University of Shizuoka

INTRODUCTION To combat invading pathogens, cells develop an adaptive immune response by changing their own genetic information. In vertebrates, the generation of genetic variation (somatic hypermutation) is an essential process for diversification and affinity maturation of antibodies that function to detect and sequester various foreign biomolecules. The activation-induced cytidine deaminase (AID) carries out hypermutation by modifying deoxycytidine bases in the variable region of the immunoglobulin locus that produces antibody. AID-generated deoxyuridine in DNA is mutagenic as it can be miss-recognized as deoxythymine, resulting in C to T mutations. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) is a prokaryotic adaptive immune system that records and degrades invasive foreign DNA or RNA. The CRISPR/Cas system cleaves and incorporates foreign DNA/RNA segments into the genomic region called the CRISPR array. The CRISPR array is transcribed to produce crispr-RNA that serves as guide RNA (gRNA) for recognition of the complementary foreign DNA/RNA in a ribonucleoprotein complex with Cas proteins, which degrade the target. The CRISPR/Cas system has been repurposed as a powerful genome editing tool, because it can be programmed to cleave specific DNA sequence by providing custom gRNAs. RATIONALE Although the precise mechanism by which AID specifically mutates the immunoglobulin locus remains elusive, targeting of AID activity is facilitated by the formation of a single-stranded DNA region, such as a transcriptional RNA/DNA hybrid (R-loop). The CRISPR/Cas system can be engineered to be nuclease-inactive. The nuclease-inactive form is capable of unfolding the DNA double strand in a protospacer adjacent motif (PAM) sequence-dependent manner so that the gRNA binds to complementary target DNA strand and forms an R-loop. The nuclease-deficient CRISPR/Cas system may serve as a suitable DNA-targeting module for AID to catalyze site-specific mutagenesis. RESULTS To determine whether AID activity can be specifically targeted by the CRISPR/Cas system, we combined dCas9 (a nuclease-deficient mutant of Cas9) from Streptococcus pyogenes and an AID ortholog, PmCDA1 from sea lamprey, to form a synthetic complex (Target-AID) by either engineering a fusion between the two proteins or attaching a SH3 (Src 3 homology) domain to the C terminus of dCas9 and a SHL (SH3 interaction ligand) to the C terminus of PmCDA1. Both of these complexes performed highly efficient site-directed mutagenesis. The mutational spectrum was analyzed in yeast and demonstrated that point mutations were dominantly induced at cytosines within the range of three to five bases surrounding the –18 position upstream of the PAM sequence on the noncomplementary strand to gRNA. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced in the Target-AID complexes. Combination of PmCDA1 with the nickase Cas9(D10A) mutant, which retains cleavage activity for noncomplementary single-stranded DNA, was more efficient in yeast but also induced deletions as well as point mutations in mammalian cells. Addition of the uracil DNA glycosylase inhibitor protein, which blocks the initial step of the uracil base excision repair pathway, suppressed collateral deletions and further improved targeting efficiency. Potential off-target effects were assessed by whole-genome sequencing of yeast as well as deep sequencing of mammalian cells for regions that contain mismatched target sequences. These results showed that off-target effects were comparable to those of conventional CRISPR/Cas systems, with a reduced risk of indel formation. CONCLUSION By expanding the genome editing potential of the CRISPR/Cas9 system by deaminase-mediated hypermutation, Target-AID demonstrated a very narrow range of targeted nucleotide substitution without the use of template DNA. Nickase Cas9 and uracil DNA glycosylase inhibitor protein can be used to boost the targeting efficiency. The reduced cytotoxicity will be beneficial for use in cells that are sensitive to artificial nucleases. Use of other types of nucleotide-modifying enzymes and/or other CRISPR-related systems with different PAM requirements will expand our genome-editing repertoire and capacity. A crippled CRISPR/Cas targets AID. In vertebrate adaptive immunity, cytosine deaminase (AID or PmCDA1) induces somatic hypermutation at single-stranded DNA regions formed during transcription. The bacterial CRISPR/Cas9 immunity system recognizes and cleaves invasive DNA in a gRNA-dependent manner. AID and nuclease-deficient CRISPR/Cas9 are engineered to form a hybrid complex (Target-AID) that performs programmable cytosine mutations in a range of a few bases surrounding the –18 position upstream of PAM sequence of the noncomplementary DNA strand.

