Site-Selective Incorporation of a Functional Group into Lys175 in the Vicinity of the Active Site of Chymotrypsin by Using Peptidyl α-Aminoalkylphosphonate Diphenyl Ester-Derivatives.

We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an N-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-PheP(OPh)2]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-ß-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-(R)-PheP(OPh)2 (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin. Ion-exchange chromatography (IEC), fluorescence spectroscopy, and LC-MS/MS were used to analyze the products from the reaction of Csin with DanD or DanE. By IEC and LC-MS/MS, the results showed that DanE reacted with Csin more effectively than DanD to produce the modified Csin (DanMCsin) bearing Dan at Lys175. DanMCsin exhibited an enzymatic activity corresponding to 1/120 of Csin against Suc-Ala-Ala-Phe-pNA. In addition, an effect of Lys175 modification on the access of the proteinaceous Bowman-Birk inhibitor to the active site of DanMCsin was investigated. In conclusion, by using a peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester, we demonstrated that a functional group could be incorporated into Lys175 in Csin.

Sociodemographic, cultural, linguistic, and test selection considerations for clinical neuropsychological assessment with Japanese and Japanese-American patients in the United States.

Objective: Japanese-Americans are the sixth largest Asian ethnicity in the United States and represent a highly heterogeneous population. Despite representing a large and diverse group, relatively little attention has been paid to clinical best practices for working with this population in the West, particularly for Japanese speakers and issei (first-generation in the United States). This paper offers guidance for providing competent neuropsychological services to Japanese-Americans. Method: Pertinent facets of Japanese culture are presented within the context of the ECLECTIC framework (education and literacy, culture and acculturation, language, economics, communication, testing situation: comfort and motivation, intelligence conceptualization, and context of immigration; Fujii, 2018). The available literature on clinical neuropsychological tests that are translated into Japanese and normed with Japanese samples was reviewed. Results: Specific recommendations for clinicians providing neuropsychological services to Japanese-Americans are presented with an aim of maximizing test fairness by addressing the following issues: comfort with the testing situation, test biases, accessibility, and validity (American Educational Research Association et al., 2014). Additional recommendations for the use of teleneuropsychology; working with geriatric, pediatric, and multiracial populations; and providing useful recommendations and feedback from clinical assessment are provided. Measures that are appropriately translated and/or adapted for use with Japanese populations are presented by cognitive domain to assist clinicians with test selection. Conclusions: This paper provides concrete recommendations for Western neuropsychologists working with patients of Japanese descent in order to address the current gap in cultural competence among clinicians when working with this heterogeneous population.

Site-selective chemical modification of chymotrypsin using peptidyl derivatives bearing optically active diphenyl 1-amino-2-phenylethylphosphonate: Stereochemical effect of the diphenyl phosphonate moiety.

Diphenyl (α-aminoalkyl)phosphonates act as mechanism-based inhibitors against serine proteases by forming a covalent bond with the hydroxy group of the active center Ser residue. Because the covalent bond was found to be broken and replaced by 2-pyridinaldoxime methiodide (2PAM), we employed a peptidyl derivative bearing diphenyl 1-amino-2-phenylethylphosphonate moiety (Phe(p) (OPh)2 ) to target the active site of chymotrypsin and to selectively anchor to Lys175 in the vicinity of the active site. Previously, it was reported that the configuration of the α-carbon of phosphorus in diphenyl (α-aminoalkyl)phosphonates affects the inactivation reaction of serine proteases, i.e., the (R)-enantiomeric diphenyl phosphonate is comparable to l-amino acids and it effectively reacts with serine proteases, whereas the (S)-enantiomeric form does not. In this study, we evaluated the stereochemical effect of the phosphonate moiety on the selective chemical modification. Epimeric dipeptidyl derivatives, Ala-(R or S)-Phe(p) (OPh)2 , were prepared by separation with RP-HPLC. A tripeptidyl (R)-epimer (Ala-Ala-(R)-Phe(p) (OPh)2 ) exhibited a more potent inactivation ability against chymotrypsin than the (S)-epimer. The enzyme inactivated by the (R)-epimer was more effectively reactivated with 2PAM than the enzyme inactivated by the (S)-epimer. Finally, N-succinimidyl (NHS) active ester derivatives, NHS-Suc-Ala-Ala- (R or S)-Phe(p) (OPh)2 , were prepared, and we evaluated their action when modifying Lys175 in chymotrypsin. We demonstrated that the epimeric NHS derivative that possessed the diphenyl phosphonate moiety with the (R)-configuration effectively modified Lys175 in chymotrypsin, whereas that with the (S)-configuration did not. These results demonstrate the utility of peptidyl derivatives that bear an optically active diphenyl phosphonate moiety as affinity labeling probes in protein bioconjugation. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 521-530, 2016.

