Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy

Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD⁺-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD⁺-bound LDH are reported at 3.0 and 3.2?Å resolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD⁺. NAD⁺ binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD⁺-bound form was approximately three times higher than that of the apo form after incubation at 80?°C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.     

The brain-derived neurotrophic factor Val66Met polymorphism modulates the effects of parental rearing on personality traits in healthy subjects

There is a growing body of data suggesting that gene-environment interaction is critical in the characterization of personality traits; however, previous studies have not taken into consideration variability in parental rearing as an environmental factor. In this study, we examined the effects of the interaction between the brain-derived neurotrophic factor (BDNF) Val66Met polymorphism and parental rearing on personality traits in 710 healthy Japanese subjects. Perceived parental rearing was assessed by the Parental Bonding Instrument (PBI), which consists of the care and protection factors. Assessment of personality traits was performed by the temperament and character inventory (TCI), which has seven dimensions, i.e. novelty seeking, harm avoidance, reward dependence, persistence, self-directedness, cooperativeness and self-transcendence. Parental rearing has significant main effects on some TCI dimensions, but no significant main effects of the BDNF genotype on the TCI scores were found. The interaction between the BDNF genotype and maternal care of the PBI had significant effects on harm avoidance and self-directedness of the TCI. Post hoc analyses showed that decreased maternal care was correlated with increased harm avoidance and decreased self-directedness, and for both personality traits the partial correlation coefficient was highest in the Met/Met genotype group and lowest in the Val/Val genotype group and the value of the Val/Met genotype group was in the middle. Data from this study suggest that the BDNF Val66Met polymorphism modulates the effects of parental rearing, especially maternal care, on harm avoidance and self-directedness in healthy subjects.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

Intercontinental union of Argentine ants: behavioral relationships among introduced populations in Europe, North America, and Asia

Many invasive ants, including the Argentine ant Linepithema humile, form expansive supercolonies, within which intraspecific aggression is absent. The behavioral relationships among introduced Argentine ant populations at within-country or within-continent scales have been studied previously, but the behavioral relationships among intercontinental populations have not been examined. The present study investigated the levels of aggression among intercontinental Argentine ant populations by transporting live ants from Europe and California to Japan and conducting aggression tests against Japanese populations. Workers from the dominant supercolonies of Europe and California did not show aggressive behavior toward workers from the dominant supercolony of Japan, whereas they fought vigorously against workers from minor supercolonies. The three massive supercolonies, together with Argentine ants from Macaronesia, may be the largest non-aggressive unit formed by a social insect species in which intraspecific aggression exists. Absence or low levels of aggression at transcontinental scale, which may have derived from low genetic variation, may help introduced Argentine ants maintain expansive supercolonies. The lack of aggression implies possible frequent exchanges of individuals among the intercontinental populations mediated by human activities.     

Oxygen-glucose deprivation-induced enhancement of extracellular ATP-P2Y purinoceptors signaling for the propagation of astrocytic calcium waves

Waves of elevated intracellular free Ca²⁺ that propagate between neighboring astrocytes (Ca²⁺ waves) are important for the communication among astrocytes. We have previously revealed that focal photolysis of a caged calcium ionophore results in an increase in the concentration of intracellular Ca²⁺ in the target astrocytes, then the increase propagates to neighboring astrocytes through gap junctions. The extracellular ATP-purinoceptors signaling pathways are not primarily responsible for the propagation of the photolytic flash-induced Ca²⁺ waves. Here we examined whether and if so how the dynamics of Ca²⁺ waves changed after treatment with sublethal simulated ischemia; oxygen-glucose deprivation (OGD). OGD treatment increased the astrocytic expression of P2Y₁ and P2Y₂ receptors early during reperfusion, resulting in an increase in the propagating waves speed. In contrast, the expression of a gap junction protein was not changed significantly by the OGD suggesting that the extracellular ATP-P2Y receptors signaling pathways were preferentially enhanced after OGD. The present method to induce Ca²⁺ waves by focal photolysis of a caged calcium ionophore may provide a valuable tool with which to analyze glial Ca²⁺ waves under not only normal but also pathologic conditions.     

Roles of Apicomplexan protein kinases at each life cycle stage

Inhibitors of cellular protein kinases have been reported to inhibit the development of Apicomplexan parasites, suggesting that the functions of protozoan protein kinases are critical for their life cycle. However, the specific roles of these protein kinases cannot be determined using only these inhibitors without molecular analysis, including gene disruption. In this report, we describe the functions of Apicomplexan protein kinases in each parasite life stage and the potential of pre-existing protein kinase inhibitors as Apicomplexan drugs against, mainly, Plasmodium and Toxoplasma.     

The I2020T Leucine-rich repeat kinase 2 transgenic mouse exhibits impaired locomotive ability accompanied by dopaminergic neuron abnormalities

ABSTRACT: BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) is the gene responsible for autosomal-dominant Parkinson's disease (PD), PARK8, but the mechanism by which LRRK2 mutations cause neuronal dysfunction remains unknown. In the present study, we investigated for the first time a transgenic (TG) mouse strain expressing human LRRK2 with an I2020T mutation in the kinase domain, which had been detected in the patients of the original PARK8 family. RESULTS: The TG mouse expressed I2020T LRRK2 in dopaminergic (DA) neurons of the substantia nigra, ventral tegmental area, and olfactory bulb. In both the beam test and rotarod test, the TG mice exhibited impaired locomotive ability in comparison with their non-transgenic (NTG) littermates. Although there was no obvious loss of DA neurons in either the substantia nigra or striatum, the TG brain showed several neurological abnormalities such as a reduced striatal dopamine content, fragmentation of the Golgi apparatus in DA neurons, and an increased degree of microtubule polymerization. Furthermore, the tyrosine hydroxylasepositive primary neurons derived from the TG mouse showed an increased frequency of apoptosis and had neurites with fewer branches and decreased outgrowth in comparison with those derived from the NTG controls. CONCLUSIONS: The I2020T LRRK2 TG mouse exhibited impaired locomotive ability accompanied by several dopaminergic neuron abnormalities. The TG mouse should provide valuable clues to the etiology of PD caused by the LRRK2 mutation.