Identification of a potent MAR element from the mouse genome and assessment of its activity in stable and transient transfections.

Matrix attachment regions are DNA sequences found throughout eukaryotic genomes that are believed to define boundaries interfacing heterochromatin and euchromatin domains, thereby acting as epigenetic regulators. When included in expression vectors, MARs can improve and sustain transgene expression, and a search for more potent novel elements is therefore actively pursued to further improve recombinant protein production. Here we describe the isolation of new MARs from the mouse genome using a modified in silico analysis. One of these MARs was found to be a powerful activator of transgene expression in stable transfections. Interestingly, this MAR also increased GFP and/or immunoglobulin expression from some but not all expression vectors in transient transfections. This effect was attributed to the presence or absence of elements on the vector backbone, providing an explanation for earlier discrepancies as to the ability of this class of elements to affect transgene expression under such conditions.

The major transcription initiation site of the SV40 late promoter is a potent thyroid hormone response element.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily, which act as transcription factors upon binding to specific DNA sequences called thyroid hormone (T3) response elements (TREs). Such elements are found in the upstream regulatory region of promoters as well as in intragenic sequences of T3-responsive genes. In this report, we demonstrate that SV40 late (SVL) promoter activity is strongly down-regulated by TR in the absence of ligand. Addition of T3 releases this repression, but does not further induce SVL promoter activity. Electrophoretic mobility shift analyses reveal a TR binding element that overlaps with the SV40 major late transcription initiation site. This element closely fits the consensus TRE, formed of two hexanucleotides organized in a tandem repeat separated by 4 nt, and is able to confer T3 responsiveness on a heterologous promoter. We further show that, although the presence of TR leads to quantitatively modified expression of an SVL-driven reporter gene, neither displacement of the site of transcription initiation nor modification of the splicing pattern of the primary transcripts occur.

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors.

Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.

Heavy metal ions are potent inhibitors of protein folding.

Environmental and occupational exposure to heavy metals such as cadmium, mercury and lead results in severe health hazards including prenatal and developmental defects. The deleterious effects of heavy metal ions have hitherto been attributed to their interactions with specific, particularly susceptible native proteins. Here, we report an as yet undescribed mode of heavy metal toxicity. Cd2+, Hg2+ and Pb2+ proved to inhibit very efficiently the spontaneous refolding of chemically denatured proteins by forming high-affinity multidentate complexes with thiol and other functional groups (IC(50) in the nanomolar range). With similar efficacy, the heavy metal ions inhibited the chaperone-assisted refolding of chemically denatured and heat-denatured proteins. Thus, the toxic effects of heavy metal ions may result as well from their interaction with the more readily accessible functional groups of proteins in nascent and other non-native form. The toxic scope of heavy metals seems to be substantially larger than assumed so far.

Allorestricted T lymphocytes with a high avidity T-cell receptor towards NY-ESO-1 have potent anti-tumor activity.

The cancer-testis antigen NY-ESO-1 has been targeted as a tumor-associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T-cell-based therapy is the induction of T cells capable of recognizing the NY-ESO-1-expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY-ESO-1(157-165) epitope known to be naturally presented with HLA-A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY-ESO-1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA-A2-positive, NY-ESO-1-expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY-ESO-1-specific T lymphocytes displayed TCRs with the highest avidity and best anti-tumor recognition activity. TCRs derived from allorestricted, NY-ESO-1-specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of NY-ESO-1-expressing hematological malignancies and solid tumors.

1,2,3,4-Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-site inhibitors of acetylcholinesterase: synthesis, pharmacological evaluation and mechanistic studies

A series of 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridines differently substituted at positions 1, 5, and 9 have been designed from the pyrano[3,2-c]quinoline derivative 1, a weak inhibitor of acetylcholinesterase (AChE) with predicted ability to bind to the AChE peripheral anionic site (PAS), at the entrance of the catalytic gorge. Fourteen novel benzonaphthyridines have been synthesized through synthetic sequences involving as the key step a multicomponent Povarov reaction between an aldehyde, an aniline and an enamine or an enamide as the activated alkene. The novel compounds have been tested against Electrophorus electricus AChE (EeAChE), human recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling. We have found that a seemingly small structural change such as a double O → NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217- and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (> 11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities.

