Casein kinase 2 is the major enzyme in brain that phosphorylates Ser129 of human alpha-synuclein: Implication for alpha-synucleinopathies

In Lewy body diseases and multiple system atrophy, α-synuclein is hyperphosphorylated at Ser129, suggesting a role in pathogenesis. Here, we report purification of the protein kinase in rat brain that phosphorylates Ser129 and its identification as casein kinase-2 (CK2). We show that most of the activity can be inhibited by heparin, an inhibitor of CK2. Phosphorylated Ser129 was detected in primary cultured neurons and inhibited by CK2 inhibitors. In some cases of Lewy body disease, CK2-like immunoreactivity was recovered in the sarkosyl-insoluble fraction, which was enriched in phosphorylated α-synuclein. Taken together, these findings suggest that CK2 may be involved in the hyperphosphorylation of α-synuclein in α-synucleinopathies.     

Role of phosphoenolpyruvate in the NADP-isocitrate dehydrogenase and isocitrate lyase reaction in Escherichia coli

Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki' of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.     

LPA4/p2y9/GPR23 mediates rho-dependent morphological changes in a rat neuronal cell line

Lysophosphatidic acid (LPA) is a potent lipid mediator that evokes a variety of biological responses in many cell types via its specific G protein-coupled receptors. In particular, LPA affects cell morphology, cell survival, and cell cycle progression in neuronal cells. Recently, we identified p2y(9)/GPR23 as a novel fourth LPA receptor, LPA(4) (Noguchi, K., Ishii, S., and Shimizu, T. (2003) J. Biol. Chem. 278, 25600-25606). To assess the functions of LPA(4) in neuronal cells, we used rat neuroblastoma B103 cells that lack endogenous responses to LPA. In B103 cells stably expressing LPA(4), we observed G(q/11)-dependent calcium mobilization, but LPA did not affect adenylyl cyclase activity. In LPA(4) transfectants, LPA induced dramatic morphological changes, i.e. neurite retraction, cell aggregation, and cadherin-dependent cell adhesion, which involved Rho-mediated signaling pathways. Thus, our results demonstrated that LPA(4) as well as LPA(1) couple to G(q/11) and G(12/13), whereas LPA(4) differs from LPA(1) in that it does not couple to G(i/o). Through neurite retraction and cell aggregation, LPA(4) may play a role in neuronal development such as neurogenesis and neuronal migration.     

Phagocytosis plays an important role in clearing dead cells caused by mono(2-ethylhexyl) phthalate administration

The role of phagocytosis in eliminating apoptotic spermatogenic cells caused by mono(2-ethylhexyl) phthalate (MEHP) was studied. Twenty-one-day-old C57Bl/6N male mice were given a single dose of 800 mg/kg MEHP in corn oil by oral gavage and sacrificed at 1, 3, 5, 7 and 9 days after initial exposure. At the same time, the role of phagocytosis in MEHP related apoptosis was examined using microinjection of annexin V into the seminiferous tubules of living mice. Results showed that mice treated with MEHP had a lower rate of testis weight gain (lower regression line) and a significant TUNEL-positive spermatogenic cell number compared to control. However, this incident was reversible, and the number of TUNEL-positive cells returned to normal after 9 days. Mice microinjected with annexin V and later treated with MEHP showed a large amount of TUNEL-positive cells compared to mice treated with MEHP only. This clearly proves that phagocytosis plays an efficient and highly important role in eliminating dead cells in the injured testis of mice treated with MEHP.     

Dynamic polymorphism of actin as activation mechanism for cell motility

Actin filament dynamics are crucial in cell motility. Actin filaments, and their bundles, networks, and gels assemble and disassemble spontaneously according to thermodynamic rules. These dynamically changing structures of actin are harnessed for some of its functions in cells. The actin systems respond to external signals, forces, or environments by biasing the fluctuation of actin assembly structures. In this study, dynamic conformation of actin molecules was studied by monitoring conformational dynamics of actin molecules at the single molecule level in real time. Actin conformation spontaneously fluctuates between multiple conformational states. Regarding myosin motility, the dynamic equilibrium of actin conformation was interpreted as between states that activates and inhibits the motility. The binding of myosin to actin filaments activates myosin motility by shifting the conformational fluctuation of actin towards the state that activates the motility. Thus, the activation mechanism based on thermal fluctuation is suggested at molecular level as well as at cellular level.     

