Regulation of food intake in the goldfish by interaction between ghrelin and orexin

Intracerebroventricular (ICV) administration of ghrelin, orexin and neuropeptide Y (NPY) stimulates food intake in goldfish. Orexin and NPY interact with each other in the regulation of feeding, while ghrelin-induced feeding has also shown to be mediated by NPY in the goldfish model. To investigate the interaction between ghrelin and orexin, we examined the effects of a selective orexin receptor-1 antagonist, SB334867, and a growth hormone secretagogue-receptor antagonist, [D-Lys(3)]-GHRP-6, on ghrelin- and orexin-A-induced feeding. Ghrelin-induced food intake was completely inhibited for 1h following ICV preinjection of SB334867, while [D-Lys(3)]-GHRP-6 attenuated orexin-A stimulated feeding. Furthermore, ICV administration of ghrelin or orexin-A at a dose sufficient to stimulate food intake increased the expression of each other's mRNA in the diencephalon. These results indicate that, in goldfish, ghrelin and orexin-A have interacting orexigenic effects in the central nervous system. This is the first report that orexin-A-induced feeding is mediated by the ghrelin signaling in any animal model.     

Assessment of prion inactivation by combined use of Bacillus-derived protease and SDS

Prions, infectious agents causing transmissible spongiform encephalopathy, retain infectivity even after undergoing routine sterilization processes. We found that MSK103 protease, identified in our previous study, effectively reduces infectivity and the level of misfolded isoform of the prion protein in scrapie-infected brain homogenates in the presence of SDS. The treatment therefore can be applied to the decontamination of thermolabile instruments.     

Novel miniature SPR immunosensor equipped with all-in-one multi-microchannel sensor chip for detecting low-molecular-weight analytes

A simple and versatile miniaturized surface plasmon resonance (SPR) immunosensor enabling parallel analysis of multiple analytes or multiple samples of an analyte has been investigated for detection of a low-molecular-weight (lmw) toxin, 2,4-dichlorophenoxyacetic acid (2,4-D). A specially designed multi-microchannel SPR sensor module, integrating an optical-prism coated with an array of thin Au-films, a multi-microchannel plate (eight channels) and a flow-cell together, has been fabricated. The sensing surface was fabricated simply by physical adsorption of a protein conjugate of 2,4-D, and an indirect competitive immunoassay principle has been applied for the quantification of 2,4-D. Multiple 2,4-D samples were analyzed in a single step and a low-detection-limit (LDL) of 0.1 ppb (ng ml(-1)) 2,4-D was established. Competence of the portable SPR immunosensor for selective detection of 2,4-D despite the presence of various structurally resemblant interferents and from river-water samples has been demonstrated. The independent all-in-one sensor module highly favors shelf-storage between multiple determinations, and reusability of a same multi-microchannel flow-module for more than 35 days with intermittent storage (4-8 degrees C) has been confirmed. The LDL of 2,4-D could be enhanced further by introducing a simple avidin-biotin interaction-based sandwich immunoassay, with which the sensor signal multiplied enormously by a factor of ca. 10 and the LDL enhanced to 0.008 ppb. The miniature SPR sensor demonstrated here for simultaneous analysis of multiple samples with reusability and good storage ability is an important consideration for the advancement of biosensor technology.     

Itaconic acid production using sago starch hydrolysate by Aspergillus terreus TN484-M1

Sago starch was hydrolyzed using either chemical agents, or enzymes at various pH and concentrations. Hydrolysis using 5000 AUN/ml (0.5%, w/v) glucoamylase exhibited the highest itaconic acid yield up to 0.36 g/g sago starch, whereas hydrolysis using nitric acid at pH 2.0 yielded 0.35 g/g sago starch. The medium was optimized and the composition was (g/l) 140 sago starch, 1.8 corn steep liquor, 1.2 MgSO(4).7H(2)O and 2.9 NH(4)NO(3). When the optimal conditions of hydrolysis and medium composition were applied to itaconic acid production in a 3-l jar fermentor, the itaconic acid production was 48.2 g/l with a yield of 0.34 g/g sago starch. This was filtered from the cultured broth and 37.1g of itaconic acid was recovered with a purity of 97.2%. This result showed that sago starch could be converted to a value-added product with only a simple pretreatment.     

Structural analysis of four and half LIM protein-2 in dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a cardiac disease characterized by dilated ventricle and systolic dysfunction. Most of the DCM patients are sporadic cases, but a certain population of DCM patients can be familial cases caused by mutations in genes for sarcomere/Z-disc components including titin/connectin. However, disease-causing mutations could be identified only in a part of the familial DCM patients, suggesting that there should be other disease causing genes for DCM. To explore a novel disease gene for DCM, we searched for mutations in FHL2, encoding for four and half LIM protein 2 (FHL2) in DCM patients, because FHL2 is known to associate with titin/connectin. A missense mutation, Gly48Ser, was identified in a patient with familial DCM. Functional analysis demonstrated that the FHL2 mutation affected the binding to titin/connectin. Because FHL2 protein is known to tether metabolic enzymes to titin/connectin, these observations suggest that the Gly48Ser mutation may be involved in the pathogenesis of DCM via impaired recruitment of metabolic enzymes to the sarcomere.     

