Suppression of dynamin GTPase decreases alpha-synuclein uptake by neuronal and oligodendroglial cells: a potent therapeutic target for synucleinopathy

ABSTRACT: BACKGROUND: The intracellular deposition of misfolded proteins is a common neuropathological hallmark of most neurodegenerative disorders. Increasing evidence suggests that these pathogenic proteins may spread to neighboring cells and induce the propagation of neurodegeneration. RESULTS: In this study, we have demonstrated that alpha-synuclein (alphaSYN), a major constituent of intracellular inclusions in synucleinopathies, was taken up by neuronal and oligodendroglial cells in both a time- and concentration-dependent manner. Once incorporated, the extracellular alphaSYN was immediately assembled into high-molecular-weight oligomers and subsequently formed cytoplasmic inclusion bodies. Furthermore, alphaSYN uptake by neurons and cells of the oligodendroglial lineage was markedly decreased by the genetic suppression and pharmacological inhibition of the dynamin GTPases, suggesting the involvement of the endocytic pathway in this process. CONCLUSIONS: Our findings shed light on the mode of alphaSYN uptake by neuronal and oligodendroglial cells and identify therapeutic strategies aimed at reducing the propagation of protein misfolding.     

SWAP70 functions as a Rac/Rop guanine nucleotide-exchange factor in rice

Rho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1.     

Efficient Production of Optically Pure d-Lactic Acid from Raw Corn Starch by Using a Genetically Modified l-Lactate Dehydrogenase Gene-Deficient and α-Amylase-Secreting Lactobacillus plantarum Strain

In order to achieve direct and efficient fermentation of optically pure d-lactic acid from raw corn starch, we constructed l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 α-amylase (AmyA). The resulting strain produced only d-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct d-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct d-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct d-lactic acid fermentation from raw starch.Poly-lactic acid (PLA) is an important agro-based plastic that can be produced from inexpensive, renewable, and abundantly available biomass resources, including starchy materials. These resources have advantages over limited oil- and fossil-based sources, as they do not result in any net carbon dioxide release to the atmosphere (7). Recently, stereocomplex PLA, which is composed of both poly-l- and -d-lactic acid, has been attracting much attention due to its high thermostability. Stereocomplex-type polymers show a melting point (ca. 230°C) that is approximately 50°C higher than that of the respective single polymers (8). Therefore, d-lactic acid, in addition to l-lactic acid, which has been the focus of production to date, is of significant importance.Lactic acid bacteria (LAB) are promising microorganisms for the efficient production of lactic acid from various sugars, such as glucose, sucrose, and lactose. However, when starchy materials are used as a carbon source, they must be saccharified by physicochemical and enzymatic treatment because most LAB cannot utilize starchy materials directly (13). This makes the whole process less economically viable. Therefore, many researchers have examined the direct production of lactic acid from starchy materials by using wild amylolytic LAB (ALAB) (6, 24, 25) or genetically modified amylase-producing LAB (15, 16). Although d-lactic acid has been produced by fermentation from pretreated substrates such as rice starch (5) and by simultaneous saccharification and fermentation from cellulose (23), there have been no reports on the direct production of d-lactic acid from starchy materials. This is due to a lack of d-lactic acid-producing ALAB and difficulties in gene manipulation of d-lactic acid-producing LAB, such as Lactobacillus delbrueckii (22).We focused on Lactobacillus plantarum, which is an industrially important strain due to its environmental flexibility and its ability to assimilate a wide range of carbohydrates (9). In recent years, several gene manipulation methods for Lactobacillus plantarum have been established (18, 19). Moreover, the complete genome sequence has been decoded for L. plantarum NCIMB 8826 (9). Based on whole-genome analysis, L. plantarum possesses two types of lactate dehydrogenase (LDH), l-LDH and d-LDH, which convert pyruvate into l- and d-lactic acid, respectively. Ferain et al. (4) reported that chromosomal deletion in the ldhL1 gene of L. plantarum NCIMB 8826 provoked an absence of l-LDH activity and produced d-lactic acid from glucose.In the present study, to produce d-lactic acid directly from starch, we constructed an l-LDH-deficient, α-amylase-secreting L. plantarum strain. The engineered strain expressed α-amylase from Streptococcus bovis 148 (AmyA) (20) and efficiently degraded raw starch with the aid of a C-terminal starch-binding domain (11). Using this strain, we achieved the direct and efficient fermentation of optically pure d-lactic acid from raw corn starch.     

