Prenatal Exposure to Histone Deacetylase Inhibitors Affects Gene Expression of Autism-Related Molecules and Delays Neuronal Maturation

Valproic acid (VPA) is a multi-target drug and an inhibitor of histone deacetylase (HDAC). We have previously demonstrated that prenatal exposure to VPA at embryonic day 12.5 (E12.5), but not at E14.5, causes autism-like behavioral abnormalities in male mouse offspring. We have also found that prenatal VPA exposure causes transient histone hyperacetylation in the embryonic brain, followed by decreased neuronal cell numbers in the prefrontal and somatosensory cortices after birth. In the present study, we examined whether prenatal HDAC inhibition affects neuronal maturation in primary mouse cortical neurons. Pregnant mice were injected intraperitoneally with VPA (500 mg/kg) and the more selective HDAC inhibitor trichostatin A (TSA; 500 µg/kg) at E12.5 or E14.5, and primary neuronal cultures were prepared from the cerebral cortices of their embryos. Prenatal exposure to VPA at E12.5, but not at E14.5, decreased total number, total length, and complexity of neuronal dendrites at 14 days in vitro (DIV). The effects of VPA weakened at 21 DIV. Exposure to TSA at E12.5, but not at E14.5, also delayed maturation of cortical neurons. In addition, real-time quantitative PCR revealed that the prenatal exposure to TSA decreased neuroligin-1 (Nlgn1), Shank2, and Shank3 mRNA levels and increased contactin-associated protein-like 2 mRNA level. The delay in neuronal maturation was also observed in Nlgn1-knockdown cells, which were transfected with Nlgn1 siRNA. These findings suggest that prenatal HDAC inhibition causes changes in gene expression of autism-related molecules linked to a delay of neuronal maturation.     

Structural stability of flagellin subunit affects the rate of flagellin export in the absence of FliS chaperone

FliS chaperone binds to flagellin FliC in the cytoplasm and transfers FliC to a sorting platform of the flagellar type III export apparatus through the interaction between FliS and FlhA for rapid and efficient protein export during flagellar filament assembly. FliS also suppresses the secretion of an anti‐σ factor, FlgM. Loss of FliS results in a short filament phenotype although the expression levels of FliC are increased considerably due to an increase in the secretion level of FlgM. Here to clarify the rate limiting step of FliC export in the absence of FliS, we isolated bypass mutants from a Salmonella ΔfliS mutant. All the bypass mutations were identified in FliC. These bypass mutations increased the export rate of FliC by ca. twofold, allowing the bypass mutant cells to produce longer filaments than the parental ΔfliS cells. Both far‐UV CD measurements and limited proteolysis revealed that the bypass mutations significantly destabilize the folded structure of FliC monomer. These results suggest that an unfolding step of FliC limits the export rate of FliC in the ΔfliS mutant, thereby producing short filaments. We propose that FliS promotes FliC docking at the FlhA platform to facilitate subsequent unfolding of FliC.     

CRISPR/Cas9 in rice can induce new mutations in later generations,leading to chimerism and unpredicted segregation of the targeted mutation

CRISPR/Cas9 is a novel tool for targeted mutagenesis and is applicable to plants, including rice. Previous reports on CRISPR/Cas9 in rice have demonstrated that target mutations are transmitted to the next generation in accordance with Mendelian law, but heritability of the target mutation and the role of inherited Cas9 gene have not been fully elucidated. Here, we targeted the rice phytoene desaturase gene, mutants of which exhibit an albino phenotype, by using CRISPR/Cas9 and analyzed segregation of target mutations. Agrobacterium-mediated methods using immature embryos successfully transformed a CRISPR/Cas9 system into five rice cultivars and subsequently induced mutation. Unpredicted segregations, with more mutants than theoretically predicted, were frequently found in T₁ plants from monoallelic T₀ mutants. Chimeric plants with both biallelic and monoallelic mutated cells were also observed in the T₁. Next, we followed segregation of a target mutation in the T₂ from monoallelic T₁ mutants. When T₁ mutants possessed Cas9, unpredicted segregations of the target mutation and chimeric plants were observed again in the T₂. When T₁ mutants did not possess Cas9, segregation of the target mutations followed Mendelian law and no chimeric plants appeared in the T₂. T₂ mutants with Cas9 had mutations different from the original ones found in T₀. Our results indicated that inherited Cas9 was still active in later generations and could induce new mutations in the progeny, leading to chimerism and unpredicted segregation. We conclude that Cas9 has to be eliminated by segregation in T₁ to generate homozygous mutants without chimerism or unpredicted segregation.     

