Synthesis and biological evaluation of truncated α-galactosylceramide derivatives focusing on cytokine induction profile

A series of truncated analogs of α-galactosylceramide with altered ceramide moiety was prepared, and evaluated for Th2-biased response in the context of IL-4/IFN-γ ratio. Phytosphingosine-modified analogs including cyclic, aromatic and ethereal compounds as well as the C-glycoside analog of OCH (2) with their cytokine inducing profile are disclosed.     

Eosinophils and Trichinella infection: toxic for the parasite and the host?

Peripheral blood and tissue eosinophilia characterize trichinellosis in humans, and present in addition to the increased total IgE levels that occur in many helminth infections. Both processes are the consequence of T-helper 2 activation. Blood and tissue eosinophilia begins with eosinophilopoiesis in the bone marrow, which is followed by the migration of eosinophils through the circulatory system, the eosinophil infiltration of tissues at the inflammatory foci and, finally, degranulation and cell death. Recently, some aspects of eosinophilia caused by Trichinella spiralis infection have been elucidated; however, the protective role of this population of cells against Trichinella parasites remains controversial. Furthermore, when eosinophils are numerous, they can be toxic for host tissues. This review discusses these issues in both human and rodent infection models.     

Facile Method for the Preparation of 7-Methyl-8-oxoguanosines as an Immunomodulator¶

Abstract

Reaction of 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-7-methylguaninium iodide (2a) with hydrogen peroxide in acetic acid gave the corresponding 7-methyl-8-oxoguanosine derivative (3a) in good yield. Deprotection of 3a easily gave 7-methyl-8-oxoguanosine (1), which is well-known as an immunomodulator. Substitution of acetyl group at the N-position of guanine ring accelerated the oxidation reaction of the 7-methylguaninium iodide.

     

Newly established in vitro system with fluorescent proteins shows that abnormal expression of downstream prion protein-like protein in mice is probably due to functional disconnection between splicing and 3' formation of prion protein pre-mRNA

We and others previously showed that, in some lines of prion protein (PrP)-knockout mice, the downstream PrP-like protein (PrPLP/Dpl) was abnormally expressed in brains partly due to impaired cleavage/polyadenylation of the residual PrP promoter-driven pre-mRNA despite the presence of a poly(A) signal. In this study, we newly established an in vitro transient transfection system in which abnormal expression of PrPLP/Dpl can be visualized by expression of the green fluorescence protein, EGFP, in cultured cells. No EGFP was detected in cells transfected by a vector carrying a PrP genomic fragment including the region targeted in the knockout mice intact upstream of the PrPLP/Dpl gene. In contrast, deletion of the targeted region from the vector caused expression of EGFP. By employing this system with other vectors carrying various deletions or point mutations in the targeted region, we identified that disruption of the splicing elements in the PrP terminal intron caused the expression of EGFP. Recent lines of evidence indicate that terminal intron splicing and cleavage/polyadenylation of pre-mRNA are functionally linked to each other. Taken together, our newly established system shows that the abnormal expression of PrPLP/Dpl in PrP-knockout mice caused by the impaired cleavage/polyadenylation of the PrP promoter-driven pre-mRNA is due to the functional dissociation between the pre-mRNA machineries, in particular those of cleavage/polyadenylation and splicing. Our newly established in vitro system, in which the functional dissociation between the pre-mRNA machineries can be visualized by EGFP green fluorescence, may be useful for studies of the functional connection of pre-mRNA machineries.     

Proteolytic processing regulates pathological accumulation in dentatorubral-pallidoluysian atrophy

