Monocyte Chemoattractant Protein-1/CCL2 Produced by Stromal Cells Promotes Lung Metastasis of 4T1 Murine Breast Cancer Cells

MCP-1/CCL2 plays an important role in the initiation and progression of cancer. Since tumor cells produce MCP-1, they are considered to be the main source of this chemokine. Here, we examined whether MCP-1 produced by non-tumor cells affects the growth and lung metastasis of 4T1 breast cancer cells by transplanting them into the mammary pad of WT or MCP-1^−/− mice. Primary tumors at the injected site grew similarly in both mice; however, lung metastases were markedly reduced in MCP-1^−/− mice, with significantly longer mouse survival. High levels of MCP-1 mRNA were detected in tumors growing in WT, but not MCP-1^−/− mice. Serum MCP-1 levels were increased in tumor-bearing WT, but not MCP-1^−/− mice. Transplantation of MCP-1^−/− bone marrow cells into WT mice did not alter the incidence of lung metastasis, whereas transplantation of WT bone marrow cells into MCP-1^−/− mice increased lung metastasis. The primary tumors of MCP-1^−/− mice consistently developed necrosis earlier than those of WT mice and showed decreased infiltration by macrophages and reduced angiogenesis. Interestingly, 4T1 cells that metastasized to the lung constitutively expressed elevated levels of MCP-1, and intravenous injection of 4T1 cells producing a high level of MCP-1 resulted in increased tumor foci in the lung of WT and MCP-1^−/− mice. Thus, stromal cell-derived MCP-1 in the primary tumors promotes lung metastasis of 4T1 cells, but tumor cell-derived MCP-1 can also contribute once tumor cells enter the circulation. A greater understanding of the source and role of this chemokine may lead to novel strategies for cancer treatment.     

Inhibition of the NAD-Dependent Protein Deacetylase SIRT2 Induces Granulocytic Differentiation in Human Leukemia Cells

Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.     

Decreased expression of VE-cadherin and claudin-5 and increased phosphorylation of VE-cadherin in vascular endothelium in nasal polyps

VE-cadherin and claudin-5 are major components of adherens and tight junctions of vascular endothelial cells and a decrease in their expression and an increase in the tyrosine-phosphorylation of VE-cadherin are associated with an increase in endothelial paracellular permeability. To clarify the mechanism underlying the development of edema in nasal polyps, we studied these molecules in polyp microvessels. Normal inferior turbinate mucosal tissues and nasal polyps from patients treated with or without glucocorticoid were stained for VE-cadherin or claudin-5 and CD31 by a double-immunofluorescence method and the immunofluorescence intensities were graded 1–3 with increasing intensity. To correct for differences in fluorescence intensity attributable to a different endothelial area being exposed in a section or to the thickness of a section, the relative immunofluorescence intensity was estimated by dividing the grade of VE-cadherin or claudin-5 by that of CD31 in each microvessel. Tyrosine-phosphorylation of VE-cadherin was examined by Western blot analysis. The relative intensities of VE-cadherin and claudin-5 in the CD31-positive microvessels significantly decreased in the following order; inferior turbinate mucosa, treated polyps and untreated polyps. The ratio of tyrosine-phosphorylated VE-cadherin to VE-cadherin was significantly higher in untreated polyps than in the inferior turbinate mucosa and treated polyps, between which no significant difference in the ratio was seen. Thus, in nasal polyps, the barrier function of endothelial adherens and tight junctions is weakened, although glucocorticoid treatment improves this weakened barrier function.     

Defective immune responses in mice lacking LUBAC‐mediated linear ubiquitination in B cells

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF‐κB pathway, which is important for B‐cell development and function. Here, we describe a mouse model (B‐HOIP^Δlinear) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF‐κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B‐HOIP^Δlinear mice due to defective activation of the IKK complex; however, B‐cell receptor (BCR)‐mediated activation of the NF‐κB and ERK pathways was unaffected. B‐HOIP^Δlinear mice show impaired B1‐cell development and defective antibody responses to thymus‐dependent and thymus‐independent II antigens. Taken together, these data suggest that LUBAC‐mediated linear polyubiquitination is essential for B‐cell development and activation, possibly via canonical NF‐κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.     

Patterns of genetic diversity of mitochondrial DNA within captive populations of the endangered itasenpara bitterling: implications for a reintroduction program

In this study, the level of genetic diversity of captive populations of the itasenpara bitterling (Acheilognathus longipinnis) was assessed to obtain information useful for successful captive breeding and reintroduction; this analysis was performed using mitochondrial DNA (mtDNA) sequence data. Comparison of the captive and wild populations showed low levels of genetic diversity within the captive population and significant genetic differentiation among the captive populations and also between the wild and captive populations, suggesting at chance effect during the founding process for the captive population and a subsequent genetic drift. Therefore, for successful reintroduction, it is important that the reintroduced population reflects all the genetic diversity available from the captive populations, and that releasing a large number of individuals that consist of all captive populations.     

Distribution of Streptococcus troglodytae and Streptococcus dentirousetti in chimpanzee oral cavities

The aim of this study was to analyze the distribution and phenotypic properties of the indigenous streptococci in chimpanzee (Pan troglodytes) oral cavities. Eleven chimpanzees (aged from 9 to 44 years, mean ± SD, 26.9 ± 12.6 years) in the Primate Research Institute of Kyoto University were enrolled in this research and brushing bacterial samples collected from them. Streptococci were isolated from the oral cavities of all chimpanzees. The isolates (n = 46) were identified as thirteen species by 16S rRNA genes analysis. The predominant species was Streptococcus sanguinis of mitis streptococci from five chimpanzees (45%). Mutans streptococci were isolated from six chimpanzees (55%). The predominant species in the mutans streptococci were Streptococcus troglodytae from four chimpanzees (36%), this species having been proposed as a novel species by us, and Streptococcus dentirousetti from three chimpanzees (27%). Streptococcus mutans was isolated from one chimpanzee (9%). However, Streptococcus sobrinus, Streptococcus macacae and Streptococcus downei, which are indigenous to human and monkey (Macaca fasciclaris) oral habitats, were not isolated. Of the mutans streptococci, S. troglodytae, S. dentirousetti, and S. mutans possessed strong adherence activity to glass surface.     

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.     

Ranging behavior of Mahale chimpanzees: a 16 year study

We have analyzed the ranging patterns of the Mimikire group (M group) of chimpanzees in the Mahale Mountains National Park, Tanzania. During 16 years, the chimpanzees moved over a total area of 25.2 or 27.4 km², as estimated by the grid-cell or minimum convex polygon (MCP) methods, respectively. Annually, the M group used an average of 18.4 km², or approximately 70 %, of the total home-range area. The chimpanzees had used 80 % of their total home range after 5 years and 95 % after 11 years. M group chimpanzees were observed more than half of the time in areas that composed only 15 % of their total home range. Thus, they typically moved over limited areas, visiting other parts of their range only occasionally. On average, the chimpanzees used 7.6 km² (in MCP) per month. Mean monthly range size was smallest at the end of the rainy season and largest at the end of the dry season, but there was much variability from year to year. The chimpanzees used many of the same areas every year when Saba comorensis fruits were abundant between August and January. In contrast, the chimpanzees used several different areas of their range in June. Here range overlap between years was relatively small. Over the 16 years of the study we found that the M group reduced their use of the northern part of their range and increased their frequency of visits to the eastern mountainous side of their home range. Changes in home-range size correlated positively with the number of adult females but not with the number of adult males. This finding does not support a prediction of the male-defended territory model proposed for some East African chimpanzee unit-groups.