An extremely stable inorganic pyrophosphatase purified from the cytosol of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius strain 7.

A highly active inorganic pyrophosphatase was purified to electrophoretical homogeneity from the cytosol of Sulfolobus acidocaldarius strain 7, an extremely thermoacidophilic archaebacterium. The enzyme has an apparent molecular mass of 80 kDa as estimated by gel permeation chromatography, and showed a 21-kDa polypeptide on SDS-PAGE, suggesting that the archaebacterial enzyme is similar to most of the eubacterial pyrophosphatases rather than eukaryotic ones. The pI = 5.1. The enzyme showed relatively high content of Pro and low content of Ser plus Thr. The optimal pH was 6.5 (at 56 degrees C). From the Arrhenius plot an activation energy of 11.2 kcal/mol was obtained between 37-95 degrees C. The specific activity was 617 mumol Pi release min-1 mg-1 at 56 degrees C. The S. acidocaldarius pyrophosphatase was extremely stable. Complete activity remained after incubation at 100 degrees C for 10 min. No dissociation into subunit or unfolding of polypeptide chain occurred in the presence of 8 M urea. Experiments using guanidine-HCl suggested that the transition between a native tetrameric state and an unfolded state is completely reversible, and essentially independent of any additional factors such as divalent metal cation or dithiothreitol.     

Effects of Controlled-Release Fertilizer on Leaf Area Index and Fruit Yield in High-Density Soilless Tomato Culture Using Low Node-Order Pinching

To further development of a simplified fertigation system using controlled-release fertilizers (CRF), we investigated the effects of differing levels of fertilizers and plant density on leaf area index (LAI), fruit yields, and nutrient use in soilless tomato cultures with low node-order pinching and high plant density during spring-summer (SS), summer-fall (SF), and fall-winter (FW) seasons. Plants were treated with 1 of 3 levels of CRF in a closed system, or with liquid fertilizer (LF) with constant electrical conductivity (EC) in a drip-draining system. Two plant densities were examined for each fertilizer treatment. In CRF treatments, LAI at pinching increased linearly with increasing nutrient supply for all cropping seasons. In SS, both light interception by plant canopy at pinching and total marketable fruit yield increased linearly with increasing LAI up to 6 m²·m⁻²; the maximization point was not reached for any of the treatments. In FW, both light interception and yield were maximized at an LAI of approximately 4. These results suggest that maximizing the LAI in SS and FW to the saturation point for light interception is important for increasing yield. In SF, however, the yield maximized at an LAI of approximately 3, although the light interception linearly increased with increasing LAI, up to 4.5. According to our results, the optimal LAI at pinching may be 6 in SS, 3 in SF, and 4 in FW. In comparing LAI values with similar fruit yield, we found that nutrient supply was 32−46% lower with the CRF method than with LF. In conclusion, CRF application in a closed system enables growers to achieve a desirable LAI to maximize fruit yield with a regulated amount of nutrient supply per unit area. Further, the CRF method greatly reduced nutrient use without decreasing fruit yield at similar LAIs, as compared to the LF method.     

Histone H3 peptides incorporating modified lysine residues as lysine-specific demethylase 1 inhibitors

Lysine-specific demethylase 1 (LSD1) is a flavin-dependent enzyme that removes methyl groups from mono- or dimethylated lysine residues at the fourth position of histone H3. We have previously reported several histone H3 peptides containing an LSD1 inactivator motif at Lys-4. In this study, histone H3 peptides having a trans-2-phenylcyclopropylamine (PCPA), a 2,5-dihydro-1H-pyrrole, and a 1,2,3,6-tetrahydropyridine moiety at Lys-4 were prepared along with related compounds possessing a shorter side chain at the fourth position. Enzymatic assays showed that PCPA peptides containing a longer side chain, which can react with FAD in the active site, are potent LSD1-selective inhibitors.     

Polymerase Chain Reaction Test for Differentiation of Five Toxin Types of Clostridium perfringens

In order to avoid the use of experimental animals, the polymerase chain reaction (PCR) method was applied to differentiate Clostridium perfringens into five toxin types. Twenty-two out of 23 strains tested produced the toxin(s) corresponding to the toxin gene(s) identified by PCR, and vice versa. Consequently, the gene typing was consistent with conventional typing by animal tests. Twenty-five strains were identified as types different from original ones by the PCR method as well as a toxin neutralization test. These findings suggest that the PCR method, which is easy and timesaving, is applicable to identify the toxin types of C. perfringens as an alternative to animal tests, and that beta-, epsilon- and iota-toxin genes might be lost by long-term preservation. The reasons why the strains lost the genes are discussed.     

