Major role of adipocyte prostaglandin E2 in lipolysis-induced macrophage recruitment

Obesity induces accumulation of adipose tissue macrophages (ATMs), which contribute to both local and systemic inflammation and modulate insulin sensitivity. Adipocyte lipolysis during fasting and weight loss also leads to ATM accumulation, but without proinflammatory activation suggesting distinct mechanisms of ATM recruitment. We examined the possibility that specific lipid mediators with anti-inflammatory properties are released from adipocytes undergoing lipolysis to induce macrophage migration. In the present study, we showed that conditioned medium (CM) from adipocytes treated with forskolin to stimulate lipolysis can induce migration of RAW 264.7 macrophages. In addition to FFAs, lipolytic stimulation increased release of prostaglandin E₂ (PGE₂) and prostaglandin D₂ (PGD₂), reflecting cytosolic phospholipase A₂ α activation and enhanced cyclooxygenase (COX) 2 expression. Reconstituted medium with the anti-inflammatory PGE₂ potently induced macrophage migration while different FFAs and PGD₂ had modest effects. The ability of CM to induce macrophage migration was abolished by treating adipocytes with the COX2 inhibitor sc236 or by treating macrophages with the prostaglandin E receptor 4 antagonist AH23848. In fasted mice, macrophage accumulation in adipose tissue coincided with increases of PGE₂ levels and COX1 expression. Collectively, our data show that adipocyte-originated PGE₂ with inflammation suppressive properties plays a significant role in mediating ATM accumulation during lipolysis.     

Recessive Mutations in COL25A1 Are a Cause of Congenital Cranial Dysinnervation Disorder

Abnormal ocular motility is a common clinical feature in congenital cranial dysinnervation disorder (CCDD). To date, eight genes related to neuronal development have been associated with different CCDD phenotypes. By using linkage analysis, candidate gene screening, and exome sequencing, we identified three mutations in collagen, type XXV, alpha 1 (COL25A1) in individuals with autosomal-recessive inheritance of CCDD ophthalmic phenotypes. These mutations affected either stability or levels of the protein. We further detected altered levels of sAPP (neuronal protein involved in axon guidance and synaptogenesis) and TUBB3 (encoded by TUBB3, which is mutated in CFEOM3) as a result of null mutations in COL25A1. Our data suggest that lack of COL25A1 might interfere with molecular pathways involved in oculomotor neuron development, leading to CCDD phenotypes.     

Discovery of 2,4-thiazolidinedione-tethered coumarins as novel selective inhibitors for carbonic anhydrase IX and XII isoforms

Different 2,4-thiazolidinedione-tethered coumarins 5a–b, 10a–n and 11a–d were synthesised and evaluated for their inhibitory action against the cancer-associated hCAs IX and XII, as well as the physiologically dominant hCAs I and II to explore their selectivity. Un-substituted phenyl-bearing coumarins 10a, 10 h, and 2-thienyl/furyl-bearing coumarins 11a–c exhibited the best hCA IX (K_Is between 0.48 and 0.93 µM) and hCA XII (K_Is between 0.44 and 1.1 µM) inhibitory actions. Interestingly, none of the coumarins had any inhibitory effect on the off-target hCA I and II isoforms. The sub-micromolar compounds from the biochemical assay, coumarins 10a, 10 h and 11a–c, were assessed in an in vitro antiproliferative assay, and then the most potent antiproliferative agent 11a was tested to explore its impact on the cell cycle phases and apoptosis in MCF-7 breast cancer cells to provide more insights into the anticancer activity of these compounds.     

Proximity of periplasmic loops in the metal-Tetracycline/H(+) antiporter of Escherichia coli observed on site-directed chemical cross-linking

Our previous study on second-site suppressor mutations of the Tn10-encoded metal-tetracycline/H(+) antiporter suggested that Leu(30) and Ala(354), located in periplasmic loop 1-2 and 11-12, respectively, are conformationally linked to each other (Kawabe, T., and Yamaguchi, A. (1999) FEBS Lett. 457, 169-173). To determine the spatial proximity of these two residues, cross-linking gel-shift assays of the L30C/A354C double mutant were performed after the mutant had been oxidized with Cu(2+)/o-phenanthroline. The results indicated that Leu(30) and Ala(354) are close to each other but that Gly(62), which is located in cytoplasmic loop 2-3, and Ala(354) are distant from each other, as a negative control. Then, a single Cys residue was introduced into each of the six periplasmic loop regions (P1-P6), and eleven double mutants were constructed. Of these eleven double Cys mutants, the L30C/A354C and L30C/T235C mutants showed a mobility shift on oxidation, indicating that P1 is spatially close to P4 as well as P6. In contrast, the other nine mutants, L30C/S92C, L30C/S156C, L30C/S296C, S92C/S296C, S92C/T235C, S92C/A354C, S156C/T235C, S156C/S296C, and S156C/A354C, showed no mobility shift under oxidized conditions on intramolecular cross-linking. The S92C and S296C mutants showed dimerization on intermolecular cross-linking, indicating that P2 and P5 are located at the periphery of the helix bundle.     

