Stereostructure-based differences in the interactions of cardiotoxic local anesthetics with cholesterol-containing biomimetic membranes

Amide-type pipecoloxylidide local anesthetics, bupivacaine, and ropivacaine, show cardiotoxic effects with the potency depending on stereostructures. Cardiotoxic drugs not only bind to cardiomyocyte membrane channels to block them but also modify the physicochemical property of membrane lipid bilayers in which channels are embedded. The opposite configurations allow enantiomers to be discriminated by their enantiospecific interactions with another chiral molecule in membranes. We compared the interactions of local anesthetic stereoisomers with biomimetic membranes consisting of chiral lipid components, the differences of which might be indicative of the drug design for reducing cardiotoxicity. Fluorescent probe-labeled biomimetic membranes were prepared with cardiolipin and cholesterol of varying compositions and different phospholipids. Local anesthetics were reacted with the membrane preparations at a cardiotoxically relevant concentration of 200 μM. The potencies to interact with biomimetic membranes and change their fluidity were compared by measuring fluorescence polarization. All local anesthetics acted on lipid bilayers to increase membrane fluidity. Chiral cardiolipin was ineffective in discriminating S(-)-enantiomers from their antipodes. On the other hand, cholesterol produced the enantiospecific membrane interactions of bupivacaine and ropivacaine with increasing its composition in membranes. In 40 mol% and more cholesterol-containing membranes, the membrane-interacting potency was S(-)-bupivacaine相似文献   

Discovery of selective indole-based prostaglandin D₂ receptor antagonist

A series of N-benzoyl-2-methylindole-3-acetic acids were synthesized and biologically evaluated as prostaglandin (PG) D? receptor antagonists. Some of the selected compounds significantly inhibited OVA-induced vascular permeability in guinea pig conjunctiva after oral dosing. Structure-activity relationship study is presented.     

IL-27-producing CD14(+) cells infiltrate inflamed joints of rheumatoid arthritis and regulate inflammation and chemotactic migration

Interleukin (IL)-27, a heterodimeric cytokine, has been reported to be involved in the pathogenesis of autoimmune diseases through mediating differentiation of Th1 or Th17 cells and immune cell activity or survival. However, the origin and effects of IL-27 in joints of rheumatoid arthritis (RA) remain unclear. In this study, we investigated the distribution and anti-inflammatory roles of IL-27 in RA synovium. The IL-27 levels in plasma of RA patients, osteoarthritis (OA) patients, or healthy volunteers (n=15 per group) were equivalent and were at most 1 ng/ml, but the IL-27 level in synovial fluid of RA patients (n=15, mean 0.13 ng/ml; range 0.017-0.37 ng/ml) was significantly higher than that in synovial fluid of OA patients (n=15, mean 0.003 ng/ml; range 0-0.033 ng/ml) and potentially lower than in plasma. We analyzed the protein level of IL-27 produced by RA fibroblast-like synoviocytes (FLSs) or mononuclear cells (MNCs) from RA or OA synovial fluid or peripheral blood and showed that IL-27 in RA joints was derived from MNCs but not from FLSs. We also found by flow cytometry that IL-27-producing MNCs were CD14(+), and that these CD14(+)IL-27(+) cells were clearly detected in RA synovium but rarely in OA synovium by immunohistochemistry. Furthermore, we demonstrated that a relatively physiological concentration of IL-27 below 10 ng/ml suppressed the production of IL-6 and CCL20 from RA FLSs induced by proinflammatory cytokines through the IL-27/IL-27R axis. In the synovial fluid of RA, the IL-27 level interestingly had positive correlation with the IFN-γ level (r=0.56, p=0.03), but weak negative correlation with the IL-17A level (r=-0.30, p=0.27), implying that IL-27 in inflammatory joints of RA induces Th1 differentiation and suppresses the development or the migration of Th17 cells. These findings indicate that circulating IL-27-producing CD14(+) cells significantly infiltrate into inflamed regions such as RA synovium and have anti-inflammatory effects in several ways: both directly through the reduction of IL-6 production, and possibly through the induction of Th1 development and the suppression of Th17 development; and indirectly by regulation of recruitment of CCR6(+) cells, such as Th17 cells, through the suppression of CCL20 production. Our results suggest that such a serial negative feedback system could be applied to RA therapy.     

