Matrix Metalloproteinase-9 Gene Polymorphism and Its Methylation in Stroke Patients.

BACKGROUND: Genetic and environmental factors, along with hypertension, diabetes mellitus and smoking cause accelerated atherosclerosis and, eventually, stroke. Matrix metalloproteinase-9 (MMP-9) are inflammatory mediators of the endoproteinase family, and their polymorphism and methylation are associated with the development of atherosclerosis and stroke. This study explores this association in the Indian population. OBJECTIVE: To study the association of MMP gene polymorphism and methylation with the risk of stroke. METHODS: A case-control study was conducted on 100 admitted patients (both genders) diagnosed with ischaemic stroke. Another 100 healthy subjects, not suffering from any chronic illness or stroke, were taken as controls. All participants were genotyped for rs3918242 (MMP-9) by polymerase chain reaction (PCR) and restriction fragment length polymorphism. Methylation of the MMP-9 gene-promoter region was assessed by methylation-specific PCR. RESULTS: The case (mean age = 61.3 ± 7.36 years old) and control (mean age = 60.68 ± 7.1 years old) groups were age-matched. Among cases, 61 patients were smokers, 55 were diabetic and 53 were hypertensive. A significant risk of ischaemic stroke was associated with the CT genotype (adjusted odds ratio [aOR] = 7.09; P < 0.001), TT genotype (aOR = 19.75; P < 0.001) and T allele (aOR = 10.71; P < 0.001). MMP-9 methylation decreased the risk of stroke (aOR = 0.23; P < 0.001). CONCLUSION: MMP-9 gene-1562C/T polymorphism (SNP rs3918242) (single-nucleotide polymorphism [SNP] rs3918242) is a potential marker to predict ischaemic stroke and constitutes a significant proportion of the general population. Its polymorphism predisposes to ischaemic stroke, while its methylation is protective.

Reciprocal regulation of alternative lineages by Rgs18 and its transcriptional repressor Gfi1b.

Appropriate diversification of cellular lineages from multi-potent progenitors is essential for normal development and homeostasis. The specification of erythroid and megakaryocytic lineages represents an especially vital developmental event whose molecular regulation remains incompletely defined. We now demonstrate the role of Rgs18, a GTPase-activating protein and transcriptional target of the repressor Gfi1b, in regulating these processes in mouse and human cells. Gfi1b stringently represses Rgs18 expression in erythroid cells, whereas, during megakaryocytic differentiation, declining Gfi1b levels facilitate a robust induction of Rgs18. Concordantly, alterations in Rgs18 expression produce disparate outcomes by augmenting megakaryocytic and potently suppressing erythroid differentiation and vice versa. These phenotypes reflect the differential impact of Rgs18 on signaling through p38 MAPK family proteins, and ERK1 and ERK2 (also known as MAPK3 and MAPK1, respectively) in the two lineages, which in turn alter the balance between the mutually antagonistic transcription factors Fli1 and Klf1. Overall, these results identify Rgs18 as a new and crucial effector of Gfi1b that regulates downstream signaling and gene expression programs to orchestrate erythro-megakaryocytic lineage choices. This dual role of Rgs18 in reciprocally regulating divergent lineages could exemplify generic mechanisms characteristic of multiple family members in different contexts.

Fine dispersion of BiFeO3 nanocrystallites over highly ordered mesoporous silica material and its photocatalytic property.

Highly crystalline pure phase multi-ferroic bismuth ferrite nanoparticles have been integrated into the ordered mesoporous silica material through one pot synthesis protocol. Here, amphiphilic tri-block copolymer Pluronic P123 is being used as structure-directing agent. High temperature heating during calcination and acid treatment eliminates the presence of probable impurity phases. The existence of large uniform ordered mesopores with hexagonal pore architecture are evidenced from the small angle powder XRD, TEM image analysis and N2 adsorption/desorption isotherms. The material has considerably small optical band gap of 2.16 eV. The large specific surface area (396 m2 g(-1)) along with high crystallinity and small optical band gap of mesoporous bismuth ferrite loaded silica nanocomposite (MBFSN-1) materials suggested their potential utility as photocatalyst. Intriguingly, it completely decomposes methyl orange dye under UV-visible light irradiation within only 1 h and together with good reusability.

Analysis of Interactions Stabilized by Fusicoccin A Reveals an Expanded Suite of Potential 14-3-3 Binding Partners.

Fusicoccin A (FC) is a diterpene glycoside that stabilizes protein-protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. Recently, FC has gained attention for its pro-apoptotic and neuroprotective properties in cell culture. Although the exact molecular mechanism(s) is (are) unresolved, 14-3-3 PPIs are central to this activity. With the goal of refining the pharmacology of this chemotype, we conducted a systematic analysis of the structural features that govern FC-induced stabilization of 14-3-3 PPIs utilizing a C-terminal phosphorylation recognition motif. This study confirmed that a C-terminal amino acid with a small alkyl group is required for the interaction of FC at canonical C-terminal 14-3-3 PPI interfaces. Using bioinformatics, this structural insight was leveraged to assemble a database of 119 candidate 14-3-3 PPIs that can serve as targets for FC. This group includes a subset of proteins with experimentally determined C-terminal phosphosites that have not been explored as potential targets of FC.

Probing the 14-3-3 Isoform-Specificity Profile of Protein-Protein Interactions Stabilized by Fusicoccin A.

