Probiotic suppression of the H. pylori-induced responses by conjugated linoleic acids in a gastric epithelial cell line

Conjugated linoleic acids (CLA) produced by Lactobacillus acidophilus was reported to decrease the activation of nuclear factor-kappa B. CLA was suggested as one of the anti-inflammatory molecular mechanisms of probiotics. In the present study, the effects of CLA on H. pylori-induced multiple responses were evaluated. IL-8, TNF-α and iNOS were measured in mRNA and/or protein levels in AGS cells after pretreatment with CLA or CLA-containing conditioned medium (CM) produced by Lactobacillus acidophilus or Lactobacillus plantarum. The increased expressions of IL-8 mRNA/protein and TNF-α mRNA were significantly suppressed by pretreatment with CM or CLA. The levels of IL-8 protein and TNF-α mRNA were suppressed by CM pretreatment better than CLA. The expression of iNOS mRNA was also significantly inhibited by CM pretreatment. These results suggest that the suppression of multiple mediators by CLA-containing CM plays a key role in the anti-inflammatory and anti-carcinogenic effects of probiotics on H. pylori infection.     

Stoichiometric expression of mtHsp40 and mtHsp70 modulates mitochondrial morphology and cristae structure via Opa1L cleavage

Deregulation of mitochondrial heat-shock protein 40 (mtHsp40) and dysfunction of mtHsp70 are associated with mitochondrial fragmentation, suggesting that mtHsp40 and mtHsp70 may play roles in modulating mitochondrial morphology. However, the mechanism of mitochondrial fragmentation induced by mtHsp40 deregulation and mtHsp70 dysfunction remains unclear. In addition, the functional link between mitochondrial morphology change upon deregulated mtHsp40/mtHsp70 and mitochondrial function has been unexplored. Our coimmunoprecipitation and protein aggregation analysis showed that both overexpression and depletion of mtHsp40 accumulated aggregated proteins in fragmented mitochondria. Moreover, mtHsp70 loss and expression of a mtHsp70 mutant lacking the client-binding domain caused mitochondrial fragmentation. Together the data suggest that the molecular ratio of mtHsp40 to mtHsp70 is important for their chaperone function and mitochondrial morphology. Whereas mitochondrial translocation of Drp1 was not altered, optic atrophy 1 (Opa1) short isoform accumulated in fragmented mitochondria, suggesting that mitochondrial fragmentation in this study results from aberration of mitochondrial inner membrane fusion. Finally, we found that fragmented mitochondria were defective in cristae development, OXPHOS, and ATP production. Taken together, our data suggest that impaired stoichiometry between mtHsp40 and mtHsp70 promotes Opa1_L cleavage, leading to cristae opening, decreased OXPHOS, and triggering of mitochondrial fragmentation after reduction in their chaperone function.     

Comparative Analysis of Disease-Linked Single Nucleotide Polymorphic Markers from Brassica rapa for Their Applicability to Brassica oleracea

Numerous studies using single nucleotide polymorphisms (SNPs) have been conducted in humans, and other animals, and in major crops, including rice, soybean, and Chinese cabbage. However, the number of SNP studies in cabbage is limited. In this present study, we evaluated whether 7,645 SNPs previously identified as molecular markers linked to disease resistance in the Brassica rapa genome could be applied to B. oleracea. In a BLAST analysis using the SNP sequences of B. rapa and B. oleracea genomic sequence data registered in the NCBI database, 256 genes for which SNPs had been identified in B. rapa were found in B. oleracea. These genes were classified into three functional groups: molecular function (64 genes), biological process (96 genes), and cellular component (96 genes). A total of 693 SNP markers, including 145 SNP markers [BRH—developed from the B. rapa genome for high-resolution melt (HRM) analysis], 425 SNP markers (BRP—based on the B. rapa genome that could be applied to B. oleracea), and 123 new SNP markers (BRS—derived from BRP and designed for HRM analysis), were investigated for their ability to amplify sequences from cabbage genomic DNA. In total, 425 of the SNP markers (BRP-based on B. rapa genome), selected from 7,645 SNPs, were successfully applied to B. oleracea. Using PCR, 108 of 145 BRH (74.5%), 415 of 425 BRP (97.6%), and 118 of 123 BRS (95.9%) showed amplification, suggesting that it is possible to apply SNP markers developed based on the B. rapa genome to B. oleracea. These results provide valuable information that can be utilized in cabbage genetics and breeding programs using molecular markers derived from other Brassica species.     

