Heat-killed Lactobacillus helveticus strain MCC1848 confers resilience to anxiety or depression-like symptoms caused by subchronic social defeat stress in mice

The gut microbiota is involved in the pathogenesis of stress-related disorders. Probiotics can benefit the central nervous system via the microbiota–gut–brain axis, which raises the possibility that probiotics are effective in managing depression. In the present study, we examined the effects of heat-killed Lactobacillus helveticus strain MCC1848 in subchronic and mild social defeat stress (sCSDS) model mice (a widely used animal model of depression). MCC1848 supplementation significantly enhanced the interaction time in the social interaction test and sucrose preference ratio in the sucrose preference test, suggesting that MCC1848 improved anxiety- or depressive-like behaviors in sCSDS mice. The gene expression profile analysis of the nucleus accumbens, which plays an important role in stress resilience, indicated that MCC1848 ameliorated sCSDS-induced gene expression alterations in signal transduction or nervous system development. These findings suggest that MCC1848 supplementation is useful as a preventive strategy for chronic-stress-induced depression.     

Development of a new E. coli strain to detect oxidative mutation and its application to the fungicide o-phenylphenol and its metabolites

Oxidative mutation is mainly induced by reactive oxygen species (ROS), such as the superoxide anion radical (O(2)(-)) and hydrogen peroxide (H(2)O(2)). However, in Escherichia coli (E. coli), ROS are eliminated by enzymes such as superoxide dismutase and catalase, which are coded by sodAB and katEG genes. In this study, to detect mutagens that induce oxidative mutation, a mutant (WP2katEGsodAB) with katEG and sodAB deleted was constructed by gene manipulation of E. coli WP2. H(2)O(2) and menadione sodium bisulfite generated mutation in WP2katEGsodAB but not in WP2. o-Phenylphenol (OPP) and its metabolites (phenylhydroquinone (PHQ) and phenyl-1,4-benzoquinone (PBQ)), which had been shown to be negative in the Ames test but reported to be carcinogenic, induced mutation in WP2katEGsodAB but not in WP2. These results suggest that the new assay may be useful for the detection of oxidative mutagens.     

Thermotropic and barotropic phase transitions in bilayer membranes of ether-linked phospholipids with varying alkyl chain lengths

The bilayer phase transitions of a series of ether-linked phospholipids, 1,2-dialkylphosphatidylcholines containing linear saturated alkyl chain (C(n)=12, 14, 16 and 18), were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the pre- and main-transitions for the ether-linked PC bilayer membranes were calculated and compared with those of a series of ester-linked PCs, 1,2-diacylphosphatidylcholines. The thermodynamic quantities of the main transition for the ether-linked PC bilayers showed distinct dependence on alkyl-chain length and were slightly different from those of the ester-linked PC bilayers. From the comparison of thermodynamic quantities for the main transition between both PC bilayers, we revealed that the attractive interaction in the gel phase for the ether-linked PC bilayers is weaker than that for the ester-linked PC bilayers. Regarding the pretransition, although changes in enthalpy and entropy for both PC bilayers were comparable to each other, the volume changes of the ether-linked PC bilayers roughly doubled those of the ester-linked PC bilayers. The larger volume change results from the smallest partial molar volume of the ether-linked PC molecule in the interdigitated gel phase. Further, we constructed the temperature-pressure phase diagrams for the ether-linked PC bilayers by using the phase-transition data. The region of the interdigitated gel phase in the phase diagrams was extended by applying pressure and by increasing the alkyl-chain length of the molecule. Comparing the phase diagrams with those for the ester-linked PC bilayers, it was proved that the phase behavior of the ester-linked PC bilayers under high temperature and pressure is almost equivalent to that of the ether-linked PC bilayers in the vicinity of ambient pressure.     

