Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila

A number of bioactive peptides are involved in regulating a wide range of animal behaviors, including food consumption. Vertebrate neuropeptide Y (NPY) is a potent stimulator of appetitive behavior. Recently, Drosophila neuropeptide F (dNPF) and short NPF (sNPF), the Drosophila homologs of the vertebrate NPY, were identified to characterize the functions of NPFs in the feeding behaviors of this insect. Dm-NPFR1 and NPFR76F are the receptors for dNPF and sNPF, respectively; both receptors are G protein-coupled receptors (GPCRs). Another GPCR (CG5811; NepYR) was indentified in Drosophila as a neuropeptide Y-like receptor. Here, we identified 2 ligands of CG5811, dRYamide-1 and dRYamide-2. Both peptides are derived from the same precursor (CG40733) and have no significant structural similarities to known bioactive peptides. The C-terminal sequence RYamide of dRYamides is identical to that of NPY family peptides; on the other hand, dNPF and sNPF have C-terminal RFamide. When administered to blowflies, dRYamide-1 suppressed feeding motivation. We propose that dRYamides are related to the NPY family in vertebrates, similar to dNPF and sNPF.     

3-D collagen-dependent cell surface expression of MT1-MMP and MMP-2 activation regardless of integrin β1 function and matrix stiffness

Matrix metalloproteinases (MMPs) play roles in spatially dynamic processes, including morphogenesis, wound healing, and tumor invasion. Three-dimensional (3-D) type I collagen stimulates cellular activation of MMP-2, however, the mechanisms underlying this are controversial. The present study investigated mechanisms for 3-D collagen-induced MMP-2 activation in highly invasive human malignant mesothelioma cells. MMP-2 was effectively activated by cells cultured in 3-D collagen but not in 2-D collagen, whereas MMP-2 activation was not regulated by the flexibility of collagen. The 3-D collagen did not largely increase the gene expression of MMP-2 and MT1-MMP. However, MT1-MMP exposed to the cell surface was much increased by 3-D collagen, and loss of MT1-MMP abolished MMP-2 activation in response to 3-D collagen. MT1-MMP and integrin β1 translocated to pericellular regions interacting with collagen-coated microbeads, however their localization was different. Importantly, inhibition of integrin β1 function and expression did not affect 3-D collagen-induced cell surface localization of MT1-MMP and MMP-2 activation. Our results strongly suggest that 3-D collagen scaffolding may provide opportunity for direct and multivalent interaction with MT1-MMP, by which MMP-2 activation occur in abundant cell surface MT1-MMP-dependent manner, rather than a manner regulated by matrix stiffness and integrin β1 function.     

The effect of transcutaneous application of carbon dioxide (CO₂) on skeletal muscle

Quasispecies is a remarkable characteristic of hepatitis C virus (HCV) and has profound roles in HCV biology and clinical practice. The understanding of HCV quasispecies behavior, in particular in acute HCV infection, is valuable for vaccine development and therapeutic interference. However, acute HCV infection is seldom encountered in clinic practice due to its silent onset. In the present study, we reported a unique case of de novo HCV infection associated with the transplantation of bone marrow from a HCV-positive donor. HCV quasispecies diversity was determined in both the donor and the recipient over a 4-year follow-up, accompanied with simultaneous measurement of HCV neutralizing antibody. Detailed genetic and phylogenetic analyses revealed a divergent quasispecies evolution, which was not related to dynamic changes of HCV neutralizing antibody. Instead, our data suggested an essential role of the fitness adaptation of founder viral population in driving such an evolutionary pattern.     

Determination of the critical region of KRAS-induced actin-interacting protein for the interaction with inositol 1,4,5-trisphosphate receptor

Concanavalin A (ConA), a Ca²⁺/Mn²⁺-dependent and mannose/glucose-binding legume lectin, has drawn a rising attention for its remarkable anti-proliferative and anti-tumor activities to a variety of cancer cells. ConA induces programmed cell death via mitochondria-mediated, P73-Foxo1a-Bim apoptosis and BNIP3-mediated mitochondrial autophagy. Through IKK-NF-κB-COX-2, SHP-2-MEK-1-ERK, and SHP-2-Ras-ERK anti-angiogenic pathways, ConA would inhibit cancer cell survival. In addition, ConA stimulates cell immunity and generates an immune memory, resisting to the same genotypic tumor. These biological findings shed light on new perspectives of ConA as a potential anti-neoplastic agent targeting apoptosis, autophagy and anti-angiogenesis in pre-clinical or clinical trials for cancer therapeutics.     