1,2,3,4-Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-site inhibitors of acetylcholinesterase: synthesis, pharmacological evaluation and mechanistic studies

A series of 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridines differently substituted at positions 1, 5, and 9 have been designed from the pyrano[3,2-c]quinoline derivative 1, a weak inhibitor of acetylcholinesterase (AChE) with predicted ability to bind to the AChE peripheral anionic site (PAS), at the entrance of the catalytic gorge. Fourteen novel benzonaphthyridines have been synthesized through synthetic sequences involving as the key step a multicomponent Povarov reaction between an aldehyde, an aniline and an enamine or an enamide as the activated alkene. The novel compounds have been tested against Electrophorus electricus AChE (EeAChE), human recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling. We have found that a seemingly small structural change such as a double O → NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217- and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (> 11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities.

Liver Glucokinase A456V induces potent hypoglycemia without dyslipidemia through a paradoxical induction of the catalytic subunit of glucose-6-phosphatase

Recent reports point out the importance of the complex GK-GKRP in controlling glucose and lipid homeostasis. Several GK mutations affect GKRP binding, resulting in permanent activation of the enzyme. We hypothesize that hepatic overexpression of a mutated form of GK, GKA456V, described in a patient with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) and could provide a model to study the consequences of GK-GKRP deregulation in vivo. GKA456V was overexpressed in the liver of streptozotocin diabetic mice. Metabolite profiling in serum and liver extracts, together with changes in key components of glucose and lipid homeostasis, were analyzed and compared to GK wild-type transfected livers. Cell compartmentalization of the mutant but not the wild-type GK was clearly affected in vivo, demonstrating impaired GKRP regulation. GKA456V overexpression markedly reduced blood glucose in the absence of dyslipidemia, in contrast to wild-type GK-overexpressing mice. Evidence in glucose utilization did not correlate with increased glycogen nor lactate levels in the liver. PEPCK mRNA was not affected, whereas the mRNA for the catalytic subunit of glucose-6-phosphatase was upregulated ~4 folds in the liver of GKA456V-treated animals, suggesting that glucose cycling was stimulated. Our results provide new insights into the complex GK regulatory network and validate liver-specific GK activation as a strategy for diabetes therapy.

Synthesis and structure - Activity relationship study of potent cytotoxic analogues of the marine alkaloid Lamellarin D

The marine alkaloid, Lamellarin D (Lam-D), has shown potent cytotoxicity in numerous cancer cell lines, and was recently identified as a potent topoisomerase I inhibitor. A library of open lactone analogs of Lam-D was prepared from a methyl 5,6-dihydropyrrolo[2,1-a]isoquinoline-3- carboxylate scaffold (1) by introducing various aryl groups through sequential and regioselective bromination, followed by Pd(0)-catalyzed Suzuki cross-coupling chemistry. The compounds were obtained in a 24-44% overall yield, and tested in a panel of three human tumor cell lines, MDA-MB- 231 (breast), A-549 (lung), and HT-29 (colon), to evaluate their cytotoxic potential. From these data the SAR study concluded that more than 75% of the open-chain Lam-D analogs tested showed cytotoxicity in a low micromolar GI50 range.

Synthesis and structure - Activity relationship study of potent cytotoxic analogues of the marine alkaloid Lamellarin D

The marine alkaloid, Lamellarin D (Lam-D), has shown potent cytotoxicity in numerous cancer cell lines, and was recently identified as a potent topoisomerase I inhibitor. A library of open lactone analogs of Lam-D was prepared from a methyl 5,6-dihydropyrrolo[2,1-a]isoquinoline-3- carboxylate scaffold (1) by introducing various aryl groups through sequential and regioselective bromination, followed by Pd(0)-catalyzed Suzuki cross-coupling chemistry. The compounds were obtained in a 24-44% overall yield, and tested in a panel of three human tumor cell lines, MDA-MB- 231 (breast), A-549 (lung), and HT-29 (colon), to evaluate their cytotoxic potential. From these data the SAR study concluded that more than 75% of the open-chain Lam-D analogs tested showed cytotoxicity in a low micromolar GI50 range.

