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Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50=5.7±0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.

Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP-based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell-autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation., Pancreatic ductal adenocarcinoma (PDAC) is a fearsome disease, with a mortality rate >95% that has remained unchanged for decades (1). GEMMs that faithfully recapitulate the histological, molecular, genetic and clinical [...]

Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) acts as a neurotransmitter and intracellular signaling molecule in the central and peripheral nervous system. NO regulates multiple processes like neuronal development, plasticity, and differentiation and is a mediator of neurotoxicity. The nNOS gene is highly complex with 12 alternative first exons, exon la-11, transcribed from distinct promoters, leading to nNOS variants with different 5'-untranslated regions. Transcriptional control of the nNOS gene is not understood in detail. To investigate regulation of nNOS gene expression by retinoic acid (RA), we used the human neuroblastoma cell line TGW-nu-I as a model system. We show that RA induces nNOS transcription in a protein synthesis-dependent fashion. We identify the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and the atypical orphan nuclear receptor DAX1 (NROB1) as critical mediators involved in RA-induced nNOS gene transcription. RA treatment increases DAX1 expression via PI3K/Akt signaling. Upregulation of DAX1 expression in turn induces nNOS transcription in response to RA. These results identify nNOS as a target gene of a novel RA/PI3K/Akt/DAX1-dependent pathway in human neuroblastoma cells and stress the functional importance of the transcriptional regulator DAX1 for nNOS gene expression in response to RA treatment. neuronal nitric oxide synthase; DAX1; phosphatidylinositol 3-kinase/ Akt signaling doi: 10.1152/ajpcell.00034.2009.

Site-and time-specific somatic gene transfer by using the avian sarcoma-leukosis retrovirus RCAS (replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor) has been shown to be a powerful tool to analyze gene function in vivo. RCAS retroviruses that express the avian subgroup A envelope transduce only mammalian cells genetically engineered to express the avian retroviral receptor, tumor virus A (TVA). Here, we generated a knockin mouse line termed [LSL-R26.sup.Tva-lacz] with concomitant conditional expression of TVA and lacZ by targeting the Rosa26 locus. A loxP-flanked transcriptional stop cassette was used for conditional activation of TVA and LacZ expression in a Cre-recombinase-dependent manner. To demonstrate the ability of this system for conditional somatic gene transfer in vivo, we directed TVA expression to the pancreas. Introduction of an RCAS vector with Bryan-RSV polymerase and subgroup A envelope [RCASBP(A)] carrying oncogenic [Kras.sup.G12D] induced focal ductal pancreatic lesions that recapitulate human pancreatic intraepithelial neoplasias that progress to pancreatic ductal adenocarcinomas. TVA-mediated infection of genetically engineered mice with endogenous expression of [Kras.su[p.G12D] in pancreatic progenitor cells by using RCASBP(A) virus carrying a short hairpin RNA directed against murine TP53, resulted in dramatically enhanced progression to invasive adenocarcinomas. These results show that conditional expression of TVA enables spatiotemporal gene expression and knockdown in a small subset of somatic cells in vivo. Therefore, it closely models carcinogenesis in humans where tumors evolve from somatic gene mutations in developmentally normal cells. Combined with the growing number of Cre expression models, RCAS-TVA-based gene expression and knockdown systems open up promising perspectives for analysis of gene function in a time-controlled and tissue-specific fashion in vitro and in vivo. pancreatic cancer | RCAS | tumor virus A | RNA interference | molecular in vivo imaging

The promotion of cell survival and regeneration in acute renal failure (ARF) is important for the restitution of renal function. Epidermal growth factor (EGF) has been implicated in the regulation of cell proliferation. We provide evidence for a direct link between EGF, nuclear factor-[kappa]B (NF-[kappa]B), and cell cycle regulation (cyclin D1). EGF was found to stimulate NF-[kappa]B-dependent gene transcription and DNA binding. In addition, EGF stimulated cyclin D1 promoter activity as well as cyclin D1 expression. Moreover, inhibition of NF-[kappa]B caused a pronounced reduction of EGF-induced cyclin D1 promoter activity. Furthermore, both EGF-mediated NF-[kappa]B activation and cyclin D1 expression were inhibited by coexpression of super I[kappa]B. Taken together, these data identify NF-[kappa]B and cyclin D1 as downstream targets of EGF and establish a molecular link between stimulation of EGF via activation of NF-[kappa]B and cyclin D1 expression in human proximal tubular cells. EGF; cyclin D1

Research has been conducted on the wild-type enzyme, an aminotransferase which utilizes S-adenosylmethionine as amino donor. The kinetic characterization of this enzyme has been carried out and the crystal structure of the R391A mutant has been determined and discussed.

