107 resultados para FLUORESCENT LAMPS


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Multiple sclerosis (MS) is a complex autoimmune disorder of the CNS with both genetic and environmental contributing factors. Clinical symptoms are broadly characterized by initial onset, and progressive debilitating neurological impairment. In this study, RNA from MS chronic active and MS acute lesions was extracted, and compared with patient matched normal white matter by fluorescent cDNA microarray hybridization analysis. This resulted in the identification of 139 genes that were differentially regulated in MS plaque tissue compared to normal tissue. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue<0.0001, ρ=0.73, by Spearman's ρ analysis); while 70 transcripts were uniquely differentially expressed (≥1.5-fold) in either acute or chronic active tissues. These results included known markers of MS such as the myelin basic protein (MBP) and glutathione S-transferase (GST) M1, nerve growth factors, such as nerve injury-induced protein 1 (NINJ1), X-ray and excision DNA repair factors (XRCC9 and ERCC5) and X-linked genes such as the ribosomal protein, RPS4X. Primers were then designed for seven array-selected genes, including transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), GSTP1, crystallin, alpha-B (CRYAB), phosphomannomutase 1 (PMM1) and tubulin β-5 (TBB5), and real time quantitative (Q)-PCR analysis was performed. The results of comparative Q-PCR analysis correlated significantly with those obtained by array analysis (r=0.75, Pvalue<0.01, by Pearson's bivariate correlation). Both chronic active and acute plaques shared the majority of factors identified suggesting that quantitative, rather than gross qualitative differences in gene expression pattern may define the progression from acute to chronic active plaques in MS.

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With an increased emphasis on genotyping of single nucleotide polymorphisms (SNPs) in disease association studies, the genotyping platform of choice is constantly evolving. In addition, the development of more specific SNP assays and appropriate genotype validation applications is becoming increasingly critical to elucidate ambiguous genotypes. In this study, we have used SNP specific Locked Nucleic Acid (LNA) hybridization probes on a real-time PCR platform to genotype an association cohort and propose three criteria to address ambiguous genotypes. Based on the kinetic properties of PCR amplification, the three criteria address PCR amplification efficiency, the net fluorescent difference between maximal and minimal fluorescent signals and the beginning of the exponential growth phase of the reaction. Initially observed SNP allelic discrimination curves were confirmed by DNA sequencing (n = 50) and application of our three genotype criteria corroborated both sequencing and observed real-time PCR results. In addition, the tested Caucasian association cohort was in Hardy-Weinberg equilibrium and observed allele frequencies were very similar to two independently tested Caucasian association cohorts for the same tested SNP. We present here a novel approach to effectively determine ambiguous genotypes generated from a real-time PCR platform. Application of our three novel criteria provides an easy to use semi-automated genotype confirmation protocol.

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Growth and metastatic spread of invasive carcinoma depends on angiogenesis, the formation of new blood vessels. Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic growth factor for a number of solid tumors, including lung, bladder, colorectal, and renal cell cancer. Cervical intraepithelial neoplasia (CIN) is the precursor to squamous cell cervical carcinoma (SCC). Mean vessel density (MVD) increases from normal cervical tissue, through low- and high-grade CIN to SCC. We evaluated PD-ECGF immunoreactivity and correlated its expression with MVD in normal, premalignant, and malignant cervical tissue. PD-ECGF expression was assessed visually within the epithelial tissues and scored on the extent and intensity of staining. MVD was calculated by counting the number of vessels positive for von Willebrand factor per unit area subtending normal or CIN epithelium or within tumor hotspots for SCC. Cytoplasmic and/or nuclear PD-ECGF immunoreactivity was seen in normal epithelium. PD-ECGF expression significantly increased with histologic grade from normal, through low- and high-grade CIN, to SCC (P < .02). A progressive significant increase in the microvessel density was also seen, ranging from a mean of 28 vessels for normal tissue to 57 for SCC (P < .0005). No correlation was found between PD-ECGF expression and MVD (P = .45). We conclude that PD-ECGF expression and MVD increase as the cervix transforms from a normal to a malignant phenotype. PD-ECGF is thymidine phosphorylase, a key enzyme in the activation of fluoropyrimidines, including 5-fluorouracil. Evaluation of PD-ECGF thymidine phosphorylase expression may be important in designing future chemotherapeutic trials in cervical cancer. Copyright (C) 2000 by W.B. Saunders Company.

