Daily Meal Supplemented with Astaxanthin-Enriched Yolk Has Mitigative Effects against Hypertension in Spontaneously Hypertensive Rats.

The aim of this study was to investigate the effects of egg yolk powder enriched with astaxanthin (ASX-E) on blood pressure in spontaneously hypertensive rats (SHR) and to verify the benefits of ASX-E as a functional food. To investigate the antihypertensive effect, SHR were fed with an ASX-E mixed diet before hypertension development. Blood pressures were determined periodically during the study by the tail-cuff method. At the end of the study, animals were euthanized, and their thoracic aortas were collected to determine vascular conductance. The thoracic aorta tension was measured with a force displacement transducer. Concentration-dependent response relationships were determined by cumulative addition of 10-9-10-4 M Carbamoylcholine (Cch). Blood pressures of the SHR in the ASX-E mixed diet group were ASX-dose-dependently lower than that of those in the control group. In SHR fed with an ASX-E mixed diet, Cch induced vasorelaxation in the thoracic aorta with endothelium lining but not without endothelium. However, the antihypertensive effect of ASX-E was not observed on blood pressures in SHR that were fed with ASX-E only after the development of hypertension. Results suggest that ASX-E protects endothelial function and thereby prevents the development of hypertension. Hence, the results of our research indicate that daily consumption of ASX-E has a potential benefit on human health.

Characterization of calcium ion sensitive region for ß-mannanase from Streptomyces thermolilacinus.

Despite the widespread industrial applications of ß-mannanase, the relations between the enzymatic properties and metal ions remain poorly understood. To elucidate the effects of metal ions on ß-mannanase, thermal stability and hydrolysis activity were characterized. The stman and tfman genes encoding ß-mannanase (EC.3.2.1.78) from Streptomyces thermolilacinus NBRC14274 and Thermobifida fusca NBRC14071 were cloned and expressed in Escherichia coli. The thermal stability of each enzyme shifted to the 7-9°C high temperature in the presence of Ca(2+) compared with that in the absence of Ca(2+). These results show that the thermal stability of StMan and TfMan was enhanced by the presence of Ca(2+). StMan, but not TfMan, required Ca(2+) for the hydrolysis activity. To identify the Ca(2+) sensitive region of StMan, we prepared eight chimeric enzymes. Based on the results of the relationship between Ca(2+) and hydrolysis activity, the region of amino-acid residues 244-349 of StMan was responsible for a Ca(2+) sensitive site.

Extracellular production and characterization of Streptomyces X-prolyl dipeptidyl aminopeptidase.

X-prolyl dipeptidyl aminopeptidases (X-PDAPs) are useful in various food industries. In this study, we performed sequence-based screening to obtain a stable X-PDAP enzyme from thermophilic Streptomyces strains. We found three genes that encoded X-PDAP from Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 X-PDAP), Streptomyces thermocyaneoviolaceus NBRC 14271 (14271 X-PDAP), and Streptomyces thermocoerulescens NBRC 14273, which were subsequently cloned and sequenced. The deduced amino acid sequences of these genes showed high similarity, with ~80% identity with each other. The isolated X-PDAPs and an X-PDAP from Streptomyces coelicolor were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among these genes, only 14270 and 14271 X-PDAPs caused overexpression and extracellular production without artificial signal peptides. We also characterized the biochemical properties of purified 14271 X-PDAP. In addition, we found that, in peptide synthesis via an aminolysis reaction, this enzyme recognized D-amino acid derivatives as acyl acceptors, similar to L-amino acid derivatives.

Extracellular production and characterization of two streptomyces L-asparaginases.

L-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four L-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.

Determination of a chloroplast degron in the regulatory domain of chlorophyllide a oxygenase.

Chlorophyll b is one of the major photosynthetic pigments of plants. The regulation of chlorophyll b biosynthesis is important for plants in order to acclimate to changing environmental conditions. In the chloroplast, chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), a Rieske-type monooxygenase. The activity of this enzyme is regulated at the level of protein stability via a feedback mechanism through chlorophyll b. The Clp protease and the N-terminal domain (designated the A domain) of CAO are essential for the regulatory mechanism. In this study, we aimed to identify the specific amino acid residue or the sequence within the A domain that is essential for this regulation. To accomplish this goal, we randomly introduced base substitutions into the A domain and searched for potentially important residues by analyzing 1,000 transformants of Arabidopsis thaliana. However, none of the single amino acid substitutions significantly stabilized CAO. Therefore, we generated serial deletions in the A domain and expressed these deletions in the background of CAO-deficient Arabidopsis mutant. We found that the amino acid sequence (97)QDLLTIMILH(106) is essential for the regulation of the protein stability. We furthermore determined that this sequence induces the destabilization of green fluorescent protein. These results suggest that this sequence serves as a degradation signal that is recognized by proteases functioning in the chloroplast.

Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement.

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.

Loss of the N-terminal domain of chlorophyllide a oxygenase induces photodamage during greening of Arabidopsis seedlings.

BACKGROUND: Chlorophyll b is a major photosynthetic pigment in green plants that is synthesized by chlorophyllide a oxygenase (CAO). The regulation of chlorophyll b biosynthesis is an important determinant for the antenna size of photosystems. Chlorophyll b synthesis is partly regulated on a transcriptional level by the expression of the CAO gene. In addition, the synthesis of chlorophyll b is strictly regulated on a protein level by the stability of the CAO enzyme. CAO consists of three domains, which are sequentially named from the N terminus as the A, B and C domains. The A domain of CAO participates in the regulation of the CAO protein stability. RESULTS: In order to clarify the physiological function of the A domain, we constructed transgenic Arabidopsis (Arabidopsis thaliana) plants which either overexpressed the complete CAO or a truncated version of CAO lacking the A domain. The transgenic plants overexpressing the A-domain-deleted CAO accumulated an excess amount of chlorophyll b during greening. The transgenic plants which lacked the A domain either died or were obviously retarded when they were exposed to continuous light immediately after etiolation. In addition, the loss of the A domain in CAO impaired another step of chlorophyll biosynthesis, namely the conversion of divinyl-protochlorophyllide a to monovinyl protochlorophyllide a under dark conditions. CONCLUSION: The A domain of CAO regulates the level of CAO, and thus prevents the excess accumulation of chlorophyll b. This function of the A domain is especially important during the greening stage of etiolated seedlings. At this stage, the plants are vulnerable to photodamages which could be caused by excessive chlorophyll b accumulation. In addition, de-regulation of the CAO level affects monovinyl-protochlorophyllide biosynthesis in darkness by unknown mechanisms. In conclusion, the A domain of CAO is essential in the control of chlorophyll biosynthesis and in the survival of seedlings during de-etiolation especially under strong illumination.

Functional analysis of N-terminal domains of Arabidopsis chlorophyllide a oxygenase.

Higher plants acclimate to various light environments by changing the antenna size of a light-harvesting photosystem. The antenna size of a photosystem is partly determined by the amount of chlorophyll b in the light-harvesting complexes. Chlorophyllide a oxygenase (CAO) converts chlorophyll a to chlorophyll b in a two-step oxygenation reaction. In our previous study, we demonstrated that the cellular level of the CAO protein controls accumulation of chlorophyll b. We found that the amino acids sequences of CAO in higher plants consist of three domains (A, B, and C domains). The C domain exhibits a catalytic function, and we demonstrated that the combination of the A and B domains regulates the cellular level of CAO. However, the individual function of each of A and B domain has not been determined yet. Therefore, in the present study we constructed a series of deleted CAO sequences that were fused with green fluorescent protein and overexpressed in a chlorophyll b-less mutant of Arabidopsis thaliana, ch1-1, to further dissect functions of A and B domains. Subsequent comparative analyses of the transgenic plants overexpressing B domain containing proteins and those lacking the B domain determined that there was no significant difference in CAO protein levels. These results indicate that the B domain is not involved in the regulation of the CAO protein levels. Taken together, we concluded that the A domain alone is involved in the regulatory mechanism of the CAO protein levels.

Clp protease controls chlorophyll b synthesis by regulating the level of chlorophyllide a oxygenase.

Chlorophyll b is one of the major light-harvesting pigments in green plants and it is essential for optimal light harvesting. Chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO) which consists of A, B and C domains. Previously, we demonstrated that the C domain alone has a catalytic function, while the A and B domains control the level of CAO protein in response to chlorophyll b accumulation. We hypothesized that the accumulation of chlorophyll b triggers the proteolytic degradation of CAO. In this study, in order to gain further insight into this regulatory mechanism we screened for mutants that have defects in the control of CAO accumulation. Seeds from a transgenic line of Arabidopsis which overexpressed a CAO-GFP fusion were mutagenized and their progenies were screened by laser-scanning confocal microscopy for mutants showing an elevated level of GFP fluorescence. One particular mutant (dca1) exhibited stronger GFP fluorescence and accumulated a GFP-CAO fusion protein at a higher level. Concomitantly, the chlorophyll a to b ratio decreased in this mutant. The mutation in the dca1 mutant was mapped to the ClpC1 gene, thereby indicating that a chloroplast Clp protease is involved in regulating chlorophyll b biosynthesis through the destabilization of CAO protein in response to the accumulation of chlorophyll b.

