Modified use of a commercial ELISA kit for deoxynivalenol determination in rice and corn silage

The analysis of deoxynivalenol (DON) in silage samples using enzyme-linked immunosorbent assay (ELISA) often leads to an overestimation. To better analyze DON in rice and corn silages using a commercially available ELISA kit, a cleanup method using a MultiSep #226 column was developed. As a result, overestimation of DON by the influence of specific cross-reaction with acetyldeoxynivalenol (AcDON) was confirmed. In samples where AcDON was not detected by liquid chromatography with mass spectrometry (LC-MS), no samples showed a significant difference (P?<?0.05) in DON amounts between ELISA with cleanup and LC-MS analysis. For the recovery study, blank silage was spiked with 0.5 or 1.0 mg/kg DON. The mean recoveries of DON determined by ELISA with cleanup and LC-MS analysis were 112 and 96 %, respectively, and the relative standard deviation for the repeatability (RSD_r) were 8.2 and 9.8 %, respectively. No samples showed a significant difference (P?<?0.05) in DON concentration determined by either ELISA or LC-MS analysis. A collaborative study to validate this rapid method was carried out using four samples, two rice and two corn silage, by 10 participating laboratories. Each sample was analyzed using blind duplicates. The mean values of DON detected were 1.5–2.3 mg/kg, RSD_r and the relative standard deviation for the reproducibility (RSD_R) were 4.1–12.7 and 7.6–23.4 %, respectively, and the HorRat values were 0.5–1.6. Therefore, the overestimation of DON by the influence of nonspecific cross-reaction with sample matrix was reduced by the cleanup method using a MultiSep #226 column, and analysis of DON in silage was improved. This use of this method for estimation of DON contamination in silage allows rapid detection at the place of use that is likely to result in improved animal health.     

Extracellular syntaxin4 triggers the differentiation program in teratocarcinoma F9 cells that impacts cell adhesion properties

The proteins in the syntaxin family are known to mediate fusion of cytoplasmic vesicles to the target membrane, yet subpopulations of certain syntaxins, including syntaxin4, translocate across the cell membrane in response to external stimuli. Here, we show that extracellularly presented syntaxin4 impacts cell behavior and differentiation in teratocarcinoma F9 cells. While undifferentiated F9 cells extruded a small subpopulation of extracellular syntaxin4 at the lateral cell membrane, the induction of differentiation with all-trans retinoic acid (RA) abolished this localized expression pattern. We found that the cells that were stimulated in a non-directional fashion by extracellular syntaxin4 displayed a flattened shape and retained a substrate-bound morphology even under a long-term, serum-starved cultivation. Such a cellular response was also elicited by a circular peptide composed of the potential functional core of syntaxin4 (AIEPQK; amino acid residues 103～108) (ST4n1). While the proliferation and metabolism were not affected in these cells, cell–cell interaction became weakened and the expression of vinculin, a regulator of both intercellular and cell-substrate adhesion molecules, was altered. We also found that the expressions of several differentiation markers were up-regulated in cells stimulated with extracellular syntaxin4 and that syntaxin3, another family member, was most prominent. Intriguingly, forced expression of syntaxin3 induced the spread morphology in F9 cells, indicating that syntaxin3 partly mediates the function of extracellular syntaxin4. These results demonstrate the involvement of a non-directional stimulation of extracellular syntaxin4 in the functional and morphological differentiation of F9 cells.     

Detection of Environmental Sources of Histoplasma capsulatum in Chiang Mai, Thailand, by Nested PCR

Histoplasmosis is a systemic mycosis caused by inhaling spores of Histoplasma capsulatum, a dimorphic fungus. This fungus grows in soil contaminated with bat and avian excreta. Each year, patients with disseminated histoplasmosis have been diagnosed in Chiang Mai, northern Thailand. No published information is currently available on the environmental sources of this fungus in Chiang Mai or anywhere else in Thailand. The aim of this study was to detect H. capsulatum in soil samples contaminated with bat guano and avian droppings by nested PCR. Two hundred and sixty-five samples were collected from the following three sources: soil contaminated with bat guano, 88 samples; soil contaminated with bird droppings, 86 samples; and soil contaminated with chicken droppings, 91 samples. Genomic DNA was directly extracted from each sample, and H. capsulatum was detected by nested PCR using a primer set specific to a gene encoding 100-kDa-like protein (HcI, HcII and HcIII, HcIV). Histoplasma capsulatum was detected in seven of 88 soil samples contaminated with bat guano, one of 21 soil samples contaminated with pigeon droppings and 10 of 91 soil samples contaminated with chicken droppings. The results indicate the possibility of the association of bat guano and chicken droppings with H. capsulatum in this area of Thailand.     

