Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes

<p>Abstract</p> <p>Background</p> <p>The melanocortin (MC) receptors have a key role in regulating body weight and pigmentation. They belong to the rhodopsin family of G protein-coupled receptors (GPCRs). The purpose of this study was to identify ancestral MC receptors in agnathan, river lamprey.</p> <p>Results</p> <p>We report cloning of two MC receptors from river lamprey. The lamprey receptors, designated MCa and MCb, showed orthology to the MC1 and MC4 receptor subtypes, respectively. The molecular clock analysis suggested that lamprey MC receptor genes were not duplicated recently and diverged from each other more than 400 MYR ago. Expression and pharmacological characterization showed that the lamprey MCa receptor was able to bind and be activated by both lamprey and human MSH peptides. The lamprey MCa receptor had relatively high affinity for ACTH derived peptides similarly to the fish MC receptors. We found that both of the lamprey MC receptors were expressed in skin, while the MCb receptor was also found in liver, heart and skeletal muscle.</p> <p>Conclusion</p> <p>This study shows presence of MC receptors in agnathans indicating early signs of specific functions of melanocortin receptor subtypes.</p

Systems Analysis of ATF3 in Stress Response and Cancer Reveals Opposing Effects on Pro-Apoptotic Genes in p53 Pathway

Stress-inducible transcription factors play a pivotal role in cellular adaptation to environment to maintain homeostasis and integrity of the genome. Activating transcription factor 3 (ATF3) is induced by a variety of stress and inflammatory conditions and is over-expressed in many kinds of cancer cells. However, molecular mechanisms underlying pleiotropic functions of ATF3 have remained elusive. Here we employed systems analysis to identify genome-wide targets of ATF3 that is either induced by an alkylating agent methyl methanesulfonate (MMS) or over-expressed in a prostate tumour cell line LNCaP. We show that stress-induced and cancer-associated ATF3 is recruited to 5,984 and 1,423 targets, respectively, in the human genome, 89% of which are common. Notably, ATF3 targets are highly enriched for not only ATF/CRE motifs but also binding sites of several other stress-inducible transcription factors indicating an extensive network of stress response factors in transcriptional regulation of target genes. Further analysis of effects of ATF3 knockdown on these targets revealed that stress-induced ATF3 regulates genes in metabolic pathways, cell cycle, apoptosis, cell adhesion, and signalling including insulin, p53, Wnt, and VEGF pathways. Cancer-associated ATF3 is involved in regulation of distinct sets of genes in processes such as calcium signalling, Wnt, p53 and diabetes pathways. Notably, stress-induced ATF3 binds to 40% of p53 targets and activates pro-apoptotic genes such as TNFRSF10B/DR5 and BBC3/PUMA. Cancer-associated ATF3, by contrast, represses these pro-apoptotic genes in addition to CDKN1A/p21. Taken together, our data reveal an extensive network of stress-inducible transcription factors and demonstrate that ATF3 has opposing, cell context-dependent effects on p53 target genes in DNA damage response and cancer development

The Dugesia ryukyuensis Database as a Molecular Resource for Studying Switching of the Reproductive System

The planarian Dugesia ryukyuensis reproduces both asexually and sexually, and can switch from one mode of reproduction to the other. We recently developed a method for experimentally switching reproduction of the planarian from the asexual to the sexual mode. We constructed a cDNA library from sexualized D. ryukyuensis and sequenced and analyzed 8,988 expressed sequence tags (ESTs). The ESTs were analyzed and grouped into 3,077 non-redundant sequences, leaving 1,929 singletons that formed the basis of unigene sets. Fifty-six percent of the cDNAs analyzed shared similarity (E-value<1E -20) with sequences deposited in NCBI. Highly redundant sequences encoded granulin and actin, which are expressed in the whole body, and other redundant sequences encoded a Vasa-like protein, which is known to be a component of germ-line cells and is expressed in the ovary, and Y-protein, which is expressed in the testis. The sexualized planarian expressed sequence tag database (http://planaria.bio.keio.ac.jp/planaria/) is an open-access, online resource providing access to sequence, classification, clustering, and annotation data. This database should constitute a powerful tool for analyzing sexualization in planarians

Acute Homeostatic Responses to Increased Fat Consumption in MCH1R Knockout Mice

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide which has been shown to regulate energy homeostasis. Using genetic knockout mice lacking the MCH1 receptor (MCH1R), we investigated how these mice adapt to metabolic changes caused by excessive caloric consumption. We show that the MCH system is one of the players mediating behavioral and metabolic responses upon increased caloric consumption. MCH1R knockout mice showed decreased tendency of food intake upon exposure to a high-fat diet. They also are resistant to gain weight upon high-fat diet by increasing fat metabolism. Therefore, the MCH system is important in regulating metabolic responses upon various environmental stimuli such as high-fat diet

The usefulness of re-attachability of anti-adhesive cross-linked gelatin film and the required physical and biological properties.

