Development and evaluation of an enzyme-linked immunosorbent assay with recombinant SAG2 for diagnosis of Toxoplasma gondii infection in cats

Cats are pivotal in the transmission of Toxoplasma gondii. To develop a sensitive and specific serodiagnostic method for feline toxoplasmosis, surface antigen 2 (SAG2) of T. gondii was expressed in Escherichia coli and its diagnostic potential evaluated in an enzyme-linked immunosorbent assay (ELISA). The ELISA with recombinant SAG2 (rSAG2) was able to differentiate very clearly between sera from cats experimentally infected with T. gondii and sera from normal cats. Serum samples collected from domestic cats in Japan were investigated by the ELISA, and the results were compared with those of a commercially available latex agglutination test (LAT) kit. Of the 192 samples screened, 42 (21.9%) were positive by ELISA. Among the 42 ELISA-positive samples, 39 were positive by LAT. There was a significant correlation between ELISA and LAT titers. All the 150 ELISA-negative samples were negative by LAT. These results indicate that the ELISA with rSAG2 expressed in E. coli should be a useful method for detection of T. gondii infection in cats.     

In situ observation of elementary growth processes of protein crystals by advanced optical microscopy

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.     

Anti-oxidation activity of ethanol extracts from natural thalli of lichens

Screening test on anti-oxidation activity using 1,1-diphenyl-2-picrylhydrazyl(DPPH) was performed for 99 ethanol extracts of 85 species of natural thalli of lichens in order to find novel anti-oxidation compounds.The 17 extracts of natural thalli showed high anti-oxidation activity.Among them,the activities of extracts from Hypogymnia vittata,Peltigera aphthosa,Nephromopsis ornata,Pseudevernia furfuracea,Cladonia vulcani and Peltigera elizabethae were higher.Extracts of Peltigera spp.showed higher activity than those of other genera.The ethanol extract of P.aphthosa had been separated into ethyl acetate-soluble and water-soluble fractions.Two anti-oxidative spots were found only in the water-soluble fractions by thin-layer chromatography.The compound in the lower spot had the same Rf value,UV spectrum,and color as authentic solorinine that was previously found as a unique quaternary ammonium compound from Peltigera spp.We now report that the hydrophilic lichen substance,solorinine showed a nearly same anti-oxidation activity(EC50=120?mol/Lol/L) as standard antioxidant Trolox(EC50=150?mol/L).     

Disruption of pheromone communication in Tecia solanivora (Lepidoptera: Gelechiidae): flight tunnel and field studies

The moth Tecia (Scrobipalpopsis) solanivora Povolny (Lepidoptera: Gelechiidae) is the most important pest of potato, Solanum spp., in Central America and adjacent South American countries. Insecticide treatments are not sufficiently effective; therefore, we investigated the feasibility of pheromone-mediated mating disruption for control of T. solanivora. Pheromone dispensers were formulated with 70 mg of the three sex pheromone compounds (E)-3-dodecenyl acetate, (Z)-3-dodecenyl acetate, and dodecyl acetate, in a ratio of 100:56:100, respectively. Male attraction to these compounds is optimal at a ratio of 100:1:20, thus the mating disruption dispensers contained an off-blend, which attracted only a few males. Nonetheless, one mating disruption dispenser suppressed male attraction to calling females in a flight tunnel and reduced male activation in response to female pheromone. Communication disruption is accordingly due to camouflage of the female signal and possibly due to a reduction of male responsiveness by sensory imbalance. Only a few males were observed in a 3-ha potato field treated with 84 g pheromone/ha, compared with an untreated control field. During 2 mo, male attraction to traps baited with calling females or synthetic pheromone was strongly reduced. This reduction confirms the potential of mating disruption for management of T. solanivora. The efficacy of the pheromone treatment can be further improved by earlier dispenser application, by increased dispenser load, and by treatment of larger fields to reduce immigration of mated females.     

Effects of a bout of traditional and original sumo training on neutrophil immune function in amateur university sumo wrestlers

In order to examine in detail the influence on the neutrophil immune function in sumo wrestlers of performing traditional and original training we examined changes in the neutrophil immune function in 17 male amateur university sumo wrestlers (aged 20.2 ± 1.5 years), before (‘Pre’) and after the training (‘Post’) for 2.5 h under fasting conditions. Assays included blood leukocyte and neutrophil counts, serum concentration of immunoglobulins, complements, myogenic enzymes and neutrophil oxidative burst activity (OBA) and phagocytic activity (PA). Myogenic enzymes, neutrophil counts, the ratio of neutrophil counts:leukocyte counts significantly increased and immunoglobulins and complements decreased in Post compared with Pre. There was a positive correlation between the change of neutrophil counts before and after the training and the change of creatine kinase (r = 0.667, p < 0.01). The Post OBA significantly increased and PA significantly decreased compared with Pre. It was concluded that sumo training causes muscular damage and an increase in the neutrophil count as a response. In this time, although OBA increased, PA decreased after training. Compensation between PA and reactive oxygen species production may exist to maintain the overall integrity of the neutrophil immune function. Copyright © 2008 John Wiley & Sons, Ltd.     

