Synthesis and biological evaluation of N-mercaptoacylproline and N-mercaptoacylthiazolidine-4-carboxylic acid derivatives as leukotriene A4 hydrolase inhibitors

We studied the synthetic modification of structurally similar N-mercaptoacyl-L-proline and (4R)-N-mercaptoacylthiazolidine-4-carboxylic acid to obtain potent leukotriene A(4) (LTA(4)) hydrolase inhibitors. An N-mercaptoacyl group, (2S)-3-mercapto-2-methylpropionyl group, was effective for both scaffolds. Additional introduction of a large substituent such as 4-isopropylbenzylthio (3f), 4-tert-butylbenzylthio (3l) or 4-cyclohexylbenzylthio group (3m) with (S)-configuration at the C(4) position of proline yielded much more potent LTA(4) hydrolase inhibitors (IC(50); 52, 31, and 34 nM, respectively) than captopril (IC(50); 630,000 nM).     

GM-CSF-mediated T-cell activation by macrophages infected with recombinant BCG that secretes major membrane protein-II of Mycobacterium leprae

The potential of Mycobacterium bovis Bacillus Calmette–Guerin (BCG) needs to be augmented to efficiently activate CD4⁺ T cells through macrophages. Mycobacterium leprae -derived recombinant major membrane protein (MMP)-II induced GM-CSF production from macrophages. A recombinant BCG-SM that secretes MMP-II more efficiently produced GM-CSF and activated interferon (IFN)-γ-producing CD4⁺ T cells than did vector control BCG when infected with macrophages. The T-cell activation by BCG-SM was dependent on the GM-CSF production by macrophages. Interleukin (IL)-10 production by macrophages stimulated with M. leprae was inhibited in a GM-CSF-dependent manner when the precursor monocytes were infected with BCG-SM. BCG inducing GM-CSF production was effective in macrophage-mediated T-cell activation partially through IL-10 inhibition.     

Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae defective in the late steps of ergosterol biosynthesis are viable but accumulate structurally altered sterols within the plasma membrane. Despite the significance of pleiotropic abnormalities in the erg mutants, little is known about how sterol alterations mechanically affect the membrane structure and correlate with individual mutant phenotypes. Here we demonstrate that the membrane order and occurrence of voids are determinants of membrane rigidity and hypersensitivity to a drug. Among five ergΔ mutants, the erg2Δ mutant exhibited the most marked sensitivity to cycloheximide. Notably, measurement of time-resolved anisotropy indicated that the erg2Δ mutation decreased the membrane order parameter (S), and dramatically increased the rotational diffusion coefficient (D_w) of 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in the plasma membrane by 8-fold, providing evidence for the requirement of ergosterol for membrane integrity. The IC₅₀ of cycloheximide was closely correlated with S/D_w in these strains, suggesting that the membrane disorder and increasing occurrence of voids within the plasma membrane synergistically enhance passive diffusion of cycloheximide across the membrane. Exogenous ergosterol partially restored the membrane properties in the upc2-1erg2Δ strain. In this study, we describe the ability of ergosterol to adjust the dynamic properties of the plasma membrane, and consider the relevance of drug permeability.     

Conservation of Silk Genes in Trichoptera and Lepidoptera

Larvae of the sister orders Trichoptera and Lepidoptera are characterized by silk secretion from a pair of labial glands. In both orders the silk filament consists of heavy (H)- and light (L)-chain fibroins and in Lepidoptera it also includes a P25 glycoprotein. The L-fibroin and H-fibroin genes of Rhyacophila obliterata and Hydropsyche angustipennis caddisflies have exon/intron structuring (seven exons in L-fibroin and two in H-fibroin) similar to that in their counterparts in Lepidoptera. Fibroin cDNAs are also known in Limnephilus decipiens, representing the third caddisfly suborder. Amino acid sequences of deduced L-fibroin proteins and of the terminal H-fibroin regions are about 50% identical among the three caddisfly species but their similarity to lepidopteran fibroins is <25%. Positions of some residues are conserved, including cysteines that were shown to link the L-fibroin and H-fibroin by a disulfide bridge in Lepidoptera. The long internal part of H-fibroins is composed of short motifs arranged in species-specific repeats. They are extremely uniform in R. obliterata. Motifs (SX)_n, GGX, and GPGXX occur in both Trichoptera and Lepidoptera. The trichopteran H-fibroins further contain charged amphiphilic motifs but lack the strings of alanines or alanine-glycine dipeptides that are typical lepidopteran motifs. On the other hand, sequences composed of a motif similar to ERIVAPTVITR surrounded by the (SX)_4-6 strings and modifications of the GRRGWGRRG motif occur in Trichoptera and not in Lepidoptera.

Propagation of Asian isolates of canine distemper virus (CDV) in hamster cell lines

Backgrounds  

The aim of this study was to confirm the propagation of various canine distemper viruses (CDV) in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance.     

Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.     

Regulation of IGF-1/PI3K/Akt signalling by the phosphoinositide phosphatase pharbin

Pharbin, a 5-phosphatase that induces arborization, is one of the phosphoinositide 5-phosphatases that is highly mutated in patients with Joubert syndrome. Pharbin can hydrolyse PI(4,5)P(2) and PI(3,4,5)P(3) and has the same substrate specificity as SHIP2 and SKIP, which negatively regulate PI3K signalling. Here, we investigated the role of pharbin in IGF-1/PI3K signalling. Ectopic expression of pharbin markedly suppressed the IGF-1-induced activation of Akt without affecting p42/44 MAP kinase phosphorylation. In contrast, pharbin silencing by RNA interference increased the IGF-1-induced phosphorylation of Akt, suggesting that pharbin negatively regulates PI3K/Akt signalling. Pharbin expression also inhibited the phosphorylation of p70 S6 kinase and 4E-BP1 as well as the subsequent protein synthesis in response to IGF-1 treatment. Taken together, these results indicate that pharbin is an important negative regulator of IGF-1/PI3K/Akt signalling and protein synthesis.     

Adipolin/C1qdc2/CTRP12 protein functions as an adipokine that improves glucose metabolism

Obesity is a major risk factor for the development of insulin resistance and type 2 diabetes. Adipose tissue secretes various bioactive molecules, referred to as adipokines, whose dysregulation can mediate changes in glucose homeostasis and inflammatory responses. Here, we identify C1qdc2/CTRP12 as an insulin-sensitizing adipokine that is abundantly expressed by fat tissues and designate this adipokine as adipolin (adipose-derived insulin-sensitizing factor). Adipolin expression in adipose tissue and plasma was reduced in rodent models of obesity. Adipolin expression was also decreased in cultured 3T3-L1 adipocytes by treatment with inducers of endoplasmic reticulum stress and inflammation. Systemic administration of adipolin ameliorated glucose intolerance and insulin resistance in diet-induced obese mice. Adipolin administration also reduced macrophage accumulation and proinflammatory gene expression in the adipose tissue of obese mice. Conditioned medium from adipolin-expressing cells diminished the expression of proinflammatory cytokines in response to stimulation with LPS or TNFα in cultured macrophages. These data suggest that adipolin functions as an anti-inflammatory adipokine that exerts beneficial actions on glucose metabolism. Therefore, adipolin represents a new target molecule for the treatment of insulin resistance and diabetes.