Deoxynucleoside triphosphate pool of mouse FM3A cell lines unaffected by mutagen treatment

The size of deoxynucleoside triphosphate pool in cultured mouse FM3A cells and mutator mutants isolated from this cell line was determined by high-pressure liquid chromatography after treatment of the cells with ultraviolet light, N-methyl-N′-nitro-N-nitrosoguanidine or mitomycin C. The results showed that, in all the FM3A cell clones, no large increase in the dATP or TTP pool was induced after treatment, while in some cases 40–50% decrease in dCTP pool was observed. It is concluded that the induction of large increase in dNTP pool is not the general effect of the mutagens.     

Antioxidative enzymes in seedlings of Nelumbo nucifera germinated under water

Dry seeds of anoxia-tolerant lotus ( Nelumbo nucifera Gaertn= Nelumbium speciosum Willd.) have green shoots with plastids containing chlorophyll, so photosynthesis starts even in seedlings germinated under water, namely hypoxia. Here we investigated antioxidative enzyme changes in N. nucifera seedlings responding to oxygen deficiency. The activity of superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were lower in seedlings germinated under water (submerged condition) in darkness (SD seedlings) than those found in seedlings germinated in air and darkness (AD seedlings). In contrast, ascorbate peroxidase (APX; EC 1.11.1.11) activity was higher in SD seedlings and the activity of catalase (EC 1.11.1.6) and monodehydroascorbate reductase (MDAR; EC 1.6.5.4) in SD seedlings was nearly the same as in AD seedlings. When SD seedlings were exposed to air, the activity of SOD, DHAR and GR increased, while the activity of catalase and MDAR decreased. Seven electrophoretically distinct SOD isozymes were detectable in N. nucifera . The levels of plastidic Cu,Zn-SODs and Fe-SOD in SD seedlings were comparable with those found in AD seedlings, which may reflect the maintenance of green plastids in SD seedlings as well as in AD seedlings. These results were substantially different from those previously found in rice seedlings germinated under water.     

Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans

To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen-induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major collagen-signaling receptor glycoprotein (GP)VI. Moreover, triplatin-1 inhibited Fc receptor gamma-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates.     

Two Phenolic Amides in the Seed Balls of Sugar Beet (Beta vulgaris L. var. saccharifera Alefeld)

Ferredoxin-dependent sulfite reductase (Fd-SiR) (EC 1.8.7.1) was purified about 1136-fold, with a yield of 11%, from fresh thalli of Porphyra yezoensis by a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Buty 1-Toyopearl chromatography, Sephadex G-100 gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous, as judged on polyacrylamide disc gel electrophoresis, with a specific activity of 100 units/mg of protein. The molecular weight of the enzyme was estimated to be 70 kilodaltons by gel filtration. On subunit analysis by SDS-PAGE, a single band corresponding to molecular weight of 65 kilodaltons appeared. The purified enzyme (Fd-SiR) showed 5-times higher ferredoxin-dependent activity than methyl viologen-linked activity. In the oxidized form, the enzyme exhibited absorption maxima at 278, 390 (Soret band), 586 (a band) and 714 (CT band) nm, indicating that siroheme is involved in the catalysis of sulfite reduction. The absorbance ratios, A₃₉₀: A₂₁₈ and A₅₈₆ :A₃₉₀, were 0.32 and 0.31, respectively. A plot of the substrate (sulfite) and electron donor (ferredoxin) concentrations versus enzymatic (Fd-SiR) activity yielded sigmoidal curves, giving Hill coefficients («) of 2.3 (for sulfite) and 2.7 (for ferredoxin), respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion suggested that it was specific against isolated Fd-SiR. Using the antiserum, dot immunoblotting was performed to determine the immunological similarity of Fd-SiRs from Porphyra yezoensis, Spirulina platensis, Brassica chinensis and Spinacia oleracea. The tests revealed that the four forms of assimilatory Fd-SiR have antigenic determinants in common.     

Mapping quantitative trait loci for root development under hypoxia conditions in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.)

Key message

Greatest potential, QTLs for hypoxia and waterlogging tolerance in soybean roots were detected using a new phenotypic evaluation method.

