General anesthetics inhibit erythropoietin induction under hypoxic conditions in the mouse brain

Background

Erythropoietin (EPO), originally identified as a hematopoietic growth factor produced in the kidney and fetal liver, is also endogenously expressed in the central nervous system (CNS). EPO in the CNS, mainly produced in astrocytes, is induced under hypoxic conditions in a hypoxia-inducible factor (HIF)-dependent manner and plays a dominant role in neuroprotection and neurogenesis. We investigated the effect of general anesthetics on EPO expression in the mouse brain and primary cultured astrocytes.

Methodology/Principal Findings

BALB/c mice were exposed to 10% oxygen with isoflurane at various concentrations (0.10–1.0%). Expression of EPO mRNA in the brain was studied, and the effects of sevoflurane, halothane, nitrous oxide, pentobarbital, ketamine, and propofol were investigated. In addition, expression of HIF-2α protein was studied by immunoblotting. Hypoxia-induced EPO mRNA expression in the brain was significantly suppressed by isoflurane in a concentration-dependent manner. A similar effect was confirmed for all other general anesthetics. Hypoxia-inducible expression of HIF-2α protein was also significantly suppressed with isoflurane. In the experiments using primary cultured astrocytes, isoflurane, pentobarbital, and ketamine suppressed hypoxia-inducible expression of HIF-2α protein and EPO mRNA.

Conclusions/Significance

Taken together, our results indicate that general anesthetics suppress activation of HIF-2 and inhibit hypoxia-induced EPO upregulation in the mouse brain through a direct effect on astrocytes.     

A role for strain differences in waveforms of ultrasonic vocalizations during male-female interaction

Male mice emit ultrasonic vocalizations (USVs) towards females during male-female interaction. It has been reported that USVs of adult male mice have the capability of attracting females. Although the waveform pattern of USVs is affected by genetic background, differences among strains with respect to USV and the effects of these differences on courtship behavior have not been analyzed fully. We analyzed USV patterns, as well as actual social behavior during USV recording, in 13 inbred mouse strains, which included laboratory and wild-derived strains. Significant effects of strain were observed for the frequency of USV emission, duration, and frequency of the waveform category. Principal component (PC) analysis showed that PC1 was related to frequency and duration, and PC2-4 were related to each waveform. In the comparison of USV patterns and behaviors among strains, wild-derived KJR mice displayed the highest scores for PC2-4, and female mice paired with KJR males did not emit rejection-related click sounds. It is assumed that the waveforms emitted by KJR males have a positive effect in male-female interaction. Therefore, we extracted waveforms in PC2-4 from the USV recordings of KJR mice to produce a sound file, "HIGH2-4". As a negative control, another sound file ("LOW2-4") was created by extracting waveforms in PC2-4 from strains with low scores for these components. In the playback experiments using these sound files, female mice were attracted to the speaker that played HIGH2-4 but not the speaker that played LOW2-4. These results highlight the role of strain differences in the waveforms of male USVs during male-female interaction. The results indicated that female mice use male USVs as information when selecting a suitable mate.     

UNC93B1 physically associates with human TLR8 and regulates TLR8-mediated signaling

Toll-like receptors (TLRs) 3, 7, 8, and 9 are localized to intracellular compartments where they encounter foreign or self nucleic acids and activate innate and adaptive immune responses. The endoplasmic reticulum (ER)-resident membrane protein, UNC93B1, is essential for intracellular trafficking and endolysosomal targeting of TLR7 and TLR9. TLR8 is phylogenetically and structurally related to TLR7 and TLR9, but little is known about its localization or function. In this study, we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants. UNC93B1 physically associated with human TLR8, similar to TLRs 3, 7, and 9, and played a critical role in TLR8-mediated signaling. Localization analyses of TLR8 tail-truncated mutants revealed that the transmembrane domain and the Toll/interleukin-1 receptor domain were required for proper targeting of TLR8 to the early endosome. Hence, although UNC93B1 participates in intracellular trafficking and signaling for all nucleotide-sensing TLRs, the mode of regulation of TLR localization differs for each TLR.     

Isolation of novel microsatellite markers for the social parasitoid wasp, Copidosoma floridanum (Hymenoptera: Encyrtidae)

We developed and characterized nine microsatellite loci for the polyembryonic wasp Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae). Eight of the nine loci were polymorphic and did not deviate from Hardy–Weinberg equilibrium. Our results will contribute to studies on the population genetic structure of C. floridanum.     

Reduction of E-cadherin by human defensin-5 in esophageal squamous cells

Barrett’s esophagus (BE) is metaplastic columnar epithelium converted from normal squamous epithelia in the distal esophagus that is thought to be a precancerous lesion of esophageal adenocarcinoma. BE is attributed to gastroesophageal reflux disease (GERD), and therefore gastric acid or bile acids are thought to be factors that cause epithelial cell damage and inflammation in the gastro-esophageal junction. The decrease of adherent junction molecules, E-cadherin has been reported to be associated with the progression of the Barrett’s carcinoma, but the initiation of BE is not sufficiently understood. BE is characterized by the presence of goblet cells and occasionally Paneth cells are observed at the base of the crypts. The Paneth cells possess dense granules, in which human antimicrobial peptide human defensin-5 (HD-5) are stored and secreted out of the cells. This study determined the roles of HD-5 produced from metaplastic Paneth cells against adjacent to squamous cells in the gastro-esophageal junction. A human squamous cell line Het-1A, was incubated with the synthetic HD-5 peptide as a model of squamous cell in the gastro-esophageal junctions, and alterations of E-cadherin were investigated. Immunocytochemistry, flowcytometry, and Western blotting showed that the expression of E-cadherin protein was decreased. And a partial recovery from the decrease was observed by treatment with a CD10/neprilysin inhibitor (thiorphan). In conclusion, E-cadherin expression in squamous cells was reduced by HD-5 using in vitro experiments. In gastro-esophageal junction, HD-5 produced from metaplastic Paneth cells may therefore accelerate the initiation of BE.     