Brownian motion, fluctuation and life

The measurements of dynamic behaviors of biomolecules in relation to their functions have been allowed using single molecule measurements. Thermal Brownian motion causes random step motion of motor proteins and structural fluctuation of protein molecules between multiple states. In hierarchic structure of life, the fluctuation is modulated. Random fluctuation is biased to directional motion and reactions as a result of interaction of proteins. The fluctuation of kinetic state of signaling proteins results in polarization and localization of cells. A recognition process in brain is also explained by the equation analogous to biochemical reaction at the molecular level. Thus dynamic processes originated from thermal motion may play an important role in activation processes in life.     

Inhibitory effects of excess sympathetic activity on parasympathetic vasodilation in the rat masseter muscle

The present study was designed to examine the effect of sympathetic tonic activity on parasympathetic vasodilation evoked by the trigeminal-mediated reflex in the masseter muscle in urethane-anesthetized rats. Sectioning of the superior cervical sympathetic trunk (CST) ipsilaterally increased the basal level of blood flow in the masseter muscle (MBF). Electrical stimulation of the peripheral cut end of the CST for 2 min using 2-ms pulses ipsilaterally decreased in a dependent manner the intensity (0.5-10 V) and frequency (0.1-5 Hz) of the MBF. The CST stimulation for 2 min at <0.5 Hz with 5 V using 2-ms pulses seems to be comparable with the spontaneous activity in the CST fibers innervating the masseter vasculature, because this stimulation restored the basal level of the MBF to the presectioned values. Parasympathetic vasodilation evoked by electrical stimulation of the central cut end of the lingual nerve in the masseter muscle was markedly reduced by CST stimulation for 2 min with 5 V using 2-ms pulses in a frequency-dependent manner (0.5-5 Hz). Intravenous administration of phentolamine significantly reduced the vasoconstriction induced by CST stimulation in a dose-dependent manner (0.1-1 mg/kg), but pretreatment with either phentolamine or propranolol failed to affect the sympathetic inhibition of the parasympathetic vasodilation. Our results suggest that 1) excess sympathetic activity inhibits parasympathetic vasodilation in the masseter muscle, and 2) alpha- and beta-adrenoceptors do not contribute to sympathetic inhibition of parasympathetic vasodilation, and thus some other types of receptors must be involved in this response.     

Reinforcement omission in concurrent fixed-interval and random-interval schedules

The present experiment examined overall and local effects of omission of reinforcers in a choice situation. Pigeons' key-pecking responses were reinforced under concurrent fixed-interval and random-interval schedules of food presentation. After some weeks of baseline sessions in which the probability of reinforcement was 1.00, approximately 25% of food presentations from the fixed-interval schedule were omitted and replaced by timeout periods. In such omission sessions, the overall relative rates of responding to the fixed-interval schedule became lower than those in the baseline sessions. On the other hand, when relative rates of responding to the fixed-interval schedule in the omission sessions were calculated separately for fixed-interval cycles preceded by timeout periods and those preceded by food presentations, the relative rates in the former type of fixed-interval cycles were higher than those in the latter type for three out of four pigeons. These results mean that relative rates of responding cannot always be regarded as reflecting a relative value of an alternative, and that the overall effect of the omission of fixed-interval reinforcers is not reducible to the local effect of omission.     

Evaluation of major membrane protein-II as a tool for serodiagnosis of leprosy

As serodiagnosis is the easiest way of diagnosing a disease, the utility of Mycobacterium leprae-derived major membrane protein-II (MMP-II), one of the immuno-dominant antigens, in the serodiagnosis of leprosy was examined. The percent positivity by an enzyme-linked immunosorbent assay for anti-MMP-II antibody was 82.4% for multi-bacillary leprosy, and the specificity of the test was 90.1%. For pauci-bacillary leprosy where cell-mediated immunity predominates, 39.0% showed positive results. These percentage values were significantly higher than these values obtained for existing phenolic glycolipid-I based methods, suggesting that MMP-II antibody detection would facilitate the diagnosis of leprosy.