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl₂ selectively destroys Fe–S center B (F_B). The function of electron acceptors in F_B-less PS I particles was studied by following the decay kinetics of P700⁺ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t _1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t _1/20.25 ms). In F_B-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in F_B-less particles, electron transfer can proceed normally at room temperature from F_X to F_A and that the charge recombination between P700⁺ and F_X ^-/A₁ ^- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of F_B. Even in the presence of 60% glycerol, F_B-less particles can transfer electrons to F_A at room temperature as efficiently as untreated particles.Abbreviations DCIP 2, 6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur center A, B and X, respectively - PMS phenazine methosulfate     

Phenotypic Effect of “Candidatus Rickettsiella viridis,” a Facultative Symbiont of the Pea Aphid (Acyrthosiphon pisum), and Its Interaction with a Coexisting Symbiont

A gammaproteobacterial facultative symbiont of the genus Rickettsiella was recently identified in the pea aphid, Acyrthosiphon pisum. Infection with this symbiont altered the color of the aphid body from red to green, potentially affecting the host''s ecological characteristics, such as attractiveness to different natural enemies. In European populations of A. pisum, the majority of Rickettsiella-infected aphids also harbor another facultative symbiont, of the genus Hamiltonella. We investigated this Rickettsiella symbiont for its interactions with the coinfecting Hamiltonella symbiont, its phenotypic effects on A. pisum with and without Hamiltonella coinfection, and its infection prevalence in A. pisum populations. Histological analyses revealed that coinfecting Rickettsiella and Hamiltonella exhibited overlapping localizations in secondary bacteriocytes, sheath cells, and hemolymph, while Rickettsiella-specific localization was found in oenocytes. Rickettsiella infections consistently altered hosts'' body color from red to green, where the greenish hue was affected by both host and symbiont genotypes. Rickettsiella-Hamiltonella coinfections also changed red aphids to green; this greenish hue tended to be enhanced by Hamiltonella coinfection. With different host genotypes, Rickettsiella infection exhibited either weakly beneficial or nearly neutral effects on host fitness, whereas Hamiltonella infection and Rickettsiella-Hamiltonella coinfection had negative effects. Despite considerable frequencies of Rickettsiella infection in European and North American A. pisum populations, no Rickettsiella infection was detected among 1,093 insects collected from 14 sites in Japan. On the basis of these results, we discuss possible mechanisms for the interaction of Rickettsiella with other facultative symbionts, their effects on their hosts'' phenotypes, and their persistence in natural host populations. We propose the designation “Candidatus Rickettsiella viridis” for the symbiont.     

DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts

Escherichia coli DNA polymerase IV (Pol IV, also known as DinB) is a Y-family DNA polymerase capable of catalyzing translesion DNA synthesis (TLS) on certain DNA lesions, and accumulating data suggest that Pol IV may play an important role in copying various kinds of spontaneous DNA damage including N²-dG adducts and alkylated bases. Pol IV has a unique ability to coexist with Pol III on the same β clamp and to positively dissociate Pol III from β clamp in a concentration-dependent manner. Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (−)-trans-anti-benzo[a]pyrene-N²-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III. Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption.     

Why animals respond to the full moon: Magnetic hypothesis

The geomagnetic field is typically about 50 μT (range 20-90 μT). Geomagnetic activity generally decreases by about 4% for the seven days leading up to a full moon, and increases by about 4% after the full moon, lasting for seven days. Animals can clearly detect the changes in magnetic field intensity that occur at full moon, as it has been shown that variations of just a few tens of nT are adequate to form a useful magnetic ‘map’. We think that moonlight increases the sensitivity of animals' magnetoreception because the radical pair model predicts that magnetoreception is light dependent. In fact, there have been some reports of changes in the sensitivity of magnetoreception with lunar phase. We propose a hypothesis that animals respond to the full moon because of changes in geomagnetic fields, and that the sensitivity of animals' magnetoreception increases at this time.     

