Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen-I (OFA-I)

Summary IgG anti-OFA-I found in melanoma patients was tested for its ability to lyse human tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC). Sera from 89 stage II melanoma patients which contained non-HLA-related IgG antibody to an OFA-I-positive melanoma cell line (M14) as tested by indirect membrane immunofluorescence (IMI) were originally chosen as possible sources of IgG anti-OFA-I. Of those tested for specific IgG activity to OFA-I by IMI, anti-OFA-I was found only in those patients immunized with OFA-I-positive tumor cells. When the same sera were tested in ADCC, no non-HLA-related activity could be demonstrated. This result was confirmed with purified IgG fractions that could, nevertheless, show anti-OFA-I reactivity in a complement-dependent cytotoxicity assay. The fact that naturally occurring IgG anti-OFA-I antibody was not readily detectable in patients' sera and that induced IgG anti-OFA-I did not participate in ADCC indicates that OFA-I-related tumor cell lysis via ADCC is an unlikely phenomenon in cancer patients.     

Macrophage activation for tumor cytotoxicity: Control of cytotoxic activity by the time interval effector and target cells are exposed to lymphokines

Macrophages continuously exposed to lymphokines (LK) and target cells throughout a 48-hr cytotoxicity assay exhibit 3-fold more tumoricidal activity than do cells optimally treated with LK before addition of tumor cells. Increased cytotoxic activity induced by continuous LK treatment was not due to direct toxic effects of LK on tumor target cells or to alterations in target cell susceptibility to cytopathic effects of LK-activated macrophages. Moreover, sensitivities of responsive macrophages to LK activation signals and time courses for onset and loss of tumoricidal activity during continuous exposure or LK pulse were identical. Analysis of macrophage or LK dose responses and time courses for development of cytotoxicity each suggest that differences in tumoricidal activity between macrophages continuously exposed or pulsed with LK were quantitative: the number of cytotoxic events was increased 2.7 ± 0.2-fold (mean ± SEM for 11 experiments) during continuous LK treatment. Optimal levels of macrophage tumoricidal activity then occur only if effector cells, target cells and activation stimuli are simultaneously present for a defined time interval: tumor cells need not be present during the initial 2 to 3 hr of culture; LK can be removed after 8 hr with little or no loss of cytotoxic activity. However, removal of LK or target cells during the critical 4- to 8-hr interval decreased levels of cytotoxicity 3-fold. Thus, nonspecific effector function by LK-activated macrophages in controlled by both the physicochemical nature of the LK mediator and the time interval effector and target cells are exposed to LK.     

Distribution of cis-Vaccenic Acid in Chinese Hamster V79-R Cells

V79-R Cells grown in lipid-free medium contained octadecenoic acids as the major fatty acids esterified to lipids. Octadecenoic acids were composed of two positional isomers, oleic and cis-vaccenic acids. The distribution of oleic and cis-vaccenic acids was altered by the addition of various fatty acids to the medium. There was no difference in the distribution of oleic and cis-vaccenic acids in phospholipids between mitochondria and microsomes. Cardiolipin contained higher amounts of palmitoleic and cis-vaccenic acids than did other lipids.     

Axial chirality in biaryl N,N-dialkylaminopyridine derivatives bearing an internal carboxy group

Axial chirality in N,N-dimethylaminopyridines as well as N,N-dipropylaminopyridines bearing an internal carboxy group were evaluated based on their racemization barriers and circular dichroism spectra. The half-life of racemization of N,N-dipropylaminopyridine derivative 2 was estimated to be 19.7 days at 20°C. Its enantiomers isolated as optically active forms showed positive-negative and negative-positive Cotton effects for (+)- 2 and (−)- 2 , respectively, from 310 to 210 nm. Furthermore, (−)- 2 was applied as a chiral nucleophilic catalyst and exhibited asymmetric induction in acylative kinetic resolution of 1-(1-naphthyl)ethane-1-ol.     

Decreased Expression of Matrix Metalloproteinase-9 and Increased Expression of Tissue Inhibitors of Matrix Metalloproteinase-1 in Paratuberculosis-Infected Cattle in the ELISA-Negative Subclinical Stage

We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. Significant decreased MMP-9 expression and increased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in control cattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheral blood cell culture supernatants from Map-infected cattle at 24 hr after Map lysate and MapPPD stimulation by gelatin zymography analysis. As a result, the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.     

Circulating Levels of Fatty Acid-Binding Protein Family and Metabolic Phenotype in the General Population

Objective

Fatty acid-binding proteins (FABPs) are a family of 14-15-kDa proteins, and some FABPs have been to be used as biomarkers of tissue injury by leak from cells. However, recent studies have shown that FABPs can be secreted from cells into circulation. Here we examined determinants and roles of circulating FABPs in a general population.

