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Two new dibenz[b,f]oxepins, empetroxepins A and B (1and 2), and seven known compounds (3–9) were isolated from an extract of the Canadian medicinal plant Empetrum nigrumthat significantly inhibited the growth of Mycobacterium tuberculosisH37Ra. The structures of 1and 2were established through analysis of NMR and MS data. The antimycobacterial activity of the plant extract was attributed primarily to the presence of two chalcone derivatives (6and 7) that exhibited selective antimycobacterial activity (IC50values of 23.8 and 32.8 μM, respectively) in comparison to mammalian (HEK 293) cells (IC50values of 109 and 249 μM, respectively).

The study of anticancer properties from natural products has regained popularity as natural molecules provide a high diversity of chemical structures with specific biological and medicinal activity. Based on a documented library of the most common medicinal plants used by the indigenous people of North America, we screened and isolated compounds with anti-breast cancer properties from Juniperus communis (common Juniper). Using bioassay-guided fractionation of a crude plant extract, we identified the diterpene isocupressic acid and the aryltetralin lignan deoxypodophyllotoxin (DPT) as potent inducers of caspase-dependent programmed cell death (apoptosis) in malignant MB231 breast cancer cells. Further elucidation revealed that DPT, in contrast to isocupressic acid, also concomitantly inhibited cell survival pathways mediated by the MAPK/ERK and NFΚB signaling pathways within hours of treatment. Our findings emphasize the potential and importance of natural product screening for new chemical entities with novel anticancer activities. Natural products research complemented with the wealth of information available through the ethnobotanical and ethnopharmacological knowledge of the indigenous peoples of North America can provide new candidate entities with desirable bioactivities to develop new cancer therapies.

S100 inflammatory proteins have been previously shown to modulate breast cancer processes. More specifically, genome-wide transcriptome studies associate S100A8 and S100A9 members to breast cancer progression and malignancy. Findings have shown that S100A8 and S100A9 can signal and regulate cancer cell behavior through both extracellular and intracellular-initiated cascades. However, functional studies exploring the effects of S100 proteins are often contradictory leaving ambiguity and a paucity of data relating to the specific function of S100A8 and S100A9 in breast cancer progression. In this study we sought to better define the functions of intracellular expressed S100A8 and S100A9 on key signaling and cellular processes driving breast cancer malignancy. We observed that extracellular treatments of the MCF7 breast cancer cell line with S100A8 and S100A9 proteins induces cell proliferation. In contrast, intracellular recombinant expression of S100A8 and S100A9 led to growth suppression. Furthermore our analysis revealed that intracellular-expressed S100A8 and S100A9 promote an epithelial-like phenotype through the induction of key markers, such as Ecadherin, integrin alpha-5 and Zona Occludens 1 (ZO-1). Concomitantly, S100A8 and S100A9 negatively regulate the activity of the promalignant Focal Adhesion Kinase-1 (FAK) signaling cascade leading to changes in cell adhesion and invasion properties. Our results uncover important differences in intracellular versus extracellular initiated S100A8 and S100A9 signaling cascades and their effects on mammary epithelial growth. Importantly, S100A8 and S100A9 appear to suppress breast cancer malignancy through an increase in mesenchymal to epithelial transitioning. Our findings shed insight into S100 protein involvement in breast cancer invasiveness and metastasis and clarify some of the controversies relating to these proteins in breast cancer processes.

The Pax-5 gene encodes a B-cell-specific activator protein (BSAP) that plays a key role in B lymphocyte differentiation and embryogenesis. The deregulation of this transcription factor is also linked to B cell malignancies and recently to other cancers. More specifically, the downstream effects of Pax-5 promote cell-cell interactions and mediate the activation of adhesion genes which result in an epithelial phenotypic behavior of human carcinoma cells. To gain a better understanding of Pax-5-mediated gene regulation, we studied available gene expression data in depth and identified several Pax-5 downstream targets. Among these, we found that Pax-5 activity is consistently inversely correlated with the expression of Focal Adhesion Kinase 1 (FAK1). FAK1 is known to enhance migration of cancer cells and promote metastatic dissemination to distant sites. Further analysis looking at genome wide profiling of Pax-5 DNAbinding points to both direct and indirect regulation of FAK1 expression by Pax-5 and its downstream targets. These findings suggest a key role for Pax-5 in phenotypic transitioning during metastasis through the regulation of FAK1 activity.

The development of gene-inactivation systems is an active and important field for both functional genomics and gene therapy. Towards this end, ribozymes (i.e. RNA enzymes), that specifically recognize and subsequently catalyze the cleavage of other target RNA molecules, are attractive molecular tools. Ribozymes represent an interesting alternative to the RNA interference (RNAi) approach for gene inactivation, especially given the fact that RNAi seems to trigger an immunological response and has demonstrated off-target effects. However, the design and optimization of a ribozymebased gene-inactivation system is not a straightforward procedure. Several aspects need to be considered in the experimental design in order to provide a suitable suppression system. In this review we present the advances in this domain made available from work using the hepatitis delta virus (HDV) ribozyme as a cis-acting RNA motif in molecular biology, as well as a trans-acting molecular scissor for the development of a gene-inactivation system. This HDV ribozyme technology possesses several unique features that are all related to the fact that it is the only catalytic cleaving RNA motif that has been discovered in humans.

