Differences in uptake kinetics of ammonium and nitrate in legumes and cereals

Influx isotherms were obtained for nitrate and ammonium from three legumes, Cajanus cajan (L.) Millsp., Cicer arietinum L. and Arachis hypogaea L. and three cereals, Sorghum bicolor (L.) Moench., Pennisetum glaucum L. and Zea mays L. The transition in influx isotherms for both nitrogen sources was found to be within the concentration range (0.05–2.5 mM) tested. There were significant differences in Km and Vmax for ammonium between legumes and cereals. The difference in the kinetic properties for nitrate uptake between the two groups of plants only became apparent at the higher concentration tested. Legumes translocated absorbed nitrate and ammonium to shoots more rapidly than cereals. Results show that there are significant differences in uptake and translocation of ammonium and nitrate between legumes and cereals.     

Disruption of the CAR1 Gene Encoding Arginase Enhances Freeze Tolerance of the Commercial Baker's Yeast Saccharomyces cerevisiae

The effect of intracellular charged amino acids on freeze tolerance in doughs was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.     

External calcium ions are requisite for fertilization of sea urchin eggs by spermatozoa with reacted acrosomes

That a small amount of external calcium ions is requisite for the fertilization by spermatozoa with reacted acrosomes was found by some simple experiments using jelly-treated sperm of the sea urchin, Hemicentrotus pulcherrimus. When eggs were inseminated with the jelly-treated sperm in artificial seawaters containing calcium at various concentrations, the percentage of fertilization decreased concomitant with the reduction in the amount of external calcium ions, 50% at 40 μM calcium and almost 0% at less than 10 μM. On the other hand, it was observed that both the morphology of the reacted acrosome and the binding capacity of the jelly-treated spermatozoa to eggs were not influenced by the calcium deficiency. These results suggest that external calcium ions are indispensable even for the fertilization processes following sperm binding to eggs after the acrosome reaction, such as penetration of reacted spermatozoa through vitelline layer and/or membrane fusion between egg and spermatozoon.     

Ammonia and other weak bases applied at any time of the hormone-dependent period inhibit 1-methyladenine-induced meiosis reinitiation of starfish oocytes

The effect of weak bases on starfish oocyte maturation induced by 1-methyladenine (1-MeAde) was investigated. Ammonium chloride, nicotine, and procaine inhibited meiosis reinitiation induced by either 1-MeAde or dithiothreitol. The inhibitory effect of the weak bases was highly dependent on the pH of sea water and increased with the increase in pH. These weak bases neither inhibited nor delayed meiosis reinitiation when given after the end of the hormone-dependent period, the period during which 1-MeAde is required for meiosis to occur, whereas they blocked hormone action when added just before the end of the hormone-dependent period.     

Toad egg-jelly as a source of divalent cations essential for fertilization

Dejellied uterine eggs of the toad Bufo bufo japonicus are not fertilizable in 1/20 De Boer's solution (1/20 DB), but are fertilized when inseminated in a uv-solubilized jelly (UVJ) or the dialyzate of UVJ (UVJD). The present study was carried out to define this fertilization-supporting activity of egg-jelly. Dejellied eggs were fertilized in a high frequency when inseminated in a medium containing the ashes obtained by heating UVJD at 600 degrees C for 16 hr. Similarly, a reconstituted salt solution (RSS), which mimics the ionic composition of UVJD, supported a high rate of fertilization. To be effective in fertilization, however, RSS had to be present at the time of insemination. Analyses of individual salts revealed that dejellied eggs are successfully fertilized in CaCl2 and/or MgCl2 at 1-5 mM, only slightly in KCl at 10 mM, but not at all in NaCl at any of the concentrations tested. The activity of UVJD was lost reversibly when divalent cations were chelated by EDTA. The fertilization of dejellied eggs is therefore possible in a medium without any organic components of egg-jelly, provided that 2-5 mM Ca2+ or Mg2+ is present. Sperm were motile in media containing cations below 20-25 mM, regardless of the ionic composition. The egg-jelly possessed cations in a concentration of about 130 mM, but most ions were lost from intact jelly on immersion of eggs in water for 2-3 min, accompanied by the acquisition of fertilizability by sperm. Examination of the behavior of salts on dialysis or gel-filtration of jelly molecules revealed that the jelly retains Ca2+ and Mg2+, and possibly K+ as well, but not Na+ and Cl-. We propose that toad egg-jelly plays a function in fertilization by retaining Ca2+ and/or Mg2+ around each egg at the level necessary for successful sperm entrance into the egg.     

Red shift in the spectrum of a chlorophyll species is essential for the drought-induced dissipation of excess light energy in a poikilohydric moss, <Emphasis Type="Italic">Bryum argenteum</Emphasis>

Some mosses are extremely tolerant of drought stress. Their high drought tolerance relies on their ability to effectively dissipate absorbed light energy to heat under dry conditions. The energy dissipation mechanism in a drought-tolerant moss, Bryum argenteum, has been investigated using low-temperature picosecond time-resolved fluorescence spectroscopy. The results are compared between moss thalli samples harvested in Antarctica and in Japan. Both samples show almost the same quenching properties, suggesting an identical drought tolerance mechanism for the same species with two completely different habitats. A global target analysis was applied to a large set of data on the fluorescence-quenching dynamics for the 430-nm (chlorophyll-a selective) and 460-nm (chlorophyll-b and carotenoid selective) excitations in the temperature region from 5 to 77 K. This analysis strongly suggested that the quencher is formed in the major peripheral antenna of photosystem II, whose emission spectrum is significantly broadened and red-shifted in its quenched form. Two emission components at around 717 and 725 nm were assigned to photosystem I (PS I). The former component at around 717 nm is mildly quenched and probably bound to the PS I core complex, while the latter at around 725 nm is probably bound to the light-harvesting complex. The dehydration treatment caused a blue shift of the PS I emission peak via reduction of the exciton energy flow to the pigment responsible for the 725 nm band.     

