In vivo antioxidative activity of propolis evaluated by the interaction with vitamins C and E and the level of lipid hydroperoxides in rats

In vivo antioxidative activity of propolis was evaluated on the basis of ameliorative effects on the oxidative stress induced by vitamin E deficiency in rats. The control group was fed vitamin E-deficient diet, and the propolis group was fed vitamin E-deficient diet supplemented with 1% of propolis for 4 and 8 weeks. Comparisons were made in tissue concentrations of vitamin C, vitamin E, and lipid hydroperoxides between these groups. No significant difference was observed in tissue vitamin E concentration between these groups after both 4 and 8 weeks. After 4 weeks, the plasma vitamin C concentration of the propolis group was significantly higher than that of the control group. After 8 weeks, the tissue concentrations of vitamin C in the kidney, stomach, small intestine, and large intestine of the propolis group were significantly higher than those of the control group. These results suggest that some components of propolis are absorbed to circulate in the blood and behave as a hydrophilic antioxidant that saves vitamin C. The concentration of lipid hydroperoxides in the large intestine of the propolis group was significantly lower than that of the control group after 8 weeks. These results suggest that propolis exerts its antioxidative effect where it is assumed to accumulate, such as on the kidney, where it is excreted, and on the gastrointestinal tract, where propolis influences these tissues even from the outside of the cell.     

Phosphorylation of egg white proteins by dry-heating in the presence of phosphate

Food proteins were phosphorylated by heating in a dry state in the presence of phosphate. When casein, whey protein isolate (WPI), and egg white proteins (EWP), which were lyophilized from their solutions in a phosphate buffer, were dry-heated at various temperatures and pH levels for 1-5 days, EWP was more highly phosphorylated than casein and WPI. Phosphorylation of EWP was promoted with a decrease of pH from 7.0 to 3.0 when the incubation temperature was raised from 55 to 100 degrees C. The phosphorus content of EWP increased from 0.08 to 0.64% by dry-heating at pH 3.0 and 85 degrees C for 5 days in the presence of phosphate. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The heat-induced polymerization of EWP by dry-heating was not affected by the presence of phosphate. Although the solubility of EWP decreased by dry-heating at pH 3.0-5.5, the phosphorylation depressed the insolubilization at low pH. The phosphate bonds in phosphorylated EWP (P-EWP) were stable at pH 2.0-10.0 and were more acid-labile and base-stable than phosphoesters of egg riboflavin-binding protein (RfBP). (31)P NMR spectral data suggested that besides phosphoesters, phosphodiester and polyphosphate bonds were introduced in P-EWP. Heat stability of EWP was improved, and calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation.     

Effects of tyrosine administration on serum biopterin in normal controls and patients with Parkinson's disease

After administration of tyrosine, total concentration of biopterin, the cofactor for tyrosine hydroxylase, was increased in the striatum, adrenal glands, and serum of rats, and in the serum of humans. Serum biopterin is lower in patients with Parkinson's disease than in normal controls. After oral administration of tyrosine, the increase in serum biopterin concentration was smaller in patients with Parkinson's disease (less than twofold) than in healthy controls (three-to sevenfold). These results suggest that tyrosine may have a regulatory role in biopterin biosynthesis and that patients with Parkinson's disease may have some abnormality in the regulation of biopterin biosynthesis.     

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV)

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats.     

Analysis of strigolactones,germination stimulants for striga and orobanche,by high-performance liquid chromatography/tandem mass spectrometry

A simple and rapid analytical method for strigolactones, germination stimulants for the root parasitic weeds witchweed (Striga spp.) and broomrape (Orobanche spp.), has been developed using high-performance liquid chromatography connected to tandem mass spectrometry (LC/MS/MS). The natural strigolactones (strigol, sorgolactone, orobanchol, and alectrol) were clearly separated and identified by LC/MS/MS. As low as 0.1 pg/microL of strigol and 0.5 pg/microL of sorgolactone could be quantified, whereas 1 pg/microL was needed for the quantification of orobanchol (S/N > 10). Using this method, it was found that red clover produces orobanchol and alectrol but not strigol. The roots of red clover seedlings were found to produce 13, 70, 58, and 65 pg of orobanchol/plant 1, 2, 3, and 4 weeks after germination, respectively.     