Bioluminescence characteristics of the fruiting body of Mycena chlorophos

Bioluminescent fungi are widely distributed on land and most belong to the class Basidomycetes. Light of about 530 nm wavelength maximum is emitted continuously. The molecular basis for the light‐emitting process remains unclear. We investigated the characteristics of the bioluminescence using cultivated fruiting bodies of M. chlorophos. Only fresh fruiting bodies exhibited long‐lasting light emission; rapid decay of light emission was observed with frozen and freeze‐dried samples. Freeze‐dried samples can be stored at room temperature under dry conditions and may be useful for the isolation of luciferin. The light emission of the fresh fruiting bodies was maintained in various buffers at varying pH; it could be stopped with pH 4 acetate buffer and could be recovered at pH 6. The isolation of luciferin from the fresh fruiting bodies might be possible by the control of buffer pH. The effect of temperature on the light emission of fruiting bodies indicated that bioluminescence in M. chlorophos may involve enzymatic reaction(s). The solubilization of bioluminescent components from the fruiting bodies could not be achieved with various surfactants. Copyright © 2011 John Wiley & Sons, Ltd.     

Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens

Cost-effective production of soluble recombinant protein in a bacterial system remains problematic with respect to expression levels and quality of the expressed target protein. These constraints have particular meaning today as "biosimilar" versions of innovator protein drugs are entering the clinic and the marketplace. A high throughput, parallel processing approach to expression strain engineering was used to evaluate soluble expression of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens. The human g-csf gene was optimized for expression in P. fluorescens and cloned into a set of periplasmic expression vectors. These plasmids were transformed into a variety of P. fluorescens host strains each having a unique phenotype, to evaluate soluble expression in a 96-well growth and protein expression format. To identify a strain producing high levels of intact, soluble Met-G-CSF product, more than 150 protease defective host strains from the Pfēnex Expression Technology? toolbox were screened in parallel using biolayer interferometry (BLI) to quantify active G-CSF binding to its receptor. A subset of these strains was screened by LC-MS analysis to assess the quality of the expressed G-CSF protein. A single strain with an antibiotic resistance marker insertion in the pfaI gene was identified that produced>99% Met-GCSF. A host with a complete deletion of the autotransporter-coding gene pfaI from the genome was constructed, and expression of soluble, active Met-GSCF in this strain was observed to be 350mg/L at the 1 liter fermentation scale.     

Temperature,recreational fishing and diapause egg connections: dispersal of spiny water fleas (<Emphasis Type="Italic">Bythotrephes longimanus</Emphasis>)

The spiny water flea (Bythotrephes longimanus) is spreading from Great Lakes coastal waters into northern inland lakes within a northern temperature-defined latitudinal band. Colonization of Great Lakes coastal embayments is assisted by winds and seiche surges, yet rapid inland expansion across the northern states comes through an overland process. The lack of invasions at Isle Royale National Park contrasts with rapid expansion on the nearby Keweenaw Peninsula. Both regions have comparable geology, lake density, and fauna, but differ in recreational fishing boat access, visitation, and containment measures. Tail spines protect Bythotrephes against young of the year, but not larger fish, yet the unusual thick-shelled diapausing eggs can pass through fish guts in viable condition. Sediment traps illustrate how fish spread diapausing eggs across lakes in fecal pellets. Trillions of diapausing eggs are produced per year in Lake Michigan and billions per year in Lake Michigamme, a large inland lake. Dispersal by recreational fishing is linked to use of baitfish, diapausing eggs defecated into live wells and bait buckets, and Bythothephes snagged on fishing line, anchor ropes, and minnow seines. Relatively simple measures, such as on-site rinsing of live wells, restricting transfer of certain baitfish species, or holding baitfish for 24 h (defecation period), should greatly reduce dispersal.     

Mamld1 knockdown reduces testosterone production and Cyp17a1 expression in mouse Leydig tumor cells

Background

MAMLD1 is known to be a causative gene for hypospadias. Although previous studies have indicated that MAMLD1 mutations result in hypospadias primarily because of compromised testosterone production around the critical period for fetal sex development, the underlying mechanism(s) remains to be clarified. Furthermore, although functional studies have indicated a transactivation function of MAMLD1 for the non-canonical Notch target Hes3, its relevance to testosterone production remains unknown. To examine these matters, we performed Mamld1 knockdown experiments.

