome by homologous recombination. Homologous recombination between subspecies of X. fastidiosa was inferred by in silico studies and was hypothesized to cause disease emergence. However, no experimental data are available on the degree to which X. fastidiosa strains are capable of competence and whether recombination can be experimentally demonstrated between subspecies. Here, using X. fastidiosa strains from different subspecies, natural competence in 11 of 13 strains was confirmed with plasmids containing antibiotic markers flanked by homologous regions and, in three of five strains, with dead bacterial cells used as source of donor DNA. Recombination frequency differed among strains and was correlated to growth rate and twitching motility. Moreover, intersubspecific recombination occurred readily between strains of subsp. fastidiosa and multiplex, as demonstrated by movement of antibiotic resistance and green fluorescent protein from donor to recipient cells and confirmed by DNA sequencing of the flanking arms of recombinant strains. Results demonstrate that natural competence is widespread among X. fastidiosa strains and could have an impact in pathogen adaptation and disease development.

scholarly journals Obtaining a fluorescently labeled endophytic strain of bacteria Herbaspirillum lusitanum P6-12 for their detection in vivo and in vitro

2021 ◽  
Vol 21 (3) ◽  
pp. 286-291
Author(s):  
Arapat Rustamovna Bagavova ◽  
◽  
Natal’ya S. Velichko ◽  
Timofey E. Pylayev ◽  
Yuliya P. Fedonenko ◽  
...  
Keyword(s):  
Recombinant Strain ◽  
Fluorescent Protein ◽  
Biochemical Properties ◽  
Growth Media ◽  
Bacterial Cells ◽  
Vector Plasmid ◽  
Green Fluorescent ◽  
Model Strain

The Herbaspirillum lusitanum P6-12 strain containing the vector plasmid pJN105TurboGFP, which encodes the synthesis of the green fluorescent protein GFP, and which has resistance to the antibiotic gentamicin, was obtained by electroporation. The constructed strain of H. lusitanum P6-12 in cultural, morphological and biochemical properties did not differ from the original typical natural strain of H. lusitanum P6-12. On solid growth media, the recombinant strain formed yellow-green colonies, fluorescent under UV irradiation. Upon inoculation with the resulting culture of plant objects, a green glow of the marked H. lusitanum P6-12 cells, actively colonizing the internal tissues of the host plant, was observed. The created strain can be used as a model strain for studying the patterns and characteristics of the behaviour of organisms in integrated systems, including for tracking bacterial cells during interaction with plants, assessing their survival, competitiveness, etc.

scholarly journals Salicylic Acid Activates Sigma Factor B by rsbU-Dependent and -Independent Mechanisms

2006 ◽  
Vol 188 (16) ◽  
pp. 5896-5903 ◽  
Author(s):  
Marco Palma ◽  
Arnold Bayer ◽  
Leon I. Kupferwasser ◽  
Tammy Joska ◽  
Michael R. Yeaman ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Parental Strain ◽  
Fluorescent Protein ◽  
Toxin Production ◽  
Dependent Manner ◽  
Alpha Toxin ◽  
Reporter System ◽  
Fibronectin Binding ◽  
Green Fluorescent

ABSTRACT Salicylic acid (SAL) may impact Staphylococcus aureus virulence by activating the sigB operon (rsbU-V-W-sigB), thus leading to reductions in alpha-toxin production and decreased fibronectin binding (L. I. Kupferwasser et al., J. Clin. Investig. 112:222-233, 2003). As these prior studies were performed in strain RN6390 (an rsbU mutant) and its rsbU-repaired variant, SH1000, the current investigation was designed to determine if the SAL effect occurs via rsbU- and/or rsbV-dependent pathways in an rsbU-intact S. aureus strain (FDA486). We thus quantified the transcription from two sigB-dependent promoters (asp23 and sarA P3) in FDA486 in response to SAL exposure in vitro, using isogenic single-knockout constructs of rsbU, rsbV, or rsbW and a green fluorescent protein reporter system. SAL induced sarA P3 and asp23 promoter activities in a dose-dependent manner in the parental strain. In contrast, sigB activation by SAL was progressively more mitigated in the rsbU and rsbV mutants. As predicted, SAL caused significant reductions in both alpha-toxin production and fibrinogen and fibronectin binding in the parental strain. The extent of these reductions, compared with the parent, was reduced in the rsb mutants (rsbV > rsbU), especially at low SAL concentrations. Since generation of the free SigB protein usually requires a sequential rsbU-V-W-sigB activation cascade, the present phenotypic and genotypic data suggest key roles for both rsbU and rsbV in SAL-mediated activation of sigB in strains with a fully intact sigB operon.

scholarly journals Oncostatin M induces IL-33 expression in liver endothelial cells in mice and expands ST2+CD4+lymphocytes

2015 ◽  
Vol 309 (7) ◽  
pp. G542-G553 ◽  
Author(s):  
Muhammad Imran Arshad ◽  
Pierre Guihard ◽  
Yannic Danger ◽  
Gregory Noel ◽  
Jacques Le Seyec ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fluorescent Protein ◽  
Vascular Endothelial Cells ◽  
Oncostatin M ◽  
Dependent Manner ◽  
Liver Sinusoidal Endothelial Cells ◽  
Liver Endothelial Cells ◽  
Green Fluorescent