N-myc Downstream-regulated Gene 1 Promotes Tumor Inflammatory Angiogenesis through JNK Activation and Autocrine Loop of Interleukin-1α by Human Gastric Cancer Cells

The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with tumor angiogenesis and malignant progression together with poor prognosis in gastric cancer. However, the underlying mechanism for the role of NDRG1 in the malignant progression of gastric cancer remains unknown. Here we examined whether and how NDRG1 could modulate tumor angiogenesis by human gastric cancer cells. We established NU/Cap12 and NU/Cap32 cells overexpressing NDRG1 in NUGC-3 cells, which show lower tumor angiogenesis in vivo. Compared with parental NU/Mock3, NU/Cap12, and NU/Cap32 cells: 1) induced higher tumor angiogenesis than NU/Mock3 cells accompanied by infiltration of tumor-associated macrophages in mouse dorsal air sac assay and Matrigel plug assay; 2) showed much higher expression of CXC chemokines, MMP-1, and the potent angiogenic factor VEGF-A; 3) increased the expression of the representative inflammatory cytokine, IL-1α; 4) augmented JNK phosphorylation and nuclear expression of activator protein 1 (AP-1). Further analysis demonstrated that knockdown of AP-1 (Jun and/or Fos) resulted in down-regulation of the expression of VEGF-A, CXC chemokines, and MMP-1, and also suppressed expression of IL-1α in NDRG1-overexpressing cell lines. Treatment with IL-1 receptor antagonist (IL-1ra) resulted in down-regulation of JNK and c-Jun phosphorylation, and the expression of VEGF-A, CXC chemokines, and MMP-1 in NU/Cap12 and NU/Cap32 cells. Finally, administration of IL-1ra suppressed both tumor angiogenesis and infiltration of macrophages by NU/Cap12 in vivo. Together, activation of JNK/AP-1 thus seems to promote tumor angiogenesis in relationship to NDRG1-induced inflammatory stimuli by gastric cancer cells.     

Par14 Protein Associates with Insulin Receptor Substrate 1 (IRS-1), Thereby Enhancing Insulin-induced IRS-1 Phosphorylation and Metabolic Actions

Pin1 and Par14 are parvulin-type peptidyl-prolyl cis/trans isomerases. Although numerous proteins have been identified as Pin1 substrates, the target proteins of Par14 remain largely unknown. Par14 expression levels are increased in the livers and embryonic fibroblasts of Pin1 KO mice, suggesting a compensatory relationship between the functions of Pin1 and Par14. In this study, the association of Par14 with insulin receptor substrate 1 (IRS-1) was demonstrated in HepG2 cells overexpressing both as well as endogenously in the mouse liver. The analysis using deletion-mutated Par14 and IRS-1 constructs revealed the N-terminal portion containing the basic domain of Par14 and the two relatively C-terminal portions of IRS-1 to be involved in these associations, in contrast to the WW domain of Pin1 and the SAIN domain of IRS-1. Par14 overexpression in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events, PI3K binding with IRS-1 and Akt phosphorylation. In contrast, treating HepG2 cells with Par14 siRNA suppressed these events. In addition, overexpression of Par14 in the insulin-resistant ob/ob mouse liver by adenoviral transfer significantly improved hyperglycemia with normalization of hepatic PEPCK and G6Pase mRNA levels, and gene suppression of Par14 using shRNA adenovirus significantly exacerbated the glucose intolerance in Pin1 KO mice. Therefore, although Pin1 and Par14 associate with different portions of IRS-1, the prolyl cis/trans isomerization in multiple sites of IRS-1 by these isomerases appears to be critical for efficient insulin receptor-induced IRS-1 phosphorylation. This process is likely to be one of the major mechanisms regulating insulin sensitivity and also constitutes a potential therapeutic target for novel insulin-sensitizing agents.     

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.     

Habitat shifting by the common brown lemur (Eulemur fulvus fulvus): a response to food scarcity

During periods of food scarcity, most primates display behavioral responses, such as dietary switching or adjustment of traveling and foraging efforts, within home ranges. In rare cases, several primate species leave their home ranges for other remote habitats to seek alternative resources; this migration-like behavior is termed “habitat shifting.” Reports of habitat shifting have concentrated on platyrrhines, but this behavior has rarely been observed among prosimians. During 1 year of observation of a troop of common brown lemurs (Eulemur fulvus fulvus) in Ankarafantsika National Park, northwestern Madagascar, habitat shifting occurred twice. To understand the causes of this behavior, I examined the seasonal availability of fruit resources in the range continuously used by the troop during the year (defined as the annual range) and compared feeding activities and vegetation types between the annual range and new areas. The troop usually stayed within a 38.7-ha annual range, defined by a 95 % fixed kernel analysis based on GPS location data collected at 5-min intervals. In April 2007, the lemurs suddenly moved to a habitat 1.0–1.5 km south of their annual range and concentrated on the consumption of Grewia triflora fruits for 2 weeks. In November 2007, they visited a habitat 0.8–1.7 km southeast of the annual range and exploited fruits of Landolphia myrtifolia. These new areas were open habitats with high densities of the respective fruit species. The density of fruiting trees was low in the annual range during these periods; thus, habitat shifting to areas with different phenological productivity appeared to be an effective response to fruit scarcity. Brown lemurs are generally categorized as a nonterritorial species, and the lemurs observed here showed no agonistic behavior in intergroup encounters during range shifting. Such nonterritoriality may allow brown lemurs to shift habitats, a behavior resulting in long-term absence from their annual range.