Cortico-Cardio-Respiratory Network Interactions during Anesthesia

General anesthetics are used during medical and surgical procedures to reversibly induce a state of total unconsciousness in patients. Here, we investigate, from a dynamic network perspective, how the cortical and cardiovascular systems behave during anesthesia by applying nonparametric spectral techniques to cortical electroencephalography, electrocardiogram and respiratory signals recorded from anesthetized rats under two drugs, ketamine-xylazine (KX) and pentobarbital (PB). We find that the patterns of low-frequency cortico-cardio-respiratory network interactions may undergo significant changes in network activity strengths and in number of network links at different depths of anesthesia dependent upon anesthetics used.     

Isolation and characterization of 13 microsatellite markers for the viviparous surfperch Ditrema temmincki (Embiotocidae) and cross-species amplification

Thirteen microsatellite loci were isolated from a size-selected genomic library of the surfperch (Ditrema temmincki Bleeker). All loci displayed a high degree of length polymorphism, as observed in the total number of alleles per locus (two to 23) and a high degree of estimated heterozygosity, ranging from 0.080 to 0.893. The primers developed for D. temmincki were also tested for their ability to amplify homologous sequences in D. viride and Neoditrema ransonetii. Distinct differences were observed among three species of surfperches, in both genetic variability and the frequency distribution of the alleles.     

Mycolyltransferase-mediated glycolipid exchange in Mycobacteria

Trehalose dimycolate (TDM), also known as cord factor, is a major surface glycolipid of the cell wall of mycobacteria. Because of its potent biological functions in models of infection, adjuvancy, and immunotherapy, it is important to determine how its biosynthesis is regulated. Here we show that glucose, a host-derived product that is not readily available in the environment, causes Mycobacterium avium to down-regulate TDM expression while up-regulating production of another major glycolipid with immunological roles in T cell activation, glucose monomycolate (GMM). In vitro, the mechanism of reciprocal regulation of TDM and GMM involves competitive substrate selection by antigen 85A. The switch from TDM to GMM biosynthesis occurs near the physiological concentration of glucose present in mammalian hosts. We further demonstrate that GMM is produced in vivo by mycobacteria growing in mouse lung. These results establish an enzymatic pathway for GMM production. More generally, these observations provide a specific enzymatic mechanism for dynamic alterations of cell wall glycolipid remodeling in response to the transition from noncellular to cellular growth environments, including factors that are monitored by the host immune system.     

Mutations causing DOK7 congenital myasthenia ablate functional motifs in Dok-7

Dok-7 is a cytoplasmic activator of muscle-specific receptor-tyrosine kinase (MuSK). Both Dok-7 and MuSK are required for neuromuscular synaptogenesis. Mutations in DOK7 underlie a congenital myasthenic syndrome (CMS) associated with small and simplified neuromuscular synapses likely due to impaired Dok-7/MuSK signaling. The overwhelming majority of patients with DOK7 CMS have at least one allele with a frameshift mutation that causes a truncation in the COOH-terminal region of Dok-7 and affects MuSK activation. Dok-7 has pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains in the NH2-terminal moiety, both of which are indispensable for MuSK activation in myotubes, but little is known about additional functional elements. Here, we identify a chromosome region maintenance 1-dependent nuclear export signal (NES) in the COOH-terminal moiety and demonstrate that the NES-mediated cytoplasmic location of Dok-7 is essential for regulating the interaction with MuSK in myotubes. The NH2-terminal PH domain is responsible for the nuclear import of Dok-7. We also show that the Src homology 2 target motifs in the COOH-terminal moiety of Dok-7 are active and crucial for MuSK activation in myotubes. In addition, CMS-associated missense mutations found in the PH or PTB domain inactivate Dok-7. Together, these findings demonstrate that, in addition to the NH2-terminal PH and PTB domains, the COOH-terminal NES and Src homology 2 target motifs play key roles in Dok-7/MuSK signaling for neuromuscular synaptogenesis. Ablation or disruption of these functional elements in Dok-7 probably underlies the neuromuscular junction synaptopathy observed in DOK7 CMS.     

Comparative study of hydrogen peroxide- and 4-hydroxy-2-nonenal-induced cell death in HT22 cells

Several studies have indicated that lipid peroxidation often occurs in response to oxidative stress, and that many aldehydic products including 4-hydroxy-2-nonenal (HNE) are formed when lipid hydroperoxides break down. In order to clarify the mechanism of oxidative stress-induced neuronal death in the nervous system, we investigated H(2)O(2)- and HNE-induced cell death pathways in HT22 cells, a mouse hippocampal cell line, under the same experimental conditions. Treatment with H(2)O(2) and HNE decreased the viability of these cells in a time- and concentration-dependent manner. In the cells treated with H(2)O(2), significant increases in the immunoreactivities of DJ-1 and nuclear factor-kappaB (NF-kappaB) subunits (p65 and p50) were observed in the nuclear fraction. H(2)O(2) also induced an increase in the intracellular concentration of Ca(2+), and cobalt chloride (CoCl(2)), a Ca(2+) channel inhibitor, suppressed the H(2)O(2)-induced cell death. In HNE-treated cells, none of these phenomena were observed; however, HNE adduct proteins were formed after exposure to HNE, but not to H(2)O(2). N-Acetyl-L-cysteine (NAC) suppressed both HNE-induced cell death and HNE-induced expression of HNE adduct proteins, whereas H(2)O(2)-induced cell death was not affected. These findings suggest that the mechanisms of cell death induced by H(2)O(2) different from those induced by HNE in HT22 cells, and that HNE adduct proteins play an important role in HNE-induced cell death. It is also suggested that the pathway for H(2)O(2)-induced cell death in HT22 cells does not involve HNE production.     

