Rapid increase of vacuolar volume in response to salt stress

Suspension-cultured cells of mangrove [Bruguiera sexangula (Lour.) Poir.] showed a rapid increase in vacuolar volume under salt stress, although there was no change in the cell volume. The rapid increase in the vacuolar volume was an active process, which followed the activation of the tonoplast H(+)-ATPase and the vacuolar acid phosphatase. The same phenomenon was observed in barley (Hordeum vulgare L. cv. Doriru) root meristematic cells under salt stress but not in pea ( Pisum sativum L.). Increases in vacuolar volume could potentially protect the cytoplasm by decreasing the cytoplasmic volume during the initial phases of salt stress.     

Age-related changes of element contents in human tendon of the iliopsoas muscle and the relationships among elements

To elucidate compositional changes of the tendons with aging, the authors investigated age-related changes of element contents in the tendon of the iliopsoas muscle by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of 14 men and 14 women, ranging in age from 4 to 102 yr. The insertion tendons of the iliopsoas were removed from the cadavers after ordinary dissection and also surgically from patients with congenital dislocation of the hip. It was found that both Mg and P in the tendon of the iliopsoas muscle increased significantly with aging, but the other elements, such as S, Ca, Na, Zn, Fe, and Si, hardly changed with aging. With regard to the relationship among element contents, extremely significant correlations were found between P and S contents, between Mg and Na contents, and between S and Zn contents. In addition, very significant correlations were found between Si and either S or Zn contents. However, there was no significant correlation between Ca and P contents. These results revealed that with regard to age-related changes of element contents and the relationships among element contents, the tendon of the iliopsoas muscle was different from the Achilles tendon, in which both Mg and P decreased significantly with aging and there were significant correlations each other among the contents of S, Mg, and P.     

Localization of sepiapterin reductase in pigment cells of Oryzias latipes

Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory.     

Age-dependent decreases of phosphorus and magnesium in human achilles’ tendons

To elucidate changes of human tendons with aging, the authors studied age-related changes of elements in human Achilles’ tendons by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. It was found that the content of calcium increased progressively with aging in the Achilles’ tendons, whereas the contents of phosphorus and magnesium decreased gradually with aging. The previous investigations demonstrated that the content of calcium and phosphorus increased progressively with aging in most, but not all, human tissues, except for the bones. In ligaments, such as the anterior cruciate ligament and the ligament of the head of the femur, which are histologically similar to the Achilles’ tendon, it was previously found that both the contents of calcium and phosphorus increased with aging in the ligaments. It should be noted that the content of phosphorus in the Achilles’ tendons decreased during the aging process. In addition, it was found that there was a very high direct correlation between phosphorus and magnesium contents in the tendons, but not between calcium and phosphorus contents.     

Correlations of calcium accumulations in arteries, veins, cartilages, ligaments, and bones in single humans

To show the relationships of calcium accumulation in the thoracic aorta to the other tissues, calcium contents were determined with a microwave-induced plasma-atomic emission spectrometer on arteries, veins, cartilages, ligaments, and bones. These tissues were resected from 18 individuals, consisting of 11 men and 7 women who died in the age range 59–91 yr. As thoracic and abdominal aortas are routinely used for radiographic examination of arterial calcification, they appear to be standard tissues of the calcium accumulation. The calcium accumulations were determined in the femoral artery, the superior and inferior venae cavae, the internal jugular vein, cartilages of the articular disk of the temporomandibular joint and the intervertebral disk, both the ligaments of the anterior cruciate ligament and the ligamentum capitis femoris, and the calcaneus, in contrast with the thoracic aorta. As calcium increased in the thoracic aorta, it increased in the femoral artery, the articular disk of the temporomandibular joint, the intervertebral disk, both ligaments of the anterior cruciate ligament, and the ligamentum capitis femoris, but it did not increase in veins, such as the superior and inferior venae cavae and the internal jugular vein. In contrast, it decreased in the calcaneus.     

Age-related changes of element contents in the human meniscus

The relative contents (RCs) of elements in the human menisci from 23 subjects in the age range between 65 and 93 yr were analyzed by inductively coupled plasma atomic emission spectrometry. The RCs of sulfur, calcium, and phosphorus in menisci increased progressively until the 80s, being the highest in the 80s, and thereafter decreased. The RCs of magnesium in menisci increased progressively until the 90s. Regarding the medial and lateral menisci, higher RCs of magnesium and iron, and a lower RC of phosphorus were found in lateral menisci in comparison with those in medial menisci. There were sexual differences in the RCs of calcium and phosphorus of medial and lateral menisci. The RCs of calcium and phosphorus were about 50% higher in women’s menisci than in men’s. Histological examinations showed that structureless mucoid masses were observed in the menisci, with very high RCs of calcium and phosphorus being detected.     

