Method for detecting hemodynamic alterations following a single gavage in rats

It is known that administering a gavage to rodents evokes a cardiac reflex, due to gastrointestinal stimulation. Consequently, it is difficult to evaluate changes in hemodynamics after a single oral dose of a pungent or astringent, which alters the circulation by increasing sympathetic activity. In the present study, we developed a method for administering a gavage without significantly affecting hemodynamics measurements. We marked a gastric tube at 10 cm from the tip, to mark the distance from the oral cavity to the stomach body of Wistar male rats. Rats were intubated under urethane anesthesia.After 10–15 min of stabilization, we measured the mean blood pressure (MBP), heart rate (HR), and blood flow (BF) in the cremaster arteriole under two different conditions; condition 1: a pointed gastric tube, room temperature distilled water, and injected at normal speed (approximately 3 ml/min); condition 2: a rounded gastric tube, 37°C distilled water, and injection at 1.0 ml/min. Under condition 1, we observed striking hemodynamic alterations, due to the somatic afferent reflex. In contrast, under condition 2, these hemodynamic changes were nearly eliminated. In addition, we could clearly detect hemodynamic changes in rats after a single gavage treatment of pungent (capsaicin) or astringent (cinnamtannin A2). We observed transient increases in the HR and MBP soon after treatment with capsaicin. Moreover, cremasteric BF was elevated with cinnamtannin A2. These results confirmed the utility of the gavage method developed in this study.     

UV-B radiation induces epithelial tumors in mice lacking DNA polymerase eta and mesenchymal tumors in mice deficient for DNA polymerase iota

DNA polymerase eta (Pol eta) is the product of the Polh gene, which is responsible for the group variant of xeroderma pigmentosum, a rare inherited recessive disease which is characterized by susceptibility to sunlight-induced skin cancer. We recently reported in a study of Polh mutant mice that Pol eta is involved in the somatic hypermutation of immunoglobulin genes, but the cancer predisposition of Polh-/- mice has not been examined until very recently. Another translesion synthesis polymerase, Pol iota, a Pol eta paralog encoded by the Poli gene, is naturally deficient in the 129 mouse strain, and the function of Pol iota is enigmatic. Here, we generated Polh Poli double-deficient mice and compared the tumor susceptibility of them with Polh- or Poli-deficient animals under the same genetic background. While Pol iota deficiency does not influence the UV sensitivity of mouse fibroblasts irrespective of Polh genotype, Polh Poli double-deficient mice show slightly earlier onset of skin tumor formation. Intriguingly, histological diagnosis after chronic treatment with UV light reveals that Pol iota deficiency leads to the formation of mesenchymal tumors, such as sarcomas, that are not observed in Polh(-/-) mice. These results suggest the involvement of the Pol eta and Pol iota proteins in UV-induced skin carcinogenesis.     

Embryonic stem cells deficient in I beta1,6-N-acetylglucosaminyltransferase exhibit reduced expression of embryoglycan and the loss of a Lewis X antigen, 4C9

Embryoglycan is a class of branched high-molecular-weight poly-N-acetyllactosamines characteristically expressed in early embryonic cells and has been shown to be involved in the intercellular adhesion of early embryonic cells in vitro. Branching of poly-N-acetyllactosamine chains is performed by beta1,6-N-acetylglucosaminylation of the galactosyl residue. We previously knocked out the gene encoding I beta1, 6-N-acetylglucosaminyltransferase (IGnT), and the resultant deficient mice were born without any abnormality, although the mice exhibited various deficits in later life. In the present investigation, we produced embryonic stem (ES) cells from IGnT-deficient embryos. The mutant ES cells exhibited a reduced capability in embryoglycan synthesis. Thus, IGnT is a major enzyme involved in the branching of poly-N-acetyllactosamine chains in embryoglycan. Since ES cells are equivalent to multipotential cells of the embryonic ectoderm in early postimplantation embryos, this result indicates that an abundance of embryoglycan in these cells is not essential for normal embryogenesis. The IGnT-deficient ES cells continued to express SSEA-1, but lacked the expression of 4C9 antigen, although the epitope of 4C9 antigen was confirmed to be Lewis X by a transfection experiment. The result establishes the distinct nature of 4C9 antigenicity, which requires either Lewis X epitope on I-branch or clustering of Lewis X epitope, best accomplished by poly-N-acetyllactosamine branching. Alpha6-integrin was newly identified as a carrier of embryoglycan. The IGnT-deficient ES cells adhered to dishes coated with laminin, which is a ligand for alpha6-integrin, significantly less than wild-type ES cells, raising the possibility that embryoglycan in ES cells enhances alpha6-integrin-dependent adhesion in vitro.     

