Immunolocalization of 8-5′ and 8-8′ linked structures of lignin in cell walls of Chamaecyparis obtusa using monoclonal antibodies

Mouse monoclonal antibodies were generated against dehydrodiconiferyl alcohol- or pinoresinol-p-aminohippuric acid (pAHA)-bovine serum albumin (BSA) conjugate as probes that specifically react with 8-5′ or 8-8′ linked structure of lignin in plant cell walls. Hybridoma clones were selected that produced antibodies that positively reacted with dehydrodiconiferyl alcohol- or pinoresinol-pAHA–BSA and negatively reacted with pAHA–BSA and guaiacylglycerol-beta-guaiacyl ether-pAHA–BSA conjugates containing 8-O-4′ linkage. Eight clones were established for each antigen and one of each clone that positively reacted with wood sections was selected. The specificity of these antibodies was examined by competitive ELISA tests using various lignin dimers with different linkages. The anti-dehydrodiconiferyl alcohol antibody reacted specifically with dehydrodiconiferyl alcohol and did not react with other model compounds containing 8-O-4′, 8-8′, or 5-5′ linkages. The anti-pinoresinol antibody reacted specifically with pinoresinol and syringaresinol and did not react with the other model compounds containing 8-O-4′, 8-5′, or 5-5′ linkages. The antibodies also did not react with dehydrodiconiferyl alcohol acetate or pinoresinol acetate, indicating that the presence of free phenolic or aliphatic hydroxyl group was an important factor in their reactivity. In sections of Japanese cypress (Chamaecyparis obtusa), labeling by the anti-dehydrodiconiferyl alcohol antibody was found in the secondary walls of phloem fibers and in the compound middle lamellae, and secondary walls of tracheids. Weak labeling by the anti-pinoresinol antibody was found in secondary walls of phloem fibers and secondary walls and compound middle lamellae of developed tracheids. These labelings show the localization of 8-5′ and 8-8′ linked structure of lignin in the cell walls.     

Genomic organization of human complement protein C8α and further examination of its linkage to C8β

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target membranes. It is composed of three nonidentical subunits (C8, C8, C8) encoded by separate genes. C8 and C8 are linked on chromosome 1p32, whereas C8 is located on 9q22.3-q32. In this study, overlapping genomic clones were isolated and used to decipher the organization of the human C8 gene. The gene contains at least 11 exons spanning 70kb of DNA. When compared to C6, C8 and C9, there is a remarkable similarity in genomic organization, consistent with amino acid sequence comparisons that suggest these proteins are ancestrally related. Regions of each protein that are structurally similar are encoded in exons of correspondingly similar lengths with highly conserved boundaries and phases. Availability of genomic sequence also facilitated a more detailed analysis of C8 and C8 linkage. Based on analysis of genomic digests with cDNA probes, the loci were previously reported to be physically linked (< 2.5kb) and in a 5- 3 orientation. In the present study, results obtained using exon-specific probes indicate the loci are not as closely linked as initially believed. Furthermore, they suggest that cDNA probes used earlier yielded misleading information because they encode exons that are distributed across large segments of genomic DNA.     

8-Aza Analogues of Deaza Purine Nucleosides. Synthesis and Biological Evaluation of 8-Aza-1-deazaadenosine and 2′-Deoxy-8-aza-1-deazaadenosine

Abstract

The syntheses of 7-amino-3-(β-D-ribofuranosyl)-3H-1,2,3-triazolo[4,5-b]pyridine (8-aza-1-deazaadenosine) (2) and 7-amino-3-(2-deoxy-β-D-erythro-pentofuranosyl)-3H-1,2,3-triazolo[4,5-b]pyridine (2′-deoxy-8-aza-1-deazaadenosine) (3) by glycosylation of the anion of 7-chloro-3H-1,2,3-triazolo[4,5-b]pyridine are described. The anomeric configuration as well as the position of glycosylation were determined by ¹H, ¹³ NMR, UV and N.O.E. difference spectroscopy. The cytotoxicity of these nucleosides against several murine and human tumor cell lines is discussed. Compounds 2 and 3 proved to be good inhibitors of adenosine deaminase.     

In Vivo Characterization of the Biodistribution Profile of Amphipol A8–35

Amphipols (APols) are polymeric surfactants that keep membrane proteins (MPs) water-soluble in the absence of detergent, while stabilizing them. They can be used to deliver MPs and other hydrophobic molecules in vivo for therapeutic purposes, e.g., vaccination or targeted delivery of drugs. The biodistribution and elimination of the best characterized APol, a polyacrylate derivative called A8–35, have been examined in mice, using two fluorescent APols, grafted with either Alexa Fluor 647 or rhodamine. Three of the most common injection routes have been used, intravenous (IV), intraperitoneal (IP), and subcutaneous (SC). The biodistribution has been studied by in vivo fluorescence imaging and by determining the concentration of fluorophore in the main organs. Free rhodamine was used as a control. Upon IV injection, A8–35 distributes rapidly throughout the organism and is found in most organs but the brain and spleen, before being slowly eliminated (10–20 days). A similar pattern is observed after IP injection, following a brief latency period during which the polymer remains confined to the peritoneal cavity. Upon SC injection, A8–35 remains essentially confined to the point of injection, from which it is only slowly released. An interesting observation is that A8–35 tends to accumulate in fat pads, suggesting that it could be used to deliver anti-obesity drugs.     