Dentatorubral-pallidoluysian atrophy is caused by polyglutamine (polyQ) expansion in atrophin-1 (ATN1). Recent studies have shown that nuclear accumulation of ATN1 and cleaved fragments with expanded polyQ is the pathological process underlying neurodegeneration in dentatorubral-pallidoluysian atrophy. However, the mechanism underlying the proteolytic processing of ATN1 remains unclear. In the present study, we examined the proteolytic processing of ATN1 aiming to understand the mechanisms of ATN1 accumulation with polyQ expansion. Using COS-7 and Neuro2a cells that express the ATN1 gene, in which ATN1 was accumulated by increasing the number of polyQs, we identified a novel C-terminal fragment containing a polyQ tract. The mutant C-terminal fragment with expanded polyQ selectively accumulated in the cells, and this was also demonstrated in the brain tissues of patients with dentatorubral-pallidoluysian atrophy. Immunocytochemical and biochemical studies revealed that full-length ATN1 and C-terminal fragments displayed individual localization. The mutant C-terminal fragment was preferentially found in the cytoplasmic membrane/organelle and insoluble fractions. Accordingly, it is assumed that the proteolytic processing of ATN1 regulates the localization of C-terminal fragments. Accumulation of the C-terminal fragment was enhanced by inhibition of caspases in the cytoplasm of COS-7 cells. Collectively, these results suggest that the C-terminal fragment plays a principal role in the pathological accumulation of ATN1 in dentatorubral-pallidoluysian atrophy.     

Prolyl 4-Hydroxylation of ��-Fibrinogen: A NOVEL PROTEIN MODIFICATION REVEALED BY PLASMA PROTEOMICS*

Plasma proteome analysis requires sufficient power to compare numerous samples and detect changes in protein modification, because the protein content of human samples varies significantly among individuals, and many plasma proteins undergo changes in the bloodstream. A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL),” is capable of these tasks. Here, we describe successful detection of novel prolyl hydroxylation of α-fibrinogen using 2DICAL, based on comparison of plasma samples of 38 pancreatic cancer patients and 39 healthy subjects. Using a newly generated monoclonal antibody 11A5, we confirmed the increase in prolyl-hydroxylated α-fibrinogen plasma levels and identified prolyl 4-hydroxylase A1 as a key enzyme for the modification. Competitive enzyme-linked immunosorbent assay of 685 blood samples revealed dynamic changes in prolyl-hydroxylated α-fibrinogen plasma level depending on clinical status. Prolyl-hydroxylated α-fibrinogen is presumably controlled by multiple biological mechanisms, which remain to be clarified in future studies.For comprehensive analysis of plasma proteins, it is necessary to compare a sufficient number of blood samples to avoid simple interindividual heterogeneity, because the protein content of human samples varies significantly among individuals. Also, the provision of sufficient power is needed to detect protein modification because many plasma proteins undergo changes in the bloodstream (1). Even though the proteomic technologies have advanced (2, 3), there remains room for improvement. Different isotope labeling and identification-based methods have been developed for quantitative proteomics technologies (4–6), but the number of samples that can be compared by the current isotope-labeling methods is limited, and identification-based proteomics is unable to capture information regarding unknown modifications.A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL)² (7), simply compares the liquid chromatography and mass spectrometry (LC-MS) data and detects a protein modification by finding changes in the mass to charge ratio (m/z) and retention time (RT). Enhanced methods for accurate MS peak alignment across multiple LC runs have enabled the successful implementation of clinical studies requiring comparison of a large number of samples (8, 9). Using 2DICAL to analyze plasma samples of pancreatic cancer patients and healthy controls, novel prolyl hydroxylation of α-fibrinogen was successfully discovered.Fibrinogen and its modification has been investigated because of its clinical importance (10, 11). On the other hand, prolyl hydroxylation has attracted attention after the discovery of the hypoxia-inducible factor 1α (HIF1α) prolyl-hydroxylase and its role in switching of HIF1α functions (12). Prolyl hydroxylation in other proteins has been energetically sought, but only a few such proteins have been identified (13). Only one study has reported prolyl hydroxylation of fibrinogen at the β chain (14).Here, we report the detection of prolyl 4-hydroxylated α-fibrinogen by plasma proteome analysis, a protein modification that dynamically changes in plasma depending on the clinical status and is a candidate plasma biomarker.     

Compartmental imbalance and aberrant immune function of blood CD123+ (plasmacytoid) and CD11c+ (myeloid) dendritic cells in atopic dermatitis

