Triplicate Cultures Research Articles

Nutrient starvation effects on the allelochemical potency of Alexandrium tamarense (Dinophyceae)

Batch culture experiments were performed to investigate potential effects of nutrient starvation on the allelochemical potency of the toxic dinoflagellate Alexandrium tamarense. Triplicate cultures with reduced nitrate (−N) or phosphate (−P) seed were compared to nutrient-replete (+N+P) cultures. Total depletion of the dissolved inorganic limiting nutrient, reduced cell quotas, changed mass ratios of C/N/P and reduced cell yield clearly indicate that treatment cultures at stationary phase were starved by either N or P, whereas growth cessation of +N+P cultures was probably due to carbon limitation and/or a direct effect of high pH. Pulsed addition of the limiting nutrient allowed −N and −P cultures to resume growth. Lytic activity of A. tamarense as quantified by a Rhodomonas bioassay was generally high (EC50 around 100 cells mL−1) and was only slightly modulated by growth phase and/or nutrient starvation. Lytic activity per cell increased with time in both +N+P and −P cultures but not −N cultures. P-starved stationary-phase cells were slightly more lytic than +N+P cultures, but this difference may be due to increased cell size and/or accumulation of extracellular compounds. In conclusion, only slight changes but no general and major increase in lytic activity in response to nutrient starvation was observed.

In Vitro and In Vivo Proof of Tolerance After Two-Step Haploidentical Bone Marrow and Kidney Transplantation of the Same Donor

In Vitro and In Vivo Proof of Tolerance After Two-Step Haploidentical Bone Marrow and Kidney Transplantation of the Same Donor

Hydrogen and volatile fatty acid production during fermentation of cellulosic substrates by a thermophilic consortium at 50 and 60 °C

The purpose of this study was to characterize the effect of temperature and cellulosic substrates on fermentative metabolites, H2 production, and community successions in an anaerobic, cellulolytic consortium, TC60. Pyrosequencing analysis indicated that the consortium was predominated by Thermoanaerobacter and Clostridium spp. Metabolite production was analyzed with four cellulosic substrates at 4kg/m3. Triplicate cultures of each substrate were incubated at 50 or 60°C. The main fermentation products (H2, CO2, ethanol, and acetate) were monitored over time. The ANOVA model for production rates showed a significant temperature effect (P<0.05) on all products. Increased temperature promoted higher H2, CO2, and ethanol yields while acetate yields were only affected prior to 24h of incubation. In addition to individual effects discerned in the model, ANOVA indicated significant interactions between the substrate and temperature. These interactions have not been previously recognized in the literature for cellulolytic and hydrogen-producing microorganisms.

Auto-activation of c-JUN Gene by Amino Acid Deprivation of Hepatocellular Carcinoma Cells Reveals a Novel c-JUN-mediated Signaling Pathway

Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. This study demonstrates that in hepatocellular carcinoma cells, expression of c-JUN, JUN-B, c-FOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, c-JUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in c-JUN expression was greater in transformed cells compared with nontransformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of c-JUN transcription was ATF4-independent. The increased expression of c-JUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of c-JUN-ATF2-activated heterodimers was increased after AA limitation, and c-JUN or ATF2 knockdown suppressed the induction of c-JUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing c-JUN protein by JNK and subsequent auto-activation of the c-JUN gene by recruitment of c-JUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the c-JUN gene can function as transcriptional amino acid-response elements.

