scholarly journals Lon recognition of the replication initiator DnaA is not confined to a single degron

2018 ◽  
Author(s):  
Jing Liu ◽  
Laura Francis ◽  
Peter Chien
Keyword(s):  
Acute Stress ◽  
Caulobacter Crescentus ◽  
Initiation Site ◽  
Active State ◽  
Lon Protease ◽  
Species Specific ◽  
Replication Initiator

SummaryDnaA initiates chromosome replication in bacteria. In Caulobacter crescentus, the Lon protease degrades DnaA to coordinate replication with nutrient availability and to halt the cell cycle during acute stress. Here we characterize the mechanism of DnaA recognition by Lon. We find that the native folded state of DnaA is crucial for its degradation, in contrast to the well-known role of Lon in degrading misfolded proteins. We fail to identify a single degradation motif (degron) sufficient for DnaA degradation, rather we show that both the ATPase domain and a species-specific N-terminal motif are important for productive Lon degradation of DnaA. Mutations in either of these determinants disrupt DnaA degradation in vitro and in vivo. DnaA switches from an inactive to active state depending on its nucleotide state and we find that locking DnaA in an active state inhibits degradation. Our working model is that Lon engages DnaA through at least two elements, one of which anchors DnaA to Lon and the other acting as an initiation site for degradation.

Conjugated dopamine: peripheral origin, distribution, and response to acute stress in the dog

1980 ◽  
Vol 58 (1) ◽  
pp. 22-27 ◽  
Author(s):  
Thomas Unger ◽  
Nguyen T. Buu ◽  
Otto Kuchel ◽  
Walter Schürch
Keyword(s):  
Acute Stress ◽  
Surgical Stress ◽  
Direct Conversion ◽  
Peripheral Tissues ◽  
Liver And Kidney ◽  
Arterial Plasma ◽  
Peripheral Origin

Conjugated catecholamines in the circulation and in peripheral tissues were measured together with free catecholamines in an attempt to investigate whether there are in vivo correlates to a possible biological role of dopamine sulfate suggested by an in vitro finding of direct conversion of dopamine sulfate to free norepinephrine by dopamine β-hydroxylase.Following the strong sympathoadrenergic stimulus of surgical stress accompanied by an increase in blood pressure and heart rate, conjugated dopamine showed a twofold rise in arterial plasma (p < 0.005) together with increases of all free catecholamines (0.005 < p < 0.02), while conjugates of noreprinephrine and epinephrine decreased in the circulation (0.01 < p < 0.05). Measurements of arteriovenous differences have shown that release of conjugated dopamine occurred from the adrenal gland during operation along with free catecholamines. However, the venous outflow of conjugated dopamine from liver and kidney did not exceed its arterial influx. Conjugated dopamine, in contrast with other conjugates, is present in adrenals, liver, small intestine, and kidney with higher concentrations than free dopamine in the adrenals (p < 0.01). After ultracentrifugation, the chromaffin granule fraction of the adrenal medulla (site of dopamine β-hydroxylase) contains large amounts of conjugated dopamine (apparently sulfate) suggesting a selective accumulation of dopamine sulfate as a readily available free norepinephrine precursor during stress.These findings establish major in vivo differences between peripheral conjugated dopamine and conjugates of norepinephrine and epinephrine. They suggest that there may be biological roles for conjugated dopamine beyond that of a dopamine metabolite.

scholarly journals Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain

2018 ◽  
Author(s):  
Séverin Ronneau ◽  
Julien Caballero-Montes ◽  
Aurélie Mayard ◽  
Abel Garcia-Pino ◽  
Régis Hallez
Keyword(s):  
Sinorhizobium Meliloti ◽  
Environmental Changes ◽  
Caulobacter Crescentus ◽  
Regulatory Domains ◽  
Act Domain ◽  
Related Proteins ◽  
Dependent Interaction

AbstractSensory and regulatory domains allow bacteria to adequately respond to environmental changes. The regulatory ACT domains are mainly found in metabolic-related proteins as well as in long (p)ppGpp synthetase/hydrolase (SD/HD) enzymes. Here, we investigate the functional role of the ACT domain of SpoT, the only (p)ppGpp SD/HD ofCaulobacter crescentus. We show that SpoT requires the ACT domain to hydrolyse ppGpp in an efficient way. In addition, ourin vivoandin vitrodata show that the phosphorylated version of EIIANtr(EIIANtr~P) interacts directly with the ACT to inhibit the hydrolase activity of SpoT. Finally, we highlight the conservation of the ACT-dependent interaction between EIIANtr~P and SpoT/Rel along with the PTSNtr-dependent regulation of (p)ppGpp accumulation upon nitrogen starvation inSinorhizobium meliloti, a plant-associated α-proteobacterium. Thus, this work suggests that α-proteobacteria might have inherited from a common ancestor, a PTSNtrdedicated to modulate (p)ppGpp levels.

