Mobility shift detection of phosphorylation on large proteins using a Phos‐tag SDS‐PAGE gel strengthened with agarose

We describe a novel technique of phosphate‐affinity SDS‐PAGE using Phos‐tag to analyze large phosphoproteins with molecular masses of more than 200 kDa. The protein phosphoisotypes were clearly separated as up‐shifted migration bands in a 3% w/v polyacrylamide gel containing 20 μM Phos‐tag and 0.5% w/v agarose. In subsequent immunoblotting, the procedure permitted the determination of the phosphoisotypes of high‐molecular‐mass proteins, such as mTOR (289 kDa), ATM kinase (350 kDa), and 53BP1 (213 kDa).     

Reduction of catechin astringency by the complexation of gallate-type catechins with pectin

The reductive effect of pectin on tea catechin astringency was investigated by using a taste sensor system and (1)H-NMR spectroscopy. The sensor analysis revealed that the astringency of gallate-type catechins (EGCg and ECg) was reduced by the addition of pectin, whereas that of non-gallate-type catechins (EGC and EC) hardly changed. Changes in the (1)H-NMR chemical shifts of the catechins and pectin in mixed solutions showed that the gallate-type catechins formed complexes with pectin more closely than the non-gallate-type catechins. These results demonstrate that complexation between the gallate-type catechins and pectin is a factor for reducing catechin astringency.     

Metofluthrin: a potent new synthetic pyrethroid with high vapor activity against mosquitoes

(1R)-trans-Norchrysanthemic acid fluorobenzyl esters are synthesized and their structure-activity relationships are discussed. These esters show outstanding insecticidal activity against mosquitoes. In particular, the 2,3,5,6-tetrafluoro-4-methoxymethylbenzyl analog (metofluthrin) exhibits the highest potency, being approximately forty times as potent as d-allethrin in a mosquito coil formulation when tested against southern house mosquitoes (Culex quinquefasciatus). Metofluthrin also exhibits a significant vapor action at room temperature.     

Biochemical and immunological demonstration of prostaglandin D2, E2, and F2 alpha formation from prostaglandin H2 by various rat glutathione S-transferase isozymes

Glutathione S-transferase isozymes purified from normal rat liver (1-1, 1-2, 2-2, 3-3, 3-4, and 4-4), liver with hyperplastic nodules (7-7), brain (Yn1Yn1), and testis (Yn1Yn2) all had prostaglandin H2-converting activity. The prostaglandin H2 E-isomerase activity was high in 1-1 (1400 nmol/min/mg protein), 1-2 (1170), and 2-2 (420), moderate in 3-3, 3-4, 4-4, Yn1Yn1, and Yn1Yn2 (52-100), and weak but significant in 7-7 (33). The prostaglandin H2 D-isomerase activity was relatively high in 1-1 (170) and 1-2 (200), moderate in 2-2 (60) and Yn1Yn2 (43), and weak but marked in 3-3 (16), 4-4 (16), and 7-7 (14). The prostaglandin H2 F-reductase activity was remarkable in 1-1 (1250), 1-2 (920), and 2-2 (390), and weakly detected in 3-3 (24), 4-4 (28), and 7-7 (14). Glutathione was absolutely required for these prostaglandin H2-converting reactions, and its stoichiometric consumption was associated with F-reductase activity but not E- and D-isomerase activities. The Km values for glutathione and prostaglandin H2 were about 200 and 10-40 microM, respectively. By immunoabsorption analyses with various antibodies specific for each isozyme, we examined its contribution to the formation of prostaglandins D2, E2, and F2 alpha from prostaglandin H2 in 100,000g supernatants of rat liver, kidney, and testis. In the liver, about 90% of the F-reductase activity (9.8 nmol/min/mg protein) was shown to be catalyzed by the 1-2 group of isozymes. The E-isomerase activity (16.5) was catalyzed about 60 and 40% by the 1-2 and 3-4 groups, respectively; and the D-isomerase activity (3.7) was catalyzed by the 1-2 group (50%) and the 3-4 group and Yn1Yn2 (15-25%). In the kidney, the E-isomerase activity (9.4) was catalyzed by 1-1, 1-2 (40%), 2-2, 3-4 group, and 7-7 (10-20%). The F-reductase activity (3.3) was mostly catalyzed by the 1-2 group (75%). In the testis, the E-isomerase activity (3.9) was catalyzed by the 1-2 group (20-30%), the 3-4 group, and Yn1Yn2 (30-60%).     