Human immunodeficiency virus type-1 (HIV-1) preferentially replicates in CD4-expressing T cells bearing a “memory” (CD45RO+) rather than a “naive” (CD45RA+/CD62L+) phenotype. Yet the basis for the higher susceptibility of these cells to HIV-1 infection remains unclear. Because the nature of the CD45 isoform itself can affect biochemical events in T cells, we set out to determine whether these isoforms could differently modulate HIV-1 long terminal repeat (LTR) activity and thereby replication. Through the use of CD4+ Jurkat T cells specifically expressing distinct CD45 isoforms (i.e.CD45RABC or CD45RO), we demonstrated that a difference in CD45 isoform expression conferred preferential replication of HIV-1 to CD45RO-expressing T cell clones following a physiological CD3/CD28 stimulation. Closer analysis indicated that higher HIV-1 LTR activation levels were consistently observed in CD45RO-positive cells, which was paralleled by more pronounced nuclear factor of activated T cells (NFAT) activation in these same cells. Specific involvement of NFAT1 was revealed in studied Jurkat clones by mobility shift analyses. In addition, preferential activation of the LTR and viral replication in CD45RO T cells was FK506- and cyclosporin A-sensitive. These results underscore the importance of NFAT in HIV-1 regulation and for the first time identify the role of the CD45 isoform in limiting productive HIV-1 replication to the human CD4 memory T cell subset.

Although protein tyrosine phosphatase (PTP) inhibitors used in combination with other stimuli can induce interleukin 2 (IL-2) production in T cells, a direct implication of nuclear factor of activated T cells (NFAT) has not yet been demonstrated. This study reports that exposure of leukemic T cells and human peripheral blood mononuclear cells to bis-peroxovanadium (bpV) PTP inhibitors markedly induce activation and nuclear translocation of NFAT. NFAT activation by bpV was inhibited by the immunosuppressive drugs FK506 and cyclosporin A, as well as by a specific peptide inhibitor of NFAT activation. Mobility shift assays showed specific induction of the NFAT1 member by bpV molecules. The bpV-mediated NFAT activation was observed to be important for the up-regulation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) and the IL-2 promoter; NFAT1 was demonstrated to be particularly important in bpV-dependent positive action on HIV-1 LTR transcription. The active participation of p56lck, ZAP-70, p21ras, and calcium in the bpV-mediated signaling cascade leading to NFAT activation was confirmed, using deficient cell lines and dominant-negative mutants. Finally, overexpression of wild-type SHP-1 resulted in a greatly diminished activation of NFAT by bpV, suggesting an involvement of SHP-1 in the regulation of NFAT activation. These data were confirmed by constitutive NFAT translocation observed in Jurkat cells stably expressing a dominant-negative version of SHP-1. The study proposes that PTP activity attenuates constitutive kinase activities that otherwise would lead to constant NFAT activation and that this activation is participating in HIV-1 LTR stimulation by PTP inhibition.

Although protein tyrosine phosphatase (PTP) inhibitors used in combination with other stimuli can induce interleukin 2 (IL-2) production in T cells, a direct implication of nuclear factor of activated T cells (NFAT) has not yet been demonstrated. This study reports that exposure of leukemic T cells and human peripheral blood mononuclear cells to bis-peroxovanadium (bpV) PTP inhibitors markedly induce activation and nuclear translocation of NFAT. NFAT activation by bpV was inhibited by the immunosuppressive drugs FK506 and cyclosporin A, as well as by a specific peptide inhibitor of NFAT activation. Mobility shift assays showed specific induction of the NFAT1 member by bpV molecules. The bpV-mediated NFAT activation was observed to be important for the up-regulation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) and the IL-2 promoter; NFAT1 was demonstrated to be particularly important in bpV-dependent positive action on HIV-1 LTR transcription. The active participation of p56lck, ZAP-70, p21ras, and calcium in the bpV-mediated signaling cascade leading to NFAT activation was confirmed, using deficient cell lines and dominant-negative mutants. Finally, overexpression of wild-type SHP-1 resulted in a greatly diminished activation of NFAT by bpV, suggesting an involvement of SHP-1 in the regulation of NFAT activation. These data were confirmed by constitutive NFAT translocation observed in Jurkat cells stably expressing a dominant-negative version of SHP-1. The study proposes that PTP activity attenuates constitutive kinase activities that otherwise would lead to constant NFAT activation and that this activation is participating in HIV-1 LTR stimulation by PTP inhibition.

