A metabolomic approach to studying mechanisms of polymeric gene delivery to retinal pigment epithelial cells

Tiivistelmä ReferatAbstract Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.

Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

Cancer cells are often associated with secondary chromosomal rearrangements, such as deletions, inversions, and translocations, which could be the consequence of unrepaired/misrepaired DNA double strand breaks (DSBs). Nonhomologous DNA end joining is one of the most common pathways to repair DSBs in higher eukaryotes. By using oligomeric DNA substrates mimicking various endogenous DSBs in a cell-free system, we studied end joining (EJ) in different cancer cell lines. We found that the efficiency of EJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of EJ proteins. Interestingly, cancer cells with lower levels of EJ possessed elevated expression of BCL2 and vice versa. Removal of BCL2 by immunoprecipitation or protein fractionation led to elevated EJ. More importantly, we show that overexpression of BCL2 or the addition of purified BCL2 led to the down-regulation of EJ. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo. Hence, our results suggest that EJ in cancer cells could be negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute toward increased chromosomal abnormalities in cancer.

A possible role for a low molecular weight peptide in regulation of testosterone production by rat Leydig cells

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of Percoll purified Leydig cell proteins from 20- and 120-day-old rats revealed a significant decrease in a low molecular weight peptide in the adult rats. Administration of human chorionic gonadotropin to immature rats resulted in a decrease in the low molecular weight peptide along with increase in testosterone production. Modulation of the peptide by human chorionic gonadotropin could be confirmed by Western blotting. The presence of a similar peptide could be detected by Western blotting in testes of immature mouse, hamster, guinea pig but not in adrenal, placenta and corpus luteum. Administration of testosterone propionate which is known to inhibit the pituitary luteinizing hormone levels in adult rats resulted in an increase in the low molecular weight peptide, as checked by Western blotting. It is suggested that this peptide may have a role in regulation of acquisition of responsiveness to luteinizing hormone by immature rat Leydig cells.

Regulation and Function of GATA Transcription Factors in Adrenocortical Tumors and Granulosa Cells