The eukaryotic transcription factor nuclear factor-[kappa]B (NF-[kappa]B)/Rel is activated by a large variety of stimuli. It has been demonstrated that NF-[kappa]B/Rel is induced during the course of cerulein pancreatitis. Here, we show that NF-[kappa]B/Rel is differentially activated in pancreatic lobules. Cerulein induces NF-[kappa]B/Rel via activation of I[kappa]B kinase (IKK), which causes degradation of I[kappa]B[alpha] but not I[kappa]B[alpha]. Tumor necrosis factor-[alpha]-mediated IKK activation leads to I[kappa]B[alpha] and I[kappa]B[beta] degradation. In contrast, oxidative stress induced by [H.sub.2][O.sub.2] activates NF-[kappa]B/Rel independent of IKK activation and I[kappa]B[alpha] degradation; instead I[kappa]B[alpha] is phosphorylated on tyrosine. [H.sub.2][O.sub.2] but not cerulein-mediated NF-[kappa]B/Rel activation can be blocked by stabilizing microtubules with Taxol. Inhibition of tubulin polymerization with nocodazole causes NF-[kappa]B/Rel activation in pancreatic lobules. These results propose three different pathways of NF-[kappa]B/Rel activation in pancreatic acinar cells. Furthermore, these data demonstrate that microtubules play a key role in IKK-independent NF-[kappa]B/Rel activation following oxidative stress. acute pancreatitis; inhibitor protein I[kappa]B; I[kappa]B kinase; inflammation; hydrogen peroxide; tyrosine phosphorylation; cytokines; cerulein

Kinetic and spectroscopic data indicated that addition of the donor substrate hydroxypyruvate to the thiamin diphosphate (ThDP)-dependent enzyme transketolase (TK) led to the accumulation of the [alpha]-carbanion/enamine of ([alpha][beta]-dihydroxyethyl) ThDP, the key reaction intermediate in enzymatic thiamin catalysis. The three-dimensional structure of this intermediate trapped in the active site of yeast TK was determined to 1.9-[Angstrom] resolution by using cryocrystallography. The electron density suggests a planar [alpha]-carbanion/enamine intermediate having the E-configuration. The reaction intermediate is firmly held in place through direct hydrogen bonds to His-103 and His-481 and an indirect hydrogen bond via a water molecule to His-69. The 4-NH2 group of the amino-pyrimidine ring of ThDP is within 3 [Angstrom] distance to the [alpha]-hydroxy oxygen atom of the dihydroxyethyl moiety but at an angle unfavorable for a strong hydrogen bond. No structural changes occur in TK on formation of the reaction intermediate, suggesting that the active site is poised for catalysis and conformational changes during the enzyme reaction are not very likely. The intermediate is present with high occupancy in both active sites, arguing against previous proposals of half-of-the-sites reactivity in yeast TK.

The functions of four histidine residues in the active site of yeast transketolase were studied by site-directed mutagenesis. The substitution of the invariant His69 with alanine produced a mutant enzyme with 1.5% of the specific activity of the wild-type enzyme and with an increased K(sub M) for the donor. Amino acid substitutions for His481 by alanine, serine and glutamine produced mutant enzymes with substantially increased K(sub M) values for the donor substrate and specific activities of 4.4%, 1.9% and 5.5% of the wild-type enzyme.

A crystallographic study of complexes of E. coli dethiobiotin synthetase and a reaction intermediate reveals that one molecule of each substrate binds to to one monomer of the enzyme. ADP and ATP analogue attach to the mononucleotide fold with the phosphate groups near the phosphate binding loop Gly8-Thr16 between beta-strand beta1 and the N-terminus of alpha-helix alpha1. Two oxygen atoms from the phosphates and oxygen atoms from the side chains of Asp54, Thr16, and Glu115 are ligands of the Mn+2 ion..

Rat prostatic acid phosphatase (rPAP) was overexpressed by recombinant baculovirus-infected insect cells. The purified protein had a specific activity of 2620 micromole.min-1.mg-1 with p-nitrophenylphosphate as the substrate, and also had phosphotyrosine phosphatase activity. The molecular mass of the secreted enzyme was 155 kDa. When recombinant rPAP was crystallized, the crystals were trigonal with cell dimensions a = 89.4 angstrom and c = 152.0 angstrom.