Interleukin (IL)-33 is crucially involved in liver pathology and drives hepatoprotective functions. However, the regulation of IL-33 by cytokines of the IL-6 family, including oncostatin M (OSM) and IL-6, is not well studied. The aim of the present study was to determine whether OSM mediates regulation of IL-33 expression in liver cells. Intramuscular administration in mice of an adenovirus encoding OSM (AdOSM) leads to increase in expression of OSM in muscles, liver, and serum of AdOSM-infected mice compared with control mice. The increase of circulating OSM markedly regulated mRNA of genes associated with blood vessel biology, chemotaxis, cellular death, induction of cell adhesion molecules, and the alarmin cytokine IL-33 in liver. Steady-state IL-33 mRNA was upregulated by OSM at an early phase (8 h) following AdOSM infection. At the protein level, the expression of IL-33 was significantly induced in liver endothelial cells [liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells] with a peak at 8 days post-AdOSM infection in mice. In addition, we found OSM-stimulated human microvascular endothelial HMEC-1 cells and human LSEC/TRP3 cells showed a significant increase in expression of IL-33 mRNA in a dose-dependent manner in cell culture. The OSM-mediated overexpression of IL-33 was associated with the activation/enrichment of CD4+ST2+cells in liver of AdOSM-infected mice compared with adenovirus encoding green fluorescent protein-treated control mice. In summary, these data suggest that the cytokine OSM regulates the IL-33 expression in liver endothelial cells in vivo and in HMEC-1/TRP3 cells in vitro and may specifically expand the target CD4+ST2+cells in liver.

scholarly journals A Novel Role for Cdk1/Cyclin B in Regulating B-Raf Activation at Mitosis

2008 ◽  
Vol 19 (7) ◽  
pp. 2907-2915 ◽  
Author(s):  
Sergiy I. Borysov ◽  
Thomas M. Guadagno
Keyword(s):  
Mapk Cascade ◽  
Cyclin B ◽  
Dependent Manner ◽  
Mapk Activity ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Subsequent Activation ◽  
Xenopus Egg Extracts

MAPK activity is important during mitosis for spindle assembly and maintenance of the spindle checkpoint arrest. We previously identified B-Raf as a critical activator of the MAPK cascade during mitosis in Xenopus egg extracts and showed that B-Raf activation is regulated in an M-phase–dependent manner. The mechanism that mediates B-Raf activation at mitosis has not been elucidated. Interestingly, activation of 95-kDa B-Raf at mitosis does not require phosphorylation of Thr-599 and Ser-602 residues (Thr-633 and Ser-636 in Xenopus B-Raf), previously shown to be essential for B-Raf activation by Ras. Instead, we provide evidence for Cdk1/cyclin B in mediating mitotic activation of B-Raf. In particular, Cdk1/cyclin B complexes associate with B-Raf at mitosis in Xenopus egg extracts and contribute to its phosphorylation. Mutagenesis and in vitro kinase assays demonstrated that Cdk1/cyclin B directly phosphorylates B-Raf at Serine-144, which is part of a conserved Cdk1 preferential consensus site (S144PQK). Importantly, phosphorylation of Ser-144 is absolutely required for mitotic activation of B-Raf and subsequent activation of the MAPK cascade. However, substitution of a phospho-mimicking amino acid at Ser-144 failed to produce a constitutive active B-Raf indicating that, in addition of Ser-144 phosphorylation, other regulatory events may be needed to activate B-Raf at mitosis. Taken together, our data reveal a novel cell cycle mechanism for activating the B-Raf/MEK/MAPK cascade.

scholarly journals An in vitro nuclear disassembly system reveals a role for the RanGTPase system and microtubule-dependent steps in nuclear envelope breakdown

2007 ◽  
Vol 178 (4) ◽  
pp. 595-610 ◽  
Author(s):  
Petra Mühlhäusser ◽  
Ulrike Kutay
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Dynamic Rupture ◽  
Vitro Assay ◽  
Nuclear Envelope Breakdown ◽  
Egg Extracts ◽  
Guanosine Triphosphatase ◽  
Green Fluorescent

During prophase, vertebrate cells disassemble their nuclear envelope (NE) in the process of NE breakdown (NEBD). We have established an in vitro assay that uses mitotic Xenopus laevis egg extracts and semipermeabilized somatic cells bearing a green fluorescent protein–tagged NE marker to study the molecular requirements underlying the dynamic changes of the NE during NEBD by live microscopy. We applied our in vitro system to analyze the role of the Ran guanosine triphosphatase (GTPase) system in NEBD. Our study shows that high levels of RanGTP affect the dynamics of late steps of NEBD in vitro. Also, inhibition of RanGTP production by RanT24N blocks the dynamic rupture of nuclei, suggesting that the local generation of RanGTP around chromatin may serve as a spatial cue in NEBD. Furthermore, the microtubule-depolymerizing drug nocodazole interferes with late steps of nuclear disassembly in vitro. High resolution live cell imaging reveals that microtubules are involved in the completion of NEBD in vivo by facilitating the efficient removal of membranes from chromatin.

scholarly journals A New Focal Adhesion Protein That Interacts with Integrin-Linked Kinase and Regulates Cell Adhesion and Spreading

2001 ◽  
Vol 153 (3) ◽  
pp. 585-598 ◽  
Author(s):  
Yizeng Tu ◽  
Yao Huang ◽  
Yongjun Zhang ◽  
Yun Hua ◽  
Chuanyue Wu
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Cytoskeleton Organization ◽  
Terminal Domain ◽  
Integrin Linked Kinase ◽  
Ch2 Domain ◽  
Green Fluorescent

Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. We report here the identification and characterization of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP). CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH2-terminal domain of ILK, form a complex in cells. Using multiple approaches (epitope-tagged CH-ILKBP, monoclonal anti–CH-ILKBP antibodies, and green fluorescent protein–CH-ILKBP), we demonstrate that CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolished the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impaired the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Finally, we show that overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibited cell adhesion and spreading. These findings reveal a novel CH-ILKBP–ILK–PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization.

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