Crystal structure of malonyl CoA-Acyl carrier protein transacylase from Xanthomanous oryzae pv. oryzae and its proposed binding with ACP

Xanthomonas oryzae pv. oryzae (Xoo) is a plant bacterial pathogen that causes bacterial blight (BB) disease, resulting in serious production losses of rice. The crystal structure of malonyl CoA-acyl carrier protein transacylase (XoMCAT), encoded by the gene fabD (Xoo0880) from Xoo, was determined at 2.3 Å resolution in complex with N-cyclohexyl-2-aminoethansulfonic acid. Malonyl CoA-acyl carrier protein transacylase transfers malonyl group from malonyl CoA to acyl carrier protein (ACP). The transacylation step is essential in fatty acid synthesis. Based on the rationale, XoMCAT has been considered as a target for antibacterial agents against BB. Protein-protein interaction between XoMCAT and ACP was also extensively investigated using computational docking, and the proposed model revealed that ACP bound to the cleft between two XoMCAT subdomains.     

Docking-based 3D-QSAR study for selectivity of DPP4, DPP8, and DPP9 inhibitors

In order to obtain information regarding the design of selective DPP4 inhibitors, a 3D-QSAR study was conducted using DPP4, DPP8, and DPP9 inhibitors including newly synthesized six- and seven-membered cyclic hydrazine derivatives (KR64300, KR64301), which were evaluated in vitro for their inhibition of DPP4, DPP8, and DPP9. In this study, a highly predictive CoMFA model based on the fast-docking for DPP4, DPP8, and DPP9 inhibitors was obtained. This reliable model showed leave-one-out cross-validation q(2) and conventional r(2) values of 0.68 and 0.96 for the DPP4 inhibitors, 0.58 and 0.98 for the DPP8 inhibitors, and 0.68 and 0.97 for the DPP9 inhibitors, respectively. The validation of the CoMFA model was confirmed by the compounds in the test set, including the synthesized six- and seven-membered cyclic hydrazines. According to this study, to obtain selective DPP4 inhibitors compared to their isozymes, the interaction of the inhibitors with the S3 site and S1' site in DPP4 must be considered. The proposed newly synthesized compounds, KR64300 and KR64301, interact well with the sites mentioned above, showing excellent selectivity.     

Novel mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system disorders. However, the FAAH inhibitors reported to date lack drug-like pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750) and N-phenyl-4-(quinolin-2-ylmethyl)piperazine-1-carboxamide (PF-622) as a novel mechanistic class of FAAH inhibitors. PF-750 and PF-622 show higher in vitro potencies than previously established classes of FAAH inhibitors. Rather unexpectedly based on the high chemical stability of the urea functional group, PF-750 and PF-622 were found to inhibit FAAH in a time-dependent manner by covalently modifying the enzyme's active site serine nucleophile. Activity-based proteomic profiling revealed that PF-750 and PF-622 were completely selective for FAAH relative to other mammalian serine hydrolases. We hypothesize that this remarkable specificity derives, at least in part, from FAAH's special ability to function as a C(O)-N bond hydrolase, which distinguishes it from the vast majority of metabolic serine hydrolases in mammals that are restricted to hydrolyzing esters and/or thioesters. The piperidine/piperazine urea may thus represent a privileged chemical scaffold for the synthesis of FAAH inhibitors that display an unprecedented combination of potency and selectivity for use as potential analgesic and anxiolytic/antidepressant agents.     

