Kenji Maehashi

Dissecting the genetic control of natural variation in salt tolerance of Arabidopsis thaliana accessions

Many accessions (ecotypes) of Arabidopsis have been collected. Although few differences exist among their nucleotide sequences, these subtle differences induce large genetic variation in phenotypic traits such as stress tolerance and flowering time. To understand the natural variability in salt tolerance, large-scale soil pot experiments were performed to evaluate salt tolerance among 350 Arabidopsis thaliana accessions. The evaluation revealed a wide variation in the salt tolerance among accessions. Several accessions, including Bu-5, Bur-0, Ll-1, Wl-0, and Zu-0, exhibited marked stress tolerance compared with a salt-sensitive experimental accession, Col-0. The salt-tolerant accessions were also evaluated by agar plate assays. The data obtained by the large-scale assay correlated well with the results of a salt acclimation (SA) assay, in which plants were transferred to high-salinity medium following placement on moderate-salinity medium for 7 d. Genetic analyses indicated that the salt tolerance without SA is a quantitative trait under polygenic control, whereas salt tolerance with SA is regulated by a single gene located on chromosome 5 that is common among the markedly salt-tolerant accessions. These results provide important information for understanding the mechanisms underlying natural variation of salt tolerance in Arabidopsis.

Primary structure of potential allergenic proteins in emu (Dromaius novaehollandiae) egg white.

The emu (Dromaius novaehollandiae) egg is considered promising as an alternative egg product. To obtain basic biochemical information on emu egg white, the major protein compositions in emu and chicken egg whites and the primary structures of potential allergenic proteins were compared. The dominant protein in emu egg white was ovotransferrin (OVT), followed by ovalbumin (OVA) and TENP protein. The OVA and ovomucoid (OVM) levels in emu egg white were estimated as significantly lower than those in chicken egg white by Western blotting and enzyme-linked immunosorbent assays using anti-chicken OVA or OVM antibodies. Lysozyme and its enzymatic activity were not detected in emu egg white. OVT, OVA, and OVM genes were also cloned, and their nucleotide and amino acid sequences were determined. The protein sequences of OVT, OVA, and OVM from emu showed lower similarities to those of chicken than other avian species, such as quail and turkey. These results emphasize the low allergenicity of emu egg white and its potential as an alternative to chicken egg white.

Molecular characterization of goose- and chicken-type lysozymes in emu (Dromaius novaehollandiae): evidence for extremely low lysozyme levels in emu egg white.

Lysozyme (LZ), a bacteriolytic enzyme, is found in the egg white of many avian eggs and plays an important role in host defense; however, LZ activity in emu (Dromaius novaehollandiae) egg white is exceptionally undetectable. We cloned and characterized emu goose-type LZ (LZG) and chicken-type LZ (LZC) genes. RT-PCR analysis revealed very low LZG gene expression levels and absence of LZC gene expression in the emu oviduct. Sequencing of full-length LZG and LZC cDNAs indicated that their amino acid sequences show high similarities to ostrich LZG and LZC, respectively, with conserved catalytic residues for enzymatic activities. Whereas recombinant emu LZG prepared using Pichia pastoris exhibited similar enzyme activity as ostrich LZG, recombinant emu LZC exhibited significantly higher lytic activity than chicken LZC. We concluded that emus have functional genes for both LZG and LZC like many other avians, and the LZG gene is expressed in oviduct probably as in other ratite, however, its expression levels in egg white were low to be detected.

Extracellular production of riboflavin-binding protein, a potential bitter inhibitor, by Brevibacillus choshinensis.

Riboflavin-binding protein (RBP) is a glycophosphoprotein found in hen eggs. We previously identified the extraordinary characteristic of RBP in reducing bitterness. For a more detailed study on the mode of action and industrial application of this characteristic, we investigated the microbial production of recombinant RBP (rRBP). We constructed a chicken RBP gene expression vector by inserting the RBP cDNA in pNCMO2, the Escherichia coli-Brevibacillus choshinensis shuttle vector. B. choshinensis HPD31 transformants produced 0.8g/l of processed and unglycosylated RBP in a soluble form in the culture supernatant. However, the expressed RBP was partially dimerized and monomeric RBP was purified by two step anion-exchange and gel-filtration chromatographies. The purified rRBP elicited bitterness reduction against quinine and caffeine, although it largely lost its riboflavin-binding ability. These results indicated that glycosylation and riboflavin-binding ability are not essential for the bitterness reduction of RBP. In addition, we assessed the usefulness of the Brevibacillus system for the expression and secretion of RBP as a new type of bitterness inhibitor.