Transcription factors play a key role in tumor development, in which dysfunction of genes regulating tissue growth and differentiation is a central phenomenon. The GATA family of transcription factors consists of six members that bind to a consensus DNA sequence (A/T)GATA(A/G) in gene promoters and enhancers. The two GATA factors expressed in the adrenal cortex are GATA-4 and GATA-6. In both mice and humans, GATA-4 can be detected only during the fetal period, whereas GATA-6 expression is abundant both throughout development and in the adult. It is already established that GATA factors are important in both normal development and tumorigenesis of several endocrine organs, and expression of GATA-4 and GATA-6 is detected in adrenocortical tumors. The aim of this study was to elucidate the function of these factors in adrenocortical tumor growth. In embryonal development, the adrenocortical cells arise and differentiate from a common pool with gonadal steroidogenic cells, the urogenital ridge. As the adult adrenal cortex undergoes constant renewal, it is hypothesized that undifferentiated adrenocortical progenitor cells reside adjacent to the adrenal capsule and give rise to daughter cells that differentiate and migrate centripetally. A diverse array of hormones controls the differentiation, growth and survival of steroidogenic cells in the adrenal gland and the gonads. Factors such as luteinizing hormone and inhibins, traditionally associated with gonadal steroidogenic cells, can also influence the function of adrenocortical cells in physiological and pathophysiological states. Certain inbred strains of mice develop subcapsular adrenocortical tumors in response to gonadectomy. In this study, we found that these tumors express GATA-4, normally absent from the adult adrenal cortex, while GATA-6 expression is downregulated. Gonadal markers such as luteinizing hormone receptor, anti-Müllerian hormone and P450c17 are also expressed in the neoplastic cells, and the tumors produce gonadal hormones. The tumor cells have lost the expression of melanocortin-2 receptor and the CYP enzymes necessary for the synthesis of corticosterone and aldosterone. By way of xenograft studies utilizing NU/J nude mice, we confirmed that chronic gonadotropin elevation is sufficient to induce adrenocortical tumorigenesis in susceptible inbred strains. Collectively, these studies suggest that subcapsular adrenocortical progenitor cells can, under certain conditions, adopt a gonadal fate. We studied the molecular mechanisms involved in gene regulation in endocrine cells in order to elucidate the role of GATA factors in endocrine tissues. Ovarian granulosa cells express both GATA-4 and GATA-6, and the TGF-β signaling pathway is active in these cells. Inhibin-α is both a target gene for, and an atypical or antagonistic member of the TGF-β growth factor superfamily. In this study, we show that GATA-4 is required for TGF-β-mediated inhibin-α promoter activation in granulosa cells, and that GATA-4 physically interacts with Smad3, a TGF-β downstream protein. Apart from the regulation of steroidogenesis and other events in normal tissues, TGF-β signaling is implicated in tumors of multiple organs, including the adrenal cortex. Another signaling pathway found often to be aberrantly active in adrenocortical tumors is the Wnt pathway. As both of these pathways regulate the expression of inhibin-α, a transcriptional target for GATA-4 and GATA-6, we wanted to investigate whether GATA factors are associated with the components of these signaling cascades in human adrenocortical tumors. We found that the expression of Wnt co-receptors LRP5 and LRP6, Smad3, GATA-6 and SF-1 was diminished in adrenocortical carcinomas with poor outcome. All of these factors drive inhibin-α expression, and their expression in adrenocortical tumors correlated with that of inhibin-α. The results support a tumor suppressor role previously suggested for inhibin-α in the mouse adrenal cortex, and offer putative pathways associated with adrenocortical tumor aggressiveness. Unraveling the role of GATA factors and associated molecules in human and mouse adrenocortical tumors could ultimately contribute to the development of diagnostic tools and future therapies for these diseases.

A new method for modulating the activity of parvalbumin-positive neurons and cerebellar Purkinje cells in vivo

Neurons can be divided into various classes according to their location, morphology, neurochemical identity and electrical properties. They form complex interconnected networks with precise roles for each cell type. GABAergic neurons expressing the calcium-binding protein parvalbumin (Pv) are mainly interneurons, which serve a coordinating function. Pv-cells modulate the activity of principal cells with high temporal precision. Abnormalities of Pv-interneuron activity in cortical areas have been linked to neuropsychiatric illnesses such as schizophrenia. Cerebellar Purkinje cells are known to be central to motor learning. They are the sole output from the layered cerebellar cortex to deep cerebellar nuclei. There are still many open questions about the precise role of Pv-neurons and Purkinje cells, many of which could be answered if one could achieve rapid, reversible cell-type specific modulation of the activity of these neurons and observe the subsequent changes at the whole-animal level. The aim of these studies was to develop a novel method for the modulation of Pv-neurons and Purkinje cells in vivo and to use this method to investigate the significance of inhibition in these neuronal types with a variety of behavioral experiments in addition to tissue autoradiography, electrophysiology and immunohistochemistry. The GABA(A) receptor γ2 subunit was ablated from Pv-neurons and Purkinje cells in four separate mouse lines. Pv-Δγ2 mice had wide-ranging behavioral alterations and increased GABA-insensitive binding indicative of an altered GABA(A) receptor composition, particularly in midbrain areas. PC-Δγ2 mice experienced little or no motor impairment despite the lack of inhibition in Purkinje cells. In Pv-Δγ2-partial rescue mice, a reversal of motor and cognitive deficits was observed in addition to restoration of the wild-type γ2F77 subunit to the reticular nucleus of thalamus and the cerebellar molecular layer. In PC-Δγ2-swap mice, zolpidem sensitivity was restored to Purkinje cells and the administration of systemic zolpidem evoked a transient motor impairment. On the basis of these results, it is concluded that this new method of cell-type specific modulation is a feasible way to modulate the activity of selected neuronal types. The importance of Purkinje cells to motor control supports previous studies, and the crucial involvement of Pv-neurons in a range of behavioral modalities is confirmed.

