TGFβ-inducible early gene-1 (TIEG1) mutations in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease. A recent study showed that male KLF10‐encoded TGFβ Inducible Early Gene‐1 knock‐out mice (TIEG^?/?) develop HCM with 13‐fold up‐regulation of PTTG1‐encoded pituitary tumor‐transforming gene 1. We hypothesized TIEG1 could be a novel candidate gene in the pathogenesis of genotype negative HCM in humans, possibly through a loss of its repression on PTTG1 expression. A cohort of 923 unrelated patients from two independent HCM centers was analyzed for mutations in TIEG's four translated exons using DHPLC and direct DNA‐sequencing. Site directed mutagenesis was performed to clone novel variants. The effect of TIEG1 mutations on SMAD7 and PTTG1 promoters was studied using transient transfection and luciferase‐assays. Altered expression of PTTG1 in cardiac tissue was studied by immunohistochemistry (IHC) to determine levels of PTTG1 protein in hypertrophic diseases. Six novel TIEG1 missense mutations were discovered in six patients (two males/four females, mean age at diagnosis 56.2 ± 23 years, MLVWT 20.8 ± 4 mm). Compared to WT TIEG1, five TIEG1 mutants significantly increased PTTG1 promoter function similar to TIEG1^?/?‐mice. By IHC, PTTG1‐protein expression was significantly increased in multiple models of hypertrophic cardiac disease, including TIEG1‐mutation positive HCM compared to normal hearts. This is the first article to associate mutations in TIEG1 to human disease with the discovery of six novel, HCM‐associated variants. Functional assays suggest a role for PTTG1 in the pathogenesis of TIEG1‐mediated HCM. Up‐regulation of PTTG1 seems to be a common pathway in hypertrophic heart disease, including TIEG1‐mediated HCM. J. Cell. Biochem. 113: 1896–1903, 2012. © 2012 Wiley Periodicals, Inc.     

The reaction of HOCl and cyanocobalamin: corrin destruction and the liberation of cyanogen chloride

Overproduction of hypochlorous acid (HOCl) has been associated with the development of a variety of disorders such as inflammation, heart disease, pulmonary fibrosis, and cancer through its ability to modify various biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl and heme destruction and free iron release from hemoglobin and RBCs. Our current findings extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl), a common pharmacological form of vitamin B12. Cyanocobalamin is a water-soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that although Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC, and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphonucleotide moiety. Additionally, it can liberate free Co, which can perpetuate metal-ion-induced oxidant stress. Taken together, these results are the first report of the generation of toxic molecular products through the interaction of Cobl with HOCl.     

IQGAP1: a regulator of intracellular spacetime relativity

Activating and inhibiting receptors of lymphocytes collect valuable information about their mikròs kósmos. This information is essential to initiate or to turn off complex signaling pathways. Irrespective of these advances, our knowledge on how these intracellular activation cascades are coordinated in a spatiotemporal manner is far from complete. Among multiple explanations, the scaffolding proteins have emerged as a critical piece of this evolutionary tangram. Among many, IQGAP1 is one of the essential scaffolding proteins that coordinate multiple signaling pathways. IQGAP1 possesses multiple protein interaction motifs to achieve its scaffolding functions. Using these domains, IQGAP1 has been shown to regulate a number of essential cellular events. This includes actin polymerization, tubulin multimerization, microtubule organizing center formation, calcium/calmodulin signaling, Pak/Raf/Mek1/2-mediated Erk1/2 activation, formation of maestrosome, E-cadherin, and CD44-mediated signaling and glycogen synthase kinase-3/adenomatous polyposis coli-mediated β-catenin activation. In this review, we summarize the recent developments and exciting new findings of cellular functions of IQGAP1.     

Proximity-Dependent Inhibition of Growth of Mannheimia haemolytica by Pasteurella multocida

Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 μm) but not when they were separated by a membrane that limited contact (pore size, 0.4 μm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures of P. multocida or from P. multocida-M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS.     

Fusarium proliferatum, an endophytic fungus from Dysoxylum binectariferum Hook.f, produces rohitukine, a chromane alkaloid possessing anti-cancer activity

Rohitukine is a chromane alkaloid possessing anti-inflammatory, anti-cancer and immuno-modulatory properties. The compound was first reported from Amoora rohituka (Meliaceae) and later from Dysoxylum binectariferum (Meliaceae) and Schumanniophyton problematicum (Rubiaceae). Flavopiridol, a semi-synthetic derivative of rohitukine is a potent CDK inhibitor and is currently in Phase III clinical trials. In this study, the isolation of an endophytic fungus, Fusarium proliferatum (MTCC 9690) from the inner bark tissue of Dysoxylum binectariferum Hook.f (Meliaceae) is reported. The endophytic fungus produces rohitukine when cultured in shake flasks containing potato dextrose broth. The yield of rohitukine was 186 μg/100 g dry mycelial weight, substantially lower than that produced by the host tissue. The compound from the fungus was authenticated by comparing the LC–HRMS and LC–HRMS/MS spectra with those of the reference standard and that produced by the host plant. Methanolic extract of the fungus was cytotoxic against HCT-116 and MCF-7 human cancer cell lines (IC₅₀ = 10 μg/ml for both cancer cell lines).     