Atopic dermatitis (AD) is a pruritic, chronically relapsing skin disease in which Th2 cells play a crucial role in cutaneous and extracutaneous immune reactions. In humans, CD11c+CD123- myeloid dendritic cells (mDC) and CD11c-CD123+ plasmacytoid DC (pDC) orchestrate the decision-making process in innate and acquired immunity. Since the number and function of these blood dendritic cell (DC) subsets reportedly reflect the host immune status, we studied the involvement of the DC subsets in the pathogenesis of AD. Patients with AD had an increased DC number and a low mDC:pDC ratio with pDC outnumbering mDC in the peripheral blood compared with normal subjects and psoriasis patients (a Th1 disease model group). The mDC:pDC ratio was correlated with the total serum IgE level, the ratio of IFN-gamma-producing blood cells:IL-4-producing blood cells, and the disease severity. In vitro allogeneic stimulation of naive CD4+ cells with atopic DC showed that the ability of pDC for Th1 induction was superior or comparable to that of mDC. In skin lesions, pDC infiltration was in close association with blood vessels expressing peripheral neural addressins. Therefore, compartmental imbalance and aberrant immune function of the blood DC subsets may deviate the Th1/Th2 differentiation and thus induce protracted allergic responses in AD.     

Cryopyrin-induced interleukin 1beta secretion in monocytic cells: enhanced activity of disease-associated mutants and requirement for ASC

Several autoinflammatory disorders are associated with missense mutations within the nucleotide-binding oligomerization domain of cryopyrin. The mechanism by which cryopyrin mutations cause inflammatory disease remains elusive. To understand the molecular bases of these diseases, we generated constructs to express three common cryopyrin disease-associated mutations, R260W, D303N, and E637G, and compared their activity with that of the wild-type protein. All cryopyrin mutant proteins tested were found to induce potent NF-kappaB activity when compared with the wild-type protein. This activation was dependent on the expression of ASC, an adaptor protein previously suggested to mediate cryopyrin signaling. When the disease-associated mutants were expressed in monocytic THP-1 cells (which express endogenous ASC), each induced spontaneous IL-1beta secretion, whereas wild-type protein did not. In the absence of stimuli, wild-type cryopyrin was unable to bind to ASC, whereas the three mutants coimmunoprecipitated with ASC, suggesting a mechanism involved in the constitutive activation of mutant proteins. The induction of cryopyrin activity by enforced oligomerization in THP-1 cells resulted in ASC binding and the secretion of IL-1beta, an effect that was abolished by the inhibition of ASC expression with small interfering RNAs. Thus, cryopyrin-mediated IL-1beta secretion requires ASC in monocytic cells. Further, these results indicate that cryopyrin disease-associated mutants are constitutively active and able to induce NF-kappaB activation and IL-1beta secretion at least in part by an increased ability to interact with ASC.     

Biochemistry and physiology of mitochondrial ion channels involved in cardioprotection

Over the past decades there has been considerable progress in understanding the multifunctional roles of mitochondrial ion channels in metabolism, energy transduction, ion transport, signaling, and cell death. Recent data have suggested that some of these channels function under physiological condition, and others may be activated in response to pathological insults and play a key role in cytoprotection. This review outlines our current understanding of the molecular identity and pathophysiological roles of the mitochondrial ion channels in the heart with particular emphasis on cardioprotection against ischemia/reperfusion injury, and future research on mitochondrial ion channels.     

Distinct roles of TLR2 and the adaptor ASC in IL-1beta/IL-18 secretion in response to Listeria monocytogenes

Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) is an adaptor molecule that has recently been implicated in the activation of caspase-1. We have studied the role of ASC in the host defense against the intracellular pathogen Listeria monocytogenes. ASC was found to be essential for the secretion of IL-1beta/IL-18, but dispensable for IL-6, TNF-alpha, and IFN-beta production, in macrophages infected with Listeria. Activation of caspase-1 was abolished in ASC-deficient macrophages, whereas activation of NF-kappaB and p38 was unaffected. In contrast, secretion of IL-1beta, IL-6, and TNF-alpha was reduced in TLR2-deficient macrophages infected with Listeria; this was associated with impaired activation of NF-kappaB and p38, but normal caspase-1 processing. Analysis of Listeria mutants revealed that cytosolic invasion was required for ASC-dependent IL-1beta secretion, consistent with a critical role for cytosolic signaling in the activation of caspase-1. Secretion of IL-1beta in response to lipopeptide, a TLR2 agonist, was greatly reduced in ASC-null macrophages and was abolished in TLR2-deficient macrophages. These results demonstrate that TLR2 and ASC regulate the secretion of IL-1beta via distinct mechanisms in response to Listeria. ASC, but not TLR2, is required for caspase-1 activation independent of NF-kappaB in Listeria-infected macrophages.