Abstract

Waterlogging is a major environmental stress limiting soybean yield in wet parts of the world. Root development is an important indicator of hypoxia tolerance in soybean. However, little is known about the genetic control of root development under hypoxia. This study was conducted to identify quantitative trait loci (QTLs) responsible for root development under hypoxia. Recombinant inbred lines (RILs) developed from a cross between a hypoxia-sensitive cultivar, Tachinagaha, and a tolerant landrace, Iyodaizu, were used. Seedlings were subjected to hypoxia, and root development was evaluated with the value change in root traits between after and before treatments. We found 230 polymorphic markers spanning 2519.2 cM distributed on all 20 chromosomes (Chrs.). Using these, we found 11 QTLs for root length (RL), root length development (RLD), root surface area (RSA), root surface area development (RSAD), root diameter (RD), and change in average root diameter (CARD) on Chrs. 11, 12, 13 and 14, and 7 QTLs for hypoxia tolerance of these root traits. These included QTLs for RLD and RSAD between markers Satt052 and Satt302 on Chr. 12, which are important markers of hypoxia tolerance in soybean; those QTLs were stable between 2 years. To validate the QTLs, we developed a near-isogenic line with the QTL region derived from Iyodaizu. The line performed well under both hypoxia and waterlogging, suggesting that the region contains one or more genes with large effects on root development. These findings may be useful for fine mapping and positional cloning of gene responsible for root development under hypoxia.

     

Probes for Catalytic Action of α-Chymotrypsin in Plastein Synthesis

A plastein was synthesized with α-chymotrypsin from a dialyzable fraction of a peptic hydrolysate of soybean protein.

The plastein was obtainable also by use of an insoluble preparation of α-chymotrypsin. This may rule out the possibility that the plastein is a product resulting from some chemical peptide-protein (enzyme) aggregation.

No appreciable amount of the plastein was produced when chymotrypsinogen was used instead of α-chymotrypsin.

The plastein synthetic, as well as the protein hydrolytic, activity of α-chymotrypsin was inhibited more or less by a hydrophobic inhibitor (n-hexane), a competitive inhibitor (benzolyl-d,l-phenylalanine), and divalent cations (Zn²⁺, Hg²⁺ and Cu²⁺); the degree of inhibition in each case was approximately similar against both the synthetic and the hydrolytic activities.

Either diisopropylphosphorylation of the β-O of Ser-195 or methylation of the 3-N of His-57 imidazole of α-chymotrypsin repressed the synthetic, as well as the hydrolytic, activity.

Based on these results a possible mechanism was discussed of the plastein synthesis by α-chymotrypsin, especially in relevance to its acylation and deacylation.     

Experimental candidiasis in liver injury

In an attempt to evaluate effects of liver injury and roles of iron metabolism on systemic fungal infection, experimental systemicCandida infection was produced in mice with galactosamine-induced liver injury. Survival rate and extent of fungal lesion are compared between mice with liver injury (Group 1) and ones without liver injury (Group 2). Median survival was 7 and 18 days in Group 1 and 2 respectively after 21 days observation. Mortality rate of Group 1 was significantly higher (P=0.05) than that of Group 2. This difference was reflected to the extent of fungal lesions in that they were extensive and disseminated, involving the multiple organs in Group 1 but predominantly localized to the kidneys in Group 2. UIBC (unbound iron binding capacity) and TIBC (total iron binding capacity), i.e., serum transferrin as well as serum iron levels were significantly lower in Group 1 as compared with those in Group 2. These results indicate that hepatic injury promotesCandida infectionin vivo and suggest that increased susceptibility toCandida in the presence of liver injury is, at least partially, attributable to low UIBC and/or TIBC.     

Detection and identification of Escherichia coli,Shigella, and Salmonella by microarrays using the gyrB gene

Commonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for identifying enteric bacteria. However, it may not always be applicable for distinguishing closely related bacteria. Therefore, we selected gyrB genes that encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as target genes. The molecular evolution rate of gyrB genes is higher than that of 16S rRNA, and gyrB genes are distributed universally among bacterial species. Microarray technology includes the methods of arraying cDNA or oligonucleotides on substrates such as glass slides while acquiring a lot of information simultaneously. Thus, it is possible to identify the enteric bacteria easily using microarray technology. We devised a simple method of rapidly identifying bacterial species through the combined use of gyrB genes and microarrays. Closely related bacteria were not identified at the species level using 16S rRNA sequence analysis, whereas they were identified at the species level based on the reaction patterns of oligonucleotides on our microarrays using gyrB genes.     

Further studies on the biosynthesis of mangiferin in Anemarrhena asphodeloides: hydroxylation of the shikimate-derived ring

The hydroxylation at C-3′ of maclurin, an intermediate in mangiferin biosynthesis, has been studied. Labelled cinnamic acid, p-coumaric acid, caffeic acid, iriflophenone and maclurin were fed to Anemarrhena asphodeloides. Cinnamic acid and p-coumaric acid were better precursors than caffeic acid for mangiferin, and iriflophenone as well as maclurin was effectively incorporated into mangiferin and isomangiferin. These results show that maclurin is biosynthesized via hydroxylation of iriflophenone derived from p-coumarate in this plant.