Abscisic acid (ABA), a plant hormone, is involved in responses to environmental stresses such as drought and high salinity, and is required for stress tolerance. ABA is synthesized de novo in response to dehydration. 9-cis-epoxycarotenoid dioxygenase (NCED) is thought to be a key enzyme in ABA biosynthesis. Here we demonstrate that the expression of an NCED gene of Arabidopsis, AtNCED3, is induced by drought stress and controls the level of endogenous ABA under drought-stressed conditions. Overexpression of AtNCED3 in transgenic Arabidopsis caused an increase in endogenous ABA level, and promoted transcription of drought- and ABA-inducible genes. Plants overexpressing AtNCED3 showed a reduction in transpiration rate from leaves and an improvement in drought tolerance. By contrast, antisense suppression and disruption of AtNCED3 gave a drought-sensitive phenotype. These results indicate that the expression of AtNCED3 plays a key role in ABA biosynthesis under drought-stressed conditions in Arabidopsis. We improved drought tolerance by gene manipulation of AtNCED3 causing the accumulation of endogenous ABA.

We made a series of improved Gateway binary vectors (pGWBs) for plant transformation. Fifteen different reporters and tags, sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, and TAP, were employed. Some vectors carry the 2x35S-Omega promoter for higher-level expression. The kanamycin- and hygromycin-resistant markers are independently available for each of the 43 types of vectors, thus an additional transformation of once-transformed plants can be carried out easily. Their small size and high-copy number in Escherichia coli make possible easier handling at plasmid preparation and sequencing. Improved pGWBs should be a powerful tool for transgenic research in plants.

Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na(+)/H(+) exchange. Yet, the functional relationship between Na(+)/H(+) exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na(+)/H(+) exchange, elevating cytosolic pH and stimulating Na(+) influx. Remarkably, although inhibition of Na(+)/H(+) exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na(+) influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na(+)/H(+) exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na(+)/H(+) exchange.

Triple tandemly repeated sequences and the corresponding complementary sequence are known to exist in the 5'-terminal regulatory region of the human gene for thymidylate synthase (TS). To examine the function of these sequences, a set of deletion mutants was prepared and used in a transient expression assay. The results showed that at least one repeated sequence and its complementary sequence were necessary for the efficient expression of the gene. As another approach to understanding the function of this unique structure, DNA polymorphism in the same region was analyzed. In addition to the TS gene with the triple tandem repeat, the TS gene with a double tandem repeat was found in genomes of normal human subjects at an estimated frequency of 19% when genomes of 21 unrelated Japanese were analyzed. The expression activity of a reporter gene linked to the promoter region of the human TS genes with the two types of repeated sequence was examined and the result showed that the expression activity of the gene with the double repeat was lower than that of the gene with the triple repeat in the transient expression assay. Thus, it appears that the unique repeated sequences in the 5'-terminal region of the human TS gene are polymorphic and contribute to the efficiency of expression of the gene.

The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the alpha2,6(SAalpha2,6Gal) linkage, while equine viruses favor SAalpha2,3Gal. However, whether a difference in relative abundance of specific SA species (N-acetylneuraminic acid [NeuAc] and N-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcalpha2,6Gal but not NeuAcalpha2,3Gal or NeuGcalpha2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcalpha2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcalpha2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.

We report that simple, synthetic organic polymer nanoparticles (NPs) can capture and clear a target peptide toxin in the bloodstream of living mice. The protein-sized polymer nanoparticles, with a binding affinity and selectivity comparable to those of natural antibodies, were prepared by combining a functional monomer optimization strategy with molecular-imprinting nanoparticle synthesis. As a result of binding and removal of melittin by NPs in vivo, the mortality and peripheral toxic symptoms due to melittin were significantly diminished. In vivo imaging of the polymer nanoparticles (or "plastic antibodies") established that the NPs accelerate clearance of the peptide from blood and accumulate in the liver. Coupled with their biocompatibility and nontoxic characteristics, plastic antibodies offer the potential for neutralizing a wide range of biomacromolecules in vivo.

Synthesis, degradation, and transport of proline (Pro) are thought to cooperatively control its endogenous levels in higher plants in response to environmental conditions. To evaluate the function of Pro degradation in the regulation of the levels of Pro and to elucidate roles of Pro in stress tolerance, we generated antisense transgenic Arabidopsis plants with an AtProDH cDNA encoding proline dehydrogenase (ProDH), which catalyzes Pro degradation. Several transgenic lines accumulated Pro at higher levels than wild-type plants, providing evidence for a key role of ProDH in Pro degradation in Arabidopsis. These antisense transgenics were more tolerant to freezing and high salinity than wild-type plants, showing a positive correlation between Pro accumulation and stress tolerance in plants.