Does Enterohemorrhagic Escherichia coli O157:H7 Enter the Viable but Nonculturable State in Salted Salmon Roe?

An outbreak caused by salted salmon roe contaminated with enterohemorrhagic Escherichia coli O157 occurred in Japan in 1998. Since about 0.75 to 1.5 viable cells were estimated to cause infection, we presumed that O157 might enter the viable but nonculturable (VNC) state in salted salmon roe and consequently that viable cell numbers might be underestimated. Although patient-originating O157 cells could not grow on agar plates after 72 h of incubation in 13% NaCl, they were resuscitated in yeast extract broth, and more than 90% of the cells were shown to be viable by fluorescent staining, suggesting that almost all of them could enter the VNC state in NaCl water. Roe-originating O157 was resistant to NaCl because it could grow on agar after 72 h of incubation in NaCl water, but about 20% of cells appeared to enter the VNC state. Therefore, germfree mice were infected with O157 to examine the resuscitation of cells in the VNC state and the retention of pathogenicity. O157 that originated in roe, but not patients, killed mice and was isolated from the intestine. However, these isolates had become sensitive to NaCl. O157 cells of roe origin incubated in normal media also killed mice and were isolated from the intestine, but they also became transiently NaCl sensitive. We therefore propose that bacterial cells might enter the VNC state under conditions of stress, such as those encountered in vivo or in high salt concentrations, and then revive when those conditions have eased. If so, the VNC state in food is potentially dangerous from a public health viewpoint and may have to be considered at the time of food inspection. Finally, the establishment of a simple recovery system for VNC cells should be established.     

Immunological properties of monoclonal antibodies to human and rat prolyl 4-hydroxylase

Monoclonal antibodies to human (8 clones) and rat (12 clones) prolyl 4-hydroxylase [EC 1.14.11.2] were prepared and characterized as regards subclass, subunit specificity, inhibition and crossreactivity. Among the antibodies to the human enzyme, four clones showed the IgG1 subclass, two IgA, one IgG2b, and one IgM. Four clones reacted with the alpha subunit of the enzyme, while the others reacted with the beta subunit. The enzymatic activity was inhibited by four clones. Five clones crossreacted with the rat enzyme. One clone inhibited the rat enzyme. Among the antibodies to the rat enzyme, seven clones showed the IgG1 subclass, four IgG2a and one IgG2b. Seven clones reacted with the alpha subunit, and four with the beta subunit. One reacted with neither subunit. The enzymatic activity was inhibited by seven clones. Seven clones crossreacted with the human enzyme. Three clones inhibited the human enzyme.     

Inhibitory effect of RNAi on Japanese encephalitis virus replication in vitro and in vivo

Flaviviruses include many insect-mediated small viruses and still cause serious problems in the world. In humans, JEV can cause acute meningioencephalomyelitis, resulting in fatality rates of 5 to 40%. RNA-interference (RNAi) as an antiviral mechanism was originally discovered in plants and then found in the specific suppression of gene expression of other organisms such as Caenorhabditis elegans, Drosophila and vertebrates. As JEV is an RNA virus, RNAi could be a reasonable approach for therapeutic purposes to use against Japanese encephalitis. In this study, we examined the effect of RNAi on JEV replication. Viral reproduction in Vero cells was decreased to 7.2% and 39.0% of control by the transfection of small interference RNAs, JCR and JN3R at 250 n M, respectively. Under the transfection of 5 microg/ml pJRi which produces stem-loop RNAi, viral reproduction was decreased to about 10% of control. Western blot analysis indicated that RNAi inhibited the translation level. We used pJRi in the animal experiment. After the inoculation of viruses at 5 x 10(3) PFU, pJRi at 1.0 and 5.0 microg/g was injected into mice i.p. JEV-infected control mice (n=5) died within 15 days. pJRi (1.0 or 5.0 microg/g)-medicated mice survived 40 or 80% at 15 days. The data clearly indicate that pJRi has highly potent inhibitory activity against JEV replication in vivo. The results in vivo and in vitro provide evidence that JEV replication was efficiently inhibited by RNAi and RNAis could be used as an antiviral drug against JEV infection.