Methods

From the database of the Tanno-Sobetsu Study, a study with a population-based cohort design, data in 2011 for 296 subjects on no medication were retrieved, and FABP1∼5 in their serum samples were assayed.

Results

Level of FABP4, but not the other isoforms, showed a gender difference, being higher in females than in males. Levels of all FABPs were negatively correlated with estimated glomerular filtration rate (eGFR), but a distinct pattern of correlation with other clinical parameters was observed for each FABP isoform; significant correlates were alanine aminotransferase (ALT), blood pressure (BP), and brain natriuretic peptide (BNP) for FABP1, none besides eGFR for FABP2, age, BP, and BNP for FABP3, age, waist circumference (WC), BP, BNP, lipid variables, high-sensitivity C-reactive protein (hsCRP), and HOMA-R for FABP4, and age, WC, BP, ALT, BNP, and HOMA-R for FABP5. FABP4 is the most strongly related to metabolic markers among FABPs. In a multivariate regression analysis, FABP4 level was an independent predictor of HOMA-R after adjustment of age, gender, WC, BP, HDL cholesterol, and hsCRP.

Conclusions

Each FABP isoform level showed a distinct pattern of correlation with clinical parameters, although levels of all FABPs were negatively determined by renal function. Circulating FABP4 appears to be a useful biomarker for detecting pre-clinical stage of metabolic syndrome, especially insulin resistance, in the general population.     

Norovirus Binding to Intestinal Epithelial Cells Is Independent of Histo-Blood Group Antigens

Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Le^b-HBGAs) on the human intestinal cell line, Caco-2, were examined by confocal laser-scanning microscopy. To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells.     

Transformation of Lipid Bodies Related to Hydrocarbon Accumulation in a Green Alga,Botryococcus braunii (Race B)

The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space; most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells.     

Studies on fouling by the freshwater mussel limnoperna fortunei and the antifouling effects of low energy surfaces

Attachment of the freshwater mussel, Limnoperna fortunei, was tested using non‐treated surfaces, viz. glass, nylon, rubber, silicone and Teflon, together with glass surfaces modified with nine kinds of silane coupling agents. Among the surfaces tested, the mussel avoided attaching to Teflon, silicone, and glass modified with 3‐bromopropyltrimethoxysilane or 3,3,3‐(trifluo‐ropropyl)‐trimethoxysilane. With respect to the relationship between the percentage attachment and the surface free energy (sfe) of the substrates, it was found that attachment was considerably reduced on the substrates which exhibited relatively low sfe, as above. The mean number of secreted byssuses per attaching mussel also decreased with decreasing substrate sfe. Furthermore, when the sfe was divided into the dispersion and polar components, the percentage mussel attachment was related to the polar component. These results suggest that effective antifouling towards L. fortunei is achieved on substrates with a low sfe polar component.     

Enzymatic Formation of β-Citraurin from β-Cryptoxanthin and Zeaxanthin by Carotenoid Cleavage Dioxygenase4 in the Flavedo of Citrus Fruit