Curcumin is a polyphenol that is commonly used for its perceived health benefits. However, the absorption efficacy of curcumin is too low to exhibit beneficial effects. We have successfully developed a highly absorptive curcumin dispersed with colloidal nano-particles, and named it THERACURMIN. The absorption efficacy of THERACURMIN was investigated and compared with that of curcumin powder. The area under the blood concentration-time curve (AUC) after the oral administration of THERACURMIN was found to be more than 40-fold higher than that of curcumin powder in rats. Then, healthy human volunteers were administered orally 30 mg of THERACURMIN or curcumin powder. The AUC of THERACURMIN was 27-fold higher than that of curcumin powder. In addition, THERACURMIN exhibited an inhibitory action against alcohol intoxication after drinking in humans, as evidenced by the reduced acetaldehyde concentration of the blood. These findings demonstrate that THERACURMIN shows a much higher bioavailability than currently available preparations. Thus, THERACURMIN may be useful to exert clinical benefits in humans at a lower dosage.

L-arginine causes insulin release from pancreatic B cells. Data from three model systems support the hypothesis that L-arginine-derived nitrogen oxides (NOs) mediate insulin release stimulated by L-arginine in the presence of D-glucose and by the hypoglycemic drug tolbutamide. The formation of NO in pancreatic B cells was detected both chemically and by the NO-induced accumulation of guanosine 3',5'-monophosphate. NG-substituted L-arginine analogs inhibited the release of both insulin and NO. Protein immunoblot and histochemical analysis with antiserum to type I NO synthase suggest that the formation of NO in pancreatic B cells is catalyzed by an NADPH- (reduced form of nicotinamide adenine dinucleotide phosphate), Ca2+/calmodulin-dependent type I NO synthase of about 150 kilodaltons.

The gene pool of influenza A viruses in aquatic birds provides all of the genetic diversity required for human and lower animals. Host range selection of the receptor binding specificity of the influenza virus hemagglutinin occurs during maintenance of the virus in different host cells that express different receptor sialo-sugar chains. In this paper, functional roles of the hemagglutinin and neuraminidase spikes of influenza viruses are described in the relation to 1) host range of influenza viruses, 2) receptor binding specificity of human and other animal influenza viruses, 3) recognition of sialyl sugar chains by Spanish influenza virus hemagglutinin, 4) highly pathogenic and potentially pandemic H5N1, H9N2, and H7N7 avian influenza viruses and molecular mechanism of host range variation of influenza viruses, 5) role of the neuraminidase spike for the host range of influenza viruses, and 6) Development of anti-influenza drugs.

Oxidative cyclizations are important transformations that occur widely during natural product biosynthesis. The transformations from acyclic precursors to cyclized products can afford morphed scaffolds, structural rigidity, and biological activities. Some of the most dramatic structural alterations in natural product biosynthesis occur through oxidative cyclization. In this Review, we examine the different strategies used by nature to create new intra(inter)molecular bonds via redox chemistry. This Review will cover both oxidation- and reduction-enabled cyclization mechanisms, with an emphasis on the former. Radical cyclizations catalyzed by P450, nonheme iron, α-KG-dependent oxygenases, and radical SAM enzymes are discussed to illustrate the use of molecular oxygen and S-adenosylmethionine to forge new bonds at unactivated sites via one-electron manifolds. Nonradical cyclizations catalyzed by flavin-dependent monooxygenases and NAD(P)H-dependent reductases are covered to show the use of two-electron manifolds in initiating cyclization reactions. The oxidative installations of epoxides and halogens into acyclic scaffolds to drive subsequent cyclizations are separately discussed as examples of "disappearing" reactive handles. Last, oxidative rearrangement of rings systems, including contractions and expansions, will be covered.

The effect of hepatocyte growth factor/scatter factor (HGF/SF) and epidermal growth factor (EGF) on cadherin-mediated adhesion of human carcinoma cells was studied. HGF/SF induced scattering of colonic adenocarcinoma HT29 and gastric adenocarcinomas MKN7 and MKN74 cells. Likewise, EGF induced scattering of HT29 and MKN7 cells. These cells expressed E-cadherin, which was concentrated at cell-cell contact sites. When the scattering of these cells was induced by HGF/SF or EGF, the E-cadherin concentration at cell-cell boundaries tended to decrease. Immunoblotting analyses, however, demonstrated that these growth factor treatments did not alter the expression of E-cadherin and E-cadherin-associated proteins, alpha- and beta-catenin and plakoglobin. beta-Catenin, plakoglobin and an unidentified 115-kDa molecule associated with E-cadherin were found to be phosphorylated at tyrosine residues, and these phosphorylations were enhanced by the growth factor treatments. These results suggest that HGF/SF and EGF may modulate the function of the cadherin-catenin system via tyrosine phosphorylation of cadherin-associated proteins.