scholarly journals Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation

2019 ◽  
Author(s):  
Adam S. B. Jalal ◽  
César L. Pastrana ◽  
Ngat T. Tran ◽  
Clare. E. Stevenson ◽  
David M. Lawson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Binding Activity ◽  
Terminal Domain ◽  
Dna Binding Activity

ABSTRACTThe tripartite ParA-ParB-parS complex ensures faithful chromosome segregation in the majority of bacterial species. ParB nucleates on a centromere-like parS site and spreads to neighboring DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the parS locus, hence the chromosome to each daughter cell. Here, we determine the co-crystal structure of a C-terminal domain truncated ParB-parS complex from Caulobacter crescentus, and show that its N-terminal domain adopts alternate conformations. The multiple conformations of the N-terminal domain might facilitate the spreading of ParB on the chromosome. Next, using ChIP-seq we show that ParBs from different bacterial species exhibit variation in their intrinsic capability for spreading, and that the N-terminal domain is a determinant of this variability. Finally, we show that the C-terminal domain of Caulobacter ParB possesses no or weak non-specific DNA-binding activity. Engineered ParB variants with enhanced non-specific DNA-binding activity condense DNA in vitro but do not spread further than wild-type in vivo. Taken all together, our results emphasize the role of the N-terminal domain in ParB spreading and faithful chromosome segregation in Caulobacter crescentus.

scholarly journals Role of RepA and DnaA Proteins in the Opening of the Origin of DNA Replication of an IncB Plasmid

2004 ◽  
Vol 186 (12) ◽  
pp. 3785-3793 ◽  
Author(s):  
T. Betteridge ◽  
J. Yang ◽  
A. J. Pittard ◽  
J. Praszkier
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Origin Of Replication ◽  
Initiator Protein ◽  
Origin Of Dna Replication ◽  
Replication Initiator Protein ◽  
Replication Initiator

ABSTRACT The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro. DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA. The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix. A 6-mer sequence, 5′-TCTTAA-3′, which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo. In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins. Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro.

Role of heme modulation in inhibition of Atheris, Atractaspis, Causus, Cerastes, Echis, and Macrovipera hemotoxic venom activity

2018 ◽  
Vol 38 (2) ◽  
pp. 216-226 ◽  
Author(s):  
VG Nielsen ◽  
N Frank
Keyword(s):  
Potential Therapeutic Target ◽  
Venomous Snake ◽  
Human Thrombin ◽  
Specific Variation ◽  
Coagulation Kinetics ◽  
Species Specific ◽  
Kinetics Of

Venomous snake bite and subsequent coagulopathy is a significant source of morbidity and mortality worldwide. The gold standard to treat coagulopathy caused by these venoms is the administration of antivenom; however, despite this therapy, coagulopathy still occurs and recurs. Of interest, our laboratory has demonstrated in vitro and in vivo that coagulopathy-inducing venom exposed to carbon monoxide (CO) is inhibited, potentially by an attached heme. The present investigation sought to determine if venoms derived from snakes of the African genera Atheris, Atractaspis, Causus, Cerastes, Echis, and Macrovipera that have no or limited antivenoms available could be inhibited with CO or with the metheme-inducing agent, O-phenylhydroxylamine (PHA). Assessing changes in coagulation kinetics of human plasma with thrombelastography, venoms were exposed in isolation to CO or PHA. Eight species were found to have procoagulant activity consistent with the generation of human thrombin, while one was likely fibrinogenolytic. All venoms were significantly inhibited by CO/PHA with species-specific variation noted. These data demonstrate indirectly that the heme is likely bound to these disparate venoms as an intermediary modulatory molecule. In conclusion, future investigation is warranted to determine if heme could serve as a potential therapeutic target to be modulated during treatment of envenomation by hemotoxic enzymes.

scholarly journals Diverse Species-Specific Phenotypic Consequences of Loss of Function Sorting Nexin 14 Mutations

2019 ◽  
Author(s):  
Dale Bryant ◽  
Marian Seda ◽  
Emma Peskett ◽  
Constance Maurer ◽  
Gideon Pomeranz ◽  
...  
Keyword(s):  
Neutral Lipids ◽  
Dermal Fibroblasts ◽  
Loss Of Function ◽  
Sorting Nexin ◽  
Species Specific ◽  
The Impact ◽  
Developmental Analysis

AbstractMutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. SCAR20 is understood to involve subcellular disruption to autophagy and lipid metabolism. Previously reported studies on the phenotypic consequences of SNX14 mutations have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. In addition, studies have investigated the biochemical roles of SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) which have demonstrated an important role during lipid biogenesis. This study investigates the impact of constitutive Snx14 mutations in laboratory species: mice and zebrafish. Loss of SNX14 in mice was found to be embryonic lethal around mid-gestation. This is due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. Zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation contrasts with other vertebrates, being both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the overall consequences of loss of function varies considerably between species. New mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Anti-obesity role of Aster glehni extract: in vivo and in vitro effects

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
HM Lee ◽  
TG Ahn ◽  
CW Kim ◽  
HJ An
Keyword(s):  
In Vitro Effects

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

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