Phenotypic stability of mature dendritic cells tuned by TLR or CD40 to control the efficiency of cytotoxic T cell priming

It is generally accepted that after stimulation immature DCs turn into mature DCs, which present exogenous antigens together with their MHC class I molecules and then activate the antigen-specific CTLs. Although both TLR and CD40 stimulation appeared to provide the same effects on DC maturation, CD40-dependent CTL activation is much more potent than CTL activation through LPS stimulation. Despite their different outcomes, the factors that lead mature DCs to different functions remain largely undefined. In this study, we defined the transient maturation and subsequent deactivation of DCs by TLR stimuli, including those by LPS and CpG-ODN. In contrast, CD40 stimulation induced stable mature DCs that elicited sufficient CTL proliferation. The deactivated DCs, which we defined as "expired DCs," were phenotypically similar to immature DCs, except for their phenotype stability, MHC class I expression level and IL-10 production. Moreover, the functions of expired DCs were comparable to those of immature DCs in terms of CTL induction and tolerogenicity. These results may provide an explanation for the role of CD40 stimulation in antigen-specific CTL induction.     

A novel I247T missense mutation in the haptoglobin 2 β-chain decreases the expression of the protein and is associated with ahaptoglobinemia

We have identified a novel base substitution at codon 247 in the -chain of the haptoglobin 2 (Hp²) allele in a Ghanaian with the Hp0 (ahaptoglobinemic) phenotype. The heterozygous TC substitution caused reduced expression of the protein when the mutant was transfected into COS7 cells. The base substitution resulted in a missense change of the non-polar amino acid isoleucine to the polar amino acid threonine at a position in the -chain that is highly conserved among several species. We had previously identified a mutation in the Hp gene promoter region for the same individual, which gives her genotype as –61CHp²/–61CHp²(I247T). Since the –61C mutation also leads to low Hp expression, the genotype represents the first and most definitive ahaptoglobinemic case reported in Africa.     

Drosophila deltex mediates suppressor of Hairless-independent and late-endosomal activation of Notch signaling

Notch (N) signaling is an evolutionarily conserved mechanism that regulates many cell-fate decisions. deltex (dx) encodes an E3-ubiquitin ligase that binds to the intracellular domain of N and positively regulates N signaling. However, the precise mechanism of Dx action is unknown. Here, we found that Dx was required and sufficient to activate the expression of gene targets of the canonical Su(H)-dependent N signaling pathway. Although Dx required N and a cis-acting element that overlaps with the Su(H)-binding site, Dx activated a target enhancer of N signaling, the dorsoventral compartment boundary enhancer of vestigial (vgBE), in a manner that was independent of the Delta (Dl)/Serrate (Ser) ligands- or Su(H). Dx caused N to be moved from the apical cell surface into the late-endosome, where it accumulated stably and co-localized with Dx. Consistent with this, the dx gene was required for the presence of N in the endocytic vesicles. Finally, blocking the N transportation from the plasma membrane to the late-endosome by a dominant-negative form of Rab5 inhibited the Dx-mediated activation of N signaling, suggesting that the accumulation of N in the late-endosome was required for the Dx-mediated Su(H)-independent N signaling.     

Evaluation of series of isobenzofuranone dimers as PKCalpha ligands: implication for the distance between the two ligand binding sites

Protein kinase C (PKC) is a family of enzymes, which play important roles in intracellular signal transduction. To examine the distance between the two ligand binding sites (C1A and C1B) of PKC, we designed and synthesized two series of isobenzofuranone dimers. Peak binding activities were observed for the C3-acyl chain dimers having a C10-C12 linker and for the C7 dimers having a C14-C16.     

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100?nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane.     

SUPERMAN regulates floral whorl boundaries through control of auxin biosynthesis

Proper floral patterning, including the number and position of floral organs in most plant species, is tightly controlled by the precise regulation of the persistence and size of floral meristems (FMs). In Arabidopsis, two known feedback pathways, one composed of WUSCHEL (WUS) and CLAVATA3 (CLV3) and the other composed of AGAMOUS (AG) and WUS, spatially and temporally control floral stem cells, respectively. However, mounting evidence suggests that other factors, including phytohormones, are also involved in floral meristem regulation. Here, we show that the boundary gene SUPERMAN (SUP) bridges floral organogenesis and floral meristem determinacy in another pathway that involves auxin signaling. SUP interacts with components of polycomb repressive complex 2 (PRC2) and fine‐tunes local auxin signaling by negatively regulating the expression of the auxin biosynthesis genes YUCCA1/4 (YUC1/4). In sup mutants, derepressed local YUC1/4 activity elevates auxin levels at the boundary between whorls 3 and 4, which leads to an increase in the number and the prolonged maintenance of floral stem cells, and consequently an increase in the number of reproductive organs. Our work presents a new floral meristem regulatory mechanism, in which SUP, a boundary gene, coordinates floral organogenesis and floral meristem size through fine‐tuning auxin biosynthesis.     

Pyrophosphate amplification reaction for measuring amino acid concentrations with high sensitivity using aminoacyl-tRNA synthetase from Escherichia coli

ABSTRACT

To measure amino acid concentrations with high sensitivity, the pyrophosphate amplification reaction conditions of histidyl-tRNA synthetase (HisRS) and tyrosyl-tRNA synthetase (TyrRS) were examined. The amount of pyrophosphate produced by reactions involving HisRS and TyrRS was amplified compared with the amount of the initial substrate L-amino acid after the addition of excess adenosine-5′-triphosphate and magnesium ions, with incubation at 50°C in an alkaline pH. The amount of pyrophosphate produced in the HisRS and TyrRS reactions was approximately 24- and 16-fold higher than the initial amount of L-His and L-Tyr, respectively. The pyrophosphate amplification reactions involving HisRS and TyrRS showed high substrate specificity for L-His and L-Tyr, respectively. Products of pyrophosphate amplification were identified as p¹, p⁴-di(adenosine) 5′-tetraphosphate, and adenosine-5′-monophosphate using high-performance liquid chromatography. A strong positive correlation was observed for 0 to 50 μM of L-His and L-Tyr in the pyrophosphate amplification reaction (R = 0.98 and R = 1.00, respectively).

Abbreviations: L-His: L-histidine; L-Tyr: L-tyrosine; aaRSs: aminoacyl-tRNA synthetases; ATP: adenosine-5′-triphosphate; aminoacyl-AMP-aaRS: aminoacyl-adenylate intermediate; Ap₄A, P¹, P⁴-di(adenosine) 5?-tetraphosphate; AMP: adenosine-5′-monophosphate; PAR: pyrophosphate amplification rate     

Epidithiodiketopiperazine as a pharmacophore for protein lysine methyltransferase G9a inhibitors: Reducing cytotoxicity by structural simplification

Chaetocin (1), a structurally complex epidithiodiketopiperazine (ETP) alkaloid produced by Chaetomium minutum, is a potent inhibitor of protein lysine methyltransferase G9a, which plays important roles in many biological processes. Here we present our synthetic investigations to identify a simple prototype G9a inhibitor structure based on structure–activity relationship (SAR) studies on chaetocin derivatives. The simple derivative PS-ETP-1 (14) was found to be a potent G9a inhibitor with greatly reduced cytotoxicity.