A Serendipitous Synthesis of 8-Dimsyl-2′-deoxyguanosine

Abstract

A serendipitous synthesis of 8-dimsyl-dG (2) has been achieved along with the known 8-benzyloxy-dG (3) in a nucleophilic substitution reaction of 8-bromo-dG (1) with in situ generated dimsyl and benzyloxy sodium. Compound 3 was directly converted into the mutagenic oxidative DNA damage product, 8-oxo-dGTP (4).     

Effects of high-fat diet feeding on Znt8-null mice: differences between β-cell and global knockout of Znt8

Genomewide association studies have linked a polymorphism in the zinc transporter 8 (Znt8) gene to higher risk of developing type 2 diabetes. Znt8 is highly expressed in pancreatic β-cells where it is involved in the regulation of zinc transport into granules. However, Znt8 is also expressed in other tissues including α-cells, where its function is as yet unknown. Previous work demonstrated that mice lacking Znt8 globally were more susceptible to diet-induced obesity (Lemaire et al., Proc Natl Acad Sci USA 106: 14872-14877, 2009; Nicolson et al., Diabetes 58: 2070-2083, 2009). Therefore, the main goal of this study was to examine the physiological impact of β-cell-specific Znt8 deficiency in mice during high-fat high-calorie (HFHC) diet feeding. For these studies, we used β-cell-specific Znt8 knockout (Ins2Cre:Znt8loxP/loxP) and whole body Znt8 knockout (Cre-:Znt8(-/-)) mice placed on a HFHC diet for 16 wk. Ins2Cre:Znt8loxP/loxP mice on HFHC diet had similar body weights throughout the study but displayed impaired insulin biosynthesis and secretion and were glucose intolerant compared with littermate control Ins2Cre mice. In contrast, Cre-:Znt8(-/-) mice became remarkably obese, hyperglycemic, hyperinsulinemic, insulin resistant, and glucose intolerant compared with littermate control Cre- mice. These data show that β-cell Znt8 alone does not considerably aggravate weight gain and glucose intolerance during metabolic stress imposed by an HFHC diet. However, global loss of Znt8 is involved in exacerbating diet-induced obesity and resulting insulin resistance, and this may be due to the loss of Znt8 activity in a tissue other than the β-cell. Thus, our data suggest that Znt8 contributes to the risk of developing type 2 diabetes through β-cell- and non-β-cell-specific effects.     

Localization of the spherocytosis gene to chromosome segment 8p11.22→8p21.1

Summary A case of hereditary spherocytosis (HS) is reported. Cytogenetic study revealed a de nove minute deletion of chromosome 8. The critical portion which affected the expression of the HS phenotype appeared to be localized to 8p11.228p21.1.     

Negative Role of RIG-I Serine 8 Phosphorylation in the Regulation of Interferon-�� Production

RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-β mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-β production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-β or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.     

The cytotoxicity of 8-O-4′ neolignans from the seeds of Crataegus pinnatifida

Nine new 8-O-4′ neolignans, named pinnatifidanin B I–IX (1–9), together with 9 known analogs (10–18) were isolated from the seeds of Crataegus pinnatifida. The structures of 1–18 were determined by spectroscopic methods, including 1D, 2D NMR, CD and HRESIMS analysis. Compounds 8–11, 17 and 18 displayed potent cytotoxic activities against human cancer cell lines, and most interestingly, none of the 6 compounds displayed inhibitory activity against human lung cell line (Mrc5). The 6 cytotoxic compounds are considered to be potential as antitumor agents, which could significantly inhibit the cancer cell growth in a dose-dependent manner and are probably safer than positive control drug.     

Down-modulation of CD8αβ is a fundamental activity of primate lentiviral Nef proteins

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αβ (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8β in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8β, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8β, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αβ is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.     

Discovery of stereospecific cytotoxicity of (8R,8′R)-trans-arctigenin against insect cells and structure-activity relationship on aromatic ring

One of the arctigenin stereoisomers, (8R,8′R)-trans-form 1, showed stereospecific cytotoxicity against insect cells, Sf9 and NIAS-AeAl-2 cells. By the comparison with other stereoisomers, the most importance of the 8′R stereochemistry for the higher activities was clarified. On the other hand, the wider range of activity level among stereoisomers against cancer cells, HL-60, was not observed. The structure-activity relationship research using derivatives bearing (8R,8′R)-trans-form was performed to show the same level of activities of 3-iodo, 4-iodo, and 3,4-methylenedioxy derivatives 28, 29, and 36 as (8R,8′R)-trans-arctigenin 1. In the examination of thiono derivatives, 4-iodo thiono and 3,4-methylenedioxy thiono derivatives 66, 67 showed similar level of activities to that of (8R,8′R)-trans-arctigenin 1. The expression of ribosomal 28S rRNA gene of Sf9 cells was increased by (8R,8′R)-trans-arctigenin 1, whereas a degradation of DNA was not observed.     

The Most Recent Dreams of Children Ages 8–11

The present study examined whether the Most Recent Dream Method is a feasible choice for dream collection from children as young as 8 years old. A quantitative analysis of 30 Most Recent Dreams from 8–11 year-old girls and 32 Most Recent Dreams from 8–11 year-old boys reveals that the method seems feasible as indicated by children's ability to respond. The basic findings for recency, length, types of characters, and other content categories show the same overall pattern of gender similarities and differences by age 10–11 as found with 12–13 year-olds and young adults. Girls' dream reports especially begin to resemble those of older girls and young women by age 10–11. These results, if confirmed by follow-up studies with larger numbers of children, suggest that reasonably representative samples of the dreams of girls from age 10 onward can be collected using the Most Recent Dream method.