Complete mitochondrial DNA sequence and phylogenetic analysis of Zhikong scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis> (Bivalvia: Pectinidae)

The complete mitochondrial genome of Zhikong scallop Chlamys farreri is 21,695 bp in length and contains 12 protein-coding genes (the atp8 gene is absent, as in most bivalves), 2 ribosomal RNA genes, and 22 transfer RNA genes. The heavy strand has an overall A+T content of 58.7%. GC and AT skews for the mt genome of C. farreri are 0.337 and ?0.184, respectively, indicating the nucleotide bias against C and A. The mitochondrial gene order of C. farreri differs drastically from the scallops Argopecten irradians, Mimachlamys nobilis and Placopecten magellanicus, which belong to the same family Pectinidae. 6623 bp non-coding nucleotides exist intergenically in the mitogenome of C. farreri, with a large continuous sequence (4763 bp) between tRNA ^Val and tRNA ^Asn . Two repeat families are found in the large continuous sequence, which seems to be a common feature of scallops. Phylogenetic analysis based on 12 concatenated amino acid sequences of protein-coding genes supports the monophyly of Pectinidae and paraphyletic Pteriomorphia with respect to Heteroconchia.     

Impaired protection against Mycobacterium bovis bacillus Calmette-Guerin infection in IL-15-deficient mice

To investigate the potential role of endogenous IL-15 in mycobacterial infection, we examined protective immunity in IL-15-deficient (IL-15(-/-)) mice after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or recombinant OVA-expressing BCG (rBCG-OVA). IL-15(-/-) mice exhibited an impaired protection in the lung on day 120 after BCG infection as assessed by bacterial growth. CD4(+) Th1 response capable of producing IFN-gamma was normally detected in spleen and lung of IL-15(-/-) mice on day 120 after infection. Although Ag-specific CD8 responses capable of producing IFN-gamma and exhibiting cytotoxic activity were detected in the lung on day 21 after infection with rBCG-OVA, the responses were severely impaired on days 70 and 120 in IL-15(-/-) mice. The degree of proliferation of Ag-specific CD8(+) T cells in IL-15(-/-) mice was similar to that in wild-type mice during the course of infection with rBCG-OVA, whereas sensitivity to apoptosis of Ag-specific CD8(+) T cells significantly increased in IL-15(-/-) mice. These results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining CD8 responses in the lung.     