Biochemical and functional characterization of transiently expressed in neural precursor (TENP) protein in emu egg white.

A protein transiently expressed in the neural precursors of developing tissues (TENP) was found to be present in emu (Dromaius novaehollandiae) egg white as one of the major proteins. Nucleotide analysis of its encoding cDNA revealed a sequence of 452 amino acids including a 19 amino acid peptide signal. Phylogenetic analysis determined that emu TENP was clustered within the bactericidal/permeability-increasing protein (BPI) superfamily together with other avian TENPs. RT-PCR analysis revealed that the emu TENP gene was highly expressed in the magnum of the oviduct, indicating that TENP is a major egg white component. Emu TENP was purified by anion exchange chromatography and ammonium sulfate fractionation. Unlike BPI, emu TENP exhibited antibacterial activity against Gram-positive bacteria, including Micrococcus luteus and Bacillus subtilis, but not against Gram-negative bacteria such as Escherichia coli and Salmonella Typhimurium. The results suggest that emu TENP is a potent novel antibacterial protein with a spectrum distinct from that of BPI.

The primary structure of a novel riboflavin-binding protein of emu (Dromaius novaehollandiae)

Emu riboflavin-binding protein (RBP) was purified from egg white and yolk, and its N-terminal amino acid sequence was determined. The molecular mass of emu RBP was estimated at approximately 48 and 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., 10 kDa larger than chicken RBP. The molecular mass of deglycosylated RBPs indicated that the content of oligosaccharide chain in emu RBP was approximately 3 times greater than that in chicken RBP. The gene encoding the RBP precursor was cloned from emu oviduct cDNA by PCR and found also in the liver and ovary cDNAs as well as oviduct cDNA. The complete cDNA consisted of an open reading frame of 714 bp encoding a protein of 238 amino acids. The amino acid sequence deduced from the cDNA sequence revealed that many essential structural features were conserved in emu RBP including 18 cysteine residues, 2 N-glycosylation sites, a clustered phosphorylation region, and riboflavin-binding sites. Two additional potential N-glycosylation sites were found in the amino acid sequences of RBPs from the emu and other sources such as the turtle and frog, which might in part account for the greater content of oligosaccharide chain of emu RBP as compared to chicken RBP.

Purification and characterization of a novel glutamyl aminopeptidase from chicken meat

A novel glutamyl aminopeptidase (aminopeptidase A, EC 3.4.11.7) was purified from chicken meat by ammonium sulfate fractionation, ethanol fractionation, heat treatment, and successive column chromatographies of DEAE-Sepharose CL-6B and Sephadex G-200. The purified enzyme migrated as a single band on SDS-PAGE. The molecular weight of this enzyme was found to be 55,000 and 550,000 by SDS-PAGE and Sephadex G-200 column chromatographies, respectively. This enzyme hydrolyzed Glu- and Asp-, but not Leu-, Arg-, and Ala-2-naphthylamide (-2NA) at all. The optimum pH and temperature for hydrolysis of Glu-2NA was 7.5. and 70°C, respectively. Reducing agents such as cysteine and dithiothreitol inhibited the activity of this enzyme at concentrations of 1 mM. However, the activation by Ca(2+) and the inhibition by amastatin were not observed.

Sweetness characterization of recombinant human lysozyme

Lysozyme, a bacteriolytic enzyme, is widely distributed in nature and is a component of the innate immune system. It is established that chicken egg lysozyme elicits sweetness. However, the sweetness of human milk lysozyme, which is vital for combating microbial infections of the gastrointestinal tract of breast-fed infants, has not been characterized. This study aimed to assess the elicitation of sweetness using recombinant mammalian lysozymes expressed in Pichia pastoris. Recombinant human lysozyme (h-LZ) and other mammalian lysozymes of mouse, dog, cat and bovine milk elicited similar sweetness as determined using a sensory test, whereas bovine stomach lysozyme (bs-LZ) did not. Assays of cell cultures showed that h-LZ activated the human sweet taste receptor hT1R2/hT1R3, whereas bs-LZ did not. Point mutations confirmed that the sweetness of h-LZ was independent of enzyme activity and substrate-binding sites, although acidic amino acid residues of bs-LZ played a significant role in diminishing sweetness. Therefore, we conclude that elicitation of sweetness is a ubiquitous function among all lysozymes including mammalian lysozymes. These findings may provide novel insights into the biological implications of T1R2/T1R3-activation by mammalian lysozyme in the oral cavity and gastrointestinal tract. However, the function of lysozyme within species lacking the functional sweet taste receptor gene, such as cat, is currently unknown.