In neurodegenerative diseases associated with AIDS, reactive astrocytosis plays a central role in the neurotoxicity of the brain parenchyma. Whereas the HIV-1 nef gene is overexpressed during restricted HIV-1 infection of human astrocytes, our previous results have demonstrated that nef expressed in human U251MG glial cells activates the sphingomyelin pathway triggered by TNF-α, increasing ceramide production. Since ceramide is an important regulatory molecule of programmed cell death induced by TNF-α, we examined whether nef could alter TNF-α-induced apoptosis in the U251MG human astrocytoma cell line. Transfection studies indicated that nef could both prevent apoptosis and promote cell proliferation in response to TNF-α stimulation. MAPK and JNK activities were further analyzed in order to elucidate signaling cascades subsequent to the upregulation of ceramide production. After TNF-α treatment, both kinases were shown to be preferentially activated in the presence of nef. These experiments strongly suggest that the HIV-1 Nef protein might modulate the sensitivity of astrocytes to inflammatory molecules, thus contributing to the development of neurodegenerative diseases associated with AIDS.

Throughout the years, most researchers have used continuous cell lines as in vitromodels to evaluate the immunopathogenesis of human immunodeficiency virus type‐1 (HIV‐1) infection. Unfortunately, the most commonly used monocytoid malignant cells have not been shown to adequately mimic primary human monocyte‐derived macrophages, at least with respect to HIV‐1 infection. The Mono Mac 1 cell line has been defined as a model system for studying biochemical, immunological, and genetic functions of human cells of the monocyte/macrophage lineage. In this study, we have investigated whether Mono Mac 1 represents an in vitroculture system for HIV‐1 infection. Flow cytometric analyses revealed that Mono Mac 1 are positive for the HIV‐1 primary receptor (CD4), as well as for the coreceptors (CXCR4, CCR5, and CCR3). Infectivity experiments conducted with recombinant luciferase‐encoding and fully infectious viruses demonstrated that Mono Mac 1 can support a highly productive infection with both macrophage‐ and dual‐tropic isolates of HIV‐1. Furthermore, differentiation of such cells led to a marked increase in virus production. Data from semiquantitative polymerase chain reaction analysis and mobility shift assays indicated that enhanced virus production in differentiated Mono Mac 1 cells was most likely related to an increase in nuclear translocation of NF‐κB. Mono Mac 1 can thus be considered as a human monocytoid cell line representing a proper in vitrosystem for studying the complex interactions between HIV‐1 and cells of the monocyte/macrophage lineage.

An extract of the endophytic fungus Fusarium acuminatum(TC2-084) isolated from the Canadian medicinal plant Geum macrophyllumexhibited significant antimycobacterial activity against Mycobacterium tuberculosisH37Ra when fermented in both malt extract and potato dextrose broths. However, significant differences observed in the NMR spectra of the respective extracts led us to further investigate both. Bioassay guided fractionation revealed that lateropyrone was solely responsible for the bioactivity observed when TC2-084 was fermented in malt extract broth, while the antimycobacterial activity of the extract derived from the isolate fermented in potato dextrose broth was augmented by the presence of enniatins B, B1, and B4 in addition to lateropyrone.

Antimycobacterial extracts of a Penicilliumsp. (isolate HL4-159-41B) and a Coniothyriumsp. (isolate HL6-097-027B) isolated from the rhizomes of the Canadian medicinal plant Aralia nudicauliswere found to contain palitantin (1) and botrallin (2), craterellin C (3), mycosporulone (4), spiromassaritone (5), and massarigenin D (6) respectively. Bioassays against Mycobacterium tuberculosisH37Ra revealed that 1-4 possess moderate antimycobacterial activity.

An extract of an unidentified endophyte from the Canadian medicinal plant Heracleum maximumexhibited a unique metabolomic profile and significant antimycobacterial activity against Mycobacterium tuberculosisH37Ra. Bioassay guided fractionation of the extract led to the isolation of phomopsolide A (1) and 6(E’)-phomopsolide A (2). This is the first report of antimycobacterial activity for 1 and 2.

1,2,3‐Triazole analogues, e.g. (III) and (V), of letrozole are synthesized by employing the “click” chemistry as the key step.

The molecular mechanisms that regulate the function of the immune system and human immunodeficiency virus type-1 (HIV-1) gene expression are diverse and complicated. However, replication of HIV-1 is controlled by many of the same regulatory signals that play a crucial role in the transcriptional regulation of the immune system. For example, the viral promoter, as is the case for the immune system, is subject to complex regulation by combinations of cellular transcription factors that may quantitatively and/or qualitatively differ depending on cell types (eg, macrophages versus T lymphocytes) and cell states (eg, undifferentiated versus differentiated or quiescent versus activated). The present review discusses the regulation of HIV-1 gene expression by nuclear factor-kappa Band nuclear factor of activated T cells, and proposes that selective interference of these two cellular transcription factors may be a route to abrogate virus replication without disrupting normal cellular functions. A better understanding of the regulation of HIV-1 gene expression is of utmost importance for the design of molecular approaches that will effectively abrogate virus replication and, ultimately, disease progression.