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The yeast transcriptional repressor Sir2p silences gene expression from the telomeric,rDNA,and silent mating-type loci and may play a role in higher order processes such as aging. Sir2p is the founding member of a large family of NAD-dependent deacetylase enzymes,named the sirtuins. These proteins are conserved from prokaryotes to eukaryotes,but most remain uncharacterized,including all seven human sirtuins. A reverse chemical genetic approach would be useful in identifying the biological function of sirtuins in a wide variety of experimental systems,but no cell-permeable small molecule inhibitors of sirtuins have been reported previously. Herein we describe a high throughput,phenotypic screen in cells that led to the discovery of a class of sirtuin inhibitors. All three compounds inhibited yeast Sir2p transcriptional silencing activity in vivo,and yeast Sir2p and human SIRT2 deacetylase activity in vitro. Such specific results demonstrate the utility and robustness of this screening methodology. Structure-activity relationship analysis of the compounds identified a key hydroxy-napthaldehyde moiety that is necessary and sufficient for inhibitory activity. Preliminary studies using one of these compounds suggest that inhibition of sirtuins interferes with body axis formation in Arabidopsis. View Publication

BACKGROUND/PURPOSE: Retinoid signaling plays an important role in many differentiation pathways. Retinoid signaling has been implicated in the induction of differentiation by pancreatic ductal cancer cell lines and in patients with pancreatic cancer. The authors wished to better understand the role of retinoid signaling in pancreatic development. METHODS: Embryonic pancreas was harvested from mice at serial gestational ages and immunohistochemical analysis was performed for retinoic acid receptors (RAR-alpha,RAR-beta,RAR-gamma),and retinoid X receptors (RXR-alpha,RXR-beta,and RXR-gamma). Also,early embryonic pancreases were cultured for 7 days with exogenous 9-cis retinoic acid (9cRA) or all-trans retinoic acid (atRA) and analyzed histologically and immunohistochemically. RESULTS: Retinoid receptors were expressed in a lineage-specific distribution,with stronger expression for many in the exocrine compartment. The receptors were not often expressed until late gestation. Exogenous 9cRA induced predominantly ducts instead of acini,plus more mature endocrine (islet) architecture. Exogenous atRA induced predominantly acini instead of ducts,with no apparent endocrine effect. CONCLUSIONS: Retinoids may have an important role in pancreatic differentiation,with a particular effect on secondary lineage selection between ductal and acinar phenotype. Because the control of ductal versus acinar differentiation has been implicated strongly in the pathogenesis of pancreatic ductal carcinoma,these results may lay the groundwork for studies in the mechanism of induced differentiation of pancreatic ductal cancer by retinoids. View Publication

Valproic acid is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen,but its mechanisms of action in any of these settings are unknown. We report that valproic acid activates Wntdependent gene expression,similar to lithium,the mainstay of therapy for bipolar disorder. Valproic acid,however,acts through a distinct pathway that involves direct inhibition of histone deacetylase (IC(50) for HDAC1 = 0.4 mm). At therapeutic levels,valproic acid mimics the histone deacetylase inhibitor trichostatin A,causing hyperacetylation of histones in cultured cells. Valproic acid,like trichostatin A,also activates transcription from diverse exogenous and endogenous promoters. Furthermore,valproic acid and trichostatin A have remarkably similar teratogenic effects in vertebrate embryos,while non-teratogenic analogues of valproic acid do not inhibit histone deacetylase and do not activate transcription. Based on these observations,we propose that inhibition of histone deacetylase provides a mechanism for valproic acid-induced birth defects and could also explain the efficacy of valproic acid in the treatment of bipolar disorder. View Publication

A method employing Percoll gradient centrifugation was developed to purify Kudoa thyrsites spores from somatic muscle tissue of Atlantic salmon Salmo salar. Highly purified spores were then used to immunize inbred BALB/c mice for derivation of hybridomas secreting Kudoa-specific monoclonal antibodies (mAbs). Analysis of mAbs by immunofluorescence microscopy and flow cytometry showed that several were specific for antigens on the surface of K. thyrsites spores whereas other mAbs reacted with polar capsules or with polar filaments of spores of K. thyrsites,K. paniformis and K. crumena. Immunoblots on spore lysates using the surface-binding mAbs showed a broad band of 46 to textgreater 220 kDa,whereas mAbs specific for antigens of polar capsules and polar filaments detected sharper bands of various molecular masses,depending on the Kudoa species. The dominant epitope of the K. thyrsites spore surface antigen was shown to be carbohydrate as determined by its sensitivity to treatment with anhydrous trifluoromethane sulfonic acid and by its resistance to treatment with Proteinase K. Immunofluorescence microscopy using the K. thyrsites-specific mAbs on isolated,intact,permeabilized plasmodia and on thin sections of somatic muscle tissue containing plasmodia revealed intense labeling of spores both within the spore-producing plasmodia and in the flesh of infected Atlantic salmon. As few as 100 spores were detected by immunoblotting,indicating that these mAbs have potential for use in developing a field-based diagnostic test. View Publication