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Cell line array (CMA) and tissue microarray (TMA) technologies are high-throughput methods for analysing both the abundance and distribution of gene expression in a panel of cell lines or multiple tissue specimens in an efficient and cost-effective manner. The process is based on Kononen's method of extracting a cylindrical core of paraffin-embedded donor tissue and inserting it into a recipient paraffin block. Donor tissue from surgically resected paraffin-embedded tissue blocks, frozen needle biopsies or cell line pellets can all be arrayed in the recipient block. The representative area of interest is identified and circled on a haematoxylin and eosin (H&E)-stained section of the donor block. Using a predesigned map showing a precise spacing pattern, a high density array of up to 1,000 cores of cell pellets and/or donor tissue can be embedded into the recipient block using a tissue arrayer from Beecher Instruments. Depending on the depth of the cell line/tissue removed from the donor block 100-300 consecutive sections can be cut from each CMA/TMA block. Sections can be stained for in situ detection of protein, DNA or RNA targets using immunohistochemistry (IHC), fluorescent in situ hybridisation (FISH) or mRNA in situ hybridisation (RNA-ISH), respectively. This chapter provides detailed methods for CMA/TMA design, construction and analysis with in-depth notes on all technical aspects including tips to deal with common pitfalls the user may encounter. © Springer Science+Business Media, LLC 2011.

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The recent advances in the understanding of the pathogenesis of ovarian cancer have been helpful in addressing issues in diagnosis, prognosis and management. The study of ovarian tumours by novel techniques such as immunohistochemistry, fluorescent in situ hybridisation, comparative genomic hybridisation, polymerase chain reaction and new tumour markers have aided the evaluation and application of new concepts into clinical practice. The correlation of novel surrogate tumour specific features with response to treatment and outcome in patients has defined prognostic factors which may allow the future design of tailored therapy based on a molecular profile of the tumour. These have also been used to design new approaches to therapy such as antibody targeting and gene therapy. The delineation of roles of c-erbB2, c-fms and other novel receptor kinases in the pathogenesis of ovarian cancer has led initially to the development of anti-c-erbB2 monoclonal antibody therapy. The discovery of BRCA1 and BRCA2 genes will have an impact in the diagnosis and the prevention of familial ovarian cancer. The important role played by recessive genes such as p53 in cancer has raised the possibility of restoration of gene function by gene therapy. Although the pathological diagnosis of ovarian cancer is still confirmed principally on morphological features, addition of newer investigations will increasingly be useful in addressing difficult diagnostic problems. The increasingly rapid pace of discovery of genes important in disease, makes it imperative that the evaluation of their contribution in the pathogenesis of ovarian cancer is undertaken swiftly, thus improving the overall management of patients and their outcome.

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The movement of molecules inside living cells is a fundamental feature of biological processes. The ability to both observe and analyse the details of molecular diffusion in vivo at the single-molecule and single-cell level can add significant insight into understanding molecular architectures of diffus- ing molecules and the nanoscale environment in which the molecules diffuse. The tool of choice for monitoring dynamic molecular localization in live cells is fluorescence microscopy, especially so combining total internal reflection fluorescence with the use of fluorescent protein (FP) reporters in offering exceptional imaging contrast for dynamic processes in the cell mem- brane under relatively physiological conditions compared with competing single-molecule techniques. There exist several different complex modes of diffusion, and discriminating these from each other is challenging at the mol- ecular level owing to underlying stochastic behaviour. Analysis is traditionally performed using mean square displacements of tracked particles; however, this generally requires more data points than is typical for single FP tracks owing to photophysical instability. Presented here is a novel approach allowing robust Bayesian ranking of diffusion processes to dis-criminate multiple complex modes probabilistically. It is a computational approach that biologists can use to understand single-molecule features in live cells.

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Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis. © 2012 Springer Basel.

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Efficient transformation of barley cv. Schooner was achieved using Agrobacterium delivery, hygromycin or bialaphos selection and embryogenic callus. Using this system, transgenic plants were generated that contained either the green fluorescent protein gene, or transgenes derived from barley yellow dwarf (BYDV) and cereal yellow dwarf (CYDV) viruses. Many of these plants contained 1-3 transgene copies that were inherited in a simple Mendelian manner. Some plants containing BYDV and/or CYDV derived transgenes showed reduced virus symptoms and rates of viral replication when challenged with the appropriate virus. The ability to transform Schooner is a significant advance for the Australian barley industry, as this elite malting variety is, and has for the last 15 years been, the most widely grown barley variety in eastern Australia.

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We report the first successful Agrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph (encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4-fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 carrying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher β-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity comparable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

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Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.

