Rrp1B gene polymorphism (1307T > C) in metastatic progression of breast cancer

Rrp1B (ribosomal RNA processing1 homolog B) is a novel candidate metastasis modifier gene in breast cancer. Functional gene assays demonstrated that a physical and functional interaction existing between Rrp1b and metastasis modifier gene SIPA1 causes reduction in the tumor growth and metastatic potential. Ectopic expression of Rrp1B modulates various metastasis predictive extra cellular matrix (ECM) genes associated with tumor suppression. The aim of this study is to determine the functional significance of single nucleotide polymorphism (SNP) in human Rrp1B gene (1307 T > C; rs9306160) with breast cancer development and progression. The study consists of 493 breast cancer cases recruited from Nizam's Institute of Medical Sciences, Hyderabad, and 558 age-matched healthy female controls from rural and urban areas. Genomic DNA was isolated by non-enzymatic method. Genotyping was done by amplification refractory mutation system (ARMS-PCR) method. Genotypes were reconfirmed by sequencing and results were analyzed statistically. We have performed Insilco analysis to know the RNA secondary structure by using online tool m fold. The TT genotype and T allele frequencies of Rrp1B1307 T > C polymorphism were significantly elevated in breast cancer (chi (2); p = < 0.008) cases compared to controls under different genetic models. The presence of T allele had conferred 1.75-fold risk for breast cancer development (OR = 1.75; 95 % CI = 1.15-2.67). The frequency of TT genotype of Rrp1b 1307T > C polymorphism was significantly elevated in obese patients (chi (2); p = 0.008) and patients with advanced disease (chi (2); p = 0.01) and with increased tumor size (chi (2); p = 0.01). Moreover, elevated frequency of T allele was also associated with positive lymph node status (chi (2); p = 0.04) and Her2 negative receptor status (chi (2); p = 0.006). Presence of Rrp1b1307TT genotype and T allele confer strong risk for breast cancer development and progression.

Effect of a natural mutation in the 5 ` untranslated region on the translational control of p53 mRNA

Tumor-suppressor protein p53, the `guardian of the genome', is critical in maintaining cellular homeostasis and genomic stability. Earlier, we have reported the discovery of internal ribosome entry sites (IRESs) within the p53 mRNA that regulate the translation of the full length and its N-terminal-truncated isoform, Delta N-p53. Polypyrimidine tract-binding protein (PTB) is an IRES trans-acting factor that positively regulates the IRES activities of both p53 isoforms by relocating from nucleus to the cytoplasm during stress conditions. Here we have demonstrated the putative contact points of PTB on the p53 IRES RNA. Studies on mutations that occur naturally in the 5' untranslated region (5' UTR) in p53 mRNA were lacking. We have investigated a naturally occurring C-to-T single-nucleotide polymorphism (SNP) first reported in human melanoma tumors. This SNP is at position 119 in the 5' UTR of p53 mRNA and we demonstrate that it has consequences on the translational control of p53. Introduction of this SNP has led to decrease in cap-independent translation from p53 5' UTR in bicistronic reporter assay. Further, the effects of this SNP on cap-independent translation have been studied in the context of p53 cDNA as well. Interestingly, the 5' UTR with this SNP has shown reduced binding to PTB that can be corroborated to its weaker IRES activity. Previously, it has been shown that G2-M checkpoint, DNA-damaging stress and oncogenic insult favor IRES-mediated translation. Under similar conditions, we demonstrate that this SNP interferes with the enhancement of the IRES activity of the 5' UTR. Taken together, the results demonstrate for the first time that SNP in the 5' UTR of the p53 mRNA might have a role in translational control of this critical tumor-suppressor gene.