KRAS-induced actin-interacting protein is required for the proper localization of inositol 1,4,5-trisphosphate receptor in the epithelial cells

Three inositol 1,4,5-trisphosphate receptor (IP₃R) subtypes are differentially expressed among tissues and function as the Ca²⁺ release channel on specialized endoplasmic reticulum (ER) membranes. The proper subcellular localization of IP₃R is crucial for its proper function, but this molecular mechanism is unclear. KRAS-induced actin-interacting protein (KRAP) was originally identified as a cancer-related molecule, and is involved in the regulation of whole-body energy homeostasis and pancreatic exocrine system. We herein identified IP₃R as an associated molecule with KRAP in vivo, and the association was validated by the co-immunoprecipitation and confocal immunostaining studies in mouse tissues including liver and pancreas. The association of KRAP with IP₃R was also observed in the human epithelial cell lines including HCT116, HeLa and HEK293 cells. Intriguingly, KRAP interacts with distinct subtypes of IP₃R in a tissue-dependent manner, i.e. IP₃R1 and IP₃R2 in the liver and IP₃R2 and IP₃R3 in the pancreas. The NH₂-terminal amino acid residues 1–610 of IP₃R are critical for the association with KRAP and KRAP–IP₃R complex resides in a specialized ER but not a typical reticular ER. Furthermore, the localization of particular IP₃R subtypes in tissues from KRAP-deficient mice is obviously disturbed, i.e. IP₃R1 and IP₃R2 in the liver and IP₃R2 and IP₃R3 in the pancreas. These findings demonstrate that KRAP physically associates with IP₃R and regulates the proper localization of IP₃R in the epithelial cells in vivo and cultured cells, and might shed light on the Ca²⁺ signaling underlying physiological cellular programs, cancer development and metabolism-related diseases.     

The effect of transcutaneous application of carbon dioxide (CO2) on skeletal muscle

In Europe, carbon dioxide therapy has been used for cardiac disease and skin problems for a long time. However there have been few reports investigating the effects of carbon dioxide therapy on skeletal muscle. Peroxisome proliferators-activated receptor (PPAR)-gamma coactivator-1 (PGC-1α) is up-regulated as a result of exercise and mediates known responses to exercise, such as mitochondrial biogenesis and muscle fiber-type switching, and neovascularization via up-regulation of vascular endothelial growth factor (VEGF). It is also known that silent mating type information regulation 2 homologs 1 (SIRT1) enhances PGC-1α-mediated muscle fiber-type switching. Previously, we demonstrated transcutaneous application of CO₂ increased blood flow and a partial increase of O₂ pressure in the local tissue known as the Bohr effect. In this study, we transcutaneously applied CO₂ to the lower limbs of rats, and investigated the effect on the fast muscle, tibialis anterior (TA) muscle. The transcutaneous CO₂ application caused: (1) the gene expression of PGC-1α, silent mating type information regulation 2 homologs 1 (SIRT1) and VEGF, and increased the number of mitochondria, as proven by real-time PCR and immunohistochemistry, (2) muscle fiber switching in the TA muscle, as proven by isolation of myosin heavy chain and ATPase staining. Our results suggest the transcutaneous application of CO₂ may have therapeutic potential for muscular strength recovery resulting from disuse atrophy in post-operative patients and the elderly population.     

Imaging mass spectrometry reveals characteristic changes in triglyceride and phospholipid species in regenerating mouse liver

After partial hepatectomy (PH), regenerating liver accumulates unknown lipid species. Here, we analyzed lipids in murine liver and adipose tissues following PH by thin-layer chromatography (TLC), imaging mass spectrometry (IMS), and real-time RT-PCR. In liver, IMS revealed that a single TLC band comprised major 19 TG species. Similarly, IMS showed a single phospholipid TLC band to be major 13 species. In adipose tissues, PH induced changes to expression of genes regulating lipid metabolism. Finally, IMS of phosphatidylcholine species demonstrated distribution gradients in lobules that resembled hepatic zonation. IMS is thus a novel and power tool for analyzing lipid species with high resolution.     

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.     

Differential heavy metal sensitivity in seven algal species from the NIES culture collection based on delayed fluorescence assays

Seafloor massive sulfide (SMS) deposits are the target of available metallic resources. The toxic impacts of leachable metals from hydrothermal ore by mining operations in marine environments are a concern. However, ecotoxicological knowledge about marine algae, and particularly open ocean species, is still limited. Here, we evaluated the toxic effects of three leachable metals (i.e. Zn, Cu, and Pb) on seven marine algae, including cyanobacteria and eukaryotes, by a delayed fluorescence method. Cyanobacterial Synechococcus and Cyanobium species were sensitive to Zn and Cu, while eukaryotic algae showed various responses to heavy metal species. The prasinophycean Bathycoccus prasinos NIES‐2670 was sensitive to all metal species; this strain is a potential test strain to detect the leachable metals. A co‐culture experiment showed that the impact on community structure varies depending on leachable metal species. This study demonstrates that surveys across multiple taxonomic groups are necessary to assess the impact of SMS‐mining operations on marine ecosystems as a whole.