This paper describes the development of the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 from a lead series of 4-anilinoquinazoline compounds. ZD1839 has suitable properties for use as a clinically effective drug and shows activity against human tumours. In particular,the use of pharmacokinetic data in the development of ZD1839 is discussed. View Publication

Many receptor-linked agents that prime or activate the NADPH oxidase in polymorphonuclear neutrophils (PMNs) elicit changes in cytosolic Ca2+ concentration and activate mitogen-activated protein (MAP) kinases. To investigate the role of Ca2+ in the activation of p38 and p42/44 MAP kinases,we examined the effects of the Ca2+-selective ionophore ionomycin on priming and activation of the PMN oxidase. Ionomycin caused a rapid rise in cytosolic Ca2+ that was due to both a release of cytosolic Ca2+ stores and Ca2+ influx. Ionomycin also activated (2 microM) and primed (20-200 nM) the PMN oxidase. Dual phosphorylation of p38 MAP kinase and phosphorylation of its substrate activating transcription factor-2 were detected at ionomycin concentrations that prime or activate the PMN oxidase,while dual phosphorylation of p42/44 MAP kinase and phosphorylation of its substrate Elk-1 were elicited at 0.2-2 microM. SB-203580,a p38 MAP kinase antagonist,inhibited ionomycin-induced activation of the oxidase (68 +/- 8%,P textless 0.05) and tyrosine phosphorylation of 105- and 72-kDa proteins; conversely,PD-98059,an inhibitor of MAP/extracellular signal-related kinase 1,had no effect. Treatment of PMNs with thapsigargin resulted in priming of the oxidase and activation of p38 MAP kinase. Chelation of cytosolic but not extracellular Ca2+ completely inhibited ionomycin activation of p38 MAP kinase,whereas chelation of extracellular Ca2+ abrogated activation of p42/44 MAP kinase. These results demonstrate the importance of changes in cytosolic Ca2+ for MAP kinase activation in PMNs. View Publication

Bisphosphonates inhibit osteoclast-mediated bone resorption by mechanisms that have only recently become clear. Whereas nitrogen-containing bisphosphonates affect osteoclast function by preventing protein prenylation (especially geranylgeranylation),non-nitrogen-containing bisphosphonates have a different molecular mechanism of action. In this study,we demonstrate that nitrogen-containing bisphosphonates (risedronate,alendronate,pamidronate,and zoledronic acid) and non-nitrogen-containing bisphosphonates (clodronate and etidronate) cause apoptosis of rabbit osteoclasts,human osteoclastoma-derived osteoclasts,and human osteoclast-like cells generated in cultures of bone marrow in vitro. Osteoclast apoptosis was shown to involve characteristic morphological changes,loss of mitochondrial membrane potential,and the activation of caspase-3-like proteases capable of cleaving peptide substrates with the sequence DEVD. Caspase-3-like activity could be visualized in unfixed,dying osteoclasts and osteoclast-like cells using a cell-permeable,fluorogenic substrate. Bisphosphonate-induced osteoclast apoptosis was dependent on caspase activation,because apoptosis resulting from alendronate,clodronate,or zoledronic acid treatment was suppressed by zVAD-fmk,a broad-range caspase inhibitor,or by SB-281277,a specific isatin sulfonamide inhibitor of caspase-3/-7. Furthermore,caspase-3 (but not caspase-6 or caspase-7) activity could be detected and quantitated in lysates from purified rabbit osteoclasts,whereas the p17 fragment of active caspase-3 could be detected in human osteoclast-like cells by immunofluorescence staining. Caspase-3,therefore,appears to be the major effector caspase activated in osteoclasts by bisphosphonate treatment. Caspase activation and apoptosis induced by nitrogen-containing bisphosphonates are likely to be the consequence of the loss of geranylgeranylated rather than farnesylated proteins,because the ability to cause apoptosis and caspase activation was mimicked by GGTI-298,a specific inhibitor of protein geranylgeranylation,whereas FTI-277,a specific inhibitor of protein farnesylation,had no effect on apoptosis or caspase activity. View Publication

The transcription factor NF-kappaB has anti-apoptotic properties and may confer chemoresistance to cancer cells. Here,we describe human pancreatic carcinoma cell lines that differ in the responsiveness to the topoisomerase-2 inhibitors VP16 (20 microM) and doxorubicin (0.3 microM): Highly sensitive T3M4 [corrected] and PT45-P1 cells,and Capan-1 and A818-4 cells that were almost resistant to both anti cancer drugs. VP16,but not doxorubicin,transiently induced NF-kappaB activity in all cell lines,whereas basal NF-kappaB binding was nearly undetectable in T3M4 [corrected] and PT45-P1 cells,but rather high in Capan-1 and A818-4 cells,as demonstrated by gel-shift and luciferase assays. Treatment with various NF-kappaB inhibitors (Gliotoxin,MG132 and Sulfasalazine),or transfection with the IkappaBalpha super-repressor,strongly enhanced the apoptotic effects of VP16 or doxorubicin on resistant Capan-1 and 818-4 cells. Our results indicate that under certain conditions the resistance of pancreatic carcinoma cells to chemotherapy is due to their constitutive NF-kappaB activity rather than the transient induction of NF-kappaB by some anti-cancer drugs. Blockade of basal NF-kappaB activity by well established drugs efficiently reduces chemoresistance of pancreatic cancer cells and offers the potential for improved therapeutic strategies. View Publication