The Chemotherapeutic Agent DMXAA as a Unique IRF3-Dependent Type-2 Vaccine Adjuvant

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent type I interferon (IFN) inducer, was evaluated as a chemotherapeutic agent in mouse cancer models and proved to be well tolerated in human cancer clinical trials. Despite its multiple biological functions, DMXAA has not been fully characterized for the potential application as a vaccine adjuvant. In this report, we show that DMXAA does act as an adjuvant due to its unique property as a soluble innate immune activator. Using OVA as a model antigen, DMXAA was demonstrated to improve on the antigen specific immune responses and induce a preferential Th2 (Type-2) response. The adjuvant effect was directly dependent on the IRF3-mediated production of type-I-interferon, but not IL-33. DMXAA could also enhance the immunogenicity of influenza split vaccine which led to significant increase in protective responses against live influenza virus challenge in mice compared to split vaccine alone. We propose that DMXAA can be used as an adjuvant that targets a specific innate immune signaling pathway via IRF3 for potential applications including vaccines against influenza which requires a high safety profile.     

Non-Specific Protein Modifications by a Phytochemical Induce Heat Shock Response for Self-Defense

Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.     

Metabolically active microbial communities in marine sediment under high-CO2 and low-pH extremes

Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO₂ in the seabed. The emission of CO₂ may critically impact the geochemical, geophysical and ecological characteristics of the deep-sea sedimentary environment. So far it remains unclear whether microbial communities that have been detected in such high-CO₂ and low-pH habitats are metabolically active, and if so, what the biogeochemical and ecological consequences for the environment are. In this study, RNA-based molecular approaches and radioactive tracer-based respiration rate assays were combined to study the density, diversity and metabolic activity of microbial communities in CO₂-seep sediment at the Yonaguni Knoll IV hydrothermal field of the southern Okinawa Trough. In general, the number of microbes decreased sharply with increasing sediment depth and CO₂ concentration. Phylogenetic analyses of community structure using reverse-transcribed 16S ribosomal RNA showed that the active microbial community became less diverse with increasing sediment depth and CO₂ concentration, indicating that microbial activity and community structure are sensitive to CO₂ venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13–30 cm in depth) characterized by high CO₂. Measurement of the potential sulfate reduction rate at pH conditions of 3–9 with and without methane in the headspace indicated that acidophilic sulfate reduction possibly occurs in the presence of methane, even at very low pH of 3. These results suggest that some members of the anaerobic methanotrophs and sulfate reducers can adapt to the CO₂-seep sedimentary environment; however, CO₂ and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community.     

Adiponectin ameliorates doxorubicin-induced cardiotoxicity through Akt protein-dependent mechanism

Accumulating evidence shows that obesity is associated with doxorubicin cardiac toxicity in the heart, but the molecular mechanisms that contribute to this pathological response are not understood. Adiponectin is an adipose-derived, cardioprotective factor that is down-regulated in obesity. Here, we investigated the effect of adiponectin on doxorubicin (DOX)-induced cardiotoxicity and assessed the mechanisms of this effect. A single dose of DOX was intraperitoneally injected into the abdomen of adiponectin knock-out (APN-KO) and wild-type (WT) mice. APN-KO mice had increased mortality and exacerbated contractile dysfunction of left ventricle compared with WT mice. APN-KO mice also showed increased apoptotic activity and diminished Akt signaling in the failing myocardium. Systemic delivery of adenoviral vector expressing adiponectin improved left ventricle dysfunction and myocardial apoptosis following DOX injection in WT and APN-KO mice but not in Akt1 heterozygous KO mice. In cultured rat neonatal cardiomyocytes, adiponectin stimulated Akt phosphorylation and inhibited DOX-stimulated apoptosis. Treatment with sphingosine kinase-1 inhibitor or sphingosine 1-phosphate receptor antagonist diminished adiponectin-induced Akt phosphorylation and reversed the inhibitory effects of adiponectin on myocyte apoptosis. Pretreatment with anti-calreticulin antibody reduced the binding of adiponectin to cardiac myocytes and blocked the adiponectin-stimulated increase in Akt activation and survival in cardiomyocytes. Interference of the LRP1/calreticulin co-receptor system by siRNA or blocking antibodies diminished the stimulatory actions of adiponectin on Akt activation and myocyte survival. These data show that adiponectin protects against DOX-induced cardiotoxicity by its ability to promote Akt signaling.