RATIONALE: Clonal hematopoiesis has been associated with increased mortality and cardiovascular disease. This condition can arise from somatic mutations in preleukemic driver genes within hematopoietic stem/progenitor cells. Approximately 40 candidate driver genes have been identified, but mutations in only 1 of these genes, TET2 (ten-eleven translocation-2), has been shown to casually contribute to cardiovascular disease in murine models. OBJECTIVE: To develop a facile system to evaluate the disease characteristics of different clonal hematopoiesis driver genes using lentivirus vector and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) methodology. Using this methodology, evaluate whether Dnmt3a (DNA [cytosine-5]-methyltransferase 3a)-a commonly occurring clonal hematopoiesis driver gene-causally contributes to cardiovascular disease. METHODS AND RESULTS: Lentivirus vectors were used to deliver Cas9 and guide RNA to introduce inactivating mutations in Tet2 and Dnmt3a in lineage-negative bone marrow cells. After implantation into lethally irradiated mice, these cells were engrafted and gave rise to labeled blood cell progeny. When challenged with an infusion of Ang II (angiotensin II), mice with inactivating mutations in Tet2 or Dnmt3a displayed greater cardiac hypertrophy, diminished cardiac function, and greater cardiac and renal fibrosis. In comparison with Tet2, inactivation of Dnmt3a did not lead to detectable expansion of the mutant hematopoietic cells during the time course of these experiments. Tet2 inactivation promoted the expression of IL (interleukin) 1β, IL-6, and Ccl5, whereas Dnmt3a inactivation promoted the expression of Cxcl1 (CXC chemokine ligand), Cxcl2, IL-6, and Ccl5 in a lipopolysaccharide-stimulated macrophage cell line. CONCLUSIONS: Experiments using lentivirus vector/CRISPR methodology provided evidence suggesting that inactivating DNMT3A mutations in hematopoietic cells contributes to cardiovascular disease. Comparative analyses showed that inactivation of Tet2 and Dnmt3 was similar in their ability to promote Ang II-induced cardiac dysfunction and renal fibrosis in mice. However, gene-specific actions were indicated by differences in kinetics of hematopoietic stem/progenitor cell expansion and different patterns of inflammatory gene expression.

In this study, we investigated the intestinal absorption of luteolin and luteolin 7-O-beta-glucoside in rats by HPLC. The absorption analysis using rat everted small intestine demonstrated that luteolin was converted to glucuronides during passing through the intestinal mucosa and that luteolin 7-O-beta-glucoside was absorbed after hydrolysis to luteolin. Free luteolin, its conjugates and methylated conjugates were present in rat plasma after dosing. This suggests that some luteolin can escape the intestinal conjugation and the hepatic sulfation/methylation. LC/MS analysis showed that the main conjugate which circulates in the blood was a monoglucuronide of the unchanged aglycone. Luteolin in propyleneglycol was absorbed more rapidly than that in 0.5% carboxymethyl cellulose. The plasma concentration of luteolin and its conjugates reached the highest level 15 min and 30 min after dosing with luteolin in propyleneglycol, respectively. HPLC analysis also allowed us to demonstrate the presence of free luteolin and its monoglucuronide in human serum after ingestion of luteolin.

Zinc is essential for the growth of the human and other animals. Bone growth retardation is a common finding in various conditions associated with zinc deficiency, suggesting a physiological role of zinc in the growth and mineralization of bone tissue. Bone zinc content is decreased by development with aging, skeletal unloading, and postmenopausal conditions. Zinc deficiency may play a pathophysiological role in the deterioration of bone metabolism. Zinc has been demonstrated to have a stimulatory effect on bone formation and mineralization; the metal directly activates aminoacyl-tRNA synthetase in osteoblastic cells, and it stimulates cellular protein synthesis. Moreover, zinc inhibits osteoclastic bone resorption by inhibiting osteoclast-like cell formation from marrow cells. Zinc may act on the process of bone-resorbing factors-induced protein kinase C activation, which is involved in Ca2+ signaling in osteoclastic cells. Zinc plays a role in the preservation of bone mass. AHZ is a zinc compound, in which zinc is chelated to β-alanyl-L-histidine. The stimulatory effect of AHZ on bone formation was more intensive than that of zinc sulfate. It is confirmed that bone-forming effect of AHZ is a greater than that of various bone-regulating hormones and other factors. The oral administration of AHZ has a fine restorative effect on osteopenia with various pathophysiological conditions. Zinc compound may be a new drug in the therapy of osteoporosis. J. Trace Elem. Exp. Med. 11:119–135, 1998. © 1998 Wiley-Liss, Inc.