A method for spatially resolved local intracellular mechanochemical sensing and organelle manipulation

Because both the chemical and mechanical properties of living cells play crucial functional roles, there is a strong need for biophysical methods to address these properties simultaneously. Here we present a novel (to our knowledge) approach to measure local intracellular micromechanical and chemical properties using a hybrid magnetic chemical biosensor. We coupled a fluorescent dye, which serves as a chemical sensor, to a magnetic particle that is used for measurement of the viscoelastic environment by studying the response of the particle to magnetic force pulses. As a demonstration of the potential of this approach, we applied the method to study the process of phagocytosis, wherein cytoskeletal reorganization occurs in parallel with acidification of the phagosome. During this process, we measured the shear modulus and viscosity of the phagosomal environment concurrently with the phagosomal pH. We found that it is possible to manipulate phagocytosis by stalling the centripetal movement of the phagosome using magnetic force. Our results suggest that preventing centripetal phagosomal transport delays the onset of acidification. To our knowledge, this is the first report of manipulation of intracellular phagosomal transport without interfering with the underlying motor proteins or cytoskeletal network through biochemical methods.     

The feruloyl esterase gene family of Fusarium graminearum is differentially regulated by aromatic compounds and hosts

Feruloyl esterases can liberate ferulic acid (FA) from plant cell wall polymers. They are expressed by plant pathogenic fungi and could play a role in pathogenicity, although this question has not been addressed yet. The fungus Fusarium graminearum is the principal causal agent of fusarium head blight (FHB) and gibberella ear rot (GER), major diseases of wheat, barley, and maize in all temperate regions of the world. The F. graminearum genome contains seven genes with strong homology to feruloyl esterase (FAE) sequences. Phylogenetic analysis showed that these included three type B, three type C, and one type D FAE genes. Expression profiling of the seven FAE genes showed complex regulation patterns unique to each gene. In F. graminearum-infected plant tissues, the FAE genes exhibited host-specific gene expression. On wheat, FAEB1 and FAED1 were strongly expressed while FAEB2, FAEB3, and FAEC1 were expressed at more modest levels. On maize, only FAEB3, FAEC1, and FAED1 were expressed and at low levels. When growing F. graminearum in liquid culture, only FAEB1 and FAEC1 were expressed. Both genes were induced by a small group of related aromatic compounds including FA, caffeic acid, and p-coumaric acid. FAEB1 was induced by xylose, while repressed by glucose and galactose. FAEC1 was constitutively expressed at low levels in the presence of those sugars. Expression of the other five FAE genes was not detected in the culture conditions used. To determine if FAE genes were important for pathogenicity of F. graminearum, mutant strains inactivated for faeB1?, faeD1? or both genes were constructed and tested on wheat plants. No statistically significant change in pathogenicity and no compensatory expression of the other FAE genes were observed in the fae gene mutants. Our results show that FAEB1 and FAED1 are not required for pathogenicity of F. graminearum on wheat.     

Altered regulation of CXCR4 expression during aging contributes to increased CXCL12-dependent chemotactic migration of CD4(+) T cells

Chemokine‐dependent migration of T lymphocytes assures recirculation of naïve T cells to secondary lymphoid organs and tissue‐specific trafficking of memory‐effector T cells. Previous studies carried out in rodents have demonstrated age‐associated modulation of the expression of chemokine receptors such as CXCR4 and CCR5; however, little is known about the molecular mechanisms that regulate receptor expression and turnover in T cells, during advancing age in humans. Our recent results demonstrating increased chemotactic migration in response to CXCL12 in CD4⁺ T cells obtained from the elderly, as compared to those from young donors, led us to hypothesize that increase in surface expression, because of altered endocytic regulation of CXCR4 on T cells during aging, might be directly responsible for increased migration toward CXCL12. Studies presented here demonstrate a significant increase in the surface expression of CXCR4 in CD4⁺ T cells from elderly human donors, relative to those from the young. Additionally, CXCL12‐mediated endocytosis of CXCR4 was differentially regulated during aging, which could be attributed to alterations in the ubiquitination of CXCR4. Thus, altered ubiquitination of CXCR4 may contribute to the increased surface expression and enhanced T‐cell migration to chemotactic stimuli in the elderly.