Syndromic and non-syndromic aneurysms of the human ascending aorta share activation of the Smad2 pathway

Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle cell apoptosis are co-localized in aneurysms of the ascending aorta of various aetiologies. Recent experimental studies reported an activation of TGF-beta in aneurysms related to Marfan (and Loeys-Dietz) syndrome. Here we investigate TGF-beta signalling in normal and pathological human ascending aortic wall in syndromic and non-syndromic aneurysmal disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic MFS (n = 15, including two mutations in TGFBR2), associated with BAV (n = 15) or degenerative forms (n = 19), were examined. We show that the amounts of TGF-beta 1 protein retained within and released by aneurysmal tissue were greater than for control aortic tissue, whatever the aetiology, contrasting with an unchanged TGF-beta 1 mRNA level. The increase in stored TGF-beta 1 was associated with enhanced LTBP-I protein and mRNA levels. These dysiregulations of the extracellular ligand are associated with higher phosphorylated Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there was no consistent association between the nuclear location of pSmad2 and extracellular TGF-beta 1 and LTBP-I staining and between their respective mRNA expressions. In parallel, decorin. was focally increased in aneurysmal media, whereas biglycan was globally decreased in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of TGF-beta retention and Smad2 signalling in syndromic and non-syndromic aneurysms of the ascending aorta. Copyright (C) 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cytotoxic effects of butanolic extract from Pfaffia paniculata (Brazilian Ginseng) on cultured human breast cancer cell line MCF-7

Roots of Pfaffia paniculata have been well documented for multifarious therapeutic values and have also been used for cancer therapy in folk medicine. This study has been performed in a human breast tumor cell line, the MCF-7 cells. These are the most commonly used model of estrogen-positive breast cancer, and it has been originally established in 1973 at the Michigan Cancer Foundation from a pleural effusion taken from a woman with metastatic breast cancer. Butanolic extract of the roots of P. paniculata showed cytotoxic effect MCF-7 cell line. as determined with crystal violet assay, cellular death with acridine orange/ethidium bromide staining, and cell proliferation with immunocytochemistry of bromodeoxyuridine (BrdU). Subcellular alterations were evaluated by electron microscopy. Cells treated With butanolic extract showed degeneration of cytoplasmic components and profound morphological and nuclear alterations. The results show that this butanolic extract indeed presents cytotoxic substances, and its fractions merit further investigations. (C) 2008 Elsevier GmbH. All rights reserved.

Evaluation of the Possibility of Removing Staining by Repolishing Composite Resins Submitted to Artificial Aging

This in vitro research verified the possibility of eliminating staining caused by coffee and red wine in five composite resins, after being submitted to thermal cycling. Thirty-six specimens were prepared and immersed in water at 37 degrees C for 24 hours. After polishing, specimen color was measured in a spectrophotometer Cintra 10 UV (Visible Spectrometer, GBC, Braeside, VIC, Australia). All specimens were submitted to thermal cycling at temperatures of 5 and 55 degrees C with a dwell time of 1 minute, for 1,000 cycles in a 75% ethanol/water solution. After thermal cycling, the specimens were immersed in water at 37 degrees C until 7 days had elapsed from the time the specimens were prepared. All specimens were then taken to the spectrophotometer for color measurement. The specimens were divided into three groups (N = 12): distilled water (control), coffee, and red wine. For the staining process to occur on only one surface, all the sides, except one, of the surfaces were isolated with white wax. The specimens were immersed in one of the solutions at 37 degrees C for 14 days. The specimens were dried and taken to the spectrophotometer for color measurement. After this, the specimens were submitted to 20 mu m wear three times, and the color was measured after each one of the wear procedures. Calculation of the color difference was made using CIEDE2000 formula. According to the methodology used in this research, it was concluded that the staining caused by coffee and red wine was superficial and one wear of 20 mu m was sufficient to remove the discoloration.