Statins are active in acute lymphoblastic leukaemia (ALL): a therapy that may treat ALL and prevent avascular necrosis

Statins are active in acute lymphoblastic leukaemia (ALL): a therapy that may treat ALL and prevent avascular necrosis

Inhibition of Anti-HIV MicroRNA Expression: A Mechanism for Opioid-Mediated Enhancement of HIV Infection of Monocytes

Inhibition of Anti-HIV MicroRNA Expression: A Mechanism for Opioid-Mediated Enhancement of HIV Infection of Monocytes

Metabolomics-based identification of apoptosis-inducing metabolites in recombinant fed-batch CHO culture media

A liquid chromatography–mass spectrometry (LC–MS) based metabolomics platform was previously established to identify and profile extracellular metabolites in culture media of mammalian cells. This presented an opportunity to isolate novel apoptosis-inducing metabolites accumulating in the media of antibody-producing Chinese hamster ovary (CHO mAb) fed-batch bioreactor cultures. Media from triplicate cultures were collected daily for the metabolomics analysis. Concurrently, cell pellets were obtained for determination of intracellular caspase activity. Metabolite profiles from the LC–MS data were subsequently examined for their degree of correlation with the caspase activity. A panel of extracellular metabolites, the majority of which were nucleotides/nucleosides and amino acid derivatives, exhibited good ( R 2 > 0.8) and reproducible correlation. Some of these metabolites, such as oxidized glutathione, AMP and GMP, were later shown to induce apoptosis when introduced to fresh CHO mAb cultures. Finally, metabolic engineering targets were proposed to potentially counter the harmful effects of these metabolites.

Immortalization of Human Carcinoma-Associated Fibroblasts from Primary Colorectal Carcinomas

Introduction: Reciprocal interplay bewteen epithelial and stromal cells contributes to the initiation, progression and metastasis of colorectal cancer. The importance of carcinoma-associated fibroblasts (CAFs) in tumor develoment is highlighted by the observation that nontumorigenic prostate epithelial cells can form tumors in nude mice when co-implanted with CAF, but not when co-implanted with fibroblasts from benign prostatic hypertrophy. The precise role of colorectal CAFs in tumor-stroma interactions has been difficult to study, in part because of the difficulty of obtaining primary CAFs in sufficient quantity for experiments. Once isolated, the CAFs can be passaged for a limited number of times before they undergo senescence. Further characterization of human colorectal CAFs would provide the basis for stromal-specific therapeutic targeting in colorectal cancers. The purpose of this study was two-fold: 1) immortalize human CAFs derived from colorectal cancers in order to begin to investigate their influence on human colorectal cancer cells and 2) document gene-specific changes attributable to the immortalization process. Methods: Freshly dissected and dissociated cells were cultured from a resected colon cancer specimen under an IRB-approved protocol. The patient had not received prior chemoradiotherapy. The cells were cultured on collagen I and the purity of the fibroblast culture was verified by immunohistochemistry. Fibroblasts are positive for vimentin and/or α-smooth muscle actin, but negative for cytokeratin. CAFs were infected with lentiviral vectors containing a CMV promotor upstream of SV40 Large T antigen and human telomerase. Total RNA was extracted using RNAqueous (Ambion) from triplicate cultures of transformed (t)CAFs and parental (p)CAFs. The purity and concentration of the RNA samples were determined using Agilent 2100 Bioanalyzer. Samples were hybridized to Affymetrix Gene Chips (HGU133 Plus 2.0). Analyses using the Partek Genomics Suite included routine quality controls, raw data background subtraction and normalization. Differentially expressed candidate genes were identified using the Benjamani-Hochberg multiple hypothesis comparisons (ANOVA, p<0.01 and p<0.005) to decrease the false discovery rate. Ingenuity Pathways analyses were performed to identify differences between the t- and p-CAFs. Results: ANOVA identified 2779 and 648 genes that were significant at the p≤0.01 and 0.005 level, respectively. The major pathways representing the genes differentially expressed bewteen the t- and p-CAFs include: 1) the transcriptional regulator TP53 2) growth factor TGFB1, 3) mitogen-activated protein kinase pathways (p38 and p-extracellularly regulated kinase), and 4) the transcriptional regulator nuclear factor-κB. As expected, one canonical pathway that was altered by the transformation, were genes involved in cell cycle and G1/S checkpoint regulation. Conclusions: The immortalization and subsequent careful characterization of human colorectal CAFs provides an invaluable tool for further study of stromal-epithelial interactions in colorectal carcinogenesis and metastasis. Although genes related to the cell cycle has been significantly altered, future studies are required to verify that the t-CAFs alone are non-tumorigenic in nude mice, similar to the p-CAFs. Additionally, immortalized normal fibroblasts derived from the normal a colonic mucosa will need to be generated as an important control.