A comprehensive analysis of interaction and localization of Arabidopsis SKP1-like (ASK) and F-box (FBX) proteins.

F-Box (FBX) proteins are encoded by a multigene family present in major lineages of eukaryotes. A number of FBX proteins are shown to be subunits of SCF complex, a type of E3 ligases composed of SKP1, CULLIN, FBX and RBX1 proteins. The Arabidopsis SKP-LIKE (ASK) proteins are also members of a family and some of them interact with FBX proteins directly. To clarify how FBX and ASK proteins combine, we carried out a large-scale interaction analysis between FBX and ASK proteins using yeast two-hybrid assay (Y2H) in Arabidopsis thaliana. FBX proteins randomly chosen from those proteins that interacted with more than one ASK protein were further analyzed for their subcellular localization and in vivo interaction with ASK proteins. Furthermore, the expression profiles of FBX and ASK genes were compared. This work reveals that FBX proteins had a preference for interacting with ASK proteins depending on the domains they contain such as the FBX-associated (FBA) domain, the Kelch domain and leucine rich repeat (LRR). In addition, it was found that a single FBX protein could form multiple SCF complexes by interacting with several ASK proteins in many cases. Furthermore, it was suggested that the variation of SCF complexes were especially abundant in tissues related to male gametophyte and seed development. More than half of the FBX proteins studied did not interact with any of the ASK proteins, implying the necessity for certain regulations for their interaction in vivo and/or distinct roles from subunits of the SCF complex.

Novel promoters that induce specific transgene expression during the green to ripening stages of tomato fruit development.

UNLABELLED: Fruit-specific promoters have been used as genetic engineering tools for studies on molecular mechanism of fruit development and advance in fruit quality and additional value by increasing functional component. Especially fruit-ripening specific promoters have been well utilized and studied in tomato; however, few studies have reported the development of promoters that act at fruit developing stages such as immature green and mature green periods. In this study, we report novel promoters for gene expression during the green to ripening stages of tomato fruit development. Genes specifically expressed at tomato fruit were selected using microarray data. Subsequent to confirmation of the expression of the selected 12 genes, upstream DNA fragments of the genes LA22CD07, Les.3122.2.A1_a_at and LesAffx.6852.1.S1_at which specifically expressed at fruit were isolated from tomato genomic DNA as promoter regions. Isolated promoter regions were fused with the GUS gene and the resultant constructs were introduced into tomato by agrobacterium-mediated transformation for evaluation of promoter activity in tomato fruit. The two promoters of LA22CD07, and LesAffx.6852.1.S1_at showed strong activity in the fruit, weak activity in the flower and undetectable activity in other tissues. Unlike well-known fruit-ripening specific promoters, such as the E8 promoter, these promoters exhibited strong activity in green fruit in addition to red-ripening fruit, indicating that the promoters are suitable for transgene expression during green to ripening stages of tomato fruit development. KEY MESSAGE: Novel fruit-specific promoters have been identified and are suitable for transgene expression during green to ripening stages of tomato fruit development.

RiceFOX: a database of Arabidopsis mutant lines overexpressing rice full-length cDNA that contains a wide range of trait information to facilitate analysis of gene function.

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named 'RiceFOX'. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice.

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.

Molecular breeding of tomato lines for mass production of miraculin in a plant factory.

A transgenic tomato line (56B, "Moneymaker") that expresses the miraculin gene driven by the CaMV 35S promoter was crossed with a dwarf tomato ("Micro-Tom") for the molecular breeding of cultivars that are suitable for miraculin production in a closed cultivation system. Plant size, miraculin accumulation, and self-pruning growth were used as selection indicators for F2 plants. Two lines were chosen for further analysis, bred to the F6 or F7 generation and cultivated in a closed cultivation system. In 56B and the two crossed lines, the concentrations of miraculin in the pericarp were 140, 367, and 343 microg/g FW, respectively. We also estimated that 26.2, 73.6, and 45.9 kg FW/m2 of tomatoes and 2.2, 16.6, and 9.8 mg/m2 of miraculin in the pericarp, respectively, could be harvested per year. These two crossed lines will be useful for the mass production of miraculin, especially in a closed cultivation system.