Immunoexpression of Ki67, proliferative cell nuclear antigen, and Bcl-2 proteins in a case of ameloblastic fibrosarcoma

Ameloblastic fibrosarcoma (AFS), regarded as the malignant counterpart of the benign ameloblastic fibroma, is an extremely rare odontogenic neoplasm with only 68 cases reported in the English literature up to 2009. It is composed of a benign odontogenic epithelium, resembling that of ameloblastoma, and a malignant mesenchymal part exhibiting features of fibrosarcoma. Due to the rarity of the lesion, little is known about its molecular pathogenesis; therefore, in the current study, we sought to evaluate the immunoexpression of Ki67, proliferative cell nuclear antigen, and Bcl-2 proteins in AFS, comparing the results obtained with its benign counterpart, as well as to report a new case of this rare entity affecting a 19-year-old female patient. The results obtained revealed that all the proteins evaluated were overexpressed in the malignant mesenchymal portion of AFS if compared with ameloblastic fibroma, suggesting that nuclear proliferative factors such as Ki67 and proliferative cell nuclear antigen, in association to histopathologic features, may be useful markers for identifying the malignancy and that, despite the lack of molecular analysis in the case reported, Bcl-2 alteration may play a role in AFS pathogenesis. (C) 2010 Elsevier Inc. All rights reserved.

Differential patterns of receptor activator of nuclear factor kappa B ligand/osteoprotegerin expression in human periapical granulomas: Possible association with progressive or stable nature of the lesions

Receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) are expressed in apical periodontitis, suggesting a role for these molecules during lesion development. However, the profiles of RANKL/OPG expression in periapical lesions remain unknown. In this study we investigated the patterns of RANKL and OPG mRNA expression by real-time polymerase chain reaction in human periapical granulomas (N = 44) and compared them with sites presenting characteristic bone resorbing activity: healthy (n = 14) and orthodontically stretched and compressed periodontal ligament (n = 26), healthy gingiva (n = 24), chronic gingivitis (n = 32), and chronic periodontitis (n = 34) samples. Both RANKL and OPG mRNA expression was higher in periapical granulomas when compared with healthy periodontal ligament. Distinct patterns of RANKL and OPG expression ratio were found in the granulomas and in different physiologic and pathologic conditions, with characteristic bone resorption activity potentially being indicative of the stable or progressive nature of the lesions. Lesions with radiographic image smaller than 5 mm showed higher RANKL/OPG expression than images greater than 5 mm. Periapical granulomas presented heterogeneous patterns of RANKL and OPG expression, ranging from samples with RANKL/OPG ratio similar to that seen in sites with minimal or absent bone resorption to samples with RANKL/OPG expression pattern comparable with active bone resorption sites.

Evaluation of the Frequency of Micronuclei in Exfoliated Cells from Oral Lesions Previously Identified by Toluidine Blue

Objective: Patients using a removable prosthesis are susceptible to a variety of oral lesions that may progress to cancer. Toluidine blue (TB) staining is used to identify premalignant lesions, but the results are still controversial. Since micronuclei (MN) are a biomarker of genetic instability, the objective of this study was to determine the frequency of MN in white lesions of the oral mucosa and to compare the results with those of the TB test. Study Design: The study included 20 removable prosthesis users with white lesions that were previously classified as toluidine positive or negative. The frequency of MN was evaluated in exfoliated cells from lesions and normal mucosa. Nuclear anomalies were also registered. Results: A significant increase (p < 0.05) in the frequency of MN was observed in exfoliated cells from lesions compared to normal mucosal cells, and no relationship was seen with TB staining. Lifestyle factors or gender did not influence the results. Conclusions: The frequency of MN is a sensitive biomarker and can be used to predict genomic instability in white oral lesions. The MN assay may serve as a good parameter in the battery of tests used to identify high-risk individuals, contributing to the identification of the biological conditions of oral lesions. Copyright (C) 2011 S. Karger AG, Basel

Biophysical characterization of interactions involving importin-alpha during nuclear import