Short chain fatty acids (SCFAs) are the major anions in the large intestine. They are produced by a bacterial fermentation of dietary fiber. SCFAs are known to have a variety of physiological and pathphysiological effects on intestine. However, the mechanisms by which intraluminal SCFAs are sensed are not known. In 2003, two orphan G protein coupled receptors (GPRs), GPR41 and GPR43, have been cloned and demonstrated to be receptors for SCFAs. Thus, we had attempted to make antibodies raised against GPR43 and GPR41 to elucidate the roles of SCFAs on colonic functions. We have also evaluated the effects of SCFAs on colonic motility to define the physiological roles on luminal SCFAs. In rat and human colon, GPR43 protein was detected by Western blot analysis in extracts of whole wall and separated mucosa, but not in muscle plus submucosa extract. By immunohistochemistry, GPR43 immunoreactivity was localized with enteroendocrine cells expressing peptide YY, whereas 5-HT immunoreactive enteroendocrine cells were not immunoreactive for GPR43. GPR41 immunoreactivity was also found in human colon. In functional studies, propionate and butyrate concentration-dependently (10 microM - 10 mM) induced phasic and tonic contractions in rat colonic circular muscle. The propionate-induced phasic contraction was attenuated by atropine, tetrodotoxin and the 5-HT(4) receptor antagonists SB204070. However, acetate did not induce phasic or tonic contractions. Propionate-induced responses were not observed in mucosal free preparations. The present results suggest that the SCFA-induced physiological effects on colonic functions might be attributable to the activation of SCFA receptors on epithelial cells in the colon.

The effect of orally administrated gamma-aminobutyric acid (GABA) on relaxation and immunity during stress has been investigated in humans. Two studies were conducted. The first evaluated the effect of GABA intake by 13 subjects on their brain waves. Electroencephalograms (EEG) were obtained after 3 tests on each volunteer as follows: intake only water, GABA, or L-theanine. After 60 minutes of administration, GABA significantly increases alpha waves and decreases beta waves compared to water or L-theanine. These findings denote that GABA not only induces relaxation but also reduces anxiety. The second study was conducted to see the role of relaxant and anxiolytic effects of GABA intake on immunity in stressed volunteers. Eight acrophobic subjects were divided into 2 groups (placebo and GABA). All subjects were crossing a suspended bridge as a stressful stimulus. Immunoglobulin A (IgA) levels in their saliva were monitored during bridge crossing. Placebo group showed marked decrease of their IgA levels, while GABA group showed significantly higher levels. In conclusion, GABA could work effectively as a natural relaxant and its effects could be seen within 1 hour of its administration to induce relaxation and diminish anxiety. Moreover, GABA administration could enhance immunity under stress conditions.

Many classes of nanoparticles have been synthesized and widely applied, however, there is a serious lack of information concerning their effects on human health and the environment. Considering that their use will increase, accurate and cost-effective measurement techniques for characterizing "nanotoxicity" are required. One major toxicological concern is that nanoparticles are easily taken up in the human body. In this study, we developed a method of evaluating the uptake potential of nanosized particles using flow cytometric light scatter. Suspended titanium dioxide (TiO2) particles (5, 23, or 5000 nm) were added to Chinese hamster ovary cells. Observation by confocal laser scanning microscopy showed that the TiO2 particles easily moved to the cytoplasm of the cultured mammalian cells, not to the nucleus. The intensity of the side-scattered light revealed that the particles were taken up in the cells dose-, time-, and size-dependently. In addition, surface-coating of TiO2 particles changed the uptake into the cells, which was accurately reflected in the intensity of the side-scattered light. The uptake of other nanoparticles such as silver (Ag) and iron oxide (Fe3O4) also could be detected. This method could be used for the initial screening of the uptake potential of nanoparticles as an index of "nanotoxicity".

The uptake and degradation of cytoplasmic material by vacuolar autophagy in plants has been studied extensively by electron microscopy and shown to be involved in developmental processes such as vacuole formation, deposition of seed storage proteins and senescence, and in the response of plants to nutrient starvation and to pathogens. The isolation of genes required for autophagy in yeast has allowed the identification of many of the corresponding Arabidopsis genes based on sequence similarity. Knockout mutations in some of these Arabidopsis genes have revealed physiological roles for autophagy in nutrient recycling during nitrogen deficiency and in senescence. Recently, markers for monitoring autophagy in whole plants have been developed, opening the way for future studies to decipher the mechanisms and pathways of autophagy, and the function of these pathways in plant development and stress responses.