Tissue manganese levels and liver pyruvate carboxylase activity in magnesium-deficient rats

To investigate the manganese status in magnesium deficiency, 40 male Wistar rats, 3 wk old, were divided into two groups and fed a magnesium deficient diet or a normal synthetic diet for 2 wk. Dietary magnesium depletion decreased magnesium levels in brain, spinal cord, lung, spleen, kidney, testis, bone, blood, and plasma, while it elevated the magnesium level in liver. In magnesium-depleted rats, calcium concentration was increased in lung, liver, spleen, kidney, and testis, while it was decreased in tibia. In magnesium-depleted rats, manganese concentration was decreased in plasma and all tissues except adrenal glands and blood. Dietary magnesium depletion diminished pyruvate carboxylase (EC 6.4.1.1) activity in the crude mitochondrial fraction of liver. Positive correlation was found between the liver manganese concentration and the pyruvate carboxylase activity. In the magnesium-depleted rats, glucose was decreased while plasma lipids (triglycerides, phospholipids, and total cholesterol) were increased. These results suggest that dietary magnesium deficiency changes manganese metabolism in rats.     

Characterization of Pseudomonas aeruginosa phage KPP21 belonging to family Podoviridae genus N4‐like viruses isolated in Japan

Bacteriophages (phages) belonging to the family Podoviridae genus N4‐like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4‐like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4‐like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.     

RAPD技术在几个苋属植物遗传分类中的应用研究

利用RAPD技术结合聚类分析,研究了亚洲和中南美洲产的苋属植物7种14个品系遗传关系。从80个10碱基长度的随机引物中筛选出8个有效引物,共扩增出103个DNA片段,平均每个引物扩增12.9个片段,其中多态性片段95个,占92．2％,依据8个有效引物扩增的DNA片段对供试材料进行了UPGMA（非加权成组算术平均法）聚类分析,结果分为4类：（1）包括A.deflexus和A.dubius各1品系的杂草苋种;（2）祖先野生种A.hybridus 1个品系;（3）包括A.hypochondriacus6品系、A.caudatus 2品系的籽粒苋种;（4）包括A.tricolor2品系和A.mangostanus 1品系的蔬菜苋种。     

Characterization of lytic enzyme open reading frame 9 (ORF9) derived from Enterococcus faecalis bacteriophage phiEF24C

In bacteriophage (phage) therapy against Gram-positive bacteria, such as Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage φEF24C, which is a SPO1-like virus infecting E. faecalis, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (orf9) of E. faecalis phage φEF24C was analyzed in silico, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein is N-acetylmuramoyl-l-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 against E. faecalis were confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn(2+) ions for its activity, contrary to the bioinformatics prediction of a Zn(2+) ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) of E. faecalis strains to 60% (6 out of 10 strains) of Enterococcus faecium strains. Fifth, N-acetylmuramoyl-l-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N- or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage φEF24C and can be a therapeutic alternative to antibiotics.     

Biochemical and immunological characterization of rat spleen prostaglandin D synthetase

Rat spleen prostaglandin D synthetase (Christ-Hazelhof, E., and Nugteren, D. H. (1979) Biochim. Biophys. Acta 572, 43-51) is very similar to rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi O. (1985) J. Biol. Chem. 260, 12410-12415) as judged by their pI (4.7-5.2), Mr (26,000-27,000), and self-inactivation during the isomerase reaction from prostaglandin H2 to prostaglandin D2. However, the amino acid compositions of these two enzymes were quite different. Furthermore, the spleen enzyme was associated with the glutathione S-transferase activity, differing from the brain enzyme. The synthetase and transferase activities of the spleen enzyme showed almost identical pH and glutathione dependencies, the optimum pH = 8.0 and Km for glutathione = 300 microM. The Km values for prostaglandin H2 and 1-chloro-2,4-dinitrobenzene (a substrate for the transferase) were about 200 microM and 5 mM, respectively. The synthetase activity was dose-dependently inhibited by 1-chloro-2,4-dinitrobenzene (IC50: approximately 5 mM) and more strongly by nonsubstrate ligands, such as bilirubin and indocyanine green (IC50: 150 and 2 microM, respectively). Both the synthetase and transferase activities of the purified enzyme dose-dependently decreased and showed identical immunotitration curves by incubation with antibody against this enzyme, but remained unchanged when treated with antibody against the brain enzyme. The antibody specific for the spleen enzyme absorbed almost all of the synthetase activity and about 10% of the transferase activity in the spleen, but not the transferase activity in the liver, heart, and testis. These results show that the two types of prostaglandin D synthetase are similar but different enzymes and that the spleen enzyme is a unique glutathione S-transferase differing from other isozymes and their subunits reported previously.