Methodology/Principal Findings

Mamld1 knockdown was performed with two siRNAs, using mouse Leydig tumor cells (MLTCs). Mamld1 knockdown did not influence the concentrations of pregnenolone and progesterone but significantly reduced those of 17-OH pregnenolone, 17-OH progesterone, dehydroepiandrosterone, androstenedione, and testosterone in the culture media. Furthermore, Mamld1 knockdown significantly decreased Cyp17a1 expression, but did not affect expressions of other genes involved in testosterone biosynthesis as well as in insulin-like 3 production. Hes3 expression was not significantly altered. In addition, while 47 genes were significantly up-regulated (fold change >2.0×) and 38 genes were significantly down-regulated (fold change <0.5×), none of them was known to be involved in testosterone production. Cell proliferation analysis revealed no evidence for compromised proliferation of siRNA-transfected MLTCs.

Conclusions/Significance

The results, in conjunction with the previous data, imply that Mamld1 enhances Cyp17a1 expression primarily in Leydig cells and permit to produce a sufficient amount of testosterone for male sex development, independently of the Hes3-related non-canonical Notch signaling.     

The ESCRT system is required for hepatitis C virus production

Background

Recently, lipid droplets have been found to be involved in an important cytoplasmic organelle for hepatitis C virus (HCV) production. However, the mechanisms of HCV assembly, budding, and release remain poorly understood. Retroviruses and some other enveloped viruses require an endosomal sorting complex required for transport (ESCRT) components and their associated proteins for their budding process.

Methodology/Principal Findings

To determine whether or not the ESCRT system is needed for HCV production, we examined the infectivity of HCV or the Core levels in culture supernatants as well as HCV RNA levels in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, expressing short hairpin RNA or siRNA targeted to tumor susceptibility gene 101 (TSG101), apoptosis-linked gene 2 interacting protein X (Alix), Vps4B, charged multivesicular body protein 4b (CHMP4b), or Brox, all of which are components of the ESCRT system. We found that the infectivity of HCV in the supernatants was significantly suppressed in these knockdown cells. Consequently, the release of the HCV Core into the culture supernatants was significantly suppressed in these knockdown cells after HCV-JFH1 infection, while the intracellular infectivity and the RNA replication of HCV-JFH1 were not significantly affected. Furthermore, the HCV Core mostly colocalized with CHMP4b, a component of ESCRT-III. In this context, HCV Core could bind to CHMP4b. Nevertheless, we failed to find the conserved viral late domain motif, which is required for interaction with the ESCRT component, in the HCV-JFH1 Core, suggesting that HCV Core has a novel motif required for HCV production.

Conclusions/Significance

These results suggest that the ESCRT system is required for infectious HCV production.     

Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges and organic fertilizers

The wide variety of bacteria in the environment permits screening for more efficient cellulases to help overcome current challenges in biofuel production. This study focuses on the isolation of efficient cellulase producing bacteria found in organic fertilizers and paper mill sludges which can be considered for use in large scale biorefining. Pure isolate cultures were screened for cellulase activity. Six isolates: S1, S2, S3, S4, E2, and E4, produced halos greater in diameter than the positive control (Cellulomonas xylanilytica), suggesting high cellulase activities. A portion of the 16S rDNA genes of cellulase positive isolates were amplified and sequenced, then BLASTed to determine likely genera. Phylogenetic analysis revealed genera belonging to two major Phyla of Gram positive bacteria: Firmicutes and Actinobacteria. All isolates were tested for the visible degradation of filter paper; only isolates E2 and E4 (Paenibacillus species) were observed to completely break down filter paper within 72 and 96 h incubation, respectively, under limited oxygen condition. Thus E2 and E4 were selected for the FP assay for quantification of total cellulase activities. It was shown that 1% (w/v) CMC could induce total cellulase activities of 1652.2±61.5 and 1456.5±30.7 μM of glucose equivalents for E2 and E4, respectively. CMC could induce cellulase activities 8 and 5.6X greater than FP, therefore CMC represented a good inducing substrate for cellulase production. The genus Paenibacillus are known to contain some excellent cellulase producing strains, E2 and E4 displayed superior cellulase activities and represent excellent candidates for further cellulase analysis and characterization.     

Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells

Background

Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.

Methodology/Principal Findings

Distinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.

Conclusions/Significance

RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.