Enhanced photodynamic effect of cobalt(III) dipyridophenazine complex on thyrotropin receptor expressing HEK293 cells

Ternary cobalt(III) complexes CoL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyridoquinoxaline (dpq in 2) and dipyridophenazine (dppz in 3) are synthesized, characterized from X-ray crystallographic, analytical and spectral techniques, and their utility in photodynamic therapy (PDT) of thyroid diseases caused by TSH receptor dysfunction is probed. The complexes display a visible spectral band within the PDT spectral window at similar to 690 nm. Photodynamic potential was estimated through DNA cleavage activity of the dpq and dppz complexes in UV-A light of 365 nm and red light of 676 nm. The reactions proceed via the hydroxyl radical pathway. The complexes retain their DNA photocleavage activity in red light under anaerobic conditions, a situation normally prevails in hypoxic tumor core. Investigation into the photocytotoxic potential of these complexes showed that the dppz complex 3 is approximately 4-fold more active in the HEK293 cells expressing human thyrotropin receptor (HEK293-hTSHR) than in the parental cell line and has an insignificant effect on an unrelated human cervical carcinoma cell line (HeLa). Photoexcitation of complex 3 in HEK293-hTSHR cells leads to damage hTSHR as evidenced from the decrease in cAMP formation both in absence and presence of hTSH and decrease in the TSHR immunofluorescence with a concomitant cytoplasmic translocation of the membrane protein, cadherin. The involvement of hTSHR is evidenced from the ability of complex 3 to bind to the extracellular domain of hTSHR (hTSHR-ECD) with a K-d value of 81 nM and from the photocleavage of hTSHR-ECD.

Functions of human papillomavirus E5 oncogene in epithelial cells and in the onset of cervical cancer

Human papillomaviruses (HPVs) are the causal agents of cervical cancer, which is the second most common cancer among women worldwide. Cellular transformation and carcinogenesis depend on the activities of viral E5, E6 and E7 proteins. Alterations in cell-cell contacts and in communication between epithelial cells take place during cervical carcinogenesis, leading to changes in cell morphology, increased cell motility and finally invasion. The aim of this thesis was to study genome-wide effects of the HPV type 16 (HPV-16) E5 protein on the expression of host cell messenger RNAs (mRNAs) and microRNAs by applying microarray technology. The results showed that the HPV-16 E5 protein alters several cellular pathways involved in cellular adhesion, motility and proliferation as well as in the extracellular matrix. The E5 protein was observed to enhance wound healing of epithelial cell monolayers by increasing cell motility in vivo. HPV-16 E5-induced alterations in the expression of cellular microRNAs and their target genes seem to favour increased proliferation and tumorigenesis. E5 was also shown to affect the expression of adherens junction proteins in HaCaT epithelial keratinocytes. In addition, a study of a membrane cytoskeletal cross-linker protein, ezrin, revealed that when activated, it localizes to adherens junctions. The results suggest that ezrin distribution to forming adherens junctions is due to Rac1 activity in epithelial cells. These studies reveal for the first time the holistic effects of HPV-16 E5 protein in promoting precancerous events in epithelial cells. The results contribute to identifyinging novel markers for cervical precancerous stages and to predicting disease behaviour.

TLR 9 Activation in Dendritic Cells Enhances Salmonella Killing and Antigen Presentation via Involvement of the Reactive Oxygen Species

Synthetic CpG containing oligodeoxynucleotide Toll like receptor-9 agonist (CpG DNA) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. It was observed that Salmonella Typhimurium growth can be inhibited by the CpG DNA treatment in the murine dendritic cells. This inhibitory effect was mediated by an increased reactive oxygen species production. In addition, it was noted that CpG DNA treatment of dendritic cells during Salmonella infection leads to an increased antigen presentation. Further this increased antigen presentation was dependent on the enhanced reactive oxygen species production elicited by Toll like receptor-9 activation. With the help of an exogenous antigen it was shown that Salmonella antigen could also be cross-presented in a better way by CpG induction. These data collectively indicate that CpG DNA enhance the ability of murine dendritic cells to contain the growth of virulent Salmonella through reactive oxygen species dependent killing.