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.

Peroxisome proliferator-activated receptor-γ activation and long-chain fatty acids alter lipogenic gene networks in bovine mammary epithelial cells to various extents

Several long-chain fatty acids (LCFA) are natural ligands of nonruminant peroxisome proliferator-activated receptor-γ (PPARG), which, along with its lipogenic target genes, is upregulated in bovine mammary tissue during lactation. Thus, PPARG might represent an important control point of bovine milk fat synthesis. We tested lipogenic gene network expression via quantitative PCR of 19 genes in bovine mammary epithelial cells cultured with 16:0, 18:0, cis-9 18:1, trans-10 18:1, trans-10,cis-12 18:2 [t10c12 conjugated linoleic acid (CLA)], 20:5, ethanol (control), and the PPARG agonist rosiglitazone (ROSI). Triplicate cultures were maintained for 12h with 50μM ROSI or 100μM LCFA. Responses common to 16:0 and 18:0 relative to the control included significantly greater expression of INSIG1 (+298%, +92%), AGPAT6 (+137%, +169%), FABP3 (+755%, +338%), and FABP4 (+171%, 157%). These were coupled with greater intracellular lipid droplet formation and mRNA of ACSS2, LPIN1, SCD, and SREBF2 in response to 16:0, and greater DGAT1 and THRSP with 18:0. Trans-10 18:1 and t10c12 CLA reduced expression of FASN (–60%, –31%), SCD (–100%, –357%), and SREBF1 (–49%, –189%). Furthermore, t10c12 CLA downregulated ACSS2, FABP3, INSIG1, SREBF2, and THRSP expression. Expression of SREBF1 was lower with cis-9 18:1 (–140%) and 20:5 (–125%) compared with the control. This latter LCFA also decreased SCD, SREBF2, and LPL expression. No effects of LCFA or ROSI on PPARG were observed, but ROSI upregulated (+39% to +269%) expression of ACACA, FASN, LPIN1, AGPAT6, DGAT1, SREBF1, SREBF2, and INSIG1. Thus, these genes are putative PPARG target genes in bovine mammary cells. This is the first report showing a direct effect of trans-10 18:1 on bovine mammary cell lipogenic gene expression. The coordinated upregulation of lipogenic gene networks in response to ROSI and saturated LCFA offers support for PPARG activation in regulating bovine milk fat synthesis.

Tetrahydrobiopterin Recycling, a Key Determinant of Endothelial Nitric-oxide Synthase-dependent Signaling Pathways in Cultured Vascular Endothelial Cells

Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated "knockdown" GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses.

Resistance of Tumor Cells to Cytolytic T Lymphocytes Involves Rho-GTPases and Focal Adhesion Kinase Activation

Tumor cells evade adaptive immunity by a variety of mechanisms, including selection of variants that are resistant to specific cytotoxic T lymphocyte (CTL) pressure. Recently, we have reported that the reorganization of the actin cytoskeleton can be used by tumor cells as a strategy to promote their resistance to CTL-mediated lysis. In this study, we further examined the functional features of a CTL-resistant tumor variant and investigated the relationship between cytoskeleton alteration, the acquisition of tumor resistance to CTL-induced cell death, Rho-GTPases, and focal adhesion kinase (FAK) pathways. Our data indicate that although the resistant cells do not display an increased migratory potential, an alteration of adhesion to the extracellular matrix was observed. When Rho-GTPases were activated in cells by the bacterial CNF1 (cytotoxic necrotizing factor 1), striking changes in the cell morphology, including actin cytoskeleton, focal adhesions, and membrane extensions, were observed. More importantly, such activation also resulted in a significant attenuation of resistance to CTL-induced cell death. Furthermore, we demonstrate that FAK signaling pathways were constitutively defective in the resistant cells. Silencing of FAK in the sensitive target cells resulted in the inhibition of immune synapse formation with specific CTLs and their subsequent lysis. Expression of the FAK mutant (Y397F) resulted in an inhibition of IGR-Heu cell adhesion and of their susceptibility to specific lysis. These results suggest that FAK activation plays a role in the control of tumor cell susceptibility to CTL-mediated lysis.