Postembryonic development of the female reproductive system in the pycnogonid Propallene longiceps (Pycnogonida,Callipallenidae)

The postembryonic development of the female reproductive system in the pycnogonid Propallene longiceps is examined. The germ cells can be detected first in the later stage of the 3rd instar and become a paired gonad covered with gonadal epithelium in the next instar. The larval gonad changes its shape: paired at the 4th instar, reversed U-shaped at the fifth, unpaired at the sixth, and paired again at the seventh. Oocytes can be distinguished, and the extension of the ovary into the walking legs begins at the 7th instar. Growing oocytes protrude outward from the ovary on cellular stalks in the pedal part. The trunk ovary becomes U-shaped, and the oviducts and genital pores start forming at the 8th instar. The disappearance of trunk ovary begins at the 9th instar, and is complete at the next adult stage. The connection between the pedal ovarian lumen and the genital pores via the oviducts is complete in the adult, and the female reproductive system becomes segmentally arranged. This study confirms that the segmental arrangement of adult female reproductive system in P. longiceps, which is unique among recently described arthropods, is a secondary state in pycnogonids attained by reducing the trunk part of ovary.     

Biosynthesis of Streptomycin

A natural precursor (L) of streptomycin which had no antibiotic potency was obtained from mycelium suspension of Streptomyces griseus in glucose solution and was transformed to streptomycin by H enzyme obtained from mycelium of the organism. This transforming reaction was carried out most effectively at slightly alkaline pH and inhibited by inorganic phosphate and ethylenediaminetetraacetate. L component was considered to be a phosphorylated compound and liberation of the phosphoric acid was essential for L component to be transformed to streptomycin. This transformation was performed not only by H enzyme but also by intestinal alkaline phosphatase, although some difference in the reaction mechanism was supposed to be between those two enzymes.     

Chemical Modification and Structure-Activity Correlation of Salinomycin

A number of the chemically modified compounds of salinomycin have been prepared and the structure-activity correlation between complexation affinity for cations, ion transport ability and antimicrobial activity have been investigated.

The results indicate that the terminal carboxylic acid, β-hydroxyketone and allylic alcohol functions in the molecule played an important role in the exhibition of biological activity of the antibiotic.

On the basis of these data, it was concluded that there is a closer parallelism between the antimicrobial and ion-transport activities of salinomycin and its derivatives.     

Synthesis and optimization of novel (3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as orally active renin inhibitors

We report synthesis and optimization of a series of (3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as renin inhibitors. Chemical modification of ${{\text{P}}_1}^{\prime }$, ${{\text{P}}_2}^{\prime }$ and P₃ portions led to a promising 3,5-disubstituted piperidine 32o showing high renin inhibitory activity and favorable oral exposure in both rats and cynomolgus monkeys with acceptable CYP and hERG current inhibition. Compound 32o exhibited a significant blood pressure lowering effect by oral administration in two hypertensive animal models, double transgenic rats and furosemide pretreated cynomolgus monkeys.     

Mechanical reproductive isolation via divergent genital morphology between Carabus insulicola and C. esakii with implications in species coexistence

The mechanical isolation hypothesis predicts that physical incompatibility between divergent reproductive morphologies hinders hybridization between populations. However, evidence for this hypothesis remains scarce. We examined this hypothesis using two parapatric carabid beetles, Carabus insulicola and C. esakii, which are of the subgenus Ohomopterus and exhibit a species-specific genital lock-and-key system. Our interspecific crossing experiment revealed that incompatibility of genital morphologies served as a strong postmating-prezygotic isolation barrier. This isolation was asymmetric: a decrease in female fitness was more costly in the cross with greater genitalic incompatibility between a C. esakii female and a C. insulicola male. These two species share a limited sympatric area, but the mechanism responsible for their coexistence is unclear given no evidence of premating isolation via male mate choice. A comparison of the present results with those of previous studies that quantified reproductive isolation between Ohomopterus species suggest that strong mechanical isolation via genitalic incompatibility plays a major role in species isolation, but that it may be less important in species coexistence.