Cloning, sequencing, and characterization of the pradimicin biosynthetic gene cluster of Actinomadura hibisca P157-2

Pradimicins are potent antifungal antibiotics having an unusual dihydrobenzo[alpha]naphthacenequinone aglycone substituted with D-alanine and sugars. Pradimicins are polyketide antibiotics produced by Actinomadura hibisca P157-2. The gene cluster involved in the biosynthesis of pradimicins was cloned and sequenced. The pradimicin gene cluster was localized to a 39-kb DNA segment and its involvement in the biosynthesis of pradimicin was proven by gene inactivation of prmA and prmB (ketosynthases alpha and beta). The pradimicin gene cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS), the enzymes involved in sugar biosynthesis and tailoring enzymes as well as two resistance proteins. The deduced proteins showed strong similarities to the previously validated gene clusters of angucyclic polyketides such as rubromycin, griseorhodin, and fredericamycin. From the pradimicin gene cluster, prmP3 encoding a component of the acetyl-CoA carboxylase complex was disrupted. The production levels of pradimicins of the resulting mutants decreased to 62% of the level produced by the wild-type strain, which indicate that the acetyl-CoA carboxylase gene would have a significant role in the production of pradimicins through supplying the extender unit precursor, malonyl-CoA.     

Human apolipoprotein(a) kringle V inhibits angiogenesis in vitro and in vivo by interfering with the activation of focal adhesion kinases

Apolipoprotein(a) [apo(a)] contains the largest numbers of kringle domains identified to date. Of these, apo(a) kringle V shows significant sequence homology with plasminogen kringle 5, which is reported to be a potent angiogenesis inhibitor. To determine the effects of apo(a) kringle V on angiogenesis, it was expressed as a soluble protein (termed rhLK8) in Pichia pastoris and its in vitro and in vivo anti-angiogenic properties were examined. rhLK8 inhibited the migration of human umbilical vein endothelial cells in vitro in a dose-dependent manner. This function was associated with the down-regulation of the activation of focal adhesion kinase and the inhibition of the consequent formation of actin stress fibers/focal adhesions. rhLK8 also inhibited new capillary formation in vivo, as assessed by the chick chorioallantoic membrane assay and the Matrigel plug assay. These results indicate that rhLK8 may be an effective angiogenesis inhibitor both in vitro and in vivo.     

Carbon Mineralization and Labile Organic Carbon Pools in the Sandy Soils of a North Florida Watershed

The large pool of actively cycling carbon (C) held in soils is susceptible to release due to changes in landuse, management, or climate. Yet, the amount and distribution of potentially mineralizable C present in soils of various types and the method by which this soil C fraction can best be quantified, are not well established. The distribution of total organic C (TOC), extractable C pools (hot-water-extractable and acid-hydrolyzable), and in vitro mineralizable C in 138 surface soils across a north Florida watershed was found to be quite heterogeneous. Thus, these C quality parameters could not statistically distinguish the eight landuses or four major soil orders represented. Only wetland and upland forest soils, with the largest and smallest C pool size, respectively, were consistently different from the soils of other landuse types. Variations in potential C mineralization were best explained by TOC (62%) and hot-water-extractable C (59%), whereas acid-hydrolyzable C (32%) and clay content (35%) were generally not adequate indicators of C bioavailability. Within certain landuse and soil orders (Alfisol, Wetland and Rangeland, all with >3% clay content), however, C mineralization and clay content were directly linearly correlated, indicating a possible stimulatory effect of clay on microbial processing of C. Generally, the sandy nature of these surface soils imparted a lack of protection against C mineralization and likely resulted in the lack of landuse/soil order differences in the soil C pools. If a single parameter is to be chosen to quantify the potential for soil C mineralization in southeastern U.S. coastal plain soils, we recommend TOC as the most efficient soil variable to measure. Author Contributions  Conceived of or designed study: Sabine Grunwald, Nick Comerford, and James Sickman—Performed research: Mi-Youn Ahn—Analyzed data: Mi-Youn Ahn, Andrew Zimmerman, and Nick Comerford—Contributed new methods or models: Andrew Zimmerman, Nick Comerford, and James Sickman—Wrote the paper: Mi-Youn Ahn, Andrew Zimmerman, and Nick Comerford.