Reinfusion of ex vivo-expanded autologous megakaryocytes together with a stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. In this study,we analyzed several serum-containing and serum-free media to identify the most suitable medium for megakaryocyte expansion. Moreover,two thrombopoietin (Tpo)-mimetic peptides were tested to evaluate whether they could replace Tpo in an expansion protocol. To analyze the effects of different media on megakaryocyte expansion,we used an in vitro liquid culture system. For this purpose,CD34(+) cells were isolated from peripheral blood and cultured for 8 days in the presence of Tpo and interleukin-3 (IL-3). The presence of megakaryocytes was analyzed by flow cytometric analysis after staining for CD41 expression. For our standard culture procedure,megakaryocyte medium (MK medium) supplemented with 10% AB plasma was used. Addition of 5% or 2.5% AB plasma yielded higher numbers of megakaryocytes,implying the presence of inhibitory factors in plasma. However,some plasma components are required for optimal megakaryocyte expansion because addition of less than 1% AB plasma or addition of human serum albumin instead of AB plasma resulted in the formation of lower numbers of megakaryocytes. Two commercially available serum-free media were also tested: Cellgro and Stemspan. If CD34(+) cells were cultured in Cellgro medium similar numbers of megakaryocytes were obtained as when CD34(+) cells were cultured in MK medium supplemented with 10% AB plasma. In MK medium with 2.5% AB plasma,higher numbers of megakaryocytes were cultured than in MK medium supplemented with 10% AB plasma. Therefore,Cellgro medium is not the best alternative medium. In cultures with Stemspan medium,higher numbers of megakaryocytes were obtained compared to MK medium with 10% AB plasma. Stemspan is thus a good alternative for MK medium. Two Tpo-mimetic peptides,AF13948 and PK1M,were tested for their ability to replace Tpo. In cultures with AF13948,comparable numbers of megakaryocytes were obtained as in the presence of Tpo,but in cultures with PK1M the number of megakaryocytes was lower. This study shows that high concentrations of plasma in medium inhibits megakaryocyte formation,but some plasma components are required for optimal megakaryocyte expansion. For an ex vivo expansion protocol,it is worthwhile to test several media,because the number of megakaryocytes differs widely with the medium used. View Publication

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However,the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However,5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood,as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted,mediated by CD8(+) lymphocytes,and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959) View Publication

The effect of resiquimod (R-848),an immune-response modifier that is similar to imiquimod,on recurrent herpes simplex virus (HSV) was evaluated using the guinea pig model of genital herpes. Guinea pigs were intravaginally infected with HSV-2 and then were randomized on day 14 to receive nothing or 0.1 mL/kg per dose of subcutaneous resiquimod,given either daily,every other day,or weekly from days 15-35. During a 3-week course of therapy,recurrences in all 3 treated groups were reduced by textgreater80%,compared with the control group. After therapy,recurrences remained significantly (Ptextless.05) decreased in all 3 groups for the next 3 weeks. The group treated weekly developed the fewest recurrences. Significant increases in interleukin-2 levels,produced by incubation of mononuclear cells with HSV-2 antigens,but not in circulating antibody also were detected in the treated groups. Resiquimod treatment may offer significant advantages to present antiviral therapies for the control of recurrent genital herpes. View Publication

The development of transcriptionally controlled systems which function in eukaryotic cells are important for achieving regulated gene expression in gene therapy. In this study we combined the components of the tetracycline-inducible system in self-contained retroviral and plasmid vectors. Regulated reporter gene expression from the autoregulatory plasmid pGTRTL in response to doxycycline (Dox) induction surpasses the expression observed from other self-contained retroviral and plasmid vectors. Induction kinetics and expression levels of luciferase and the therapeutic molecule,truncated soluble complement receptor 1 (sCR1) were characterised in a mouse fibroblast and a human neuroblastoma cell line. The regulatory characteristics of the plasmids were shown to be optimal for gene therapy applications,as there was a rapid reduction in expression levels following removal of Dox. Co-transfection of cells with an autoregulatory plasmid and a Dox inducible enhanced green fluorescent protein (EGFP) plasmid demonstrated the feasibility of using this plasmid combination to achieve parallel regulation of two genes of interest in a single cell under the control of Dox. These novel autoregulatory plasmids display the requirements for gene therapy applications in chronic conditions which are remitting/relapsing such as rheumatoid arthritis or multiple sclerosis,where novel protein therapeutics and combination therapies are needed. Gene Therapy (2000) 7,2061-2070. View Publication