Introduction of silencing-inducing transgene against Fgf19 does not affect expression of Tbx5 and beta3-tubulin in the developing chicken retina

Fgf19 is known to be expressed in the developing chicken eye but its functions during retinal development have remained elusive. Since Fgf19 is expressed in the dorsal portion of the optic cup, it is intriguing to know whether FGF19 is required for expression of dorso-ventral morphogenetic genes in the eye. To clarify this, expression patterns of Tbx5 and Vax were examined in the developing eye after in ovo RNA interference targeted against Fgf19 . Quantitative polymerase chain reaction (PCR) analysis showed that the short-hairpin RNAs (shRNAs) targeted against Fgf19 could reduce its expression in the eye to less than 50% of a relative amount of mRNA, compared with contralateral or untreated control eyes. However, no obvious alteration in expression domains of Tbx5 or Vax was observed. Misexpression of Tbx5 or Tbx5 -RNAi did not alter the Fgf19 expression either. Furthermore, although Fgf19 is expressed in the central retina before neurogenesis occurs, β3-tubulin, a marker for early retinal differentiation was still detected in the central retina after knockdown of Fgf19 . Thus, knockdown of Fgf19 supports no obvious regulations between Fgf19 and Tbx5 , or exhibits no phenotypes that perturb early retinal differentiation.     

Superoxide scavenging activity of pirfenidone-iron complex

Pirfenidone (PFD) is focused on a new anti-fibrotic drug, which can minimize lung fibrosis etc. We evaluated the superoxide () scavenging activities of PFD and the PFD-iron complex by electron spin resonance (ESR) spectroscopy, luminol-dependent chemiluminescence assay, and cytochrome c reduction assay. Firstly, we confirmed that the PFD-iron complex was formed by mixing iron chloride with threefold molar PFD, and the complex was stable in distillated water and ethanol. Secondary, the PFD-iron complex reduced the amount of produced by xanthine oxidase/hypoxanthine without inhibiting the enzyme activity. Thirdly, it also reduced the amount of released from phorbor ester-stimulated human neutrophils. PFD alone showed few such effects. These results suggest the possibility that the scavenging effect of the PFD-iron complex contributes to the anti-fibrotic action of PFD used for treating idiopathic pulmonary fibrosis.     

A monoclonal antibody (1D12) defines novel distribution patterns of prion protein (PrP) as granules in nucleus

A monoclonal antibody (mAb) panel to bovine prion protein (PrP) was studied by immunoblotting and immunohistochemistry for scrapie and bovine spongiform encephalopathy. A mAb panel recognized both normal (PrP^C) and abnormal (PrP^Sc) isoforms of PrP in murine, ovine and bovine brain tissues. Interestingly, an anti-bovine PrP mAb, 1D12, prepared by immunizing PrP gene-knockout mice with a synthetic polypeptides corresponding to codons 153-166 of the bovine PrP gene showed novel patterns of reactivity for prion-uninfected neuronal cells. An enzyme-linked immunosorbent assay-mapping of the mAb epitopes resulted in a reaction of monoclonal 1D12 to YEDRY and M corresponding to amino acids 156-160 and 165 of bovine PrP. Several patterns of bovine PrP^C distribution in PrP-deficient neuronal cells (HpL3-4) transfected with bovine PrP were observed after different fixation methods. Stained cell surface was observed after formalin fixation by immunofluorescent assay of 1D12 with confocal microscopy, whereas granules in nucleus were stained after acetone fixation. No reactivity in the nucleus was observed to HpL3-4, or HpL3-4mPrP cells expressing mouse PrP. This is the first paper that has reported the detection of the PrP^C at both cell surface and nuclei of prion-uninfected cell line.     

Applicability of new spin trap agent, 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide, in biological system

Electron spin resonance using spin-trapping is a useful technique for detecting direct reactive oxygen species, such as superoxide (). However, the widely used spin trap 2,2-dimethyl-3,4-dihydro-2H-pyrrole N-oxide (DMPO) has several fundamental limitations in terms of half-life and stability. Recently, the new spin trap 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO) was developed by us. We evaluated the biological applicability of DPhPMPO to analyze in both cell-free and cellular systems. DPhPMPO had a larger rate constant for and formed more stable spin adducts for than DMPO in the xanthine/xanthine oxidase (X/XO) system. In the phorbol myristate acetate-activated neutrophil system, the detection potential of DPhPMPO for was significantly higher than that of DMPO (k_DMPO = 13.95 M⁻¹ s⁻¹, k_DPhPMPO = 42.4 M⁻¹ s⁻¹). These results indicated that DPhPMPO is a potentially good candidate for trapping in a biological system.