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco

The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres.     

Involvement of AtNAP1 in the regulation of chlorophyll degradation in Arabidopsis thaliana

In plants, chlorophyll is actively synthesized from glutamate in the developmental phase and is degraded into non-fluorescent chlorophyll catabolites during senescence. The chlorophyll metabolism must be strictly regulated because chlorophylls and their intermediate molecules generate reactive oxygen species. Many mechanisms have been proposed for the regulation of chlorophyll synthesis including gene expression, protein stability, and feedback inhibition. However, information on the regulation of chlorophyll degradation is limited. The conversion of chlorophyll b to chlorophyll a is the first step of chlorophyll degradation. In order to understand the regulatory mechanism of this reaction, we isolated a mutant which accumulates 7-hydroxymethyl chlorophyll a (HMChl), an intermediate molecule of chlorophyll b to chlorophyll a conversion, and designated the mutant hmc1. In addition to HMChl, hmc1 accumulated pheophorbide a, a chlorophyll degradation product, when chlorophyll degradation was induced by dark incubation. These results indicate that the activities of HMChl reductase (HAR) and pheophorbide a oxygenase (PaO) are simultaneously down-regulated in this mutant. We identified a mutation in the AtNAP1 gene, which encodes a subunit of the complex for iron–sulfur cluster formation. HAR and PaO use ferredoxin as a reducing power and PaO has an iron-sulfur center; however, there were no distinct differences in the protein levels of ferredoxin and PaO between wild type and hmc1. The concerted regulation of chlorophyll degradation is discussed in relation to the function of AtNAP1.     

A case of giardiasis expressing severe systemic symptoms and marked hypereosinophilia

An 88-year-old Japanese woman was referred to our hospital due to a one-month history of face edema, aphagia, shortness of breath, and skin rush over almost her entire skin. She had no abdominal symptoms. Her peripheral blood count showed a white blood cell (WBC) count of 27.1 × 10⁹/L with 82.1% eosinophils. Serum non-specific Immunoglobulin E was within a normal range. Soluble interleukin-2 receptor was elevated to 4200 U/mL. At first, her eosinophil count was so high that we suspected she had an eosinophilic leukemia or hypereosinophilic syndrome. After admission, cysts of Giardia duodenalis (G. duodenalis) were detected in the patient's feces by microscopic analysis, then she was diagnosed with giardiasis, and 750 mg per day of metronidazole was administered for seven days. Her WBC count decreased to 6.0 × 10⁹/L with 10% eosinophils, and her systemic symptoms improved. At that time her serum IL-5 was within a normal range. A few months later, the patient again complained of skin rush, and G. duodenalis was once again found in her feces. Her serum IL-5 was elevated to 751 pg/mL. Metronidazole was administered for two weeks, and her eosinophil count decreased. G. duodenalis is a protozoan parasite, and it is one of the most common waterborne transmission gastrointestinal parasites in the world. G. duodenalis rarely causes hypereosinophilia. To our knowledge, this is the first case report of giardiasis with extreme hypereosinophilia and severe systemic symptoms.     

Effects of retinol binding protein-4 on vascular endothelial cells

The study was designed to investigate the effect of retinol binding protein (RBP)-4 on the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, which mediate the effects of insulin in vascular endothelial cells. The effects of RBP4 on nitric oxide (NO) and insulin-stimulated endothelin-1 (ET-1) secretion and on phosphorylation (p) of Akt, endothelial NO synthetase (eNOS), and extracellular signal-regulated kinase (ERK)1/2 were investigated in bovine vascular aortic endothelial cells (BAECs). RBP4 showed an acute vasodilatatory effect on aortic rings of rats within a few minutes. In BAECs, RBP4-treatment for 5 min significantly increased NO production, but inhibited insulin-stimulated ET-1 secretion. RBP4-induced NO production was not inhibited by tetraacetoxymethylester (BAPTA-AM), an intracellular calcium chelator, but was completely abolished by wortmannin, a PI3K inhibitor. RBP4 significantly increased p-Akt and p-eNOS production, and significantly inhibited p-ERK1/2 production. Triciribine, an Akt inhibitor, and wortmannin significantly inhibited RBP4-induced p-Akt and p-eNOS production. Inhibition of Akt1 by small interfering RNA decreased p-eNOS production enhanced by RBP4 in human umbilical vein endothelial cells. In conclusion, RBP4 has a robust acute effect of enhancement of NO production via stimulation of part of the PI3K/Akt/eNOS pathway and inhibition of ERK1/2 phosphorylation and insulin-induced ET-1 secretion, probably in the MAPK pathway, which results in vasodilatation.