Structure–activity relationships of bacterial outer-membrane permeabilizers based on polymyxin B heptapeptides

A series of cationic cyclic heptapeptides based on polymyxin B have been synthesized for use as permeabilizers of the outer membrane of Gram-negative bacteria. Only analogs with the Dab²-d-Phe³-Leu⁴-Xxx⁵ sequence (Xxx = Dab or Orn) showed a synergistic bactericidal effect when combined with conventional antibiotics, indicating that the Dab² residue plays a critical role in permeation of the outer membrane of Gram-negative bacteria.     

Cell wall proteome of wheat roots under flooding stress using gel-based and LC MS/MS-based proteomics approaches

Cell wall proteins (CWPs) are important both for maintenance of cell structure and for responses to abiotic and biotic stresses. In this study, a destructive CWP purification procedure was adopted using wheat seedling roots and the purity of the CWP extract was confirmed by minimizing the activity of glucose-6-phosphate dehydrogenase, a cytoplasmic marker enzyme. To determine differentially expressed CWPs under flooding stress, gel-based proteomic and LC-MS/MS-based proteomic techniques were applied. Eighteen proteins were found to be significantly regulated in response to flood by gel-based proteomics and 15 proteins by LC MS/MS-based proteomics. Among the flooding down-regulated proteins, most were related to the glycolysis pathway and cell wall structure and modification. However, the most highly up-regulated proteins in response to flooding belong to the category of defense and disease response proteins. Among these differentially expressed proteins, only methionine synthase, β-1,3-glucanases, and β-glucosidase were consistently identified by both techniques. The down-regulation of these three proteins suggested that wheat seedlings respond to flooding stress by restricting cell growth to avoid energy consumption; by coordinating methionine assimilation and cell wall hydrolysis, CWPs played critical roles in flooding responsiveness.     

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel‐based and a LC MS/MS‐based proteomics method. Two‐day‐old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two‐phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel‐based proteomics, four and eight protein spots were identified as up‐ and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up‐ and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low‐abundance proteins could be identified by the LC MS/MS‐based method. Three homologues of plasma membrane H⁺‐ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H⁺‐ATPase protein.     

Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase,an Enzyme Involved in the Metabolism of Daidzein,from Equol-Producing Lactococcus Strain 20-92

Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.Isoflavones are flavonoids present in various plants and are known to be abundant in soybeans and legumes. These compounds have been called phytoestrogens because their chemical structure is similar to that of the female sex hormone, estrogen. Isoflavones have an ability to bind to estrogen receptors and show protection against or improvement in several sex hormone-dependent diseases, such as breast cancer, prostate cancer, menopausal disorder, lower bone density, and hypertension, due to their weak agonistic or antagonistic effects (1, 19, 27).Daidzein is one of the main soy isoflavonoids produced from daidzin by the glucosidase of intestinal bacteria (17). Equol is a metabolite produced from daidzein by the enterobacterial microflora (5). Recently, equol has attracted a great deal of attention because its estrogenic activity is more potent than that of other isoflavonoids, including daidzein (27). It is well known that individual variation exists in the ability of these enteric microflora to produce equol and that less than half the human population is capable of producing equol after ingesting soy isoflavones (3). Therefore, to increase the production of equol in the enteric environment of each individual, the development of probiotics using safe bacteria which have the ability to produce equol from daidzein is ongoing.Lactococcus strain 20-92 (Lactococcus 20-92; 30a) is an equol-producing lactic acid bacterium isolated from the feces of healthy humans by Uchiyama et al. (30). This bacterium is spherical and Gram positive and is a strain of L. garvieae. The application of Lactococcus 20-92 in probiotics is advantageous because L. garvieae is not pathogenic or toxic to humans.To date, other bacterial strains that are capable of transforming daidzein to dihydrodaidzein or equol have been isolated (9, 21, 22, 23, 29, 32, 36, 37). Daidzein is thought to be metabolized by human intestinal bacteria to equol or to O-desmethylangolensin via dihydrodaidzein and tetrahydrodaidzein (14, 15, 22, 32); however, neither the enzymes involved in the metabolism of daidzein to equol nor even the metabolic pathway has been clarified fully for equol-producing bacteria.In this study, we purified an enzyme from Lactococcus 20-92 that assisted in the conversion of daidzein to dihydrodaidzein. Furthermore, we cloned the L-DZNR gene and expressed the active recombinant enzyme in E. coli.