Stomatal Blue Light Response Is Present in Early Vascular Plants

Light is a major environmental factor required for stomatal opening. Blue light (BL) induces stomatal opening in higher plants as a signal under the photosynthetic active radiation. The stomatal BL response is not present in the fern species of Polypodiopsida. The acquisition of a stomatal BL response might provide competitive advantages in both the uptake of CO₂ and prevention of water loss with the ability to rapidly open and close stomata. We surveyed the stomatal opening in response to strong red light (RL) and weak BL under the RL with gas exchange technique in a diverse selection of plant species from euphyllophytes, including spermatophytes and monilophytes, to lycophytes. We showed the presence of RL-induced stomatal opening in most of these species and found that the BL responses operated in all euphyllophytes except Polypodiopsida. We also confirmed that the stomatal opening in lycophytes, the early vascular plants, is driven by plasma membrane proton-translocating adenosine triphosphatase and K⁺ accumulation in guard cells, which is the same mechanism operating in stomata of angiosperms. These results suggest that the early vascular plants respond to both RL and BL and actively regulate stomatal aperture. We also found three plant species that absolutely require BL for both stomatal opening and photosynthetic CO₂ fixation, including a gymnosperm, C. revoluta, and the ferns Equisetum hyemale and Psilotum nudum.Stomata regulate gas exchange between plants and the atmosphere (Zeiger, 1983; Assmann, 1993; Roelfsema and Hedrich, 2005; Shimazaki et al., 2007; Kim et al., 2010). Acquisition of stomata was a key step in the evolution of terrestrial plants by allowing uptake of CO₂ from the atmosphere and accelerating the provision of nutrients via the transpiration stream within the plant (Hetherington and Woodward, 2003; McAdam and Brodribb, 2013). Stomatal aperture is regulated by changes in the turgor of guard cells, which are induced by environmental factors and endogenous phytohormones. Light is a major factor in the promotion of stomatal opening, and the opening is mediated via two distinct light-regulated pathways that are known as photosynthesis- and blue light (BL)-dependent responses under photosynthetic active radiation (PAR; Vavasseur and Raghavendra, 2005; Shimazaki et al., 2007; Lawson et al., 2014).The photosynthesis-dependent stomatal opening is induced by a continuous high intensity of light, and the action spectrum for the stomatal opening resembles that of photosynthetic pigments in leaves (Willmer and Fricker, 1996). Both mesophyll and guard cells possess photosynthetically active chloroplasts, and their photosynthesis has been suggested to contribute to stomatal opening in leaves. The decrease in the concentration of intercellular CO₂ (Ci) caused by photosynthetic CO₂ fixation or some unidentified mediators and metabolites from mesophyll cells is supposed to elicit stomatal opening, although the exact nature of the events is unclear (Wong et al., 1979; Vavasseur and Raghavendra, 2005; Roelfsema et al., 2006; Mott et al., 2008; Lawson et al., 2014).BL-dependent stomatal opening requires a strong intensity of PAR as a background: weak BL solely scarcely elicits stomatal opening, but the same intensity of BL induces the fast and large stomatal opening in the presence of strong red light (RL; Ogawa et al., 1978; Shimazaki et al., 2007). Since such stomatal opening requires BL under the RL or PAR, we call the opening reaction a BL-dependent response of stomata. BL-dependent stomatal response takes place and proceeds in natural environments because the sunlight contains both BL and RL and facilitates photosynthetic CO₂ fixation (Assmann, 1988; Takemiya et al., 2013a). In this stomatal response, BL and PAR (BL, RL, and other wavelengths of light) seem to act as a signal and an energy source, respectively.The BL-dependent stomatal opening is initiated by the absorption of BL by phototropin1 and phototropin2 (Kinoshita et al., 2001), the plant-specific BL receptors, in guard cells followed by activation of the plasma membrane proton-translocating adenosine triphosphatase (H+-ATPase; Kinoshita and Shimazaki, 1999). Two newly identified proteins, protein phosphatase1 and blue light signaling1 (BLUS1), mediate the signaling between phototropins and H+-ATPase (Takemiya et al., 2006, 2013a, 2013b). The activated H+-ATPase evokes a plasma membrane hyperpolarization, which drives K⁺ uptake through the voltage-gated, inward-rectifying K⁺ channels (Assmann, 1993; Shimazaki et al., 2007; Kim et al., 2010; Kollist et al., 2014). The accumulation of K⁺ causes water uptake and increases turgor pressure of guard cells, and finally results in stomatal opening. The BL-dependent opening is enhanced by RL, and BL at low intensity is effective in the presence of RL (Ogawa et al., 1978; Iino et al., 1985; Shimazaki et al., 2007; Suetsugu et al., 2014). These stomatal responses by RL and BL are commonly observed in a number of seed plants so far examined.Fine control of stomatal aperture to various environmental factors has been characterized in many angiosperms. Although morphological and mechanical diversity of stomata is widely documented, little is known about the functional diversity (Willmer and Fricker, 1996; Hetherington and Woodward, 2003). Our previous study indicated that BL-dependent stomatal response is absent in the major fern species of Polypodiopsida, including Adiantum capillus-veneris, Pteris cretica, Asplenium scolopendrium, and Nephrolepis auriculata, but the stomata of these species open by PAR including RL (Doi et al., 2006). When the epidermal peels isolated from A. capillus-veneris are treated with photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1dimethylurea (Doi and Shimazaki, 2008), the response is completely inhibited, but the responses in the seed plants of Vicia faba and Commelina communis are relatively insensitive to 3-(3,4-dichlorophenyl)-1,1dimethylurea (Schwartz and Zeiger, 1984). These findings suggest that there is functional diversity in light-dependent stomatal response in different lineages of land plants. In accord with this notion, the different sensitivities of stomatal response to abscisic acid and CO₂ have been reported among the plant species of angiosperm, gymnosperm, ferns, and lycophytes (Mansfield and Willmer, 1969; Brodribb and McAdam, 2011), although the exact responsiveness to abscisic acid and CO₂ is still debated (Chater et al., 2011, 2013; Ruszala et al., 2011; McAdam and Brodribb, 2013).To address the origin and distribution of stomatal light responses, we investigated the presence of a stomatal response using a gas exchange method and various lineages of vascular plants, including euphyllophytes and lycophytes. Unexpectedly, all plant lineages except Polypodiopsida in monilophytes exhibited a stomatal response to BL in the presence of RL, suggesting that the response was present in the early evolutionary stage of vascular plants. We also report the stomatal opening in response to RL in these plant species.     