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein-protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. Recently, FC has emerged as an important chemical probe of human 14-3-3 PPIs involved in cancer and neurobiology. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of 14-3-3 isoforms on FC activity remains underexplored. This is a relevant question for the continued development of FC variants because there are seven isoforms of 14-3-3 in humans. Despite their sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions in vivo. Herein, we interrogate the isoform-specificity profile of FC in vitro using recombinant 14-3-3 isoforms and a library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal recognition domains of client proteins that are characterized targets of FC in vivo. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3σ. Together, these data support the feasibility of developing FC variants with enhanced isoform selectivity.

Cooperative Stimulation of Megakaryocytic Differentiation by Gfi1b Gene Targets Kindlin3 and Talin1.

Understanding the production and differentiation of megakaryocytes from progenitors is crucial for realizing the biology and functions of these vital cells. Previous gene ablation studies demonstrated the essential role of the transcriptional repressor Gfi1b (growth factor independence 1b) in the generation of both erythroid and megakaryocytic cells. However, our recent work has demonstrated the down-regulation of this factor during megakaryocytic differentiation. In this study we identify two new gene targets of Gfi1b, the cytoskeletal proteins Kindlin3 and Talin1, and demonstrate the inverse expression and functions of these cytoskeletal targets relative to Gfi1b, during megakaryocytic differentiation. Both kindlin3 and talin1 promoters exhibit dose dependent Gfi1b and LSD1 (lysine specific demethylase 1; a Gfi1b cofactor) enrichment in megakaryocytes and repression in non-hematopoietic cells. Accordingly the expression of these genes is elevated in gfi1b mutant and LSD1 inhibited hematopoietic cells, while during megakaryocytic differentiation, declining Gfi1b levels fostered the reciprocal upregulation of these cytoskeletal factors. Concordantly, manipulation of Kindlin3 and Talin1 expression demonstrated positive correlation with megakaryocytic differentiation with over-expression stimulating, and inhibition diminishing, this process. Co-operativity between these factors and integrins in promoting differentiation was further underscored by physical interactions between them and integrinß3/CD61 and by stimulation of differentiation by the Talin1 head domain, which is necessary and sufficient for integrin activation. Therefore this study demonstrates the significance of Gfi1b regulated Kindlin3-Talin1 expression in driving megakaryocytic differentiation and highlights the contribution of cytoskeletal agents in the developmental progression of these platelet progenitors.

Tunable Electrical Conductivity and Magnetic Property of the Two Dimensional Metal Organic Framework [Cu(TPyP)Cu2(O2CCH3)4].

The coordination chemistry between copper acetate [Cu2(OAc)4] and 5,10,15,20-tetra-4-pyridyl-21H,23H-porphine (porphyrin, H2TPyP) is found to give rise to either a 2D metal-organic framework (MOF) [Cu(TPyP)Cu2(O2CCH3)4] or a 3D MOF [Cu(TPyP)CuCl2]·2.5TCE·7H2O], depending on the choice of solvent. The 2D MOF can be made into a film, which was doped with 7,7,8,8-tetracyanoquinodimethane (TCNQ), and the electrical conductivity of the thin film was increased by 3 orders of magnitude with respect to that of the undoped Cu-MOF. The formation of a charge-transfer complex between TCNQ and the 2D Cu-MOF also imparts stronger paramagnetic properties than for the undoped MOF.

Differential transcriptional regulation of meis1 by Gfi1b and its co-factors LSD1 and CoREST.

Gfi1b (growth factor independence 1b) is a zinc finger transcription factor essential for development of the erythroid and megakaryocytic lineages. To elucidate the mechanism underlying Gfi1b function, potential downstream transcriptional targets were identified by chromatin immunoprecipitation and expression profiling approaches. The combination of these approaches revealed the oncogene meis1, which encodes a homeobox protein, as a direct and prominent target of Gfi1b. Examination of the meis1 promoter sequence revealed multiple Gfi1/1b consensus binding motifs. Distinct regions of the promoter were occupied by Gfi1b and its cofactors LSD1 and CoREST/Rcor1, in erythroid cells but not in the closely related megakaryocyte lineage. Accordingly, Meis1 was significantly upregulated in LSD1 inhibited erythroid cells, but not in megakaryocytes. This lineage specific upregulation in Meis1 expression was accompanied by a parallel increase in di-methyl histone3 lysine4 levels in the Meis1 promoter in LSD1 inhibited, erythroid cells. Meis1 was also substantially upregulated in gfi1b-/- fetal liver cells along with its transcriptional partners Pbx1 and several Hox messages. Elevated Meis1 message levels persisted in gfi1b mutant fetal liver cells differentiated along the erythroid lineage, relative to wild type. However, cells differentiated along the megakaryocytic lineage, exhibited no difference in Meis1 levels between controls and mutants. Transfection experiments further demonstrated specific repression of meis1 promoter driven reporters by wild type Gfi1b but neither by a SNAG domain mutant nor by a DNA binding deficient one, thus confirming direct functional regulation of this promoter by the Gfi1b transcriptional complex. Overall, our results demonstrate direct yet differential regulation of meis1 transcription by Gfi1b in distinct hematopoietic lineages thus revealing it to be a common, albeit lineage specific, target of both Gfi1b and its paralog Gfi1.