Development of sensitive chemical and immunochemical methods for detecting sulfated sialic acids and their application to glycoconjugates from sea urchin sperm and eggs

Sulfated sialic acid (SiaS) is a unique sialic acid (Sia) derivative in which an additional anionic group is attached to a carboxylated monosaccharide. Very little is known about the occurrence and biologic function of SiaS, due to the limitations of analytical methods to detect it in minute amounts. In this study, to develop methods and probes for detecting and pursuing the functions of SiaS, we developed sensitive chemical and immunochemical detection methods. First, we synthesized as model compounds 4-methylumbelliferyl glycosides of 8-O- and 9-O-sulfated Sia consisting of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (Kdn). Second, we applied fluorometric high performance liquid chromatography (HPLC) analysis to these synthetic glycosides. After acid hydrolysis of the samples, the liberated SiaS were labeled with a fluorescent reagent, 1,2-diamino-4,5-methylenedioxybenzene, and analyzed on fluorometric HPLC. We established an optimal elution condition for successful separation of 8-O- and 9-O-sulfated Neu5Ac, Neu5Gc, and Kdn on HPLC. Third, we generated a monoclonal antibody (mAb) 2C4 against SiaS using sea urchin egg components as the immunogen. mAb.2C4 recognizes both 8-O-sulfated Neu5Ac (Neu5Ac8S) and Neu5Gc8S, whereas the previously prepared mAb.3G9 only recognizes Neu5Ac8S. Finally, using the fluorometric HPLC and monoclonal antibodies, we demonstrated that glycoconjugates from sea urchin sperm exclusively contained Neu5Ac8S, whereas those from eggs contained Neu5Gc8S. Furthermore, we clarified the quantitative differences in the SiaS content in eggs and sperm from two different species of sea urchins. Immunostaining using mAb.2C4 showed that Neu5Gc8S is localized in the cortical granules in unfertilized eggs, whereas it is localized in the outer surface of the fertilization layer as well as in the inner surface of fertilized eggs. Thus, 8-O-sulfation is dependent on the species, gametic cell-type, site-localization of the eggs, and glycoconjugates.     

Trafficking of QD-conjugated MPO-ANCA in murine systemic vasculitis and glomerulonephritis model mice

In systemic vasculitis, the serum level of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA) is significantly elevated with the progression of disease. We have established a model of murine systemic vasculitis by administration of MPO-ANCA and fungal mannoprotein to C57BL/6 mice. We examined the role of MPO and MPO-ANCA in the pathogenesis of glomerulonephritis and systemic vasculitis in this model using quantum dots (QDs). We demonstrated that QD-conjugated MPO-ANCA (ANCA-QD) visualized the translocation of MPO on the neutrophil membrane surface after stimulation with proinflammatory cytokines. We also observed that MPO translocation on neutrophils in both patients with rapid progressive glomerulonephritis and these model mice without any stimulation, suggesting that MPO translocation is certain to contribute to the development of glomerular lesion. In addition, blood flow on the kidney surface vessel was significantly decelerated in both SCG/Kj mice and this model, suggesting that ANCA induces the damage of blood vessel. These results indicate that MPO-ANCA and surface-translocated MPO on the activated neutrophils coordinately plays essential roles in the initial steps of the glomerulonephritis.     

Kinase-interacting substrate screening is a novel method to identify kinase substrates

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.     

Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation

Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D₃, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D₃ administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition.     

Tamoxifen induces masculinization of genetic females and regulates P450 aromatase and Müllerian inhibiting substance mRNA expression in Japanese flounder (Paralichthys olivaceus)

Japanese flounder, Paralichthys olivaceus, provides an excellent model to elucidate the roles of sex steroid hormones in gonadal sex differentiation because the sex is easily altered by sex steroid treatments or water temperature control during the sex differentiation. We have previously shown that high water temperature, an aromatase inhibitor (fadrozole), or 17alpha-methyltestosterone treatment causes the sex-reversal from genetic females to phenotypic males and suppression of mRNA expression of ovary-type P450 aromatase (P450arom), which is a steroidogenic enzyme responsible for the conversion of androgens to estrogens, in Japanese flounder. In the present study, we demonstrate that treatment of the genetic females with anti-estrogen (tamoxifen) leads to their masculinization, suppresses P450arom mRNA expression, and induces mRNA expression of Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta (TGF-beta) superfamily, while it has no effect on mRNAs expression of estrogen receptor-alpha (ERalpha) and ERbeta. In contrast, 17beta-estradiol counteracted masculinization of the genetic females by tamoxifen or high water temperature treatment, up-regulated P450arom mRNA expression, and down-regulated MIS mRNA expression. These results strongly suggest that estrogen signaling through ERs dramatically influences the gonadal sex differentiation by regulating P450arom and MIS mRNA expression.