Cdk5--p39 is a labile complex with the similar substrate specificity to Cdk5--p35

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase that plays important roles in various neuronal activities, including neuronal migration, synaptic activity, and neuronal cell death. Cdk5 is activated by association with a neuron-specific activator, p35 or its isoform p39, but little is known about the kinase activity of Cdk5--p39. In fact, kinase-active Cdk5--p39 was not prepared from rat brain extracts nor from HEK293 cells expressing Cdk5 and p39 by immunoprecipitation in the presence of non-ionic detergent, under conditions with which active Cdk5--p35 could be isolated. p39 dissociated from Cdk5 in the presence of detergent, indicating that p39 has a lower binding affinity for Cdk5 than p35. We developed a method for purifying kinase-active Cdk5--p39 from Sf9 cells infected with baculovirus encoding Cdk5 and p39. The purified Cdk5--p39 complex showed similar substrate specificity to that of Cdk5--p35, but with opposite sensitivity to detergent. Cdk5--p39 was inactivated by Triton X-100, whereas Cdk5--p35 was activated. The N-terminal deletion from p35 and p39, the amino acid sequences of which are different, did not change the stability or substrate specificity of either Cdk5 complex. The different stability between Cdk5--p35 and Cdk5--p39 suggests their distinct roles under different regulation mechanisms in neurons.     

Heat attenuates sensitivity of mammalian cells to capsaicin

The transient receptor potential (TRP) channels are thermo‐sensors, and transient receptor potential vanilloid (TRPV)1 and V4 are widely expressed in primary afferent neurons and nonneuronal cells. Although heat acclimation is considered as changes of thermoregulatory responses by thermo‐effectors to heat, functional changes of TRP channels in heat acclimation has not been fully elucidated. Here, we investigated whether heat acclimation induces capsaicin tolerance. NIH3T3 cells were incubated at 39.5°C. We determined the expression level of TRPV1 and TRPV4 messenger RNA (mRNA), performed cellular staining of TRPV1 and TRPV4, and investigated actin assembly and activation of the extracellular signal‐regulated kinase (ERK). Exposure to moderate heat decreased the levels of TRPV1 but not TRPV4 mRNA. It also induced stress fiber formation and the intensity of TRPV1 seemed to be decreased by chronic heat stimuli. In addition, heat acclimation attenuated the capsaicin‐induced activation of ERK. Heat acclimation may induce capsaicin tolerance via the downregulation of TRPV1.     

On-chip constructive cell-network study (II): on-chip quasi-in vivo cardiac toxicity assay for ventricular tachycardia/fibrillation measurement using ring-shaped closed circuit microelectrode with lined-up cardiomyocyte cell network

Backgrounds

Conventional in vitro approach using human ether-a-go-go related gene (hERG) assay has been considered worldwide as the first screening assay for cardiac repolarization safety. However, it does not always oredict the potential QT prolongation risk or pro-arrhythmic risk correctly. For adaptable preclinical strategiesto evaluate global cardiac safety, an on-chip quasi-in vivo cardiac toxicity assay for lethal arrhythmia (ventricular tachyarrhythmia) measurement using ring-shaped closed circuit microelectrode chip has been developed.

Results

The ventricular electrocardiogram (ECG)-like field potential data, which includes both the repolarization and the conductance abnormality, was acquired from the self-convolutied extracellular field potentials (FPs) of a lined-up cardiomyocyte network on a circle-shaped microelectrode in an agarose microchamber. When Astemisol applied to the closed-loop cardiomyocyte network, self-convoluted FP profile of normal beating changed into an early afterdepolarization (EAD) like waveform, and then showed ventricular tachyarrhythmias and ventricular fibrilations (VT/Vf). QT-prolongation-like self-convoluted FP duration prolongation and its fluctuation increase was also observed according to the increase of Astemizole concentration.

Conclusions

The results indicate that the convoluted FPs of the quasi-in vivo cell network assay includes both of the repolarization data and the conductance abnormality of cardiomyocyte networks has the strong potential to prediction lethal arrhythmia.     

Effects of phospholipid unsaturation on the bilayer nonpolar region: a molecular simulation study

Molecular dynamics simulations of two monounsaturated phosphatidylcholine (PC) bilayers made of 1-palmitoyl-2-oleoyl-PC (POPC; cis-unsaturated) and 1-palmitoyl-2-elaidoyl-PC (PEPC; trans-unsaturated) were carried out to investigate the effect of a double bond in the PC beta-chain and its conformation on the bilayer core. Four nanosecond trajectories were used for analyses. A fully saturated 1,2-dimyristoyl-PC (DMPC) bilayer was used as a reference system. In agreement with experimental data, this study shows that properties of the PEPC bilayer are more similar to those of the DMPC than to the POPC bilayer. The differences between POPC and PEPC bilayers may be attributed to the different ranges of angles covered by the torsion angles beta10 and beta12 of the single bonds next to the double bond in the oleoyl (O) and elaidoyl (E) chains. Broader distributions of beta10 and beta12 in the E chain than in the O chain make the E chain more flexible. In effect, the packing of chains in the PEPC bilayer is similar to that in the DMPC bilayer, whereas that in the POPC bilayer is looser than that in the DMPC bilayer. The effect of the cis-double bond on torsions at the beginning of the O chain (beta4 and beta5) is similar to that of cholesterol on these torsions in a myristoyl chain.     