Src Homology 3-interacting Domain of Rv1917c of Mycobacterium tuberculosis Induces Selective Maturation of Human Dendritic Cells by Regulating PI3K-MAPK-NF-kappa B Signaling and Drives Th2 Immune Responses

Mycobacterium tuberculosis, an etiological agent of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Pathogenic mycobacteria survive in the host by subverting host innate immunity. Dendritic cells (DCs) are professional antigen-presenting cells that are vital for eliciting immune responses to infectious agents, including pathogenic mycobacteria. DCs orchestrate distinct Th responses based on the signals they receive. In this perspective, deciphering the interactions of the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family of proteins of M. tuberculosis with DCs assumes significant pathophysiological attributes. In this study, we demonstrate that Rv1917c (PPE34), a representative member of the proline-proline-glutamic-major polymorphic tandem repeat family, interacts with TLR2 and triggers functional maturation of human DCs. Signaling perturbations implicated a critical role for integrated cross-talk among PI3K-MAPK and NF-kappa B signaling cascades in Rv1917c-induced maturation of DCs. However, this maturation of DCs was associated with a secretion of high amounts of anti-inflammatory cytokine IL-10, whereas Th1-polarizing cytokine IL-12 was not induced. Consistent with these results, Rv1917c-matured DCs favored secretion of IL-4, IL-5, and IL-10 from CD4(+) T cells and contributed to Th2-skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, the Rv1917c-skewed Th2 immune response involved induced expression of cyclooxygenase-2 (COX-2) in DCs. Taken together, these results indicate that Rv1917c facilitates a shift in the ensuing immunity toward the Th2 phenotype and could aid in immune evasion by mycobacteria.

A heuristic clustering algorithm using union of overlapping pattern-cells

Relative geometric arrangements of the sample points, with reference to the structure of the imbedding space, produce clusters. Hence, if each sample point is imagined to acquire a volume of a small M-cube (called pattern-cell), depending on the ranges of its (M) features and number (N) of samples; then overlapping pattern-cells would indicate naturally closer sample-points. A chain or blob of such overlapping cells would mean a cluster and separate clusters would not share a common pattern-cell between them. The conditions and an analytic method to find such an overlap are developed. A simple, intuitive, nonparametric clustering procedure, based on such overlapping pattern-cells is presented. It may be classified as an agglomerative, hierarchical, linkage-type clustering procedure. The algorithm is fast, requires low storage and can identify irregular clusters. Two extensions of the algorithm, to separate overlapping clusters and to estimate the nature of pattern distributions in the sample space, are also indicated.

A Comparative Study of the Effect of Metallic Au and ReO3 Nanoparticles on the Performance of Silicon Solar Cells

The influence of gold (similar to 35 nm diameter) as well as ReO3 (similar to 17 nm diameter) nanoparticles placed atop silicon photovoltaic devices on absorption and photocurrent generation has been investigated. The nanoparticles improve the power transmission into the semiconductor and consequently, the photocurrent response at wavelengths corresponding to plasmon absorption. An increase in short circuit current up to 4.5% under simulated solar irradiation was observed with the ReO3 nanoparticles, while the gold nanoparticles showed enhancements up to 6.5%. The increase in photocurrent is observed at wavelengths corresponding to the maxima in the surface plasmon resonance absorption spectra. (C) 2010 The Japan Society of Applied Physics

Photo-activated cytotoxicity of a pyrenyl-terpyridine copper(II) complex in HeLa cells