In this study, the pathway of β-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of β-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of β-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of β-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved β-cryptoxanthin and zeaxanthin at the 7,8 or 7′,8′ position. But other carotenoids tested in this study (lycopene, α-carotene, β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of β-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of β-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of β-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.Carotenoids, a diverse group of pigments widely distributed in nature, fulfill a variety of important functions in plants and play a critical role in human nutrition and health (Schwartz et al., 1997; Cunningham and Gantt, 1998; Havaux, 1998; Krinsky et al., 2003; Ledford and Niyogi, 2005). The pathway of carotenoid biosynthesis has been well documented in various plant species, including Arabidopsis (Arabidopsis thaliana; Park et al., 2002), tomato (Lycopersicon esculentum; Isaacson et al., 2002), pepper (Capsicum annuum; Bouvier et al., 1998), citrus (Citrus spp.; Kato et al., 2004, 2006; Rodrigo et al., 2004; Rodrigo and Zacarías, 2007; Kato, 2012; Zhang et al., 2012a), and apricot (Prunus armenaica; Kita et al., 2007). Genes encoding the enzymes in the carotenoid biosynthetic pathway have been cloned, and their expression profiles have also been characterized (Fig. 1). As carotenoids contain a series of conjugated double bonds in the central chain, they can be oxidatively cleaved in a site-specific manner (Mein et al., 2011). The oxidative cleavage of carotenoids not only regulates their accumulation but also produces a range of apocarotenoids (Walter et al., 2010). In higher plants, many different apocarotenoids derive from the cleavage of carotenoids and have important metabolic functions, such as plant hormones, pigments, aroma and scent compounds, as well as signaling compounds (Fig. 1). A well-known example is abscisic acid, which is a C₁₅ compound derived from the cleavage of the 11,12 double bond of 9-cis-violaxanthin and 9′-cis-neoxanthin (Schwartz et al., 1997; Tan et al., 1997; Cutler and Krochko, 1999; Chernys and Zeevaart, 2000; Giuliano et al., 2003).Open in a separate windowFigure 1.Carotenoid and apocarotenoid metabolic pathway in plants. GGPP, Geranylgeranyl diphosphate. Enzymes, listed here from top to bottom, are named according to the designation of their genes: PSY, phytoene synthase; PDS, Phytoene desaturase; ZDS, ζ-carotene desaturase; ZISO, 15-cis-ζ-carotene isomerase; CRTISO, carotenoid isomerase; LCYb, lycopene β-cyclase; LCYe, lycopene ε-cyclase; HYe, ε-ring hydroxylase; HYb, β-ring hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin deepoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase.Carotenoid cleavage dioxygenases (CCDs) are a group of enzymes that catalyze the oxidative cleavage of carotenoids (Ryle and Hausinger, 2002). CCDs are nonheme iron enzymes present in plants, bacteria, and animals. In plants, CCDs belong to an ancient and highly heterogenous family (CCD1, CCD4, CCD7, CCD8, and 9-cis-epoxycarotenoid dioxygenases [NCEDs]). The similarity among the different members is very low apart from four strictly conserved His residues and a few Glu residues (Kloer and Schulz, 2006; Walter et al., 2010). In Arabidopsis, the CCD family contains nine members (CCD1, NCED2, NCED3, CCD4, NCED5, NCED6, CCD7, CCD8, and NCED9), and orthologs in other plant species are typically named according to their homology with an Arabidopsis CCD (Huang et al., 2009). In our previous study, the functions of CitCCD1, CitNCED2, and CitNCED3 were investigated in citrus fruits (Kato et al., 2006). The recombinant CitCCD1 protein cleaved β-cryptoxanthin, zeaxanthin, and all-trans-violaxanthin at the 9,10 and 9′,10′ positions and 9-cis-violaxanthin at the 9′,10′ position. The recombinant CitNCED2 and CitNCED3 proteins cleaved 9-cis-violaxanthin at the 11,12 position to form xanthoxin, a precursor of abscisic acid (Kato et al., 2006). To date, information on the functions of other CCDs in citrus fruits remains limited, while the functions of CCD7 and CCD8, as well as NCED5, NCED6, and NCED9, in Arabidopsis have been characterized (Kloer and Schulz, 2006; Walter et al., 2010). In Arabidopsis, CCD7 cleaves all-trans-β-carotene at the 9′,10′ position to form all-trans-β-apo-10′-carotenal. All-trans-β-apo-10′-carotenal is further shortened by AtCCD8 at the 13,14 position to produce β-apo-13-carotenone (Alder et al., 2012). NCED5, NCED6, and NCED9 cleave 9-cis-violaxanthin at the 11,12 position to form xanthoxin (Tan et al., 2003). Compared with other CCDs, the function of CCD4 is poorly understood. In Chrysanthemum morifolium, CmCCD4a contributed to the white color formation by cleaving carotenoids into colorless compounds (Ohmiya et al., 2006). Recently, it has been reported that CsCCD4, CmCCD4a, and MdCCD4 could cleave β-carotene to yield β-ionone (Rubio et al., 2008; Huang et al., 2009).β-Citraurin, a C₃₀ apocarotenoid, is a color-imparting pigment responsible for the reddish color of citrus fruits (Farin et al., 1983). In 1936, it was first discovered in Sicilian oranges (Cual, 1965). In citrus fruits, the accumulation of β-citraurin is not a common event; it is only observed in the flavedos of some varieties during fruit ripening. The citrus varieties accumulating β-citraurin are considered more attractive because of their red-orange color (Ríos et al., 2010). Although more than 70 years have passed since β-citraurin was first identified, the pathway of its biosynthesis is still unknown. As its structure is similar to that of β-cryptoxanthin and zeaxanthin, β-citraurin was presumed to be a degradation product of β-cryptoxanthin or zeaxanthin (Oberholster et al., 2001; Rodrigo et al., 2004; Ríos et al., 2010; Fig. 1). To date, however, the specific cleavage reaction producing β-citraurin has not been elucidated. In this study, we found that the CitCCD4 gene was involved in the synthesis of β-citraurin, using two citrus varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. To confirm the role of the CitCCD4 gene further, functional analyses of the CitCCD4 enzyme were performed in vivo and in vitro. Additionally, the regulation of β-citraurin content and CitCCD4 gene expression in response to ethylene and red light-emitting diode (LED) light treatments was also examined. This study, to our knowledge, is the first to investigate the biosynthesis of β-citraurin in citrus fruits. The results might provide new strategies to enhance the nutritional and commercial qualities of citrus fruits.