A Major Latex-Like Protein Is a Key Factor in Crop Contamination by Persistent Organic Pollutants

This is the first report, to our knowledge, to reveal important factors by which members of the Cucurbitaceae family, such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), pumpkin (Cucurbita pepo), squash (C. pepo), and zucchini (C. pepo), are selectively polluted with highly toxic hydrophobic contaminants, including organochlorine insecticides and dioxins. Xylem sap of C. pepo ssp. pepo, which is a high accumulator of hydrophobic compounds, solubilized the hydrophobic compound pyrene into the aqueous phase via some protein(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of xylem sap of two C. pepo subspecies revealed that the amount of 17-kD proteins in C. pepo ssp. pepo was larger than that in C. pepo ssp. ovifera, a low accumulator, suggesting that these proteins may be related to the translocation of hydrophobic compounds. The protein bands at 17 kD contained major latex-like proteins (MLPs), and the corresponding genes MLP-PG1, MLP-GR1, and MLP-GR3 were cloned from the C. pepo cultivars Patty Green and Gold Rush. Expression of the MLP-GR3 gene in C. pepo cultivars was positively correlated with the band intensity of 17-kD proteins and bioconcentration factors toward dioxins and dioxin-like compounds. Recombinant MLP-GR3 bound polychlorinated biphenyls immobilized on magnetic beads, whereas recombinant MLP-PG1 and MLP-GR1 did not. These results indicate that the high expression of MLP-GR3 in C. pepo ssp. pepo plants and the existence of MLP-GR3 in their xylem sap are related to the efficient translocation of hydrophobic contaminants. These findings should be useful for decreasing the contamination of fruit of the Cucurbitaceae family as well as the phytoremediation of hydrophobic contaminants.Numerous agricultural fields and crops have been contaminated with persistent organic pollutants (POPs), including dioxins, such as polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs); dioxin-like compounds, such as coplanar polychlorinated biphenyls (PCBs) and the insecticide dichlorodiphenyltrichloroethane; drins, such as aldrin, dieldrin, and endrin; and chlordane (Hashimoto, 2005; Uegaki et al., 2006; Hilber et al., 2008). POPs show carcinogenicity, teratogenicity, immunotoxicity, and estrogenicity toward humans and wildlife after accumulation through the food chain. Despite the fact that the use of PCBs and these insecticides was prohibited several decades ago, environmental and crop contamination remains a problem due to their high hydrophobicity and chemical stability.Members of the Cucurbitaceae family, such as cucumber (Cucumis sativus), watermelon (Citrullas lanatus), melon (Cucumis melo), pumpkin (Cucurbita pepo), and zucchini (C. pepo), are some of the major crops in the world. Previous studies reported that members of the Cucurbitaceae family, particularly C. pepo, which includes pumpkin and zucchini, accumulated higher levels of PCDDs and PCDFs (Hülster et al., 1994; Inui et al., 2008), 2,2-bis(p-chlorophenyl) 1,1-dichloroethylene (p,p′-DDE; White et al., 2003), PCBs (Aslund et al., 2008; Inui et al., 2008), chlordane (Mattina et al., 2004), and drins (Otani et al., 2007) compared with the levels in other plant species. Thus, it appears that the Cucurbitaceae family has unique mechanisms of POP uptake and translocation. Lunney et al. (2004) reported that the shoots of pumpkin and zucchini plants showed much higher concentrations of dichlorodiphenyltrichloroethane than those of tall fescue (Festuca arundinacea), alfalfa (Medicago sativa), and ryegrass (Lolium multiflorum), whereas concentrations in roots were similar among these plants. Likewise, significant differences were found between C. pepo ssp. pepo and ssp. ovifera in concentrations of dioxins and dioxin-like compounds in the aerial parts, whereas the concentrations in their roots were similar (Inui et al., 2011). These results suggest that the mechanisms causing the high accumulation of POPs in C. pepo plants mainly occur during translocation from the roots to the aerial parts.The transport of substances such as nutrients and signal molecules over long distances in higher land plants is mediated by the vascular bundles, which consist of phloem and xylem strands. In addition to inorganic salts, organic nutrients such as amino acids, sugars, and organic acids are translocated through the xylem from the roots to the aerial parts (Satoh, 2006). Furthermore, the fact that POPs such as chlordane, dieldrin, and PCBs were detected in xylem sap of C. pepo suggests that their accumulation in the aerial parts of plants occurs during the translocation from roots to aerial parts in xylem sap (Mattina et al., 2004; Murano et al., 2010b; Greenwood et al., 2011). A recent study revealed that there were protein-like materials with the ability to dissolve dieldrin in xylem sap (Murano et al., 2010a). However, these materials have yet to be identified, and the mechanisms underlying the high transport ability and high accumulation of POPs in C. pepo plants are not fully understood.In this study, to clarify the molecular mechanisms of the efficient uptake and high accumulation of POPs by C. pepo plants, xylem sap proteins related to the transport of POPs in xylem sap were identified. The aim of this research is to provide a means of preventing cucumber, melon, watermelon, pumpkin, and zucchini fruits from being contaminated by POPs.     

