Detection of ruminant meat and bone meal in feeds by sandwich ELISA with monoclonal antibodies

A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies directed against a synthetic peptide with an amino-acid sequence related to the C-terminus of bovine myoglobin and the whole molecule of sodium dodecyl sulphate (SDS)-denatured bovine myoglobin was adapted for detecting bovine myoglobin in contaminated feeds. The ELISA employed bovine meat extract of a known myoglobin concentration as a calibration standard and had an limit of detection (LOD) of 3.54 ng/ml and an limit of quantification (LOQ) of 11.0 ng/ml corresponding to 0.022% and 0.067% (wt/wt) bovine meat-and-bone-meal (MBM) mixed in 20-fold-diluted feed extracts, respectively. A cut-off threshold of 20.6 ng/ml bovine myoglobin was set to simplify ELISA and facilitate quick assessment of test results without a tedious calibration process. The ELISA was able to detect bovine MBM in artificially prepared model feeds, mixed botanical feeds, mixed botanical feeds with skimmed milk, fish meal, pork meal and pork/chicken meal at 0.1% (wt/wt). It was also able to detect sheep MBM in test feeds, but showed no reactivity to swine MBM, chicken MBM, skimmed milk or gelatine of bovine origin. The advantages of this method are the quick and easy extraction protocol of proteins from test feeds, using 100 mM sodium sulphide and 0.6% sodium dodecyl sulphate in the extraction solution and the effective detection of bovine and sheep MBM at 0.1% (wt/wt).     

Effect of the d‐glucose analog,d‐allose,on the growth of Arabidopsis roots

Although d ‐glucose increased the root growth of Arabidopsis seedlings, d ‐allose (a d ‐glucose epimer at the third carbon atom) inhibited the root growth at concentrations >0.1 mmol L^?1 and the inhibition increased with increasing d ‐allose concentrations. Allitol (a reduction product of d ‐allose) did not show any significant effect on the growth. The addition of d ‐glucose into the growth medium of Arabidopsis reversed the d ‐allose‐induced growth inhibition, which suggests that the inhibition is not caused by the toxicity of the accumulation of d ‐allose and/or its metabolites in the seedlings. d ‐Allose is phosphorylated by hexokinase, using ATP and phosphate, to allose‐6‐phosphate, with no known capacity for further metabolism. The addition of phosphate into the growth medium did not affect the d ‐allose‐induced growth inhibition and d ‐allose did not reduce the ATP level in the roots. These results suggest that the inhibition is not due to phosphate starvation and ATP depletion. d ‐Mannoheptulose, a specific competitive inhibitor of hexokinase, defeated the d ‐allose‐induced growth inhibition. Hexokinase is known to have a sugar‐sensing function and possibly triggers a signal cascade, resulting in the change of several gene expressions. Therefore, the phosphorylation of d ‐allose by hexokinase might trigger a signal cascade, resulting in the inhibition of Arabidopsis root growth. This is probably a useful model system for studies of the hexokinase‐mediated sugar‐sensing function and for developing new types of weed‐control agents.     

Detection of Human Herpesviruses (HHVs) in Semen of Human Male Infertile Patients

Recently we demonstrated an ectopic expression of the human herpesvirus 1 thymidine kinase (HHV1-TK) gene by functioning of an intrinsic endogenous promoter in the transgenic rat (TG-rat), suggesting that HHV1 infection in humans induces expression of the TK gene with the ectopic promoter in the testis and results in accumulation of HHV1-TK protein, triggering male infertility similar to that in the TG-rat. Hence, in this study, we started to investigate a relationship between infection of herpesvirus and human male infertility. Semen was donated by Chinese male infertile patients (153 men, aged 21–49 years) with informed consent, followed by DNA preparation and analysis by PCR and DNA sequencing. Semen volume, sperm number and density, and sperm motility were examined. DNAs of HHV1, HHV4, HHV5 and HHV6 were confirmed by PCR, electrophoresis and DNA sequencing. Finally, virus DNA was identified in 59 patients (39%). The number of carriers was 39 (25%) for HHV1, 6 (4%) for HHV4, 33 (22%) for HHV5 and 3 (2%) for HHV6, respectively. Moreover, double-infection was found in 22 out of 59 specimens (37%), most of which were double-infection of HHV1 and HHV5 (15 out of 22 carriers). Though slight severity was present in some of the carriers, the relationship between virus infection and sperm impairment was not conclusive. Accordingly, it is essential to examine whether the viral HHV1-TK gene is expressed in the testis of the infertile human HHV carrier.     

Viral-derived DNA invasion and individual variation in an Indonesian population of large flying fox Pteropus vampyrus

Here, we performed next-generation sequencing (NGS) on six large flying foxes (Pteropus vampyrus) collected in Indonesia. Seventy-five virus species in the liver tissue of each specimen were listed. Viral homologous sequences in the bat genome were identified from the listed viruses. This finding provides collateral evidence of viral endogenization into the host genome. We found that two of the six specimens bore partial sequences that were homologous to the plant pathogens Geminiviridae and Luteoviridae. These sequences were absent in the P. vampyrus chromosomal sequences. Hence, plant viral homologous sequences were localized to the hepatocytes as extrachromosomal DNA fragments. Therefore, this suggests that the bat is a potential carrier or vector of plant viruses. The present investigation on wild animals offered novel perspectives on viral invasion, variation, and host interaction.     

Investigation into the dosage of dietary sphingomyelin concentrate in relation to the improvement of epidermal function in hairless mice

We previously found that dietary sphingomyelin (SPM) concentrate from bovine milk improved epidermal function. In this study, we investigated the dosage of dietary SPM concentrate from bovine milk in relation to the improvement of epidermal function. Thirteen-week-old hairless male mice were separated into four experimental groups, each fed one of four types of experimental diet: the control group, the low SPM group, the medium SPM group and the high SPM group. The mice were each fed the experimental diet for 6 weeks. The stratum corneum hydration and the transepidermal water loss (TEWL) were measured using a Corneometer and a Tewameter at 3 weeks and 6 weeks. After the feeding period, ceramides in the stratum corneum were analyzed. We found that the stratum corneum hydration in all the SPM groups was significantly higher than that in the control group, whereas TEWL in all the SPM groups was significantly lower than that in the control group. Ceramides increased significantly in mice fed the medium SPM diet and statistically tended to increase in mice fed the high SPM diet. Our results indicate that a daily intake of 17 mg SPM concentrate is enough to improve epidermal function in hairless mice.