Metabolomic screening applied to rice FOX Arabidopsis lines leads to the identification of a gene-changing nitrogen metabolism.

Plant metabolomics developed as a powerful tool to examine gene functions and to gain deeper insight into the physiology of the plant cell. In this study, we screened Arabidopsis lines overexpressing rice full-length (FL) cDNAs (rice FOX Arabidopsis lines) using a gas chromatography-time-of-flight mass spectrometry (GC-TOF/MS)-based technique to identify rice genes that caused metabolic changes. This screening system allows fast and reliable identification of candidate lines showing altered metabolite profiles. We performed metabolomic and transcriptomic analysis of a rice FOX Arabidopsis line that harbored the FL cDNA of the rice ortholog of the Lateral Organ Boundaries (LOB) Domain (LBD)/Asymmetric Leaves2-like (ASL) gene of Arabidopsis, At-LBD37/ASL39. The investigated rice FOX Arabidopsis line showed prominent changes in the levels of metabolites related to nitrogen metabolism. The transcriptomic data as well as the results from the metabolite analysis of the Arabidopsis At-LBD37/ASL39-overexpressor plants were consistent with these findings. Furthermore, the metabolomic and transcriptomic analysis of the Os-LBD37/ASL39-overexpressing rice plants indicated that Os-LBD37/ASL39 is associated with processes related to nitrogen metabolism in rice. Thus, the combination of a metabolomics-based screening method and a gain-of-function approach is useful for rapid characterization of novel genes in both Arabidopsis and rice.

Systematic approaches to using the FOX hunting system to identify useful rice genes.

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23,000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis. This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.

Light-dependent polyploidy control by a CUE protein variant in Arabidopsis.

Endoreduplication is a special cell cycle that increases ploidy without cell and nuclear division. In plants endoreduplication is essential for development. We isolated a dominant Arabidopsis mutant from activation tagging lines that had increased polyploidy in darkness. This mutant, ipd1-1D (increased polyploidy level in darkness 1-1D), shows longer hypocotyls and increased ploidy levels only in dark-grown seedlings. The corresponding gene encodes a protein that contains a CUE domain variant. IPD1 is specifically expressed in mitotically dividing cells. Furthermore we show that blue and far-red light can suppress the ploidy increase in ipd1-1D and also suppress the reporter expression in IPD1-promoter beta-glucuronidase transgenic plants. These results suggest that IPD1 regulates the endocycle leading to hypocotyl elongation and this function is controlled by blue and far-red light.

The FOX hunting system: an alternative gain-of-function gene hunting technique.

We have developed a novel gain-of-function system that we have named the FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system). We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. About 10 000 independent full-length Arabidopsis cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Each transgenic Arabidopsis contained on average 2.6 cDNA clones and was monitored under various categories such as morphological changes, fertility and leaf color. We found 1487 possible morphological mutants from 15 547 transformants. When 115 pale green T(1) mutants were analyzed, 59 lines represented the mutant phenotypes in more than 50% of the T(2) progeny. Characterization of two leaf color mutants revealed the significance of this approach. We also document mutants from several categories and their corresponding full-length cDNAs.

Identification of a novel cis-element exhibiting cytokinin-dependent protein binding in vitro in the 5'-region of NADPH-protochlorophyllide oxidoreductase gene in cucumber.

Cytokinins and light activate the transcription of the cucumber NADPH-protochlorophyllide reductase (POR) gene. We have previously reported that 2.3 kb of the 5'-region of this gene contains a cis-element that is responsive to cytokinin. In this study, to identify the cytokinin-responsive cis-element corresponding to chlorophyll biosynthesis and chloroplast development, we performed transient expression assays in etiolated cucumber cotyledons. A 5'-deletional analysis indicated that a 411-bp fragment (-451 to -40 bp) contained at least one of the cis-elements related to cytokinin-responsiveness. Gel mobility shift assays also detected cytokinin-enhanced binding in this region. DNase I footprinting analysis, using a 150-bp fragment (-490 to -340 bp) as the probe, identified the cytokinin-enhanced protected sequence as 5'-ATATTAGTGATAT-3'. More detailed gel mobility shift and competition analyses identified 5'-TATTAG-3' as the sequence critical for cytokinin-enhanced binding. Mutations in the identified sequence in the transient expression assay caused a reduced but retained cytokinin-responsiveness, as well as low reporter activity of untreated control. These results suggest that the identified sequence is a novel cis-element exhibiting cytokinin-dependent protein binding in vitro, which may function effectively when interacting with other cytokinin-related elements. The effects of this element on the chloroplast development are discussed in relation to other cytokinin-related elements.

