Capped diaminopropionamide-glycine dipeptides are inhibitors of CC chemokine receptor 2 (CCR2)

A new series of CCR2 antagonists has been discovered that incorporates intramolecular hydrogen bonding as a strategy for rigidifying the scaffold. The structure-activity relationship was established through initial systematic modification of substitution pattern and chain length, followed by independent optimization of three different substituents (benzylamine, carboxamide, and benzamide). Several of the acyclic compounds display 10-30 nM binding affinity for CCR2. Moreover, these antagonists are able to block both MCP-1-induced Ca(2+) flux and monocyte chemotaxis, and are selective for binding to CCR2 over CCR1 and CCR3.     

A critical role for cortactin phosphorylation by Abl-family kinases in PDGF-induced dorsal-wave formation

Proper regulation of cell morphogenesis and migration by adhesion and growth-factor receptors requires Abl-family tyrosine kinases [1-3]. Several substrates of Abl-family kinase have been identified, but they are unlikely to mediate all of the downstream actions of these kinases on cytoskeletal structure. We used a human protein microarray to identify the actin-regulatory protein cortactin as a novel substrate of the Abl and Abl-related gene (Arg) nonreceptor tyrosine kinases. Cortactin stimulates cell motility [4-6], and its upregulation in several cancers correlates with poor prognosis [7]. Even though cortactin can be tyrosine phosphorylated by Src-family kinases in vitro [8], we show that Abl and Arg are more adept at binding and phosphorylating cortactin. Importantly, we demonstrate that platelet-derived growth-factor (PDGF)-induced cortactin phosphorylation on three tyrosine residues requires Abl or Arg. Cortactin triggers F-actin-dependent dorsal waves in fibroblasts after PDGF treatment and thus results in actin reorganization and lamellipodial protrusion [9]. We provide evidence that Abl/Arg-mediated phosphorylation of cortactin is required for this PDGF-induced dorsal-wave response. Our results reveal that Abl-family kinases target cortactin as an effector of cytoskeletal rearrangements in response to PDGF.     

CC chemokine receptor-3 (CCR3) antagonists: improving the selectivity of DPC168 by reducing central ring lipophilicity

DPC168, a benzylpiperidine-substituted aryl urea CCR3 antagonist evaluated in clinical trials, was a relatively potent inhibitor of the 2D6 isoform of cytochrome P-450 (CYP2D6). Replacement of the cyclohexyl central ring with saturated heterocycles provided potent CCR3 antagonists with improved selectivity against CYP2D6. The favorable preclinical profile of DPC168 was maintained in an acetylpiperidine derivative, BMS-570520.     

Synthesis and structure-activity relationships of 16-modified analogs of 2-methoxyestradiol

A series of 16-modified 2-methoxyestradiol analogs were synthesized and evaluated for antiproliferative activity toward HUVEC and MDA-MB-231 cells, and for susceptibility to conjugation. In addition, the estrogenicity of these analogs was accessed by measuring cell proliferation of the estrogen-dependent cell line MCF7 in response to compound treatment. It was observed that antiproliferative activity dropped as the size of the 16 substituent increased. Selected analogs tested in glucuronidation assays had similar rates of clearance to 2-methoxyestradiol, but had enhanced clearance in sulfonate conjugation assays.     

Crossing paths with Notch in the hyper-network

The development of complex and diverse metazoan morphologies is coordinated by a surprisingly small number of evolutionarily conserved signaling mechanisms. These signals can act in parallel but often appear to function as an integrated hyper-network. The nodes defining this complex molecular circuitry are poorly understood, but the biological significance of pathway cross-talk is profound. The importance of such large-scale signal integration is exemplified by Notch and its ability to cross-talk with all the major pathways to influence cell differentiation, proliferation, survival and migration. The Notch pathway is, thus, a useful paradigm to illustrate the complexity of pathway cross-talk: its pervasiveness, context dependency, and importance in development and disease.     

A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti-effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000.     

Composting rapidly reduces levels of extractable oxytetracycline in manure from therapeutically treated beef calves