Background:To overcome the unfavorable issues associated with conventional anti-adhesive HA/CMC film, we developed an anti-adhesive thermally cross-linked gelatin film.Objective:We tried to clarify the re-attachability of the film and the required properties concerning the film thickness, stiffness and anti-adhesion effect.Methods:To determine the optimal thickness, 5 kinds of the thickness of gelatin film and the conventional film were analyzed by the tensile test, shearing test, buckling test and tissue injury test. Finally, using the optimal film thickness, we tried to clarify the anti-adhesion effect of the reattached film.Results:The tensile and shearing test showed gelatin films ≥30 μm thick had greater tensile strength and a smaller number of film fractures, than the conventional film. The buckling and tissue injury test showed gelatin films ≥60 μm thick had higher buckling strength and worse injury scores than the conventional film. The anti-adhesive effect of re-attached gelatin film using optimal thickness (30-40 μm) found the anti-adhesion score was significantly better than that of the control.Conclusions:Provided it has an optimal thickness, gelatin film can be reattached with enough physical strength not to tear, safety stiffness not to induce tissue injury, and a sufficient anti-adhesion effect

Effects of Fiber Diameter and Spacing Size of an Artificial Scaffold on the In Vivo Cellular Response and Tissue Remodeling.

By mimicking the extracellular matrix, nonwoven fabrics can function as scaffolds for tissue engineering application ideally, and they have been characterized regarding their fiber diameter and fiber spacing (spacing size) in vitro. We chronologically examined the in vivo effects of these fabrics on the cellular response and tissue remodeling. Four types of nonwoven polyglycolic acid fabrics (Fabric-0.7, Fabric-0.9, Fabric-3, and Fabric-16 with fiber diameters of 0.7, 0.9, 3.0, and 16.2 μm and spacing sizes of 2.0, 19.3, 19.0, and 825.4 μm, respectively) were implanted into the rat dorsum and subjected to histologic and immunohistochemical analyses from day 3 to 70. With Fabric-0.7, inflammatory cells (mainly M1 macrophages) and myofibroblasts with collagen type III accumulated mainly on the surface of the fabric and did not infiltrate inside the fabric initially, likely due to the narrow fiber space. Massive formation of collagen type I then appeared with the degradation of the fabrics, and finally, the remodeled tissue turned into a dense scar. With Fabric-0.9 and Fabric-3, inflammatory cells (predominantly M2 macrophages) were seen in all layers of the fabric initially. A mild increase in collagen type I was then seen, with few myofibroblasts, and the remodeled tissue ultimately showed a relatively little scar with an adequate thickness of the tissue induced by the fabrics. With Fabric-16, inflammatory cells (predominantly M1 macrophages) infiltrated into all layers of the fabric initially along with many myofibroblasts, especially in the hole. Lately, massive formation of collagen type I was noted due to the slow degradation of the fabric, with the shrinking of the fabric substantially, and the remodeled tissue finally turned to a dense scar. These findings suggest that optimizing the spacing size as well as the fiber diameter of artificial scaffolds may control the cellular response and tissue remodeling and facilitate favorable tissue regeneration without scar formation

Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, the key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical disease phenotypes, including disease onset in heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD models with PKD1 mutations in cynomolgus monkeys. As in humans and mice, near-complete PKD1 depletion induces severe cyst formation mainly in collecting ducts. Importantly, unlike in mice, PKD1 heterozygote monkeys exhibit cyst formation perinatally in distal tubules, possibly reflecting the initial pathology in humans. Many monkeys in these models survive after cyst formation, and cysts progress with age. Furthermore, we succeed in generating selective heterozygous mutations using allele-specific targeting. We propose that our models elucidate the onset and progression of ADPKD, which will serve as a critical basis for establishing new therapeutic strategies, including drug treatments