Change in Hair Cycle and Hair Length in Nude Mice by Administration of Deuterium Oxide

D₂O increased hair length in Balb/c nu/nu (nude) mice in our previous study although it has an antimitotic effect in cells. To investigate the mechanism of the effect on the hair length, we examined the change by the administration of D₂O in the duration of the hair cycle and the proliferating activity of the hair matrix in relation with hair length in nude mice. The results showed that 20 or 30% D₂O administration did not change the gross structure of the hairs, the proliferative activity and keratinization of the hair matrix cells, but elongated the hair cycle. The duration of the hair cycle increased by the administration of D₂O in a dose-dependent manner over the examined range and these effects were reversible by discontinuation of D₂O. The change in the hair length correlated with the change in the hair-existing phase particularly. We also showed that the mast cell density in the skin, which is related to the hair cycle, increased in the deuterated mice at anagen VI stage which nearly corresponds to the hair-existing phase. The increase in the mast cell density may be related to the increase in the hair cycle duration. These findings indicate that the increase in hair length may be due to the increase in the duration of the hair cycle, in particular, an increase in the hair-existing phase. This study thus suggests that D₂O slows not only short-term cycles such as circadian clock or ultradian clock, but also the hair cycle which is a long-term cycle.     

Mitochondria-specific RNA-modifying enzymes responsible for the biosynthesis of the wobble base in mitochondrial tRNAs. Implications for the molecular pathogenesis of human mitochondrial diseases

Human mitochondrial (mt) tRNA(Lys) has a taurine-containing modified uridine, 5-taurinomethyl-2-thiouridine (taum5s2U), at its anticodon wobble position. We previously found that the mt tRNA(Lys), carrying the A8344G mutation from cells of patients with myoclonus epilepsy associated with ragged-red fibers (MERRF), lacks the taum5s2U modification. Here we describe the identification and characterization of a tRNA-modifying enzyme MTU1 (mitochondrial tRNA-specific 2-thiouridylase 1) that is responsible for the 2-thiolation of the wobble position in human and yeast mt tRNAs. Disruption of the yeast MTU1 gene eliminated the 2-thio modification of mt tRNAs and impaired mitochondrial protein synthesis, which led to reduced respiratory activity. Furthermore, when MTO1 or MSS1, which are responsible for the C5 substituent of the modified uridine, was disrupted along with MTU1, a much more severe reduction in mitochondrial activity was observed. Thus, the C5 and 2-thio modifications act synergistically in promoting efficient cognate codon decoding. Partial inactivation of MTU1 in HeLa cells by small interference RNA also reduced their oxygen consumption and resulted in mitochondria with defective membrane potentials, which are similar phenotypic features observed in MERRF.     

Characteristics of hepatitis B virus isolates of genotype G and their phylogenetic differences from the other six genotypes (A through F)

Eight hepatitis B virus (HBV) isolates of genotype G were recovered from patients and sequenced over the entire genome. Six of them had a genomic length of 3,248 bp and two had genomic lengths of 3,239 bp (USG15) and 3,113 bp (USG18) due to deletions. The 10 HBV/G isolates, including the 8 sequenced isolates as well as the original isolate (AF160501) and another isolate (B1-89), had a close sequence homology of 99.3 to 99.8% among themselves (excluding USG18 with a long deletion) but of <88.7% to any of the 68 HBV isolates of the other six genotypes with the full-length sequence known. The eight HBV/G isolates possessed an insertion of 36 bp in the core gene and two stop codons in the precore region, as did the AF160501 and B1-89 isolates. The 10 HBV/G isolates clustered on a branch separate from those bearing the other six genotypes (A through F [A-F]) in the phylogenetic tree constructed from full-length sequences of 78 HBV isolates as well as in those constructed from the core, polymerase, X, and envelope genes. Despite two stop codons in the precore region that prohibited the translation of the HBV e antigen (HBeAg), all of the eight patients with HBV/G infection possessed the HBeAg in serum. By restriction fragment length polymorphism of the surface gene, all of the eight patients were found to be coinfected with HBV of genotype A (HBV/A), which would be responsible for the expression of HBeAg in them. It is worthy of examination to determine how coinfection occurs and whether HBV/G needs HBV/A for replication.