Oxytetracycline (OTC) is a broad-spectrum antibiotic used in livestock production. The widespread use and relative persistence of OTC may encourage development of antibiotic-resistant bacteria. The objective of this study was to determine whether composting would substantially reduce the concentration of OTC found in manure from medicated animals. The effect of OTC on composting was also investigated. Five beef calves were medicated for 5 days with 22 mg/kg/day of OTC. Approximately 23% of the OTC fed to the calves was recovered in the manure. Manure samples collected from calves prior to and after medication were mixed with straw and woodchips, and aliquots of the subsequent mixtures were treated in laboratory composters for 35 days. In addition, aliquots of the OTC-containing mixture were incubated at 25 degrees C or sterilized followed by incubation at 25 degrees C. The presence of OTC did not appear to affect composting processes. Within the first six days of composting, levels of extractable OTC in the compost mixture decreased from 115+/-8 microg/g dry weight to less than 6+/-1 microg/g dry weight (a 95% reduction). In contrast, levels of extractable OTC in room temperature incubated and sterilized mixtures decreased only 12-25% after 37 and 35 days, respectively. Levels of total heterotrophic bacteria and OTC-resistant bacteria in the finished compost mixture were roughly 30-fold higher and 10-fold lower, respectively, than levels in the mixture prior to composting. Although the basis of the OTC disappearance during composting is not known, the preponderence of OTC-sensitive bacteria and the decrease of OTC-resistant bacteria in the finished compost suggests that OTC residues have been rendered biologically inactive or unavailable.     

Spermatinamine, the first natural product inhibitor of isoprenylcysteine carboxyl methyltransferase, a new cancer target

Isoprenylcysteine methyltransferase (Icmt) catalyzes the carboxyl methylation of oncogenic proteins in the final step of a series of post-translational modifications. The inhibition of Icmt provides an attractive and novel anticancer target. A natural product high-throughput screening campaign was conducted to discover inhibitors of Icmt. The Australian marine sponge, Pseudoceratina sp., yielded spermatinamine, a novel alkaloid with a bromotyrosyl-spermine-bromotyrosyl sequence, as the bioactive constituent. Its structure was determined by 1D and 2D NMR spectroscopy. Spermatinamine is the first natural product inhibitor of Icmt.     

Metabolic flux analysis in a nonstationary system: fed-batch fermentation of a high yielding strain of E. coli producing 1,3-propanediol

Metabolic fluxes estimated from stable-isotope studies provide a key to understanding cell physiology and regulation of metabolism. A limitation of the classical method for metabolic flux analysis (MFA) is the requirement for isotopic steady state. To extend the scope of flux determination from stationary to nonstationary systems, we present a novel modeling strategy that combines key ideas from isotopomer spectral analysis (ISA) and stationary MFA. Isotopic transients of the precursor pool and the sampled products are described by two parameters, D and G parameters, respectively, which are incorporated into the flux model. The G value is the fraction of labeled product in the sample, and the D value is the fractional contribution of the feed for the production of labeled products. We illustrate the novel modeling strategy with a nonstationary system that closely resembles industrial production conditions, i.e. fed-batch fermentation of Escherichia coli that produces 1,3-propanediol (PDO). Metabolic fluxes and the D and G parameters were estimated by fitting labeling distributions of biomass amino acids measured by GC/MS to a model of E. coli metabolism. We obtained highly consistent fits from the data with 82 redundant measurements. Metabolic fluxes were estimated for 20 time points during course of the fermentation. As such we established, for the first time, detailed time profiles of in vivo fluxes. We found that intracellular fluxes changed significantly during the fed-batch. The intracellular flux associated with PDO pathway increased by 10%. Concurrently, we observed a decrease in the split ratio between glycolysis and pentose phosphate pathway from 70/30 to 50/50 as a function of time. The TCA cycle flux, on the other hand, remained constant throughout the fermentation. Furthermore, our flux results provided additional insight in support of the assumed genotype of the organism.     

Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers

Actinobacillus succinogenes is a promising candidate for industrial succinate production. However, in addition to producing succinate, it also produces formate and acetate. To understand carbon flux distribution to succinate and alternative products we fed A. succinogenes [1-(13)C]glucose and analyzed the resulting isotopomers of excreted organic acids, proteinaceous amino acids, and glycogen monomers by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The isotopomer data, together with the glucose consumption and product formation rates and the A. succinogenes biomass composition, were supplied to a metabolic flux model. Oxidative pentose phosphate pathway flux supplied, at most, 20% of the estimated NADPH requirement for cell growth. The model indicated that NADPH was instead produced primarily by the conversion of NADH to NADPH by transhydrogenase and/or by NADP-dependent malic enzyme. Transhydrogenase activity was detected in A. succinogenes cell extracts, as were formate and pyruvate dehydrogenases, which the model suggested were contributing to NADH production. Malic enzyme activity was also detected in cell extracts, consistent with the flux analysis results. Labeling patterns in amino acids and organic acids showed that oxaloacetate and malate were being decarboxylated to pyruvate. These are the first in vivo experiments to show that the partitioning of flux between succinate and alternative fermentation products can occur at multiple nodes in A. succinogenes. The implications for designing effective metabolic engineering strategies to increase A. succinogenes succinate production are discussed.