1,2,3,4-Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-site inhibitors of acetylcholinesterase: synthesis, pharmacological evaluation and mechanistic studies

A series of 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridines differently substituted at positions 1, 5, and 9 have been designed from the pyrano[3,2-c]quinoline derivative 1, a weak inhibitor of acetylcholinesterase (AChE) with predicted ability to bind to the AChE peripheral anionic site (PAS), at the entrance of the catalytic gorge. Fourteen novel benzonaphthyridines have been synthesized through synthetic sequences involving as the key step a multicomponent Povarov reaction between an aldehyde, an aniline and an enamine or an enamide as the activated alkene. The novel compounds have been tested against Electrophorus electricus AChE (EeAChE), human recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling. We have found that a seemingly small structural change such as a double O → NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217- and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (> 11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities.

1,2,3,4-Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-site inhibitors of acetylcholinesterase: synthesis, pharmacological evaluation and mechanistic studies

A series of 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridines differently substituted at positions 1, 5, and 9 have been designed from the pyrano[3,2-c]quinoline derivative 1, a weak inhibitor of acetylcholinesterase (AChE) with predicted ability to bind to the AChE peripheral anionic site (PAS), at the entrance of the catalytic gorge. Fourteen novel benzonaphthyridines have been synthesized through synthetic sequences involving as the key step a multicomponent Povarov reaction between an aldehyde, an aniline and an enamine or an enamide as the activated alkene. The novel compounds have been tested against Electrophorus electricus AChE (EeAChE), human recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling. We have found that a seemingly small structural change such as a double O → NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217- and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (> 11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities.

1,2,3,4-Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-site inhibitors of acetylcholinesterase: synthesis, pharmacological evaluation and mechanistic studies

A series of 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridines differently substituted at positions 1, 5, and 9 have been designed from the pyrano[3,2-c]quinoline derivative 1, a weak inhibitor of acetylcholinesterase (AChE) with predicted ability to bind to the AChE peripheral anionic site (PAS), at the entrance of the catalytic gorge. Fourteen novel benzonaphthyridines have been synthesized through synthetic sequences involving as the key step a multicomponent Povarov reaction between an aldehyde, an aniline and an enamine or an enamide as the activated alkene. The novel compounds have been tested against Electrophorus electricus AChE (EeAChE), human recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling. We have found that a seemingly small structural change such as a double O → NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217- and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (> 11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities.

Elucidation of odour-potent compounds and sensory profiles of Vidal blanc and Riesling icewines from the Niagara Peninsula : effect of harvest date and crop level

I t was hypothesized that the freeze/thaw cycles endured by icewine grapes would change their chemical composition, resulting in unique chemical fingerprint and sensory properties, and would be affected by harvest date (HD) and crop level (CL). The objectives were: 1) to identify odour-active compounds using gas chromatographic and sensory analysis; 2) to determine the effect of CL and HD on these compounds; 3) to determine the icewine sensory profiles; 4) to correlate analytical and sensory results for an overall icewine profile. CharmAnalysis™ determined the Top 15 odour-potent compounds in Vidal and Riesling icewine and table wines; 24 and 23 compounds, respectively. The majority of the compounds had the highest concentrations in the icewines compared to table wines. These compounds were used as the foundation for assessing differences in icewine chemical profiles from different HD and CL. Vidal and Riesling icewine were made from grapes picked at different HD; HI : 19 December; H2: 29 December; H3: 18 January; H4: 11 February (Vidal only). HI wines differed from H3 and H4 wines in both Vidal and Riesling for aroma compounds and sensory profiles. - Three·CL [control (fully cropped), cluster thin at fruit set to one basal cluster/shoot (TFS), and cluster thin at veraison to one basal cluster/shoot (TV)] were evaluated for Riesling and Vidal cultivars over two seasons. Vidal icewines had the highest concentration of aroma compounds in the control and TV icewines in 2003 and in TFS icewines in 2004. In Riesling, most aroma compounds had the highest concentration in the TV icewines and the lowest concentration in the TFS wine for both years. The thinned treatments were associated with almost all of the sensory attributes in both cultivars, both years. HD and CL affected the chemical variables, aroma compounds and sensory properties of Vidal and Riesling icewines and freeze/thaw events changed their sensory profile. The most odour-potent compounds were p-damascenone, cis-rose oxide, 1- octen-3-ol, 4-vinylguaiacol, ethyl octanoate, and ethyl hexanoate. The role of Pdamascenone as a marker compound for icewine requires further investigation. This research provides a strong foundation for the understanding the odour-active volatiles and sensory profiles important to icewine.