Diclofenac Enhances Proinflammatory Cytokine-Induced Aquaporin-4 Expression in Cultured Astrocyte

Acute encephalopathy is a generic term for acute brain dysfunction occurring after infection. Acute encephalopathy induced by influenza virus results in high mortality, and most cases of influenza-associated encephalopathy (IAE) result in brain edema. Administration of diclofenac sodium (DCF), a non-steroidal anti-inflammatory drug (NSAID), is associated with a significant increased mortality rate of IAE. These previous clinical findings proposed further investigation of DCF administration and brain edema to clarify how DCF aggravates IAE. Aquaporin-4 (AQP4) is the predominant water channel protein in the mammalian brain, and is mainly expressed in astrocytes. AQP4 plays an important role in brain water homeostasis. Therefore, we investigated a possible association between DCF and AQP4 production in astrocytes. We stimulated cultured rat astrocytes with three cytokines, interleukin-1β, tumor necrosis factor α, and interferon γ, and then treated with DCF. DCF enhanced proinflammatory cytokine-induced AQP4 gene and protein expression in astrocytes, whereas DCF alone did not change the AQP4 gene expression. The addition of nuclear factor-kappa B (NF-κB) inhibitor abrogated AQP4 gene and protein expression completely in astrocytes treated with cytokines alone and in those also treated with DCF. In conclusion, this study demonstrated that AQP4 is upregulated in astrocyte by proinflammatory cytokines, and that the addition of DCF further augments AQP4 production. This effect is mediated via NF-κB signaling. The enhancement of AQP4 production by DCF may explain the significantly increased mortality rates in IAE patients treated with DCF.     

TNF inhibitor suppresses bone metastasis in a breast cancer cell line

In the evolution of cancer, tumor necrosis factor-alpha (TNF-α) plays a paradoxical role. High doses induce significant anticancer effects, but conversely, physiologic and pathologic levels of TNF-α may be involved in cancer promotion, tumor growth, and metastasis.Infliximab is a chimeric murine monoclonal antibody that binds with high affinity to soluble and membrane TNF-α and inhibits binding of TNF-α to its receptors. In the present study, we investigated the effect of infliximab, a TNF-α antagonist, on breast cancer aggressiveness and bone metastases.Infliximab greatly reduced cell motility and bone metastases in a metastatic breast cancer cell line, MDA-MB-231. The mechanism of bone metastasis inhibition involved decreased expression of CXC chemokine receptor 4 (CXCR4) and increased expression of decorin, which is the prototype of an expanding family of small leucine-rich proteoglycans. These results suggest a novel role for TNF-α inhibition in the reduction or prevention of bone metastases in this breast cancer model. Our study suggests that inhibition of TNF-α using infliximab may become a preventive therapeutic option for breast cancer.     

Internal selection against the evolution of left-right reversal

Among metazoan species, left-right reversals in primary asymmetry have rarely gone to fixation. This suggests that a general mechanism suppresses the evolution of polarity reversal. Most metazoans appear externally symmetric and reproduce by external fertilization or copulation with genitalia located in the midline. Thus, reversal should generate little exogenous disadvantage when interacting with the external environment or in mating with the common wild-type. Accordingly, an endogenously caused fitness reduction may be responsible for the general absence of reversed species. However, how this selection operates is little understood. Phenotypic changes associated with reversal are usually inseparable from zygotic pleiotropy. By exploiting hermaphroditism and the maternal inheritance of left-right polarity, we generated dextral and sinistral snails that share the same zygotic genotype. Before hatching, these sinistrals developed lethal morphological anomalies more frequently than dextrals. Their shell shape at maturity differed from the mirror image of the dextral shell. These interchiral differences demonstrate pleiotropy in maternal effects of the polarity or linked genes. Variation in interchiral differences between parental crosses suggests the presence of epistatic variation in relative performance of sinistrals. Our results show that internal selection operates against polarity reversal, and we suggest that this is due to changes in blastomere configuration.     