The N-terminal domain of chlorophyllide a oxygenase confers protein instability in response to chlorophyll B accumulation in Arabidopsis.

Plants acclimate to variations in light intensity by changing the antenna size of photosystems. This acclimation allows them to undergo efficient photosynthesis and creates a protective strategy to minimize photodamage. Chlorophyll b synthesis by chlorophyllide a oxygenase (CAO) is a key regulatory step in the control of antenna size. Recently, we found that higher plant CAOs consist of three domains (A, B, and C domains) and confirmed that the C domain possesses catalytic function. To investigate the function of the A domain, we fused various combinations of these three domains with green fluorescent protein (GFP) and introduced them into Arabidopsis thaliana. When a full-length CAO-GFP fusion protein was introduced into a chlorophyll b-less chlorina1-1 mutant, chlorophyll b accumulated to almost the same levels as in the chlorophyll b-containing Columbia wild type, but the CAO-GFP could not be detected by immunoblotting. By contrast, when a GFP-C domain fusion was introduced into chlorina1-1 or Columbia wild type, a large amount of GFP-C domain protein accumulated and the chlorophyll a/b ratio decreased drastically from 3.6 to 2.2 in Columbia wild type. When an A domain-GFP was introduced into Columbia wild type, A domain-GFP levels were very low. Conversely, a large amount of the protein accumulated when it was introduced into the chlorina1-1 mutant. These results indicate that the A domain may sense the presence of chlorophyll b and regulate the accumulation of CAO protein in the chloroplasts.

Synthesis of chlorophyll b: localization of chlorophyllide a oxygenase and discovery of a stable radical in the catalytic subunit.

BACKGROUND: Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly. RESULTS: Fluorescence of a CAO-GFP fusion protein, transiently expressed in young pea leaves, was found at the periphery of mature chloroplasts and on thylakoid membranes by confocal fluorescence microscopy. However, when membranes from partially degreened cells of Chlamydomonas reinhardtii cw15 were resolved on sucrose gradients, full-length CAO was detected by immunoblot analysis only on the chloroplast envelope inner membrane. The electron paramagnetic resonance spectrum of CAO included a resonance at g = 4.3, assigned to the predicted mononuclear iron center. Instead of a spectrum of the predicted Rieske iron-sulfur center, a nearly symmetrical, approximately 100 Gauss peak-to-trough signal was observed at g = 2.057, with a sensitivity to temperature characteristic of an iron-sulfur center. A remarkably stable radical in the protein was revealed by an isotropic, 9 Gauss peak-to-trough signal at g = 2.0042. Fragmentation of the protein after incorporation of 125I- identified a conserved tyrosine residue (Tyr-422 in Chlamydomonas and Tyr-518 in Arabidopsis) as the radical species. The radical was quenched by chlorophyll a, an indication that it may be involved in the enzymatic reaction. CONCLUSION: CAO was found on the chloroplast envelope and thylakoid membranes in mature chloroplasts but only on the envelope inner membrane in dark-grown C. reinhardtii cells. Such localization provides further support for the envelope membranes as the initial site of Chl b synthesis and assembly of LHCs during chloroplast development. Identification of a tyrosine radical in the protein provides insight into the mechanism of Chl b synthesis.

Random mutagenesis targeted to the psbAII gene of Synechocystis sp. PCC 6803 to identify functionally important residues in the D1 protein of the photosystem II reaction center.

More than one hundred mutants of Synechocystis sp. PCC 6803 impaired in photoautotrophic growth were generated by in vitro random PCR mutagenesis targeted to a region of the psbAII gene corresponding to a 210 amino acid (Ser148-Ala357) segment of the D1 protein. The 90 random mutants that could translate the full-length D1 protein carried 1-9 (on average 3.0) amino acid substitutions in the targeted region. Mutations were often found in the obligate photoheterotrophic strains at specific residues that have been reported or speculated to be important in the function of PSII, such as Y161, H198, H272, E333 and H337. This verifies the usefulness of the present method to identify functionally important residues in PSII. Other residues that were often mutated in the strains with impaired photoautotrophy included non-charged residues around the lumenal edges of transmembrane helices C, D and E, such as I192 and N296. Eleven mutants carried a single-point mutation in residues, such as Q165, Q187, W278, A294 and N298, and these identified the functional importance of these residues, most of which were on the donor side of PSII. A preliminary characterization of some of the mutants obtained in this study is provided.