Purification and characterization of three isoforms of chrysophsin, a novel antimicrobial peptide in the gills of the red sea bream, Chrysophrys major.

We report here the isolation of three isoforms of a novel C-terminally amidated peptide from the gills of red sea bream, Chrysophrys (Pagrus) major. Peptide sequences were determined by a combination of Edman degradation, MS and HPLC analysis of native and synthetic peptides. Three peptides, named chrysophsin-1, chrysophsin-2, and chrysophsin-3, consist of 25, 25, and 20 amino acids, respectively, and are highly cationic, containing an unusual C-terminal RRRH sequence. The alpha-helical structures of the three chrysophsin peptides were predicted from their secondary structures and were confirmed by CD spectroscopy. The synthetic peptides displayed broad-spectrum bactericidal activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Bacillus subtilis, and fish and crustacean pathogens. The three peptides were also hemolytic. Immunohistochemical analysis showed that chrysophsins were localized in certain epithelial cells lining the surface of secondary lamellae and eosinophilic granule cell-like cells at the base of the secondary lamellae in red sea bream gills. Their broad ranging bactericidal activities, combined with their localization in certain cells and eosinophilic granule cell-like cells in the gills, suggest that chrysophsins play a significant role in the innate defense system of red sea bream gills.     

Amyloid β-Mediated Zn2+ Influx into Dentate Granule Cells Transiently Induces a Short-Term Cognitive Deficit

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn²⁺ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn²⁺ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn²⁺ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn²⁺ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn²⁺ levels in the dentate granule cell layer in ACSF without Zn²⁺, but in ACSF containing Zn²⁺. The increase in intracellular Zn²⁺ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn²⁺ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn²⁺ may play a key role for transiently Aβ-induced cognition deficits.     

Discovery of novel serine palmitoyltransferase inhibitors as cancer therapeutic agents

We pursued serine palmitoyltransferase (SPT) inhibitors as novel cancer therapeutic agents based on a correlation between SPT inhibition and growth suppression of cancer cells. High-throughput screening and medicinal chemistry efforts led to the identification of structurally diverse SPT inhibitors 4 and 5. Both compounds potently inhibited SPT enzyme and decreased intracellular ceramide content. In addition, they suppressed cell growth of human lung adenocarcinoma HCC4006 and acute promyelocytic leukemia PL-21, and displayed good pharmacokinetic profiles. Reduction of 3-ketodihydrosphingosine, the direct downstream product of SPT, was confirmed under in vivo settings after oral administration of compounds 4 and 5. Their anti-tumor efficacy was observed in a PL-21 xenograft mouse model. These results suggested that SPT inhibitors might have potential to be effective cancer therapeutics.     

Increase in the thermostability of Bacillus sp. strain TAR-1 xylanase using a site saturation mutagenesis library

Site saturation mutagenesis library is a recently developed technique, in which any one out of all amino acid residues in a target region is substituted into other 19 amino acid residues. In this study, we used this technique to increase the thermostability of a GH10 xylanase, XynR, from Bacillus sp. strain TAR-1. We hypothesized that the substrate binding region of XynR is flexible, and that the thermostability of XynR will increase if the flexibility of the substrate binding region is decreased without impairing the substrate binding ability. Site saturation mutagenesis libraries of amino acid residues Tyr43–Lys115 and Ala300–Asn325 of XynR were constructed. By screening 480 clones, S92E was selected as the most thermostable one, exhibiting the residual activity of 80% after heat treatment at 80°C for 15 min in the hydrolysis of Remazol Brilliant Blue-xylan. Our results suggest that this strategy is effective for stabilization of GH10 xylanase.

Abbreviations: DNS: 3,5-dinitrosalicylic acid; RBB-xylan: Remazol Brilliant Blue-xylan     

Novel Defense by Metallothionein Induction Against Cognitive Decline: From Amyloid β<Subscript>1–42</Subscript>-Induced Excess Zn<Superscript>2+</Superscript> to Functional Zn<Superscript>2+</Superscript> Deficiency

The role of metallothioneins (MTs) in cognitive decline associated with intracellular Zn²⁺ dysregulation remains unclear. Here, we report that hippocampal MT induction defends cognitive decline, which was induced by amyloid β_1–42 (Aβ_1–42)-mediated excess Zn²⁺ and functional Zn²⁺ deficiency. Excess increase in intracellular Zn²⁺, which was induced by local injection of Aβ_1–42 into the dentate granule cell layer, attenuated in vivo perforant pathway LTP, while the attenuation was rescued by preinjection of MT inducers into the same region. Intraperitoneal injection of dexamethasone, which increased hippocampal MT proteins and blocked Aβ_1–42-mediated Zn²⁺ uptake, but not Aβ_1–42 uptake, into dentate granule cells, also rescued Aβ_1–42-induced impairment of memory via attenuated LTP. The present study indicates that hippocampal MT induction blocks rapid excess increase in intracellular Zn²⁺ in dentate granule cells, which originates in Zn²⁺ released from Aβ_1–42, followed by rescuing Aβ_1–42-induced cognitive decline. Furthermore, LTP was vulnerable to Aβ_1–42 in the aged dentate gyrus, consistent with enhanced Aβ_1–42-mediated Zn²⁺ uptake into aged dentate granule cells, suggesting that Aβ_1–42-induced cognitive decline, which is caused by excess intracellular Zn²⁺, can more frequently occur along with aging. On the other hand, attenuated LTP under functional Zn²⁺ deficiency in dentate granule cells was also rescued by MT induction. Hippocampal MT induction may rescue cognitive decline under lack of cellular transient changes in functional Zn²⁺ concentration, while its induction is an attractive defense strategy against Aβ_1–42-induced cognitive decline.     

Analysis of polyamine biosynthetic- and transport ability of human indigenous Bifidobacterium

Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium.

Abbreviations: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion     

Spontaneous intracranial hypotension is diagnosed by a combination of lipocalin-type prostaglandin D synthase and brain-type transferrin in cerebrospinal fluid

Background

Spontaneous intracranial hypotension (SIH) is caused by cerebrospinal fluid (CSF) leakage. Definitive diagnosis can be difficult by clinical examinations and imaging studies.

An optimal stopping approach for onset of fish migration

Comprehending life history of migratory fish, onset of migration in particular, is a key biological and ecological research topic that still has not been clarified. In this paper, we propose a simple mathematical model for the onset of fish migration in the context of a stochastic optimal stopping theory, which is a new attempt to our knowledge. Finding the criteria of the onset of migration reduces to solving a variational inequality of a degenerate elliptic type. As a first step of the new mathematical modeling, mathematical and numerical analyses with particular emphasis on whether the model is consistent with the past observation results of fish migration are examined, demonstrating reasonable agreement between the theory and observation results. The present mathematical model thus potentially serves as a simple basis for analyzing onset of fish migration.