Feeding-induced changes of melanin-concentrating hormone (MCH)-like immunoreactivity in goldfish brain

Intracerebroventricular (ICV) injection of melanin-concentrating hormone (MCH) influences feeding behavior in the goldfish and exerts an anorexigenic action in goldfish brain, unlike its orexigenic action in mammals. Despite a growing body of knowledge concerning MCH function in mammals, the role of MCH in appetite has not yet been well studied in fish. The aim of the present study was to investigate the involvement of endogenous MCH in the feeding behavior of the goldfish. We examined the distribution of MCH-like immunoreactivity (MCH-LI) in the goldfish brain and the effect of feeding status upon this distribution. Neuronal cell bodies containing MCH-LI were localized specifically to four areas of the hypothalamus. Nerve fibers with MCH-LI were found mainly in the neurohypophysis, with a few in the telencephalon, mesencephalon, and diencephalon. The number of neuronal cell bodies containing MCH-LI in the dorsal area adjoining the lateral recess of the third ventricle in the posterior and inferior lobes of the hypothalamus showed a significant decrease in fasted fish compared with that in normally fed fish, although other areas showed no evident differences. We also administered an antiserum against fish MCH (anti-MCH serum) by ICV injection and examined its immunoneutralizing effect on food intake by using an automatic monitoring system. Cumulative food intake was significantly increased by ICV injection of the anti-MCH serum. These results indicate that MCH potentially functions as an anorexigenic neuropeptide in the goldfish brain, and that the further study of the evolutionary background of the MCH system and its role in appetite is warranted. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (K.M. and A.T.) and by a research grant from the Toyama Marine Biotechnology Association (K.M.).     

Paeoniflorin, a monoterpene glycoside, attenuates lipopolysaccharide-induced neuronal injury and brain microglial inflammatory response

Chronic activation of microglial cells endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Paeoniflorin (PF), a water-soluble monoterpene glycoside found in the root of Paeonia lactiflora Pall, has a wide range of pharmacological functions, such as anti-oxidant, anti-inflammatory, and anti-cancer effects. Neuroprotective potential of PF has also been demonstrated in animal models of neuropathologies. Here, we have examined the efficacy of PF in the repression of inflammation-induced neurotoxicity and microglial inflammatory response. In organotypic hippocampal slice cultures, PF significantly blocked lipopolysaccharide (LPS)-induced hippocampal cell death and productions of nitric oxide (NO) and interleukin (IL)-1β. PF also inhibited the LPS-stimulated productions of NO, tumor necrosis factor-α, and IL-1β from primary microglial cells. These results suggest that PF possesses neuroprotective activity by reducing the production of proinflammatory factors from activated microglial cells.     

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.     

Environmental pressure rather than ongoing hybridization is responsible for an altitudinal cline in the morphologies of two oaks

在相关类群之间的接触区,表型变异可能源于遗传和/或环境梯度。本研究旨在阐明两种栎属植物[Quercus crispula (QCC)和 Q. mongolicoides (QCM)],在其接触区沿海拔梯度形成叶片形态表型变异的原因。对于源自接触区48个个体和QCC和QCM的每个参照 种群24个个体样本,我们测定其6种叶片形态特征,同时记录13个核微卫星位点的基因型。我们通过构建模型解释表型变异(叶片形态)与环境(海拔)和遗传(参照种群世系)梯度的关系。研究结果表明,形态和遗传标记均能较好地区分参照种群中的两个品种。我们能够确认形态和遗传标记的作用。接触区种群内的个体具有略偏向QCM分支的中间世系,其形态分布与参照种群中两个变种的形态分布重叠。海拔会显著影响叶片形态性状,而遗传对叶片形态性状无显著影响。接触区种群的世系和种间杂合性分布与F₂或后代杂交种中的分布相似。这些结果表明,在两种栎属植物QCC和QCM之间的接触区,并没有发生杂交,但环境压力通过表型可塑性和/或功能基因的变异,导致了其在形态性状上的海拔梯度效应。     

Proteomic analysis of microvesicles derived from human colorectal cancer ascites

The presence of malignant ascites in the peritoneal cavity is a poor prognostic indicator of low survival rate. Various cancer cells, including those of colorectal cancer (CRC), release microvesicles (exosomes) into surrounding tissues and peripheral circulation including malignant ascites. Although recent progress has revealed that microvesicles play multiple roles in tumor progression, the protein composition and the pathological function of malignant ascites-derived microvesicles are still unknown. Here, we report the first global proteomic analyses of highly purified microvesicles derived from human CRC ascites. With 1-D SDS-PAGE and nano-LC-MS/MS analyses, we identified a total of 846 microvesicular proteins from ascites of three CRC patients with high confidence; 384 proteins were identified in at least two patients. We identified proteins that might function in tumor progression via disruption of epithelial polarity, migration, invasion, tumor growth, immune modulation, and angiogenesis. Furthermore, we identified several potential diagnostic markers of CRC including colon-specific surface antigens. Our proteomic analyses will help to elucidate diverse functions of microvesicles in cancer progression and will aid in the development of novel diagnostic tools for CRC.     

Fenton reaction induced cancer in wild type rats recapitulates genomic alterations observed in human cancer

Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC) after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis.     

Target-site preferences of Sleeping Beauty transposons

The Sleeping Beauty (SB) transposon is a Tc1/mariner family transposon that has applications in vertebrate animals for gene transfer, gene-tagging, and human gene therapy. In this study, we analyzed the target-site preferences of the SB transposon. At the genomic level, integration of SB transposons with respect to genes (exons and introns) and intergenic regions appears fairly random but not on a micro-scale. Although there appears to be a consensus sequence around the vicinity of the target sites, the primary sequence is not the determining factor for target selection. When integrations were examined over a limited topography, the sites used most often for integration did not match the consensus sequence. Rather, a unique deformation inherent in the sequence may be a recognition signal for target selection. The deformation is characterized by an angling of the target site such that the axis around the insertion site is off-center, the rotation of the helix (twisting) is non-uniform and there is an increase in the distance between the central base-pairs. Our observations offer several hypothetical insights into the transposition process. Our observations suggest that particular deformations of the double helix predicted by the V(step) algorithm can distinguish TA sites that vary by about 16-fold in their preferences for accommodating insertions of SB transposons.