Mutation analysis of the SLC4A11 gene in Indian families with congenital hereditary endothelial dystrophy 2 and a review of the literature

Purpose: Congenital hereditary endothelial dystrophy 2 (CHED2) is an autosomal recessive disorder caused by mutations in the solute carrier family 4, sodium borate transporter, member 11 (SLC4A11) gene. The purpose of this study was to identify the genetic cause of CHED2 in six Indian families and catalog all known mutations in the SLC4A11 gene. Methods: Peripheral blood samples were collected from individuals of the families with CHED2 and used in genomic DNA isolation. PCR primers were used to amplify the entire coding region including intron-exon junctions of SLC4A11. Amplicons were subsequently sequenced to identify the mutations. Results: DNA sequence analysis of the six families identified four novel (viz., p.Thr262Ile, p.Gly417Arg, p.Cys611Arg, and p.His724Asp) mutations and one known p.Arg869His homozygous mutation in the SLC4A11 gene. The mutation p.Gly417Arg was identified in two families. Conclusions: This study increases the mutation spectrum of the SLC4A11 gene. A review of the literature showed that the total number of mutations in the SLC4A11 gene described to date is 78. Most of the mutations are missense, followed by insertions-deletions. The present study will be helpful in genetic diagnosis of the families reported here.

Characterization of an Evolutionarily Conserved Metallophosphoesterase That Is Expressed in the Fetal Brain and Associated with the WAGR Syndrome

Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2',3'-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events.

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea)

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C-4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-C-14]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparision of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

The requirements for Ca2+, protein phosphorylation and concurrent protein synthesis for zeatin signaling of acidic chitinase transcript accumulation in Cucumis sativus L

We have previously reported that both Ca2+ and staurosporine-sensitive protein kinase(s) are involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content (Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induction of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark-grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe (Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exogenous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumulation of CACHT mRNA was inhibited by a plasma membrane Ca2+ channel blocker verapamil. Treatment of cotyledons with exogenous CaCl2 and calcium ionophore A23187 in the presence and absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of staurosporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involvement of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment. Taken together, these results suggest that Ca2+ influx from extracellular space, protein phosphorylation, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced CACHT transcript accumulation.

Pathogen-specific TLR2 Protein Activation Programs Macrophages to Induce Wnt-beta-Catenin Signaling

Innate immunity recognizes and resists various pathogens; however, the mechanisms regulating pathogen versus non-pathogen discrimination are still imprecisely understood. Here, we demonstrate that pathogen-specific activation of TLR2 upon infection with Mycobacterium bovis BCG, in comparison with other pathogenic microbes, including Salmonella typhimurium and Staphylococcus aureus, programs macrophages for robust up-regulation of signaling cohorts of Wnt-beta-catenin signaling. Signaling perturbations or genetic approaches suggest that infection-mediated stimulation of Wnt-beta-catenin is vital for activation of Notch1 signaling. Interestingly, inducible NOS (iNOS) activity is pivotal for TLR2-mediated activation of Wnt-beta-catenin signaling as iNOS(-/-) mice demonstrated compromised ability to trigger activation of Wnt-beta-catenin signaling as well as Notch1-mediated cellular responses. Intriguingly, TLR2-driven integration of iNOS/NO, Wnt-beta-catenin, and Notch1 signaling contributes to its capacity to regulate the battery of genes associated with T(Reg) cell lineage commitment. These findings reveal a role for differential stimulation of TLR2 in deciding the strength of Wnt-beta-catenin signaling, which together with signals from Notch1 contributes toward the modulation of a defined set of effector functions in macrophages and thus establishes a conceptual framework for the development of novel therapeutics.

Effect of 80 kDa protein of porcine follicular fluid on gonadotropin stimulated progesterone production in rat granulosa cells in vitro

We reported the presence of a 80 kDa polypeptide in porcine follicular fluid that inhibited the binding of 125I-radiolabelled hFSH as well as hCG to the rat ovarian gonadotropin receptors. In the present study, the biological activity of the receptor binding inhibitor is determined using an in vitro bioassay procedure. Granulosa cells isolated from PMSG primed immature rat ovaries respond to exogenously added gonadotropins in terms of progesterone production. Addition of fractions containing the gonadotropin receptor binding inhibitory activity inhibited progesterone production stimulated by the gonadotropins in a dose-dependent fashion. The receptor binding inhibitory activity was also capable of inhibiting progesterone production stimulated by PMSG, which has both FSH- and LH-like activities in rats. In contrast, progesterone production stimulated by dbcAMP was not inhibited by the receptor binding inhibitor. This result indicates that the site of action of the inhibitor is proximal to the formation of the cAMP. The above observations point out to a possible role for this factor in modulating gonadotropin activity at the ovarian level.

