scholarly journals A novel mode of DnaA–DnaA interaction promotes ADP dissociation for reactivation of replication initiation activity

2019 ◽  
Vol 47 (21) ◽  
pp. 11209-11224
Author(s):  
Ryo Sugiyama ◽  
Kazutoshi Kasho ◽  
Kenya Miyoshi ◽  
Shogo Ozaki ◽  
Wataru Kagawa ◽  
...  
Keyword(s):  
Structural Model ◽  
Complex Dynamics ◽  
Replication Initiation ◽  
Nucleotide Exchange ◽  
Timely Initiation ◽  
Adp Release ◽  
In Vivo Analysis ◽  
Model Dimer

Abstract ATP-DnaA is temporally increased to initiate replication during the cell cycle. Two chromosomal loci, DARS (DnaA-reactivating sequences) 1 and 2, promote ATP-DnaA production by nucleotide exchange of ADP-DnaA for timely initiation. ADP-DnaA complexes are constructed on DARS1 and DARS2, bearing a cluster of three DnaA-binding sequences (DnaA boxes I−III), promoting ADP dissociation. Although DnaA has an AAA+ domain, which ordinarily directs construction of oligomers in a head-to-tail manner, DnaA boxes I and II are oriented oppositely. In this study, we constructed a structural model of a head-to-head dimer of DnaA AAA+ domains, and analyzed residues residing on the interface of the model dimer. Gln208 was specifically required for DARS-dependent ADP dissociation in vitro, and in vivo analysis yielded consistent results. Additionally, ADP release from DnaA protomers bound to DnaA boxes I and II was dependent on Gln208 of the DnaA protomers, and DnaA box III-bound DnaA did not release ADP nor require Gln208 for ADP dissociation by DARS–DnaA complexes. Based on these and other findings, we propose a model for DARS–DnaA complex dynamics during ADP dissociation, and provide novel insight into the regulatory mechanisms of DnaA and the interaction modes of AAA+ domains.

The Hierarchic Order of Intermediate Filament Structure and Assembly

1985 ◽  
Vol 43 ◽  
pp. 722-725
Author(s):  
U. Aebi ◽  
E.C. Glavaris ◽  
R. Eichner
Keyword(s):  
Tissue Specificity ◽  
Structural Model ◽  
Eukaryotic Cells ◽  
Cdna Sequencing ◽  
Filament Structure ◽  
Keratin Filaments ◽  
Isoelectric Points ◽  
Immunological Crossreactivity

Five different classes of intermediate-sized filaments (IFs) have been identified in differentiated eukaryotic cells: vimentin in mesenchymal cells, desmin in muscle cells, neurofilaments in nerve cells, glial filaments in glial cells and keratin filaments in epithelial cells. Despite their tissue specificity, all IFs share several common attributes, including immunological crossreactivity, similar morphology (e.g. about 10 nm diameter - hence ‘10-nm filaments’) and the ability to reassemble in vitro from denatured subunits into filaments virtually indistinguishable from those observed in vivo. Further more, despite their proteinchemical heterogeneity (their MWs range from 40 kDa to 200 kDa and their isoelectric points from about 5 to 8), protein and cDNA sequencing of several IF polypeptides (for refs, see 1,2) have provided the framework for a common structural model of all IF subunits.

scholarly journals Drosophila Hormone Receptor 38 Functions in Metamorphosis: A Role in Adult Cuticle Formation

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1465-1475 ◽  
Author(s):  
T Kozlova ◽  
G V Pokholkova ◽  
G Tzertzinis ◽  
J D Sutherland ◽  
I F Zhimulev ◽  
...  
Keyword(s):  
Pupal Stage ◽  
Transcription Unit ◽  
P Element ◽  
Specific Response ◽  
Orphan Receptors ◽  
Mrna Isoforms ◽  
A Cell ◽  
In Vivo Analysis

Abstract DHR38 is a member of the steroid receptor superfamily in Drosophila homologous to the vertebrate NGFI-B-type orphan receptors. In addition to binding to specific response elements as a monomer, DHR38 interacts with the USP component of the ecdysone receptor complex in vitro, in yeast and in a cell line, suggesting that DHR38 might modulate ecdysone-triggered signals in the fly. We characterized the molecular structure and expression of the Dhr38 gene and initiated an in vivo analysis of its function(s) in development. The Dhr38 transcription unit spans more than 40 kb in length, includes four introns, and produces at least four mRNA isoforms differentially expressed in development; two of these are greatly enriched in the pupal stage and encode nested polypeptides. We characterized four alleles of Dhr38: a P-element enchancer trap line, l(2)02306, which shows exclusively epidermal staining in the late larval, pre-pupal and pupal stages, and three EMS-induced alleles. Dhr38 alleles cause localized fragility and rupturing of the adult cuticle, demonstrating that Dhr38 plays an important role in late stages of epidermal metamorphosis.

scholarly journals TBC-2 Regulates RAB-5/RAB-7-mediated Endosomal Trafficking inCaenorhabditis elegans

2010 ◽  
Vol 21 (13) ◽  
pp. 2285-2296 ◽  
Author(s):  
Laëtitia Chotard ◽  
Ashwini K. Mishra ◽  
Marc-André Sylvain ◽  
Simon Tuck ◽  
David G. Lambright ◽  
...  
Keyword(s):  
Rab Gtpase ◽  
Endosomal Trafficking ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Late Endosomes ◽  
Arginine Finger ◽  
Endosome Maturation ◽  
Hops Complex

During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7–positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(−) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(−) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.

scholarly journals The Scaffold Protein Cybr Is Required for Cytokine-Modulated Trafficking of Leukocytes In Vivo