Terpyridine copper(II) complexes Cu(L)(2)](NO3)(2) where L is (4'-phenyl)-2 2' 6' 2 `'-terpyridine (ph-tpy in 1) and 4-(1 pyrenyl)]-2 2' 6' 2'-terpyridine (py-tpy in 2) are prepared characterized and their photocytotoxic activity studied The crystal structure of complex 1 shows distorted octahedral CuN6 coordination geometry The 1 2 electrolytic and one-electron paramagnetic complexes show a visible band near 650 nm in DMF-H2O The complexes show emission band at 352 nm for 1 and 425 nm for 2 when excited at 283 and 346 nm respectively The Cu(II)-Cu(I) redox couple is observed near -0 2 V versus SCE in DMF-0 1 m TBAP The complexes are avid partial-intercalative binders to calf thymus DNA giving binding constant (K-b) values of similar to 10(6) M-1 Complex 2 with its photoactive pyrenyl moiety exhibits significant photocleavage of pUC19 DNA in red light via singlet oxygen pathway Complex 2 also exhibits significant photo-activated cytotoxicity in HeLa cancer cells in visible light giving IC50 value of 11 9 mu M while being non-toxic in dark with an IC50 value of 130 5 mu M (C) 2010 Elsevier Ltd All rights reserved

Mesoporous Carbon and Poly(3,4-ethylenedioxythiophene) Composite as Catalyst Support for Polymer Electrolyte Fuel Cells

In situ polymerization of 3,4-ethylenedioxythiophene with sol-gel-derived mesoporous carbon (MC) leading to a new composite and its subsequent impregnation with Pt nanoparticles for application in polymer electrolyte fuel cells (PEFCs) is reported. The composite exhibits good dispersion and utilization of platinum nanoparticles akin to other commonly used microporous carbon materials, such as carbon black. Pt-supported MC-poly(3,4-ethylenedioxythiophene) (PEDOT) composite also exhibits promising electrocatalytic activity toward oxygen reduction reaction, which is central to PEFCs. The PEFC with Pt-loaded MC-PEDOT support exhibits 75% of enhancement in its power density in relation to the PEFC with Pt-loaded pristine MC support while operating under identical conditions. It is conjectured that Pt-supported MC-PEDOT composite ameliorates PEFC performance/durability on repetitive potential cycling. (C) 2010 The Electrochemical Society. DOI: 10.1149/1.3486172] All rights reserved.

Signaling different pathways of cell death: Abrin induced programmed necrosis in U266B1 cells

Abrin is a type II ribosome-inactivating protein comprising of two subunits, A and B. Of the two, the A-subunit harbours the RNA-N-glycosidase activity and the B subunit is a galactose specific lectin that enables the entry of the protein inside the cell. Abrin inhibits protein synthesis and has been reported to induce apoptosis in several cell types. Based on these observations abrin is considered to have potential for the construction of immunotoxin in cell targeted therapy. Preliminary data from our laboratory however showed that although abrin inhibited the protein synthesis in all cell types, the mode of cell death varied. The aim of the present study was therefore to understand different death pathways induced by abrin in different cells. We used the human B cell line, U266B1 and compared it with the earlier studied T cell line Jurkat, for abrin-mediated inhibition of protein translation as well as cell death. While abrin triggered programmed apoptosis in Jurkat cells in a caspase-dependent manner, it induced programmed necrosis in U266B1 cells in a caspase-independent manner, even when there was reactive oxygen species production and loss of mitochondrial membrane potential. The data revealed that abrin-mediated necrosis involves lysosomal membrane permeabilization and release of cathepsins from the lysosomes. Importantly, the choice of abrin-mediated death pathway in the cells appears to depend on which of the two events occurs first: lysosomal membrane permeabilization or loss of mitochondrial membrane potential that decides cell death by necrosis or apoptosis. (C) 2010 Elsevier Ltd. All rights reserved.