scholarly journals The V Antigen of Yersinia pestisRegulates Yop Vectorial Targeting as Well as Yop Secretion through Effects on YopB and LcrG

1998 ◽  
Vol 180 (13) ◽  
pp. 3410-3420 ◽  
Author(s):  
Matthew L. Nilles ◽  
Kenneth A. Fields ◽  
Susan C. Straley
Keyword(s):  
Hela Cells ◽  
Regulatory Protein ◽  
Eukaryotic Cell ◽  
Negative Regulator ◽  
Eukaryotic Cells ◽  
Cell Contact ◽  
Environmental Signals ◽  
Culture Model ◽  
Tissue Culture Model

ABSTRACT Yersinia pestis expresses a set of secreted proteins called Yops and the bifunctional LcrV, which has both regulatory and antihost functions. Yops and LcrV expression and the activity of the type III mechanism for their secretion are coordinately regulated by environmental signals such as Ca2+ concentration and eukaryotic cell contact. In vitro, Yops and LcrV are secreted into the culture medium in the absence of Ca2+ as part of the low-Ca2+ response (LCR). The LCR is induced in a tissue culture model by contact with eukaryotic cells that results in Yop translocation into cells and subsequent cytotoxicity. The secretion mechanism is believed to indirectly regulate expression oflcrV and yop operons by controlling the intracellular concentration of a secreted negative regulator. LcrG, a secretion-regulatory protein, is thought to block secretion of Yops and LcrV, possibly at the inner face of the inner membrane. A recent model proposes that when the LCR is induced, the increased expression of LcrV yields an excess of LcrV relative to LcrG, and this is sufficient for LcrV to bind LcrG and unblock secretion. To test this LcrG titration model, LcrG and LcrV were expressed alone or together in a newly constructed lcrG deletion strain, a ΔlcrG2mutant, of Y. pestis that produces low levels of LcrV and constitutively expresses and secretes Yops. Overexpression of LcrG in this mutant background was able to block secretion and depress expression of Yops in the presence of Ca2+ and to dramatically decrease Yop expression and secretion in growth medium lacking Ca2+. Overexpression of both LcrG and LcrV in the ΔlcrG2 strain restored wild-type levels of Yop expression and Ca2+ control of Yop secretion. Surprisingly, when HeLa cells were infected with the ΔlcrG2 strain, no cytotoxicity was apparent and translocation of Yops was abolished. This correlated with an altered distribution of YopB as measured by accessibility to trypsin. These effects were not due to the absence of LcrG, because they were alleviated by restoration of LcrV expression and secretion alone. LcrV itself was found to enter HeLa cells in a nonpolarized manner. These studies supported the LcrG titration model of LcrV’s regulatory effect at the level of Yop secretion and revealed a further role of LcrV in the deployment of YopB, which in turn is essential for the vectorial translocation of Yops into eukaryotic cells.

Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa

1992 ◽  
Vol 12 (4) ◽  
pp. 1568-1577
Author(s):  
J V Paietta
Keyword(s):  
Gene Expression ◽  
Neurospora Crassa ◽  
Structural Gene ◽  
Control Point ◽  
Regulatory Protein ◽  
Negative Regulator ◽  
Temperature Sensitive ◽  
Cys3 Protein

The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. The N. crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant. Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis. The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions. In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.

Induction of deep layer cortical neurons in vitro

Development ◽  
1997 ◽  
Vol 124 (4) ◽  
pp. 915-923 ◽  
Author(s):  
A.P. Bohner ◽  
R.M. Akers ◽  
S.K. McConnell
Keyword(s):  
Cortical Neurons ◽  
Short Range ◽  
Deep Layer ◽  
Ventricular Zone ◽  
Cell Contact ◽  
Low Density ◽  
Environmental Signals ◽  
Multipotent Progenitors ◽  
Short Time

Transplantation studies suggest that the laminar fates of cerebral cortical neurons are determined by environmental signals encountered just before mitosis. In ferret, E29 progenitor cells normally produce neurons of layers 5 and 6. When transplanted during S-phase into an older ventricular zone, E29 progenitors produce neurons that change their fates and migrate to layer 2/3; however, cells transplanted later in the cell cycle migrate to their normal deep-layer positions even in an older environment (McConnell and Kaznowski, 1991). Here we utilize three culture systems to investigate the nature of the environmental signals involved in laminar specification. E29 cells were first cultured at low density to ascertain whether cell contact and/or short-range cues are required for deep layer specification. Neurons transplanted after a short time in low-density culture failed to adopt their normal fates and migrated instead to the upper layers. When crude cell contacts were restored by pelleting E29 cells together, most transplanted neurons cells became specified to their normal deep layer fates. Finally, E29 cells were transplanted after being cultured in explants that maintained the architecture of the cerebral wall. Explants allowed normal deep layer specification to occur, as transplanted cells migrated to layers 5 and 6. These results suggest that short-range cues induce multipotent progenitors to produce deep layer neurons.