2006 ◽  
Vol 26 (14) ◽  
pp. 5249-5258 ◽  
Author(s):  
Vincenzo Coppola ◽  
Colleen A. Barrick ◽  
Sara Bobisse ◽  
Maria Cecilia Rodriguez-Galan ◽  
Michela Pivetta ◽  
...  
Keyword(s):  
Immune System ◽  
Cytotoxic T Lymphocyte ◽  
Leukemia Virus ◽  
Physiological Role ◽  
Scaffold Protein ◽  
Nucleotide Exchange ◽  
Lymphocyte Migration ◽  
And Migration

ABSTRACT Trafficking and cell adhesion are key properties of cells of the immune system. However, the molecular pathways that control these cellular behaviors are still poorly understood. Cybr is a scaffold protein highly expressed in the hematopoietic/immune system whose physiological role is still unknown. In vitro studies have shown it regulates LFA-1, a crucial molecule in lymphocyte attachment and migration. Cybr also binds cytohesin-1, a guanine nucleotide exchange factor for the ARF GTPases, which affects actin cytoskeleton remodeling during cell migration. Here we show that expression of Cybr in vivo is differentially modulated by type 1 cytokines during lymphocyte maturation. In mice, Cybr deficiency negatively affects leukocytes circulating in blood and lymphocytes present in the lymph nodes. Moreover, in a Th1-polarized mouse model, lymphocyte trafficking is impaired by loss of Cybr, and Cybr-deficient mice with aseptic peritonitis have fewer cells than controls present in the peritoneal cavity, as well as fewer leukocytes leaving the bloodstream. Mutant mice injected with Moloney murine sarcoma/leukemia virus develop significantly larger tumors than wild-type mice and have reduced lymph node enlargement, suggesting reduced cytotoxic T-lymphocyte migration. Taken together, these data support a role for Cybr in leukocyte trafficking, especially in response to proinflammatory cytokines in stress conditions.

scholarly journals In vitro and in vivo evaluation of diamond-coated strips

2016 ◽  
Vol 87 (3) ◽  
pp. 455-459 ◽  
Author(s):  
Roberta Lione ◽  
Francesca Gazzani ◽  
Chiara Pavoni ◽  
Stefano Guarino ◽  
Vincenzo Tagliaferri ◽  
...  
Keyword(s):  
Contact Point ◽  
Sem Analysis ◽  
Tribological Test ◽  
In Vivo Evaluation ◽  
Wear Performance ◽  
Abrasive Grains ◽  
Abrasive Power ◽  
In Vivo Analysis

ABSTRACT Objective: To test in vitro and in vivo the wear performance of diamond-coated strips by means of tribological testing and scanning electronic microscope (SEM). Materials and Methods: To evaluate the in vitro wear performance, a tribological test was performed by a standard tribometer. The abrasive strips slid against stationary, freshly extracted premolars fixed in resin blocks, at a 2-newton load. At the end of the tribological test, the residual surface of the strip was observed by means of SEM analysis, which was performed every 50 meters until reaching 300 meters. For the in vivo analysis, the strip was used for 300 seconds, corresponding to 250 meters. Results: The strips presented a fenestrated structure characterized by diamond granules alternating with voids. After the first 50 meters, it was possible to observe tooth material deposited on the surface of the strips and a certain number of abrasive grains detached. The surface of the strip after 250 meters appeared smoother and therefore less effective in its abrasive power. After 300 seconds of in vivo utilization of the strip, it was possible to observe the detachment of diamond abrasive grains, the near absence of the grains and, therefore, loss of abrasive power. Conclusions: Under ideal conditions, after 5 minutes (300 meters) of use, the strip loses its abrasive capacity by about 60%. In vivo, a more rapid loss of abrasive power was observed due to the greater load applied by the clinician in forcing the strip into the contact point.

The control of trophoblastic growth in the guinea pig

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 405-418
Author(s):  
E. B. Ilgren
Keyword(s):  
Guinea Pig ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
The Other ◽  
In Vivo Analysis

The growth of mouse trophectoderm depends upon the presence of the inner cell mass. Whether this applies to other species of mammals is not known. To investigate this problem, the guinea pig was selected for two reasons. Firstly, the growth of guinea-pig trophoblast resembles that of man. Secondly, earlier studies suggest that the proliferation of guinea-pig trophectoderm may not be under ICM control. Therefore, in the present study, the guinea-pig blastocyst was cut microsurgically to yield two tissue fragments. These contained roughly equal numbers of trophectodermal cells, one fragment being composed only of trophectoderm and the other containing ICM tissue as well. Subsequently, the growth of these mural and polar fragments was followed in vitro since numerous technical difficulties make an in vivo analysis of this problem impracticable. In a manner similar to the mouse, the isolated mural trophectoderm of the guinea pig stopped dividing and became giant. In contrast, guinea-pig polar fragments formed egg-cylinder-like structures. The latter contained regions structurally similar to two presumptive polar trophectodermal derivatives namely the ectoplacental and extraembryonic ectodermal tissues. These findings suggest that guinea-pig trophectodermal growth may occur in a manner similar to the mouse and thus be under ICM control.

scholarly journals Structure Based Design and Synthesis of Peptide Inhibitor of Human LOX-12: In Vitro and In Vivo Analysis of a Novel Therapeutic Agent for Breast Cancer

PLoS ONE ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. e32521 ◽  
Author(s):  
Abhay kumar Singh ◽  
Ratnakar Singh ◽  
Farhat Naz ◽  
Shyam Singh Chauhan ◽  
Amit Dinda ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Therapeutic Agent ◽  
Peptide Inhibitor ◽  
Design And Synthesis ◽  
In Vivo Analysis ◽  
Novel Therapeutic
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