Angelica archengelica extract induced perturbation of rat skin and tight junctional protein (ZO-1) of HaCaT cells

Background and purpose of the study: Herbal enhancers compared to the synthetic ones have shown less toxis effects. Coumarins have been shown at concentrations inhibiting phospoliphase C-Y (Phc-Y) are able to enhance tight junction (TJ) permeability due to hyperpoalation of Zonolous Occludense-1 (ZO-1) proteins. The purpose of this study was to evaluate the influence of ethanolic extract of Angelica archengelica (AA-E) which contain coumarin on permeation of repaglinide across rat epidermis and on the tight junction plaque protein ZO-1 in HaCaT cells. Methods: Transepidermal water loss (TEWL) from the rat skin treated with different concentrations of AA-E was assessed by Tewameter. Scanning and Transmission Electron Microscopy (TEM) on were performed on AA-E treated rat skin portions. The possibility of AA-E influence on the architecture of tight junctions by adverse effect on the cytoplasmic ZO-1 in HaCaT cells was investigated. Finally, the systemic delivery of repaglinide from the optimized transdermal formulation was investigated in rats. Results: The permeation of repaglinide across excised rat epidermis was 7-fold higher in the presence of AA-E (5% w/v) as compared to propylene glycol:ethanol (7:3) mixture. The extract was found to perturb the lipid microconstituents in both excised and viable rat skin, although, the effect was less intense in the later. The enhanced permeation of repaglinide across rat epidermis excised after treatment with AA-E (5% w/v) for different periods was in concordance with the high TEWL values of similarly treated viable rat skin. Further, the observed increase in intercellular space, disordering of lipid structure and corneocyte detachment indicated considerable effect on the ultrastructure of rat epidermis. Treatment of HaCaT cell line with AA-E (0.16% w/v) for 6 hrs influenced ZO-1 as evidenced by reduced immunofluorescence of anti-TJP1 (ZO-1) antibody in Confocal Laser Scanning Microscopy studies (CLSM) studies. The plasma concentration of repaglinide from transdermal formulation was maintained higher and for longer time as compared to oral administration of repaglinide. Major conclusion: Results suggest the overwhelming influence of Angelica archengelica in enhancing the percutaneous permeation of repaglinide to be mediated through perturbation of skin lipids and tight junction protein (ZO-1).

Familial Diarrhea Syndrome Caused by an Activating GUCY2C Mutation

BACKGROUND Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis. METHODS We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells. RESULTS We identified a heterozygous missense mutation (c.2519G -> T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members. CONCLUSIONS Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohn's disease is warranted. (Funded by Helse Vest Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.)

A Coordinated Interdependent Protein Circuitry Stabilizes the Kinetochore Ensemble to Protect CENP-A in the Human Pathogenic Yeast Candida albicans

Unlike most eukaryotes, a kinetochore is fully assembled early in the cell cycle in budding yeasts Saccharomyces cerevisiae and Candida albicans. These kinetochores are clustered together throughout the cell cycle. Kinetochore assembly on point centromeres of S. cerevisiae is considered to be a step-wise process that initiates with binding of inner kinetochore proteins on specific centromere DNA sequence motifs. In contrast, kinetochore formation in C. albicans, that carries regional centromeres of 3-5 kb long, has been shown to be a sequence independent but an epigenetically regulated event. In this study, we investigated the process of kinetochore assembly/disassembly in C. albicans. Localization dependence of various kinetochore proteins studied by confocal microscopy and chromatin immunoprecipitation (ChIP) assays revealed that assembly of a kinetochore is a highly coordinated and interdependent event. Partial depletion of an essential kinetochore protein affects integrity of the kinetochore cluster. Further protein depletion results in complete collapse of the kinetochore architecture. In addition, GFP-tagged kinetochore proteins confirmed similar time-dependent disintegration upon gradual depletion of an outer kinetochore protein (Dam1). The loss of integrity of a kinetochore formed on centromeric chromatin was demonstrated by reduced binding of CENP-A and CENP-C at the centromeres. Most strikingly, Western blot analysis revealed that gradual depletion of any of these essential kinetochore proteins results in concomitant reduction in cellular protein levels of CENP-A. We further demonstrated that centromere bound CENP-A is protected from the proteosomal mediated degradation. Based on these results, we propose that a coordinated interdependent circuitry of several evolutionarily conserved essential kinetochore proteins ensures integrity of a kinetochore formed on the foundation of CENP-A containing centromeric chromatin.