Effects of manganese toxicity on photosynthesis of white birch (Betula platyphylla var. japonica) seedlings

The effects of manganese (Mn) toxicity on photosynthesis in white birch ( Betula platyphylla var. japonica ) leaves were examined by the measurement of gas exchange and chlorophyll fluorescence in hydroponically cultured plants. The net photosynthetic rate at saturating light and ambient CO₂ (C_a) of 35 Pa decreased with increasing leaf Mn concentrations. The carboxylation efficiency, derived from the difference in CO₂ assimilation rate at intercellular CO₂ pressures attained at C_a of 13 Pa and O Pa, decreased with greater leaf Mn accumulation. Net photosynthetic rate at saturating light and saturating CO₂ (5%) also declined with leaf Mn accumulation while the maximum quantum yield of O₂ evolution at saturating CO₂ was not affected. The maximum efficiency of PSII photochemistry (F_v/F_m) was little affected by Mn accumulation in white birch leaves over a wide range of leaf Mn concentrations (2–17 mg g₋₁ dry weight). When measured in the steady state of photosynthesis under ambient air at 430 μmol quanta m₋₂ s₋₁, the levels of photochemical quenching (qP) and the excitation capture efficiency of open PSII (F'^v/F'^m) declined with Mn accumulation in leaves. The present results suggest that excess Mn in leaves affects the activities of the CO² reduction cycle rather than the potential efficiency of photochemistry, leading to increases in Q_A reduction state and thermal energy dissipation, and a decrease in quantum yield of PSII in the steady state.     

Analysis of Genetic Relationships and Antimicrobial Susceptibility of Verotoxin-Producing Escherichia coli Strains Isolated in Nagasaki Prefecture,Japan in 1996

A total of 19 Escherichia coli O157 isolates were obtained in Nagasaki Prefecture, in the southwestern part of Japan, between 1990 and 1996. Pulsed-field gel electrophoresis (PFGE) and computer-assisted analysis were applied to determine genetic relationships among these strains. Fragment patterns of the isolates in Nagasaki, as determined by PFGE, were compared with those of isolates in other areas where large outbreaks and sporadic cases of E. coli O157 infection occurred. Similarity values of all the strains isolated in Nagasaki Prefecture were over 0.65 except for E. coli O26. Some strains were identical to the strains isolated from the areas where large outbreaks occurred. All strains were susceptible to ampicillin, fosfomycin, minocycline, amikacin, ofloxacin and sulfamethoxazole-trimethoprim.     

Reproductive interference between two serious pests,oriental fruit flies <Emphasis Type="Italic">Bactrocera carambolae</Emphasis> and <Emphasis Type="Italic">B. dorsalis</Emphasis> (Diptera: Tephritidae), with very wide but partially overlapping host ranges

Bactrocera carambolae and B. dorsalis (Diptera: Tephritidae) are extremely destructive pests of fruits and vegetables in the Asia-Pacific region. Earlier reports have described that B. carambolae and B. dorsalis, respectively, use mainly star fruit and mango, suggesting a certain level of host partitioning which can be ascribed neither to differences in larval food qualities nor host-specific parasitoid mortality. This study specifically examined reproductive interference (antagonistic sexual interaction) between B. carambolae and B. dorsalis as a potential factor strongly affecting their host partitioning. We observed mating behaviors, especially interspecific courtships and mating, by cohabiting the conspecific and heterospecific pairs together. Consequently, we quantified their effects on the reproductive success of females. Males of both species frequently courted their own females, but they also courted females of other species. Courtship refusal by females was not selective in males of either species. This incomplete discrimination of both sexes led to frequent occurrences of interspecific sexual interactions in both species, but only B. carambolae females showed reduced reproductive success. These results suggest that B. dorsalis, superior in reproductive interference, can occupy high-quality mango, whereas B. carambolae, inferior in reproductive interference, must use low-quality star fruit.     

Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cell-free translation system

Biotinylation is useful for the detection, purification and immobilization of proteins. It is performed by chemical modification, although position-specific and quantitative biotinylation is rarely achieved. We developed a position-specific biotinylation method using biotinylated non-natural amino acids. We showed that biotinylated p-aminophenylalanine derivatives were incorporated into a protein more efficiently than biotinylated lysine derivatives in a cell-free translation system. In addition, the biotinylated p-aminophenylalanines overcame the serious position-dependency observed for biotinylated lysines. The present method will be useful for detection and purification of proteins along with comprehensive exploration of surface-exposed residues and oriented immobilization of proteins.     

Contribution of the Cell Wall Component Teichuronopeptide to pH Homeostasis and Alkaliphily in the Alkaliphile Bacillus lentus C-125

A teichuronopeptide (TUP) is one of major structural components of the cell wall of the facultative alkaliphilic strain Bacillus lentus C-125. A mutant defective in TUP synthesis grows slowly at alkaline pH. An upper limit of pH for growth of the mutant was 10.4, while that of the parental strain C-125 was 10.8. Gene tupA, directing synthesis of TUP, was cloned from C-125 chromosomal DNA. The primary translation product of this gene is likely a cytoplasmic protein (57. 3 kDa) consisting of 489 amino acid residues. Introduction of the tupA gene into the TUP-defective mutant complemented the mutation responsible for the pleiotropic phenotypes of the mutant, leading to simultaneous disappearance of the defect in TUP synthesis, the diminished ability for cytoplasmic pH homeostasis, and the low tolerance for alkaline conditions. These results demonstrate that the acidic polymer TUP in the cell wall plays a role in pH homeostasis in this alkaliphile.