Development of a Serial Order in Speech Constrained by Articulatory Coordination

Universal linguistic constraints seem to govern the organization of sound sequences in words. However, our understanding of the origin and development of these constraints is incomplete. One possibility is that the development of neuromuscular control of articulators acts as a constraint for the emergence of sequences in words. Repetitions of the same consonant observed in early infancy and an increase in variation of consonantal sequences over months of age have been interpreted as a consequence of the development of neuromuscular control. Yet, it is not clear how sequential coordination of articulators such as lips, tongue apex and tongue dorsum constrains sequences of labial, coronal and dorsal consonants in words over the course of development. We examined longitudinal development of consonant-vowel-consonant(-vowel) sequences produced by Japanese children between 7 and 60 months of age. The sequences were classified according to places of articulation for corresponding consonants. The analyses of individual and group data show that infants prefer repetitive and fronting articulations, as shown in previous studies. Furthermore, we reveal that serial order of different places of articulations within the same organ appears earlier and then gradually develops, whereas serial order of different articulatory organs appears later and then rapidly develops. In the same way, we also analyzed the sequences produced by English children and obtained similar developmental trends. These results suggest that the development of intra- and inter-articulator coordination constrains the acquisition of serial orders in speech with the complexity that characterizes adult language.     

Initial Responses of Articular Tissues in a Murine High-Fat Diet-Induced Osteoarthritis Model: Pivotal Role of the IPFP as a Cytokine Fountain

Obesity and high body mass index are associated with a higher incidence of osteoarthritis (OA). The aim of this study is to investigate the involvement of the infrapatellar fat pad (IPFP) in the sub-acute effect of a high fat diet (HFD) on the development of knee-OA. C57BL/6J male mice were fed either a HFD or a normal diet beginning at seven weeks of age. Tissue sections were evaluated with immunohistological analysis. The IPFP was excised, and mRNA expression profiles were compared using real-time RT-PCR analysis. Osteoarthritic changes were initiated in the HFD group after eight weeks of the HFD. Increased synovial cell number and angiogenesis at the anterior edge of the tibial plateau were exhibited prior to osteophyte formation. Quantitative histological analysis indicated that osteophyte volume was significantly increased in the HFD group after eight weeks, along with an increase in the IPFP volume, the size of individual adipocytes and the number of vessels in the IPFP. Histomorphometrical analysis revealed osteophyte area was significantly associated with IPFP area, individual adipocyte area and vascular area. Real-time RT-PCR analysis demonstrated elevated mRNA expression of inflammatory cytokines, growth factor, and adipokines in the IPFP after eight weeks of the HFD. These findings are in parallel with increased expression of the CD68 macrophage marker after eight weeks of the HFD. Expression levels of the adipokines were significantly correlated with expression of TNF-α, VEGF and TGF-β. Immunohistological analysis revealed that the Nampt protein was highly expressed in the IPFP especially around the site of osteophyte formation. Apoptosis and proliferation of chondrocytes were both enhanced at the site of osteophyte formation, indicating higher cell turnover at this region. These observations suggest the IPFP plays a pivotal role in the formation of osteophytes and functions as a secretory organ in response to a HFD.     

Palmitic Acid Induces Osteoblastic Differentiation in Vascular Smooth Muscle Cells through ACSL3 and NF-κB,Novel Targets of Eicosapentaenoic Acid

Free fatty acids (FFAs), elevated in metabolic syndrome and diabetes, play a crucial role in the development of atherosclerotic cardiovascular disease, and eicosapentaenoic acid (EPA) counteracts many aspects of FFA-induced vascular pathology. Although vascular calcification is invariably associated with atherosclerosis, the mechanisms involved are not completely elucidated. In this study, we tested the hypothesis that EPA prevents the osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC) induced by palmitic acid (PA), the most abundant long-chain saturated fatty acid in plasma. PA increased and EPA abolished the expression of the genes for bone-related proteins, including bone morphogenetic protein (BMP)-2, Msx2 and osteopontin in human aortic smooth muscle cells (HASMC). Among the long-chain acyl-CoA synthetase (ACSL) subfamily, ACSL3 expression was predominant in HASMC, and PA robustly increased and EPA efficiently inhibited ACSL3 expression. Importantly, PA-induced osteoblastic differentiation was mediated, at least in part, by ACSL3 activation because acyl-CoA synthetase (ACS) inhibitor or siRNA targeted to ACSL3 completely prevented the PA induction of both BMP-2 and Msx2. Conversely, adenovirus-mediated ACSL3 overexpression enhanced PA-induced BMP-2 and Msx2 expression. In addition, EPA, ACSL3 siRNA and ACS inhibitor attenuated calcium deposition and caspase activation induced by PA. Notably, PA induced activation of NF-κB, and NF-κB inhibitor prevented PA-induction of osteoblastic gene expression and calcium deposition. Immunohistochemistry revealed the prominent expression of ACSL3 in VSMC and macrophages in human non-calcifying and calcifying atherosclerotic plaques from the carotid arteries. These results identify ACSL3 and NF-κB as mediators of PA-induced osteoblastic differentiation and calcium deposition in VSMC and suggest that EPA prevents vascular calcification by inhibiting such a new molecular pathway elicited by PA.