Carrier concentration dependence of donor activation energy in n-type GaN epilayers grown on Si (111) by plasma-assisted MBE

The n-type GaN layers were grown by plasma-assisted MBE and either intentionally doped with Si or unintentionally doped. The optical characteristics of a donor level in Si-doped, GaN were studied in terms of photoluminescence (PL) spectroscopy as a function of electron concentration. Temperature dependent PL measurements allowed us to estimate the activation energy of a Si-related donor from temperature-induced decay of PL intensity. PL peak positions, full width at half maximum of PL and activation energies are found to be proportional to the cube root of carrier density. The involvement of donor levels is supported by the temperature-dependent electron concentration measurements. (C) 2012 Elsevier Ltd. All rights reserved.

Self-assembly of three new coordination complexes: Formation of 2-D square grid, 1-D chain and tape structures

Three distinct coordination complexes, viz., [Co(imi)(2)(tmb)(2)] (1) [where imi = imidazole], {[Ni(tmb)(2)(H2O)(3)]center dot 2H(2)O}(n) (2) and [Cu-2(mu-tmb)(4)(CH3OH)(2)] (3), have been synthesized hydrothermally by the reactions of metal acetates,2,4,6-trimethylbenzoic acid (Htmb) and with or without appropriate amine. The Ni analogue of 1 and the Co analogue of 2 have also been synthesized. X-ray single-crystal diffraction suggests that complex 1 represents discrete mononuclear species and complex 2 represents a 1D chain coordination polymer in which the Ni(H) ions are connected by the bridging water molecules. Complex 3 represents a neutral dinuclear complex. In 1, the central metal ions are associated by the carboxylate moiety and imidazole ligands, whereas the central metal atom is coordinated to the carboxylate moiety and the respective solvent molecules in 2 and 3. In 3, the four 2,4,6-trimethylbenzoate moieties act as a bridge connecting two copper (11) ions and the 0 atoms of methanol coord geometry, with the methanol molecule at the apical position. In all the three structures the central metal atom sits on a crystallographic inversion centre. In all the cases, the coordination entities are further organized via hydrogen bonding interactions to generate multifarious supramolecular networks. Complexes 1, 2 and 3 have also been characterized by spectroscopic (UV/Vis and IR) and thermal analysis (TGA). In addition, the complexes were found to exhibit antimicrobial activity. The magnetic susceptibility measurements, measured from 8 to 300 K, revealed antiferromagnetic interactions between the Co(II) ions in compound 1 and the Ni(II) ions in la, respectively.

Purification of RNA polymerase from mycobacteria for optimized promoter-polymerase interactions

In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNA polymerase (RNAP) with high specific activity is necessary to carry out variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low promoter specificity in promoter-polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of sigma(A) required for house-keeping transcription. We describe an in vivo reconstitution of RNAP holoenzyme with sigma(A) and its purification, which resulted in holoenzyme with stoichiometric sigma(A) content. The reconstituted holoenzyme showed enhanced promoter-specific binding and promoter-specific-transcription activity compared to the enzyme isolated using standard procedure. Such in vivo reconstitution of stoichiometric holoenzyme could facilitate promoter-specific transcription assays, especially in organisms which encode a large number of sigma factors.