Extraction methods for quantitation of gentamicin residues from tissues using fluorescence polarization immunoassay

Sodium hydroxide digestion of unhomogenized kidney and skeletal muscle for 20 min at 70 degrees C was a superior method for extracting gentamicin from tissues, compared with simple homogenization, trichloroacetic acid precipitation of homogenized tissue, and sodium hydroxide digestion of homogenized tissue. Fluorescence polarization immunoassay was used to quantitate gentamicin. Sodium hydroxide digestion of unhomogenized tissue allowed for the recovery of 90.0 +/- 5.9% (means +/- SD) from renal cortex and 79.9 +/- 3.5% from skeletal muscle. The limit of sensitivity was 17.4 ng/g kidney tissue, 15.8 ng/g digested muscle, and 39.0 ng/g digested heart. The within-assay coefficient of variation (CV) at 100 ng/g kidney was 9.2%; at 500 ng/g kidney, the CV was 2.5%; and at 2000 ng/g kidney, the CV was 1.5%. The between-assay coefficient of variation was less than 7.5% for all concentrations from kidney, and the 99% confidence interval at 100 ng/g kidney was 71.7-112.4 ng gentamicin/g kidney. The within-assay coefficient of variation (CV) at 100 ng/g muscle was 15%; at 500 ng/g muscle, the CV was 2.6%; and at 2000 ng/g muscle, the CV was 2.3%. The between-assay coefficient of variation was less than 15% for all concentrations from muscle, and the 99% confidence interval at 100 ng/g muscle was 72.5-136.8 ng gentamicin/g muscle. Gentamicin-free milk could be distinguished from milk containing gentamicin concentrations of 10 ng/mL milk with 95% confidence, and from milk containing concentrations of 30 ng gentamicin/mL milk with 99% confidence. Quantitative results at or below the tolerance level can be obtained within 90 min of sample acquisition using these extraction and assay methods.     

Experimental infections of mice and pigs with Streptococcus suis type 2.

Five inbred strains of mice were tested for their susceptibility to Streptococcus suis type 2 including the type strain, two isolates from meningitis in pigs and two isolates from tonsils of clinically healthy pigs. C57BL/6, ICR and ddY strain mice showed lower susceptibility to all strains of S. suis type 2 than BALB/c and SS strain mice. The type strain and the isolates from diseased pigs produced septicaemia and meningitis in BALB/c and SS mice inoculated with 10(8) colony forming unit of the bacteria and 60 to 100% of these infected mice died. On the other hand, mice inoculated with the isolates from healthy pigs showed mild clinical signs but none of them died. In BALB/c mice which died or developed nervous signs, the purulent meningo-encephalitis, myocarditis, ophthalmitis, labyrinthitis and otitis media were observed. S. suis type 2 antigen was demonstrated in these lesions by immunoperoxidase staining using rabbit S. suis type 2 antiserum. These results were similar to those in the experimentally infected pigs with these virulent and avirulent strains against mice. These results indicate that BALB/c and SS strains of mice are useful as an experimental model of S. suis type 2 infections in pigs, and that there are virulent and avirulent strains against mice and pigs among the strains of S. suis type 2.     

Molecular epidemiological survey of benign Theileria parasites of cattle in Japan: detection of a new type of major piroplasm surface protein gene

Benign Theileria species of cattle are found in most parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a maker for epidemiological and phylogenetical studies of benign Theileria species. Parasites with Ikeda- or Chitose-type MPSP genes are dominant in Japan, but we report here mixed infection cases of Theileria parasites with an additional MPSP type parasite infecting cattle in Abashiri District, Hokkaido. The MPSP gene sequence found in the additional type was closely related to MPSP genes of Theileria parasites found in Southeast Asian countries, including Thailand (Narathiwat) and Indonesia (Java). Theileria parasites from the blood sample were also distinguishable from the Ikeda or Chitose type parasites by the small subunit (SSU) rRNA gene sequence analysis, and they are grouped into the SSU rRNA types C/D found in Korea, North America, and Spain. The present finding of mixed infections of cattle with three different types of Theileria makes epidemiological feature of bovine theileriosis in Japan more complex. We have designed a set of primers specific to this MPSP type in order to conduct further epidemiological study.     

PCR-based discrimination of Toxoplasma gondii from pigs at an abattoir in Okinawa, Japan

To determine the prevalence of the 3 primary clonal lineages of Toxoplasma gondii (strain types I, II, and III) in pigs in Okinawa Prefecture, we analyzed lymph node samples that had been collected at an abattoir by PCR analysis using primers specific for the Toxoplasma gondii SAG2 locus. This study revealed the presence of this parasite in 57 out of 101 samples examined. Restriction fragment length polymorphism (RFLP) in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Genotypes I and II were equally predominant, accounting for 22 (44.9%) and 23 (46.9%) of 49 SAG2-positive samples, respectively, while the type III strain was found in only 4 (8.2%) of the 49 samples. The other 8 samples were indistinguishable by PCR-RFLP analysis. Polymorphisms for the 3 genotypes were confirmed at the sequence level for several samples using the sequences from the RH strain, the Beverley strain, and the C56 strain as references. On the other hand, the dihydropteroate synthase gene, which is responsible for sulfonamide resistance, was amplified in 40 of 54 SAG2-positive samples by PCR with the specific primers, and further RFLP and sequence analysis revealed that none of them carried the drug-resistant form of the dhps gene. This is the first report of genotyping of T. gondii distributed in Japan.