Antibiotic Overproduction in Streptomyces coelicolor A3(2) Mediated by Phosphofructokinase Deletion

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by (13)C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.

Common 677C→T mutation of the 5,10-methylenetetrahydrofolate reductase gene affects follicular estradiol synthesis

To investigate the influence of the 5,10-methylenetetrahydrofolate reductase 677C-->T mutation on the E(2) synthesis in human granulosa cells (GCs). In vitro cell culture study. Research laboratory of a university hospital. Follicular fluids (n = 139) and GCs (n = 66) were obtained from patients undergoing controlled ovarian hyperstimulation for IVF with or without ICSI. Granulosa cells were cultured for a total of 5 days. On day 3, the cells either were stimulated with recombinant (r-) FSH or r-LH (80 IU/L for 48 h) or were sham stimulated. Estradiol and protein content were measured in the pooled follicular fluids of each individual. At the end of each GC-culturing period, the concentrations of E(2) were measured in the supernatants of triplicate cultures by immunoassays. The 5,10-methylenetetrahydrofolate reductase 677C-->T genotype was determined by RFLP analysis. The E(2)-protein ratio of homozygous T/T carriers was significantly lower compared with that of homozygous C/C individuals. Furthermore, basal and r-FSH- as well as r-LH-stimulated E(2) synthesis of GC obtained from homozygous T/T patients was significantly reduced, compared with GC from heterozygous C/T and homozygous C/C subjects. Decreased E(2) in follicular fluid and decreased E(2) synthesis of GC from homozygous T/T individuals suggest that reduced follicular E(2) is a result of impaired E(2) production of human GC.

A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-κB activation

Nod1 and Nod2 are intracellular proteins that are involved in host recognition of specific bacterial molecules and are genetically associated with several inflammatory diseases. Nod1 and Nod2 stimulation activates NF-kappaB through RICK, a caspase-recruitment domain-containing kinase. However, the mechanism by which RICK activates NF-kappaB in response to Nod1 and Nod2 stimulation is unknown. Here we show that RICK is conjugated with lysine-63-linked polyubiquitin chains at lysine 209 (K209) located in its kinase domain upon Nod1 or Nod2 stimulation and by induced oligomerization of RICK. Polyubiquitination of RICK at K209 was essential for RICK-mediated IKK activation and cytokine/chemokine secretion. However, RICK polyubiquitination did not require the kinase activity of RICK or alter the interaction of RICK with NEMO, a regulatory subunit of IkappaB kinase (IKK). Instead, polyubiquitination of RICK was found to mediate the recruitment of TAK1, a kinase that was found to be essential for Nod1-induced signaling. Thus, RICK polyubiquitination links TAK1 to IKK complexes, a critical step in Nod1/Nod2-mediated NF-kappaB activation.