Expression and interaction analysis of Arabidopsis Skp1-related genes.

Specific protein degradation has been observed in several aspects of development and differentiation in many organisms. One example of such proteolysis is regulated by protein polyubiquitination that is promoted by the SCF complex consisting of Skp1, cullin, and an F-box protein. We examined the activities of the Arabidopsis Skp1-related proteins (ASKs). Among 19 annotated ASK genes, we isolated 16 of the corresponding cDNAs (ASK1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19), and examined their gene products for interactions with 24 representatives of F-box proteins carrying various classes of the C-terminal domains using the yeast two-hybrid system. As a result, we found diverse binding specificities: ASK1, ASK2, ASK11 and ASK12 interacted well with COI1, FKF1, UFO-like protein, LRR-containing F-box proteins, and other F-box proteins with unknown C-terminal motifs. We also observed specific interaction between F-box proteins and ASK3, ASK9, ASK13, ASK14, ASK16 and ASK18. In contrast, we detected no interaction between any of the 12 ASK proteins and F-box proteins containing CRFA, CRFB or CRFC domains. Both histochemical and RT-PCR analysis of eight ASK genes expression revealed unique expression patterns for the respective genes.

Novel phosphine-catalyzed zipper cyclization of aliphatic diyne-dione and yne-dione systems.

[reaction: see text] An unexpected tri-n-butylphosphine-catalyzed zipper cyclization of diyne[bond]diones (1a-d) or yne-diones (1e and 1f) is described. Bicyclic ketones (2a, 2b, 2c, 2e, and 2f) with five- or six-membered rings fused to the five-membered ring were obtained from both aliphatic diyne-diones (1a-c) and yne-diones (1e and 1f) having tetra- or pentamethylene spacers. The bicyclic products (2) were produced with high diastereoselectivity.

Classification and expression analysis of Arabidopsis F-box-containing protein genes.

F-box proteins regulate diverse cellular processes, including cell cycle transition, transcriptional regulation and signal transduction, by playing roles in Skp1p-cullin-F-box protein (SCF) complexes or non-SCF complexes. F-box proteins are encoded by a large gene family. Our database search revealed that at least 568 F-box protein genes are present in the Arabidopsis thaliana (Arabidopsis) genome. Domain search analysis using SMART and Pfam-A databases revealed that 67 of the F-box proteins contained Kelch repeats and 29 contained leucine-rich repeats (LRRs). Interestingly only two F-box proteins contained WD40 repeats that are found in many F-box proteins of other organisms. Kelch repeats, LRRs and WD40 repeats are implicated in protein-protein interactions. This analysis also resulted in the finding of several unique functional domains; however, 448 of the F-box proteins did not contain any known domains. Therefore, these proteins were used to search the Pfam-B database to find novel domains, and three putative ones were found. These domain search analyses led us to classify the Arabidopsis F-box proteins into at least 19 groups based on their domain structures. Macro array analysis showed that several F-box protein genes are expressed in a tissue-specific manner.

Identification of two differentially regulated isoforms of protochlorophyllide oxidoreductase (POR) from tobacco revealed a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms.

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Here, we identified two distinct POR cDNAs from tobacco. Both POR isoforms are encoded by a respective single copy gene in tobacco genome. The overall deduced amino acid sequences of two tobacco cDNAs, designated here POR1 and POR2, displayed significant identities ( approximately 75%), but showed different patterns of light and developmental regulation. In contrast to the previously isolated POR isoforms of Arabidopsis thaliana and barley, the expression of both tobacco POR isoforms were not negatively regulated by light and persisted in matured green tissues. Furthermore, the expression of both genes appeared to be regulated by a diurnal regulation. These results show a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms. Furthermore, phylogenetic analysis including tobacco revealed that POR gene family is differentially represented by angiosperms, most of which is probably caused by independent gene duplication in individual plant. Present results imply a modification of the previous concept that chlorophyll biosynthesis and chloroplast differentiation in angiosperms are ubiquitously controlled by unique functions of two POR isoforms.