Inactivation of staphylococcal enterotoxin-A with an electrolyzed anodic solution.

Electrolyzed anodic NaCl solutions [EW+], prepared by the electrolysis of 0.1% NaCl, have been shown to instantly inactivate most pathogens that cause food-borne disease. Elimination of food-borne pathogens does not necessarily guarantee food safety because enterotoxins produced by pathogens may remain active. We have tested whether EW+ can inactivate Staphylococcal enterotoxin A (SEA), one of the major enterotoxins responsible for food poisoning. Fixed quantities of SEA were mixed with increasing molar ratios of EW+, and SEA was evaluated by reversed-phase passive latex agglutination (RPLA) test, immunoassay, native polyacrylamide gel electrophoresis (PAGE), and amino acid analysis after 30 min incubations. Exposure of 70 ng, or 2.6 pmol, of SEA in 25 microL of PBS to a 10-fold volume of EW+, or ca. 64.6 x 10(3)-fold molar excess of HOCl in EW+, caused a loss of immuno-reactivity between SEA and a specific anti-SEA antibody. Native PAGE indicated that EW+ caused fragmentation of SEA, and amino acid analysis indicated a loss in amino acid content, in particular Met, Tyr, Ile, Asn, and Asp. Staphylococcal enterotoxin-A excreted into culture broth was also inactivated by exposure to an excess molar ratio of EW+. Thus, EW+ may be a useful management tool to ensure food hygiene by food processing industries.     

Cartilage acidic protein-1B (LOTUS), an endogenous Nogo receptor antagonist for axon tract formation

Neural circuitry formation depends on the molecular control of axonal projection during development. By screening with fluorophore-assisted light inactivation in the developing mouse brain, we identified cartilage acidic protein-1B as a key molecule for lateral olfactory tract (LOT) formation and named it LOT usher substance (LOTUS). We further identified Nogo receptor-1 (NgR1) as a LOTUS-binding protein. NgR1 is a receptor of myelin-derived axon growth inhibitors, such as Nogo, which prevent neural regeneration in the adult. LOTUS suppressed Nogo-NgR1 binding and Nogo-induced growth cone collapse. A defasciculated LOT was present in lotus-deficient mice but not in mice lacking both lotus- and ngr1. These findings suggest that endogenous antagonism of NgR1 by LOTUS is crucial for normal LOT formation.     

Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus

Genomic imprinting causes parental origin-specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.     

Detection of the EGFP sequence in breast muscle of 3‐year‐old chicken after transfection using sonoporation

In our continuing effort to generate transgenic chickens, sonoporation was chosen to insert an exogenous gene into the chicken genome. An EGFP expression vector (pCAG‐EGFPac) and microbubbles were injected into the central disc of stage‐X blastoderm or the germinal crescent of stage‐4 embryos, followed by ultrasonic vibration. Nineteen chicks out of 108 treated embryos hatched, six females and six males out of these 19 chicks grew to sexual maturity and two females and three males lived for 3 years. Genomic DNA from 17 out of 35 gonads from embryos and chicks that died before sexual maturity was EGFP‐positive by PCR. No EGFP sequence was detected in the genomic DNA of 322 embryos from six sexually mature females and the semen from four sexually mature males by PCR. When genomic DNA was obtained from various tissues of five 3‐year‐old chickens, the EGFP sequence was amplified from the genomic DNA of the breast muscle of a female (No. 85). The above sequence was subjected to DNA sequencing and verified to be the EGFP sequence. These results showed that sonoporation is an effective tool for the transduction of exogenous genes into chicken embryos for the generation of transgenic chickens.     

Changes in oxidative status in periparturient dairy cows in hot conditions

We investigated the changes in the plasma concentrations of oxidative stress markers in periparturient dairy cows in hot conditions. This study was performed on 10 cows from day ?5 to day 10 after parturition. The experimental duration was divided into three periods: period 1 (5 days before parturition); period 2 (5 days after parturition); and period 3 (from day 6 to day 10 after parturition). The average rectal temperature in the cows during the experimental period was more than 39°C. Plasma aspartate aminotransferase activities in periods 2 and 3 were higher than that in period 1 (P < 0.05). Blood urea nitrogen concentration in period 3 was lower than those in periods 1 and 2 (P < 0.05). The total ascorbic acid concentration in the plasma in period 2 was greater than those in periods 1 and 3 (P < 0.05). Thiobarbituric acid reactive substances (TBARS) concentrations in periods 2 and 3 were greater than that in period 1 (P < 0.05). TBARS concentration was positively correlated with aspartate aminotransferase activity (P < 0.05). These results suggested that oxidative stress increased in cows after parturition under hot climatic conditions.     

Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro

Fifty-four polyphenols isolated from tea leaves were evaluated for their inhibitory activities against pancreatic lipase, the key enzyme of lipid absorption in the gut. (-)-Epigallocatechin 3-O-gallate (EGCG), which is one of major polyphenols in green tea, showed lipase inhibition with an IC50 of 0.349 microM. Moreover, flavan-3-ol digallate esters, such as (-)-epigallocatechin-3,5-digallate, showed higher activities of inhibition on lipase with an IC50 of 0.098 microM. On the other hand, nonesterified flavan-3-ols, such as (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigallocatechin, showed zero and/or the lowest activities against pancreatic lipase (IC50 > 20 microM). These data suggested that the presence of galloyl moieties within the structure was required for enhancement of pancreatic lipase inhibition. It is well-known that flavan-3-ols are polymerized by polyphenol oxidase and/or heating in a manufacturing process of oolong tea. Oolonghomobisflavans A and B and oolongtheanin 3'-O-gallate, which are typical in oolong tea leaves, showed strong inhibitory activities with IC50 values of 0.048, 0.108, and 0.068 microM, respectively, even higher than that of EGCG. The oolong tea polymerized polyphenols (OTPP) were prepared for the assay from oolong tea extract, from which the preparation effectively subtracted the zero and/or less-active monomeric flavan-3-ols by preparative high-performance liquid chromatography. The weight-average molecular weight (Mw) and number-average molecular-weight (Mn) values of OTPP were 2017 and 903, respectively, by using gel permeation choromatography. OTPP showed a 5-fold stronger inhibition against pancreatic lipase (IC50 = 0.28 microg/mL) by comparison with that of the tannase-treated OTPP (IC50 = 1.38 microg/mL). These data suggested that the presence of galloyl moieties within their chemical structures and/or the polymerization of flavan-3-ols were required for enhancement of pancreatic lipase inhibition.     

Effect of the strain combination of the donor and recipient on the production efficiency of W‐bearing sperm in mixed‐sex germline chimeric chickens

Effect of the strain combination of the donor and recipient on production efficiency of W‐bearing sperm in mixed‐sex chimeric testes was analyzed. The combinations of the donors and recipients were White Leghorn (WL) and Rhode Island Red (RIR), and vice versa. Generated mixed‐sex chimeras that had the male phenotype at sexual maturity were classified into four groups: (1) a female WL donor and a male RIR recipient; (2) a male WL donor and a female RIR recipient; (3) a female RIR donor and a male WL recipient; (4) a male RIR donor and a female WL recipient. The mean number of W‐bearing sperm detected by in situ hybridization among 10 000 sperm observed were 147, 165, 30 and 45 in groups 1, 2, 3 and 4, respectively. The numbers in groups 1 and 2 were both significantly higher than those of groups 3 and 4 (P < 0.05). The combination of a WL donor and a RIR recipient produced W‐bearing sperm more efficiently than the reverse combination.     

Molecular cloning and sequences of interleukin-10 in the Djungarian (Phodopus sungorus), Chinese (Cricetulus griseus), and Syrian (Mesocricetus auratus) hamster

Interleukin 10 (IL-10) genes of Djungarian, Chinese, and Syrian hamsters were cloned. The clones of IL-10 consisted of 537 bp nucleotides and 178 amino acids in full length, and the nucleotide and amino acid sequences exhibited a high degree of homology with those of the mouse and human. Since the number and position of signal sequences, N-glycosylations and cysteine sites in the IL-10 amino acid sequences of the hamsters were the same as those of the mouse, we suggest that the IL-10 molecular structures of the hamster are closer to that of the mouse than human.     

Detection of Cryptosporidium sp. Oocyst and Giardia sp. cyst in faucet water samples from cattle and goat farms in Taiwan

A survey on the presence of Cryptosporidium oocyst and Giardia cyst in livestock drinking water as well as the urban tap water throughout Taiwan was carried out. Water examination for the presence of the protozoa was conducted by filtering through a PTFE membrane followed by immunomagnetic separation (IMS) and immunostaining the sediment with commercially available monoclonal antibody against Cryptosporidium and Giardia. Of the 55 different water samples from various sources examined, 2 were found to contain both of Cryptosporidium oocyst and Giardia cyst, 1 was found to contain Cryptosporidium oocyst only. These protozoa-positive water samples, originating from underground well and from the mountain spring, were also used as drinking water for livestock. However, no Cryptosporidium oocyst was found in the city tap water. This is the first report of Cryptosporidium oocyst and Giardia cyst being found in the drinking water for livestock.