Cloning and functional expression of a degradation-resistant novel isoform of p27Kip1

2000 ◽  
Vol 353 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Katsuya HIRANO ◽  
Mayumi HIRANO ◽  
Ying ZENG ◽  
Junji NISHIMURA ◽  
Keiichi HARA ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Growth Arrest ◽  
Negative Regulator ◽  
G1 Phase ◽  
Serum Starvation ◽  
Cell Contact ◽  
The Novel ◽  
Specific Expression

p27Kip1 is an inhibitor of cyclin-dependent kinases. It has been implicated as having a role in the induction of growth arrest at the G1 phase of the cell cycle in response to anti-mitogenic signals such as cell contact and serum starvation. Proteasome-mediated degradation plays an important role in the rapid inactivation of p27Kip1, causing quiescent cells to re-enter the cell cycle. Although the existence of a second isoform has been suggested, no such isoform was isolated. Through screening of a cDNA library derived from growth-arrested confluent porcine endothelial cells, we obtained clones for a novel isoform of p27Kip1 in addition to the original isoform. The novel isoform differed from the original isoform at the C-terminus. The tissue-specific expression of the original and novel isoforms was demonstrated at the mRNA and protein levels. An in vitro degradation assay demonstrated this novel isoform to be resistant to proteasome-mediated destruction. The expression as a fusion protein with green fluorescent protein revealed this isoform to be targeted to the nucleus by a bipartite nuclear-localization signal with a C-terminal part different from that of the original isoform. The expression of the novel isoform caused the growth arrest of HeLa cells and an accumulation of cells in the G0/G1 phase, and this effect was similar to that seen with the original isoform. The present study suggests that the novel isoform functions as a negative regulator of the cell cycle, and may play a distinct role. The novel isoform was named p27Kip1R because of its resistance to degradation.

scholarly journals Molecular Determinants for PspA-Mediated Repression of the AAA Transcriptional Activator PspF

2005 ◽  
Vol 187 (9) ◽  
pp. 3238-3248 ◽  
Author(s):  
Sarah Elderkin ◽  
Patricia Bordes ◽  
Susan Jones ◽  
Mathieu Rappas ◽  
Martin Buck
Keyword(s):  
Regulatory Protein ◽  
Membrane Integrity ◽  
Transcriptional Activator ◽  
Negative Regulator ◽  
E Coli ◽  
Molecular Determinants ◽  
Negative Effect

ABSTRACT The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the σ54 subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of σ54 AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own expression. The PspA protein is distributed between the cytoplasm and the inner membrane fraction. In addition to its transcriptional inhibitory role, PspA assists maintenance of the proton motive force and protein export. Several lines of in vitro evidence indicate that PspA-PspF interactions inhibit the ATPase activity of PspF, resulting in the inhibition of PspF-dependent gene expression. In this study, we characterize sequences within PspA and PspF crucial for the negative effect of PspA upon PspF. Using a protein fragmentation approach, we show that the integrity of the three putative N-terminal α-helical domains of PspA is crucial for the role of PspA as a negative regulator of PspF. A bacterial two-hybrid system allowed us to provide clear evidence for an interaction in E. coli between PspA and PspF in vivo, which strongly suggests that PspA-directed inhibition of PspF occurs via an inhibitory complex. Finally, we identify a single PspF residue that is a binding determinant for PspA.

scholarly journals The actin nucleation factor JMY is a negative regulator of neuritogenesis

2011 ◽  
Vol 22 (23) ◽  
pp. 4563-4574 ◽  
Author(s):  
Elif Nur Firat-Karalar ◽  
Peter P. Hsiue ◽  
Matthew D. Welch
Keyword(s):  
Cell Lines ◽  
Regulatory Protein ◽  
Neuronal Cell ◽  
Full Length ◽  
Negative Regulator ◽  
Neurite Formation ◽  
A Minor ◽  
And Function ◽  
In Cells

Junction-mediating and regulatory protein (JMY) is a p53 cofactor that was recently shown to nucleate actin assembly by a hybrid mechanism involving tandem actin monomer binding and Arp2/3 complex activation. However, the regulation and function of JMY remain largely uncharacterized. We examined the activity of JMY in vitro and in cells, its subcellular distribution, and its function in fibroblast and neuronal cell lines. We demonstrated that recombinant full-length JMY and its isolated WASP homology 2 domain, connector, and acidic region (WWWCA) have potent actin-nucleating and Arp2/3-activating abilities in vitro. In contrast, the activity of full-length JMY, but not the isolated WWWCA domain, is suppressed in cells. The WWWCA domain is sufficient to promote actin-based bead motility in cytoplasmic extracts, and this activity depends on its ability to activate the Arp2/3 complex. JMY is expressed at high levels in brain tissue, and in various cell lines JMY is predominantly cytoplasmic, with a minor fraction in the nucleus. Of interest, silencing JMY expression in neuronal cells results in a significant enhancement of the ability of these cells to form neurites, suggesting that JMY functions to suppress neurite formation. This function of JMY requires its actin-nucleating activity. These findings highlight a previously unrecognized function for JMY as a modulator of neuritogenesis.