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A series of aza-boron-diquinomethene (aza-BODIQU) complexes with different aryl-substituents (B1–B6) were synthesized and characterized. Their photophysical properties were investigated systematically via spectroscopic and theoretical methods. All complexes exhibit strong 1π–π* absorption bands and intense fluorescent emission bands in the visible spectral region at room temperature. The fluorescence spectra in solution show the mirror image features of the S0→S1 absorption bands, which can be assigned to the 1π–π*/1ICT (intramolecular charge transfer) emitting states. Except for B6, all complexes exhibit high photoluminescence quantum yields (ΦPL = 0.47–0.93). The spectroscopic studies and theoretical calculations indicate that the photophysical properties of these aza-BODIQUs can be tuned by the appended aryl-substituents, which would be useful for rational design of boron–fluorine complexes with high emission quantum yield for organic light-emitting applications.

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A cell classification algorithm that uses first, second and third order statistics of pixel intensity distributions over pre-defined regions is implemented and evaluated. A cell image is segmented into 6 regions extending from a boundary layer to an inner circle. First, second and third order statistical features are extracted from histograms of pixel intensities in these regions. Third order statistical features used are one-dimensional bispectral invariants. 108 features were considered as candidates for Adaboost based fusion. The best 10 stage fused classifier was selected for each class and a decision tree constructed for the 6-class problem. The classifier is robust, accurate and fast by design.

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Heparan sulfate proteoglycans (HSPGs) are complex and labile macromolecular moieties on the surfaces of cells that control the activities of a range of extracellular proteins, particularly those driving growth and regeneration. Here, we examine the biosynthesis of heparan sulfate (HS) sugars produced by cultured MC3T3-E1 mouse calvarial pre-osteoblast cells in order to explore the idea that changes in HS activity in turn drive phenotypic development during osteogenesis. Cells grown for 5 days under proliferating conditions were compared to cells grown for 20 days under mineralizing conditions with respect to their phenotype, the forms of HS core protein produced, and their HS sulfotransferase biosynthetic enzyme levels. RQ-PCR data was supported by the results from the purification of day 5 and day 20 HS forms by anionic exchange chromatography. The data show that cells in active growth phases produce more complex forms of sugar than cells that have become relatively quiescent during active mineralization, and that these in turn can differentially influence rates of cell growth when added exogenously back to preosteoblasts.

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The standard method of labelling proliferating cells uses the thymidine analogue, bromodeoxyuridine (BrdU), which incorporates into the DNA during S-phase of the cell cycle. A disadvantage of this method is that the immunochemical processing requires pre-treatment of the cells and tissue with heat or acid to reveal the antigen. This pre-treatment reduces reliability of the method and degrades the specimen, reducing the ability for multiple immuno-fluorescence labelling at high resolution. We report here the utility of a novel thymidine analogue, ethynyl deoxyuridine (EdU), detected with a fluorescent azide via the “click” chemistry reaction (the Huisgen 1,3-dipolar cycloaddition reaction of an organic azide to a terminal acetylene). The detection of EdU requires no heat or acid treatment and the incorporated EdU is covalently conjugated to fluorescent probe. The reaction is quick and compatible with fluorescence immunochemistry and other fluorescent probes. We show here that EdU is non-toxic in vitro and in vivo and can be used in place of BrdU to label cells during neurogenesis and the progeny identified at least 30 days later. The fluorescent labelling of EdU, markedly improves the detection of proliferating cells and allows concurrent high resolution fluorescence immunochemistry.

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Large, osseous, segmental defects heal poorly. Muscle has a propensity to form bone when exposed to an osteogenic stimulus such as that provided by transfer and expression of cDNA encoding bone morphogenetic protein-2 (BMP-2). The present study evaluated the ability of genetically modified, autologous muscle to heal large cranial defects in rats. Autologous grafts (8 mm � 2 mm) were punched from the biceps femoris muscle and transduced intraoperatively with recombinant adenovirus vector containing human BMP-2 or green fluorescent protein cDNA. While the muscle biopsies were incubating with the vector, a central parietal 8 mm defect was surgically created in the calvarium of the same animal. The gene-activated muscle graft was then implanted into the cranial defect. After 8 weeks, crania were examined radiographically, histologically, and by micro-computed tomography and dual energy X-ray absorptiometry. Although none of the defects were completely healed in this time, muscle grafts expressing BMP-2 deposited more than twice as much new bone as controls. Histology confirmed the anatomical integrity of the newly formed bone, which was comparable in thickness and mineral density to the original cranial bone. This study confirms the in vivo osteogenic properties of genetically modified muscle and suggests novel strategies for healing bone. � 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1095–1102, 2012