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-alpha/beta heterodimer. Importin-alpha contains the NLS binding site, whereas importin-beta mediates the translocation through the nuclear pore. We characterized the interactions involving importin-alpha during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-alpha is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-beta (stoichiometry, 1:1; K-D = 1.1 x 10(-8) m) increases the affinity for NLSs; the importin-alpha/beta complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K-D = 3.5 x 10(-8) m and 4.8 x 10(-8) m, respectively) comparable with those of a truncated importin-alpha lacking the autoinhibitory domain (T-antigen NLS, K-D = 1.7 x 10(-8) m; nucleoplasmin NLS, K-D = 1.4 x 10(-8) m). The autoinhibitory domain (as a separate peptide) binds the truncated importin-alpha, and the crystal structure of the complex resembles the structure of full-length importin-alpha. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-alpha and provide a quantitative description of the binding and regulatory steps during nuclear import.

Comparison of differentiated dendritic cell infiltration of autoimmune and osteoarthritis synovial tissue

Objective. Infiltration of rheumatoid arthritis (RA) synovial tissue (ST) by differentiated dendritic cells (DC) is a consistent feature in patients with active disease. However, mononuclear cells (MNC), including DC, may be nonspecifically chemoattracted to inflammatory sites regardless of etiology, Therefore, to evaluate the specificity of ST infiltration by differentiated DC, synovial biopsies from patients with RA, spondylarthropathy (SpA), osteoarthritis (OA), and gout were examined. Methods. Formalin-fixed ST sections were analyzed by double immunohistochemical staining for vascularity and infiltration by differentiated DC, lymphocytes, and macrophages. Results, DC containing nuclear RelB were found in perivascular MNC aggregates from patients with all arthritides studied. Infiltration by differentiated DC was similar in RA and SpA ST, but reduced in OA ST. Differentiated DC were always observed in close association with lymphocytes, and the correlation between these variables suggested that the infiltration of inflammatory sites by DC and lymphocytes was associated. Conclusion, Perivascular infiltration by DC, lymphocytes, and macrophages is nonspecifically related to inflammation, but signals present in RA and SpA ST lead to more intense cellular infiltration and accumulation.

The ratio of messenger RNA levels of receptor activator of nuclear factor kappa B ligand to osteoprotegerin correlates with bone remodeling indices in normal human cancellous bone but not in osteoarthritis

skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor KB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA, (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.

Nuclear PTEN expression and clinicopathologic features in a population-based series of primary cutaneous melanoma

Germline mutations of the PTEN tumor-suppressor gene, on 10q23, cause Cowden syndrome, an inherited hamartoma syndrome with a high risk of breast, thyroid and endometrial carcinomas and, some suggest, melanoma. To date, most studies which strongly implicate PTEN in the etiology of sporadic melanomas have depended on cell lines, short-term tumor cultures and noncultured metastatic melanomas. The only study which reports PTEN protein expression in melanoma focuses on cytoplasmic expression, mainly in metastatic samples. To determine how PTEN contributes to the etiology or the progression of primary cutaneous melanoma, we examined cytoplasmic and nuclear PTEN expression against clinical and pathologic features in a population-based sample of 150 individuals with incident primary cutaneous melanoma. Among 92 evaluable samples, 30 had no or decreased cytoplasmic PTEN protein expression and the remaining 62 had normal PTEN expression. In contrast, 84 tumors had no or decreased nuclear expression and 8 had normal nuclear PTEN expression. None of the clinical features studied, such as Clark's level and Breslow thickness or sun exposure, were associated with cytoplasmic PTEN expressional levels. An association with loss of nuclear PTEN expression was indicated for anatomical site (p = 0.06) and mitotic index (p = 0.02). There was also an association for melanomas to either not express nuclear PTEN or to express p53 alone, rather than both simultaneously (p = 0.02). In contrast with metastatic melanoma, where we have shown previously that almost two-thirds of tumors have some PTEN inactivation, only one-third of primary melanomas had PTEN silencing. This suggests that PTEN inactivation is a late event likely related to melanoma progression rather than initiation. Taken together with our previous observations in thyroid and islet cell tumors, our data suggest that nuclear-cytoplasmic partitioning of PTEN might also play a role in melanoma progression. (C) 2002 Wiley-Liss, Inc.