Transamidation of Wheat Flour Inhibits the Response to Gliadin of Intestinal T Cells in Celiac Disease

Celiac disease is characterized by activation of HLA-DQ2/DQ8-restricted intestinal gluten-specific CD4(+) T cells. In particular, gluten becomes a better T-cell antigen following deamidation catalyzed by tissue transglutaminase. To date, the only available therapy is represented by adherence to a gluten-free diet. Here, we examined a new enzyme strategy to preventively abolish gluten activity. Enzyme modifications of the immunodominant alpha-gliadin peptide p56-68 were analyzed by mass spectrometry, and peptide binding to HLA-DQ2 was simulated by modeling studies. Wheat flour was treated with microbial transglutaminase and lysine methyl ester; gliadin was subsequently extracted, digested, and deamidated. Gliadin-specific intestinal T-cell lines (iTCLs) were generated from biopsy specimens from 12 adult patients with celiac disease and challenged in vitro with different antigen preparations. Tissue transglutaminase-mediated transamidation with lysine or lysine methyl ester of p56-68 or gliadin in alkaline conditions inhibited the interferon gamma expression in iTCLs; also, binding to DQ2 was reduced but not abolished, as suggested by in silico analysis. Lysine methyl ester was particularly effective in abrogating the activity of gliadin. Notably, a block in the response was observed when iTCLs were challenged with gliadin extracted from flour pretreated with microbial transglutaminase and lysine methyl ester. Transamidation of wheat flour with a food-grade enzyme and an appropriate amine donor can be used to block the T cell-mediated gliadin activity. Considering the crucial role of adaptive immunity in celiac disease, our findings highlight the potential of the proposed treatment to prevent cereal toxicity.

Oncogenic KRAS Activates Hedgehog Signaling Pathway in Pancreatic Cancer Cells

Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAF(E600), but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression.

A Role of Lipophilic Peptidoglycan-related Molecules in Induction of Nod1-mediated Immune Responses

Nod1 is an intracellular protein that is involved in recognition of bacterial molecules and whose genetic variation has been linked to several inflammatory diseases. Previous studies suggested that the recognition core of Nod1 stimulatory molecules is gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), but the identity of the major Nod1 stimulatory molecule produced by bacteria remains unknown. Here we show that bacteria produce lipophilic molecules capable of stimulating Nod1. Analysis of synthetic compounds revealed stereoselectivity of the DAP residue and that conjugation of lipophilic acyl residues specifically enhances the Nod1 stimulatory activity of the core iE-DAP. Furthermore, we demonstrate that lipophilic molecules induce and/or enhance the secretion of innate immune mediators from primary mouse mesothelial cells and human monocytic MonoMac6 cells, and this effect is mediated through Nod1. These results provide insight into the mechanism of immune recognition via Nod1, which might be useful in the design and testing of novel immunoregulators.

Molecular and Functional Analyses of a Novel Class I Secretory Nuclease from the Human Pathogen, Leishmania donovani

The primitive protozoan pathogen of humans, Leishmania donovani, resides and multiplies in highly restricted micro-environments within their hosts (i.e. as promastigotes in the gut lumen of their sandfly vectors and as amastigotes in the phagolysosomal compartments of infected mammalian macrophages). Like other trypanosomatid parasites, they are purine auxotrophs (i.e. lack the ability to synthesize purines de novo) and therefore are totally dependent upon salvaging these essential nutrients from their hosts. In that context, in this study we identified a unique 35-kDa, dithiothreitol-sensitive nuclease and showed that it was constitutively released/secreted by both promastigote and amastigote developmental forms of this parasite. By using several different molecular approaches, we identified and characterized the structure of LdNuc(s), a gene that encodes this new 35-kDa class I nuclease family member in these organisms. Homologous episomal expression of an epitope-tagged LdNuc(s) chimeric construct was used in conjunction with an anti-LdNuc(s) peptide antibody to delineate the functional and biochemical properties of this unique 35-kDa parasite released/secreted enzyme. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that this "secretory" enzyme could hydrolyze a variety of synthetic polynucleotides as well as several natural nucleic acid substrates, including RNA and single- and double-stranded DNA. Based on these cumulative observations, we hypothesize that within the micro-environments of its host, this leishmanial "secretory" nuclease could function at a distance away from the parasite to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, this enzyme might play an important role(s) in facilitating the survival, growth, and development of this important human pathogen.