scholarly journals Reproducible In Vitro Tissue Culture Model to Study Basic Mechanisms of Calcific Aortic Valve Disease: Comparative Analysis to Valvular Interstitials Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 474
Author(s):  
Andreas Weber ◽  
Melissa Pfaff ◽  
Friederike Schöttler ◽  
Vera Schmidt ◽  
Artur Lichtenberg ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Aortic Valve ◽  
Aortic Valve Disease ◽  
Valve Disease ◽  
Natural Conditions ◽  
Calcific Aortic Valve Disease ◽  
Culture Model ◽  
3D Environments ◽  
Tissue Culture Model

The hallmarks of calcific aortic valve disease (CAVD), an active and regulated process involving the creation of calcium nodules, lipoprotein accumulation, and chronic inflammation, are the significant changes that occur in the composition, organization, and mechanical properties of the extracellular matrix (ECM) of the aortic valve (AV). Most research regarding CAVD is based on experiments using two-dimensional (2D) cell culture or artificially created three-dimensional (3D) environments of valvular interstitial cells (VICs). Because the valvular ECM has a powerful influence in regulating pathological events, we developed an in vitro AV tissue culture model, which is more closely able to mimic natural conditions to study cellular responses underlying CAVD. AV leaflets, isolated from the hearts of 6–8-month-old sheep, were fixed with needles on silicon rubber rings to achieve passive tension and treated in vitro under pro-degenerative and pro-calcifying conditions. The degeneration of AV leaflets progressed over time, commencing with the first visible calcified domains after 14 d and winding up with the distinct formation of calcium nodules, heightened stiffness, and clear disruption of the ECM after 56 d. Both the expression of pro-degenerative genes and the myofibroblastic differentiation of VICs were altered in AV leaflets compared to that in VIC cultures. In this study, we have established an easily applicable, reproducible, and cost-effective in vitro AV tissue culture model to study pathological mechanisms underlying CAVD. The valvular ECM and realistic VIC–VEC interactions mimic natural conditions more closely than VIC cultures or 3D environments. The application of various culture conditions enables the examination of different pathological mechanisms underlying CAVD and could lead to a better understanding of the molecular mechanisms that lead to VIC degeneration and AS. Our model provides a valuable tool to study the complex pathobiology of CAVD and can be used to identify potential therapeutic targets for slowing disease progression.

scholarly journals Chemerin, a Novel Regulator of Follicular Steroidogenesis and Its Potential Involvement in Polycystic Ovarian Syndrome

Endocrinology ◽  
2012 ◽  
Vol 153 (11) ◽  
pp. 5600-5611 ◽  
Author(s):  
Qi Wang ◽  
Ji Young Kim ◽  
Kai Xue ◽  
Jia-yin Liu ◽  
Arthur Leader ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Polycystic Ovarian Syndrome ◽  
Regulatory Protein ◽  
Hydroxysteroid Dehydrogenase ◽  
Negative Regulator ◽  
Estradiol Secretion ◽  
Chain Cleavage ◽  
Steroidogenic Acute Regulatory ◽  
Ovarian Syndrome

Abstract Polycystic ovarian syndrome (PCOS) is a heterogeneous syndrome associated with follicle growth arrest, minimal granulosa cell proliferation, dysregulated sex hormone profile, hyperthecosis, and insulin resistance. Using a 5α-dihydrotestosterone (DHT)-induced rat model that recapitulates the reproductive and metabolic phenotypes of human PCOS, we have examined the steroidogenic capability of granulosa cells from DHT-treated rats. Gene expression of several key steroidogenic enzymes including p450 side-chain cleavage enzyme (p450scc), aromatase, steroidogenic acute regulatory protein, hydroxysteroid dehydrogenase-17β, and hydroxysteroid dehydrogenase-3β were markedly lower in DHT-treated rats than the controls, although the responsiveness of their granulosa cells to FSH was higher. Expression of the adipokine chemerin and its receptor, chemokine receptor-like 1, was evident in control and DHT-treated rats, with significantly higher ovarian mRNA abundances and protein contents of chemerin and its receptor. Recombinant chemerin decreases basal estradiol secretion in granulosa cells from DHT-treated rats. When the inhibitory role of chemerin on steroidogenesis was further examined in vitro, chemerin suppressed FSH-induced progesterone and estradiol secretion in cultured preantral follicles and granulosa cells. Chemerin also inhibits FSH-induced aromatase and p450scc expression in granulosa cells. Overexpression of nuclear receptors NR5a1 and NR5a2 promotes p450scc and aromatase expression, respectively, which is suppressed by chemerin. These findings suggest that chemerin is a novel negative regulator of FSH-induced follicular steroidogenesis and may contribute to the pathogenesis of PCOS.

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