Nuclear RelB(+) cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions

Differentiated dendritic cells (DC) have been identified by the presence of nuclear RelB (nRelB) and HLA-DR, and the absence of CD20 or high levels of CD68, in lymph nodes and active rheumatoid arthritis synovial tissue. The current studies aimed to identify conditions in which nRelB is expressed in human tissues, by single and double immunohistochemistry of formalin-fixed peripheral and lymphoid tissue. Normal peripheral tissue did not contain nRelB(+) cells. nRelB(+) DC were located only in T- or B-cell areas of lymphoid tissue associated with normal organs or peripheral tissues, including tonsil, colon, spleen and thymus, or in association with T cells in inflamed peripheral tissue. Inflamed sites included skin delayed-type hypersensitivity reaction, and a wide range of tissues affected by autoimmune disease. Nuclear RelB(+) -HLA-DR- follicular DC were located in B-cell follicles in lymphoid organs and in lymphoid-like follicles of some tissues affected by autoimmune disease. Lymphoid tissue T-cell areas also contained nRelB(-) -HLA-DR+ cells, some of which expressed CD123 and/or CD68. Nuclear RelB(+) cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions.

RelB nuclear translocation mediated by C-terminal activator reigons of Epstein Barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells

Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitt's lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-kappaB activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative IkappaBalpha S32/36A, an NF-kappaB inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-kappaB subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.

Evolution and developmental expression of nuclear receptor genes in the ascidian Herdmania

Nuclear receptors are a superfamily of metazoan transcription factors that have been shown to be involved in a wide range of developmental and physiological processes. A PCR-based survey of genomic DNA and developmental cDNAs from the ascidian Herdmania identifies eight members of this multigene family. Sequence comparisons and phylogenetic analyses reveal that these ascidian nuclear receptors are representative of five of the six previously defined nuclear receptor subfamilies and are apparent homologues of retinoic acid [NR1B], retinoid X [NR2B], peroxisome proliferator-activated [NR1C], estrogen related [NR3B], neuron-derived orphan (NOR) [NR4A3], nuclear orphan [NR4A], TR2 orphan [NR2C1] and COUP orphan [NR2F3] receptors. Phylogenetic analyses that include the ascidian genes produce topologically distinct trees that suggest a redefinition of some nuclear receptor subfamilies. These trees also suggest that extensive gene duplication occurred after the vertebrates split from invertebrate chordates. These ascidian nuclear receptor genes are expressed differentially during embryogenesis and metamorphosis.

Read-only-memory-based quantum computation: Experimental explorations using nuclear magnetic resonance and future prospects

Read-only-memory-based (ROM-based) quantum computation (QC) is an alternative to oracle-based QC. It has the advantages of being less magical, and being more suited to implementing space-efficient computation (i.e., computation using the minimum number of writable qubits). Here we consider a number of small (one- and two-qubit) quantum algorithms illustrating different aspects of ROM-based QC. They are: (a) a one-qubit algorithm to solve the Deutsch problem; (b) a one-qubit binary multiplication algorithm; (c) a two-qubit controlled binary multiplication algorithm; and (d) a two-qubit ROM-based version of the Deutsch-Jozsa algorithm. For each algorithm we present experimental verification using nuclear magnetic resonance ensemble QC. The average fidelities for the implementation were in the ranges 0.9-0.97 for the one-qubit algorithms, and 0.84-0.94 for the two-qubit algorithms. We conclude with a discussion of future prospects for ROM-based quantum computation. We propose a four-qubit algorithm, using Grover's iterate, for solving a miniature real-world problem relating to the lengths of paths in a network.

Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41 -, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.