PKCδ Knockout Mice Are Protected from Dextromethorphan-Induced Serotonergic Behaviors in Mice: Involvements of Downregulation of 5-HT<Subscript>1A</Subscript> Receptor and Upregulation of Nrf2-Dependent GSH Synthesis

We investigated whether a specific serotonin (5-HT) receptor-mediated mechanism was involved in dextromethorphan (DM)-induced serotonergic behaviors. We firstly observed that the activation of 5-HT_1A receptor, but not 5-HT_2A receptor, contributed to DM-induced serotonergic behaviors in mice. We aimed to determine whether the upregulation of 5-HT_1A receptor induced by DM facilitates the specific induction of certain PKC isoform, because previous reports suggested that 5-HT_1A receptor activates protein kinase C (PKC). A high dose of DM (80 mg/kg, i.p.) induced a selective induction of PKCδ out of PKCα, PKCβI, PKCβII, PKCξ, and PKCδ in the hypothalamus of wild-type (WT) mice. More importantly, 5-HT_1A receptor co-immunoprecipitated PKCδ in the presence of DM. Consistently, rottlerin, a pharmacological inhibitor of PKCδ, or PKCδ knockout significantly protected against increases in 5-HT_1A receptor gene expression, 5-HT turnover rate, and serotonergic behaviors induced by DM. Treatment with DM resulted in an initial increase in nuclear factor erythroid-2-related factor 2 (Nrf2) nuclear translocation and DNA-binding activity, γ-glutamylcysteine (GCL) mRNA expression, and glutathione (GSH) level. This compensative induction was further potentiated by rottlerin or PKCδ knockout. However, GCL mRNA and GSH/GSSG levels were decreased 6 and 12 h post-DM. These decreases were attenuated by PKCδ inhibition. Our results suggest that interaction between 5-HT_1A receptor and PKCδ is critical for inducing DM-induced serotonergic behaviors and that inhibition of PKCδ attenuates the serotonergic behaviors via downregulation of 5-HT_1A receptor and upregulation of Nrf2-dependent GSH synthesis.     

The Class-A GPCR Dopamine D2 Receptor Forms Transient Dimers Stabilized by Agonists: Detection by Single-Molecule Tracking

Whether class-A G-protein coupled receptors (GPCRs) exist and work as monomers or dimers has drawn extensive attention. A class-A GPCR dopamine D2 receptor (D2R) is involved in many physiological and pathological processes and diseases, indicating its critical role in proper functioning of neuronal circuits. In particular, D2R homodimers might play key roles in schizophrenia development and amphetamine-induced psychosis. Here, using single-molecule imaging, we directly tracked single D2R molecules in the plasma membrane at a physiological temperature of 37?°C, and unequivocally determined that D2R forms transient dimers with a lifetime of 68?ms in its resting state. Agonist addition prolonged the dimer lifetime by a factor of ~1.5, suggesting the possibility that transient dimers might be involved in signaling.     

Sodium hydroxide based non-detergent decellularizing solution for rat lung

Lung transplantation is the last option for the treatment of end stage chronic lung disorders. Because the shortage of donor lung organs represents the main hurdle, lung regeneration has been considered to overcome this hurdle. Recellularization of decellularized organ scaffold is a promising option for organ regeneration. Although detergents are ordinarily used for decellularization, other approaches are possible. Here we used high alkaline (pH12) sodium hydroxide (NaOH)-PBS solution without detergents for lung decellularization and compared the efficacy on DNA elimination and ECM preservation with detergent based decellularization solutions CHAPS and SDS. Immunohistochemical image analysis showed that cell components were removed by NaOH solution as well as other detergents. A Collagen and GAG assay showed that the collagen reduction of the NaOH group was comparable to that of the CHAPS and SDS groups. However, DNA reduction was more significant in the NaOH group than in other groups (p < 0.0001). The recellularization of HUVEC revealed cell attachment was not inferior to that of the SDS group. Ex vivo functional analysis showed 100% oxygen ventilation increased oxygen partial pressure as artificial hemoglobin vesicle-PBS solution passed through regenerated lungs in the SDS or NaOH group. It was concluded that the NaOH-PBS based decellularization solution was comparable to ordinal decellularizaton solutions and competitive in cost effectiveness and residues in the decellularized scaffold negligible, thus providing another potential option to detergent for future clinical usage.