Clustered negative charges on the lipid membrane surface induce beta-sheet formation of prion protein fragment 106-126

The conformational conversion of prion protein (PrP) from an alpha-helix-rich normal cellular isoform (PrPC) to a beta-sheet-rich pathogenic isoform (PrPSc) is a key event in the development of prion diseases, and it takes place in caveolae, cavelike invaginations of the plasma membrane. A peptide homologous to residues 106-126 of human PrP (PrP106-126) is known to share several properties with PrPSc, e.g., the capability to form a beta-sheet and toxicity against PrPC-expressing cells. PrP106-126 is thus expected to represent a segment of PrP that is involved in the formation of PrPSc. We have examined the effect of lipid membranes containing negatively charged ganglioside, an important component of caveolae, on the secondary structure of PrP106-126 by circular dichroism. The peptide forms an alpha-helical or a beta-sheet structure on the ganglioside-containing membranes. The beta-sheet content increases with an increase of the peptide:lipid ratio, indicating that the beta-sheet formation is linked with self-association of the positively charged peptide on the negatively charged membrane surface. Analogous beta-sheet formation is also induced by membranes composed of negatively charged and neutral glycerophospholipids with high and low melting temperatures, respectively, in which lateral phase separation and clustering of negatively charged lipids occur as shown by Raman spectroscopy. Since ganglioside-containing membranes also exhibit lateral phase separation, clustered negative charges are concluded to be responsible for the beta-sheet formation of PrP106-126. In caveolae, clustered ganglioside molecules are likely to interact with the residue 106-126 region of PrPC to promote the PrPC-to-PrPSc conversion.     

A progestin and an estrogen regulate early stages of oogenesis in fish

Using two species of teleost fish, Japanese huchen (Hucho perryi) and common carp (Cyprinus carpio), we investigated whether sex steroids are involved in early oogenesis in vitro. Ovarian fragments were cultured to examine the effects of a progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP), and an estrogen, estradiol-17 beta (E2). DHP and E2 significantly promoted DNA synthesis in ovarian germ cells, as judged by 5-bromo-2-deoxyuridine (BrdU) incorporation into these cells. Furthermore, to detect the initiation of the first meiotic division of early oogenesis, we assessed ultrastructurally the occurrence of synaptonemal complexes (SCs) and analyzed by immunohistochemistry the expression of a meiosis-specific marker, Spo11. In huchen, a higher percentage of oocytes with SC was seen in DHP-treated ovarian fragments than in control or E2-treated ovarian fragments. Spo11 was expressed in germ cells after DHP treatment of carp ovarian explants. These data suggest that the progression of germ cells through early oogenesis involves two sex steroids: E2, which acts directly on oogonial proliferation, and DHP, which acts directly on the initiation of the first meiotic division of oogenesis.     

Layered Structure of Bacterial and Archaeal Communities and Their In Situ Activities in Anaerobic Granules

The microbial community structure and spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by 16S rRNA gene-based molecular techniques and microsensors for CH₄, H₂, pH, and the oxidation-reduction potential (ORP). The 16S rRNA gene-cloning analysis revealed that the clones related to the phyla Alphaproteobacteria (detection frequency, 51%), Firmicutes (20%), Chloroflexi (9%), and Betaproteobacteria (8%) dominated the bacterial clone library, and the predominant clones in the archaeal clone library were affiliated with Methanosaeta (73%). In situ hybridization with oligonucleotide probes at the phylum level revealed that these microorganisms were numerically abundant in the granule. A layered structure of microorganisms was found in the granule, where Chloroflexi and Betaproteobacteria were present in the outer shell of the granule, Firmicutes were found in the middle layer, and aceticlastic Archaea were restricted to the inner layer. Microsensor measurements for CH₄, H₂, pH, and ORP revealed that acid and H₂ production occurred in the upper part of the granule, below which H₂ consumption and CH₄ production were detected. Direct comparison of the in situ activity distribution with the spatial distribution of the microorganisms implied that Chloroflexi contributed to the degradation of complex organic compounds in the outermost layer, H₂ was produced mainly by Firmicutes in the middle layer, and Methanosaeta produced CH₄ in the inner layer. We determined the effective diffusion coefficient for H₂ in the anaerobic granules to be 2.66 × 10⁻⁵ cm² s⁻¹, which was 57% in water.