S-glutathiolation of Ras mediates redox-sensitive signaling by angiotensin II in vascular smooth muscle cells

Angiotensin II (AII) increases production of reactive oxygen species from NAD(P)H oxidase, a response that contributes to vascular hypertrophy. Here we show in cultured vascular smooth muscle cells that S-glutathiolation of the redox-sensitive Cys(118) on the small GTPase, Ras, plays a critical role in AII-induced hypertrophic signaling. AII simultaneously increased the Ras activity and the S-glutathiolation of Ras (GSS-Ras) detected by biotin-labeled GSH or mass spectrometry. Both the increase in activity and GSS-Ras was labile under reducing conditions, suggesting the essential nature of this thiol modification to Ras activation. Overexpression of catalase, a dominant-negative p47(phox), or glutaredoxin-1 decreased GSS-Ras, Ras activation, p38, and Akt phosphorylation and the induction of protein synthesis by AII. Furthermore, expression of a Cys(118) mutant Ras decreased AII-mediated p38 and Akt phosphorylation as well as protein synthesis. These results show that H(2)O(2) from NAD(P)H oxidase forms GSS-Ras on Cys(118) and increases its activity leading to p38 and Akt phosphorylation, which contributes to the induction of protein synthesis. This study suggests that GSS-Ras is a redox-sensitive signaling switch that participates in the cellular response to AII.     

FliZ induces a kinetic switch in flagellar gene expression

FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica. To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.     

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.     

Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.     

A novel inverse relationship between metformin-triggered AMPK-SIRT1 signaling and p53 protein abundance in high glucose-exposed HepG2 cells

AMP-activated protein kinase (AMPK) and the NAD(+)-dependent histone/protein deacetylase sirtuin 1 (SIRT1) are metabolic sensors that can increase each other's activity. They are also both activated by the antidiabetic drug metformin and downregulated in the liver under conditions of nutrient excess (e.g., hyperglycemia, high-fat diet, obesity). In these situations, the abundance of the tumor suppressor p53 is increased; however, the relevance of this to the changes in AMPK and SIRT1 is not known. In the present study we investigated this question in HepG2 cells under high glucose conditions. Metformin induced activation of AMPK and SIRT1 and decreased p53 protein abundance. It also decreased triglyceride accumulation and cytosolic oxidative stress (a trigger for p53 accumulation) and increased the deacetylation of p53 at a SIRT1-targeted site. The decrease in p53 abundance caused by metformin was abolished by inhibition of murine double minute 2 (MDM2), a ubiquitin ligase that mediates p53 degradation, as well as by overexpression of a dominant-negative AMPK or a shRNA-mediated knockdown of SIRT1. In addition, overexpression of p53 decreased SIRT1 gene expression and protein abundance, as well as AMPK activity in metformin-treated cells. It also diminished the triglyceride-lowering action of metformin, an effect that was rescued by incubation with the SIRT1 activator SRT2183. Collectively, these findings suggest the existence of a novel reciprocal interaction between AMPK/SIRT1 and p53 that may have implications for the pathogenesis and treatment of metabolic diseases.     

Temporal variation in sex allocation in the mealybug Planococcus citri: adaptation, constraint, or both?

Sex ratio theory has been very successful in predicting under which circumstances parents should bias their investment towards a particular offspring sex. However, most examples of adaptive sex ratio bias come from species with well-defined mating systems and sex determining mechanisms, while in many other groups there is still an on-going debate about the adaptive nature of sex allocation. Here we study the sex allocation in the mealybug Planococcus citri, a species in which it is currently unclear how females adjust their sex ratio, even though experiments have shown support for facultative sex ratio adjustment. Previous work has shown that the sex ratio females produce changes over the oviposition period, with males being overproduced early and late in the laying sequence. Here we investigate this complex pattern further, examining both the robustness of the pattern and possible explanations for it. We first show that this sex allocation behaviour is indeed consistent across lines from three geographical regions. Second, we test whether females produce sons first in order to synchronize reproductive maturation of her offspring, although our data provide little evidence for this adaptive explanation. Finally we test the age at which females are able to mate successfully and show that females are able to mate and store sperm before adult eclosion. Whilst early-male production may still function in promoting protandry in mealybugs, we discuss whether mechanistic constraints limit how female allocate sex across their lifetime.     

Dual-color bioluminescent assay using infected HepG2 cells sheds new light on Chlamydia pneumoniae and human cytomegalovirus effects on human cholesterol 7α-hydroxylase (CYP7A1) transcription

Human bones, recovered from excavations, are an important biological archive of information. In particular, the analysis of the collagen fraction is useful for paleodietary reconstruction, via light stable isotopes, and for (14)C dating. Generally, collagen extraction procedures do not prevent loss of integrity of proteins. As a consequence, information about the state-of-remains preservation is unavailable. Here we describe a "soft" nondestructive CH(3)COOH-based method to recover collagen from archaeological bones, and also to obtain material for successive isotopic analyses. Our isotopic measurements on the extracts indicate that the CH(3)COOH-based method of extraction may be routinely employed in the context of paleodiet studies. In addition, we propose that biochemical characterization by denaturant electrophoresis and Western blot on CH(3)COOH extracts may be used as a bone collagen quality indicator.     

Antiproteinuric treatment reduces urinary loss of vitamin D-binding protein but does not affect vitamin D status in patients with chronic kidney disease

Vitamin D deficiency is common in chronic kidney disease (CKD). Increased urinary loss of vitamin D binding protein (VDBP), the main transporter of 25-hydroxyvitamin D(3) in the circulation, has been postulated to contribute to vitamin D deficiency in proteinuria. To test this hypothesis we analyzed urinary and plasma levels of VDBP, 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) from proteinuric patients, before and after antiproteinuric interventions. We performed a post-hoc analysis of a clinical trial in CKD patients (n=13, creatinine clearance median 60 (range 25-177) ml/min) subjected to the following study periods: washout (no antiproteinuric treatment, 4 weeks), lisinopril 40mg QD (ACEi, 6 weeks), or indomethacin 75mg BID (NSAID, 4 weeks) in randomized sequence. Healthy subjects screened for donation (n=10) served as controls. Plasma and urine VDBP levels were measured by ELISA, 25-hydroxyvitamin D(3) levels by LC-MS and 1,25-dihydroxyvitamin D(3) levels by radioimmunoassay. In CKD patients urinary VDBP excretion was strongly increased (median (range) 5413 (155-211,027) μg/24h) as compared to healthy controls (64 (23-111) μg/24h, p<0.001). Both NSAID and ACEi significantly decreased urinary VDBP excretion, in proportion to proteinuria reduction. Plasma VDBP, 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) levels, however, were similar between patients and controls and not affected by antiproteinuric intervention. Urinary VDBP excretion is markedly increased in proteinuria and responds to antiproteinuric treatment. Urinary VDBP loss is not associated with plasma VDBP or vitamin D(3) levels, suggesting that urinary loss of VDBP does not affect vitamin D status.