Epigenetic status of the H19 locus in human oocytes following in vitro maturation

Imprinting is an epigenetic modification that is reprogrammed in the germ line and leads to the monoallelic expression of some genes. Imprinting involves DNA methylation. Maternal imprint is reset during oocyte growth and maturation. In vitro maturation (IVM) of oocytes may, therefore, interfere with imprint acquisition and/or maintenance. To evaluate if maturing human oocytes in vitro would be hazardous at the epigenetic level, we first determined the methylation profile of the H19 differentially methylated region (DMR). The methylation status of the H19 DMR seems particularly vulnerable to in vitro culture conditions. We analyzed oocytes at different stages of maturation following IVM, germinal vesicle (GV), metaphase I (MI), and metaphase II (MII), using the bisulfite mutagenesis technique. Our results indicated that the unmethylated specific maternal profile for the H19 DMR was stably established at the GV stage. The majority of MI-arrested oocytes exhibited an altered pattern of methylation, the CTCF-binding site being methylated in half of the DNA strands analyzed. Of the 20 MII oocytes analyzed, 15 showed the normal unmethylated maternal pattern, while 5 originating from two different patients exhibited a methylated pattern. These findings highlight the need for extended analysis on MII-rescued oocytes to appreciate the epigenetic safety of the IVM procedure, before it becomes a routine and practical assisted reproductive procedure.     

Purification,immobilization, and biochemical characterization of l‐arginine deiminase from thermophilic Aspergillus fumigatus KJ434941: Anticancer activity in vitro

l ‐Arginine deiminase (ADI) has a powerful anticancer activity against various tumors, via arginine depletion, arresting the cell cycle at G₁ phase. However, the current clinically tried bacterial ADI displayed a higher antigenicity and lower thermal stability. Thus, our objective was to purify and characterize this enzyme from thermophilic fungi, to explore its catalytic and antigenic properties for therapeutic uses. ADI was purified from thermophilic Aspergillus fumigatus KJ434941 to its electrophoretic homogeneity by 5.1‐fold, with molecular subunit 50 kDa. The purified ADI was PEGylated and covalently immobilized on dextran to explore its catalytic properties. The specific activity of free ADI, PEG‐ADI, and Dex‐ADI was 26.7, 21.5, and 18.0 U/mg, respectively. At 50°C, PEG‐ADI displays twofold resistance to thermal denaturation (t_1/2 13.9 h), than free ADI (t_1/2 6.9 h), while at 70°C, the thermal stability of PEG‐ADI was increased by 1.7‐fold, with similar stability to Dex‐ADI with the free one. Kinetically, free ADI had the higher catalytic affinity to arginine, followed by PEG‐ADI and Dex‐ADI. Upon proteolysis for 30 min, the residual activity of native ADI, PEG‐ADI, and Dex‐AD was 8.0, 32.0, and 20.0% for proteinase K and 10.0, 52.0, and 90.0% for acid protease, respectively. The anticancer activity of the ADIs was assessed against HCT, HEP‐G2, and MCF7, in vitro. The free and PEG‐ADI exhibits a similar cytotoxic efficacy for the tested cells, lower than Dex‐ADI. The free ADI had IC₅₀ value 22.0, 16.6, and 13.9 U/mL, while Dex‐ADI had 3.98, 5.18, and 4.43 U/mL for HCT, MCF7, and HEPG‐2, respectively. The in vitro anticancer activity of ADI against HCT, MCF7, and HEPG‐2 was increased by five‐, three‐, and threefold upon covalent modification by dextran. The biochemical and hematological parameters of the experimented animals were not affected by ADIs dosing, with no signs of anti‐ADI immunoglobulins in vivo. The in vivo half‐life time of free ADI, PEG‐ADI, and Dex‐ADI was 29.7, 91.1, 59.6 h, respectively. The present findings explored a novel thermostable, less antigenic ADI from thermophilic A. fumigatus, with further molecular and crystallographic analyses, this enzyme will be a powerful candidate for clinical trials. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:396–405, 2015     

The CYPome (Cytochrome P450 complement) of Aspergillus nidulans

The cytochromes P450 (CYPs) are found in all biological kingdoms and genome sequencing projects continue to reveal an ever increasing number. The principle aim of this paper is to identify the complete CYPome of Aspergillus nidulans from the genome sequence version AN.3 deposited at the Broad institute, assign the appropriate CYP nomenclature and define function where possible. The completed analysis revealed a total of 111 CYP genes, 3 of which were previously unknown and 8 pseudogenes, representing 89CYP families, 21 of which are unique. We have identified 28 potential gene clusters associated with one or more CYP genes and discussed those with putative PKS and NRPS associated function. The chromosomal location of the genes, predicted cellular location of the proteins and possible function(s) are discussed.