Glucose-6-phosphate dehydrogenase cytochemistry using a copper ferrocyanide method and its application to rapidly frozen cells

We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett’s brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells. Accepted: 14 July 1999     

Regulation of progenitor cell survival by a novel chromatin remodeling factor during neural tube development

During development, progenitor cell survival is essential for proper tissue functions, but the underlying mechanisms are not fully understood. Here we show that ERCC6L2, a member of the Snf2 family of helicase-like proteins, plays an essential role in the survival of developing chick neural cells. ERCC6L2 expression is induced by the Sonic Hedgehog (Shh) signaling molecule by a mechanism similar to that of the known Shh target genes Ptch1 and Gli1. ERCC6L2 blocks programmed cell death induced by Shh inhibition and this inhibition is independent of neural tube patterning. ERCC6L2 knockdown by siRNA resulted in the aberrant appearance of apoptotic cells. Furthermore, ERCC6L2 cooperates with the Shh signal and plays an essential role in the induction of the anti-apoptotic factor Bcl-2. Taken together, ERCC6L2 acts as a key factor in ensuring the survival of neural progenitor cells.     

Isolation and spectral characterization of thermally generated multi-Z-isomers of lycopene and the theoretically preferred pathway to di-Z-isomers

Lycopene has a large number of geometric isomers caused by E/Z isomerization at arbitrary sites within the 11 conjugated double bonds, offering varying characteristics related to features such as antioxidant capacity and bioavailability. However, the geometric structures of only a few lycopene Z-isomers have been thoroughly identified from natural sources. In this study, seven multi-Z-isomers of lycopene, (9Z,13′Z)-, (5Z,13Z,9′Z)-, (9Z,9′Z)-, (5Z,13′Z)-, (5Z,9′Z)-, (5Z,9Z,5′Z)-, and (5Z,9Z)-lycopene, were obtained from tomato samples by thermal isomerization, and then isolated by elaborate chromatography, and fully assigned using proton nuclear magnetic resonance. Moreover, the theoretically preferred pathway from (all-E)-lycopene to di-Z-isomers was examined with a computational approach using a Gaussian program. Fine-tuning of the HPLC separation conditions led to the discovery of novel multi-Z-isomers, and whose formation was supported by advanced theoretical calculations.     

Exclusion of assembled MreB by anionic phospholipids at cell poles confers cell polarity for bidirectional growth

Cell polarity determines the direction of cell growth in bacteria. MreB actin spatially regulates peptidoglycan synthesis to enable cells to elongate bidirectionally. MreB densely localizes in the cylindrical part of the rod cell and not in polar regions in Escherichia coli. When treated with A22, which inhibits MreB polymerization, rod‐shaped cells became round and MreB was diffusely distributed throughout the cytoplasmic membrane. A22 removal resulted in restoration of the rod shape. Initially, diffuse MreB started to re‐assemble, and MreB‐free zones were subsequently observed in the cytoplasmic membrane. These MreB‐free zones finally became cell poles, allowing the cells to elongate bidirectionally. When MreB was artificially located at the cell poles, an additional pole was created, indicating that artificial localization of MreB at the cell pole induced local peptidoglycan synthesis. It was found that the anionic phospholipids (aPLs), phosphatidylglycerol and cardiolipin, which were enriched in cell poles preferentially interact with monomeric MreB compared with assembled MreB in vitro. MreB tended to localize to cell poles in cells lacking both aPLs, resulting in production of Y‐shaped cells. Their findings indicated that aPLs exclude assembled MreB from cell poles to establish cell polarity, thereby allowing cells to elongate in a particular direction.     

Inhibitory effect of the alkyl side chain of caffeic acid analogues on lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages

Caffeic acid esters, one of the components of propolis, are known to show a variety of biological effects such as anti-tumor, anti-oxidant, and anti-inflammatory activities. Although, the anti-inflammatory activities of caffeic acid esters have been studied by analyzing their structure, the detailed mechanisms of their activities remain unclear. Thus, in this study, we examined the function of the ester functional group and the alkyl side chain (alcoholic part) and transformed caffeic acid to several derivatives. The inhibitory effect of these derivatives on NO production in murine macrophage RAW264.7 cells was dependent on the length and size of the alkyl moiety, and undecyl caffeate was the most potent inhibitor of NO production. In addition, individual experiments using undecanol, caffeic acid, undecanol plus caffeic acid, and undecyl caffeate showed that the connection between caffeic acid and the alkyl chain is critical for activity. Amide and ketone derivatives showed that not only the ester functional group but also the amide and ketone functional groups exhibit an inhibitory effect on NO production.     

Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

Molecular Basis for E-cadherin Recognition by Killer Cell Lectin-like Receptor G1 (KLRG1)

The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca²⁺-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K_d ∼7–12 μm), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1⁺NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.Natural killer (NK)³ cells play a critical role in the innate immune system because of their ability to kill other cells. For example, NK cells can kill virus-infected cells and tumor cells without presensitization to a specific antigen, and they produce various cytokines, including interferon-γ and tumor necrosis factor-α (1). NK cells are controlled by both inhibitory and activating receptors that are expressed on their surfaces (2). The killer cell Ig-like receptor, Ly49, CD94/NKG2, and paired Ig-like type 2 receptor families include both inhibitory and activating members and thus are designated as paired receptor families. On the other hand, some inhibitory receptors, including KLRG1 (killer cell lectin-like receptor G1), and activating receptors, such as NKG2D, also exist. The integration of the signals from these receptors determines the final functional outcome of NK cells.These inhibitory and activating receptors can also be divided into two structurally different groups, the Ig-like receptors and the C-type lectin-like receptors, based on the structural aspects of their extracellular regions. The Ig-like receptors include killer cell Ig-like receptors and the leukocyte Ig-like receptors, and the C-type lectin-like receptors include CD94/NKG2(KLRD/KLRC), Ly49(KLRA), NKG2D(KLRK), NKR-P1(KLRB), and KLRG1. Many of these immune receptors recognize major histocompatibility complex class I molecules or their relatives (2–4), but there are still many orphan receptors expressed on NK cells. KLRG1 was one such orphan receptor; however, E-cadherin was recently found to be a ligand of KLRG1 (5, 6). Although major histocompatibility complex-receptor interactions have been extensively examined, the molecular basis of non-major histocompatibility complex ligand-receptor recognition is poorly understood.KLRG1 is a type II membrane protein, with one C-type lectin domain in the extracellular region, one transmembrane region, and one immunoreceptor tyrosine-based inhibitory motif. KLRG1 is expressed on a subset of mature NK cells in spleen, lungs, and peripheral blood during normal development. KLRG1 expression is induced on the surface of NK cells during viral responses (7, 8). NK cells expressing KLRG1 produce low levels of interferon-γ and cytokines and have a slow in vivo turnover rate and low proliferative responsiveness to interleukin-15 (9). Furthermore, KLRG1 is recognized as a marker of some T cell subsets, as follows. KLRG1 defines a subset of T cells, short lived effector CD8 T cells (SLECs), which are mature effector cells that express high levels of KLRG1 and cannot be differentiated into long lived memory CD8 T cells. In addition, memory precursor effector cells express low levels of KLRG1 and harbor the potential to become long lived memory CD8 T cells (10). Since SLECs exhibit stronger effector function than memory precursor effector cells, it is potentially beneficial, in terms of preventing harmful excess cytotoxicity, that SLECs express KLRG1 at a higher level to inhibit the immune response. Taken together, the expression of KLRG1 during the viral response and normal development might confer the inhibition of effector function and the regulation of NK and T cell proliferation (9).E-cadherin plays a pivotal role in Ca²⁺-dependent cell-cell adhesion and also contributes to tissue organization and development (11–14). E-cadherin is primarily expressed on epithelial cells, and its extracellular region consists of several domains that include cadherin motifs (15, 16). These domains mediate Ca²⁺-dependent homophilic interactions to facilitate cell adhesion. When E-cadherins form cis- or trans-homodimers, they utilize their N-terminal regions as an interface, which can dock with domain 1 of another E-cadherin to form strand exchange (17). Therefore, the N-terminal region plays important roles in homophilic binding and cell adhesion.KLRG1 recognizes E-cadherins (and other class I cadherins), which are widely expressed in tissues and form tight adhesive cell-cell junctions, and Ito et al. (5) demonstrated that E-cadherin binding by KLRG1 inhibits NK cytotoxicity. Further, Gründermann et al. (6) showed that the E-cadherin-KLRG1 interaction inhibits the antigen-induced proliferation and induction of the cytolytic activity of CD8 T cells. Therefore, it is plausible that E-cadherin recognition by KLRG1, expressed on the surfaces of NK cells and T cells, may raise their activation thresholds by transducing inhibitory signals. Such an inhibition would prevent the excess injury of normal cells, which might result in inflammatory autoimmune diseases. KLRG1 may also have an important role in monitoring and removing cancer cells that lose E-cadherin expression. A recent report demonstrated that N-terminal domains 1 and 2 of E-cadherin are critical for KLRG1 recognition (18); however, despite accumulating evidence supporting the functional importance of the E-cadherin-KLRG1 interaction, the molecular basis of this interaction is poorly understood. Here, we report that the N-terminal region of E-cadherin, comprising the dimer interface, is the binding site for KLRG1. This suggests that KLRG1 does not recognize the dimeric form of E-cadherin but rather recognizes the monomeric form, which is exposed on the cell surfaces of disrupted or infected cells. This may suppress excess immune responses.