DNA damage by ethylbenzenehydroperoxide formed from carcinogenic ethylbenzene by sunlight irradiation

Ethylbenzene, widely used in human life, is a non-mutagenic carcinogen. Sunlight-irradiated ethylbenzene caused DNA damage in the presence of Cu2+, but unirradiated ethylbenzene did not. A Cu+ -specific chelator bathocuproine inhibited DNA damage and catalase showed a little inhibitory effect. The scopoletin assay revealed that peroxides and H(2)O(2) were formed in ethylbenzene exposed to sunlight. These results suggest that Cu+ and alkoxyl radical mainly participate in DNA damage, and H(2)O(2) partially does. When catalase was added, DNA damage at thymine and cytosine was inhibited. Ethylbenzenehydroperoxide, identified by GC/MS analysis, induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and caused DNA damage at consecutive guanines, as observed with cumenehydroperoxide. Equimolar concentrations of H(2)O(2) and acetophenone were produced by the sunlight-irradiation of 1-phenylethanol, a further degraded product of ethylbenzene. These results indicate a novel pathway that oxidative DNA damage induced by the peroxide and H(2)O(2) derived from sunlight-irradiated ethylbenzene may lead to expression of the carcinogenicity.     

Optimal covalent immobilization of glucose oxidase-containing liposomes for highly stable biocatalyst in bioreactor

The glucose oxidase-containing liposomes (GOL) were prepared by entrapping glucose oxidase (GO) in the liposomes composed of phosphatidylcholine (PC), dimyristoyl L-alpha-phosphatidylethanolamine (DMPE), and cholesterol (Chol) and then covalently immobilized in the glutaraldehyde-activated chitosan gel beads. The immobilized GOL gel beads (IGOL) were characterized to obtain a highly stable biocatalyst applicable to bioreactor. At first, the glutaraldehyde concentration used in the gel beads activation as well as the immobilizing temperature and time were optimized to enhance the immobilization yield of the GOL to the highest extent. The liposome membrane composition and liposome size were then optimized to obtain the greatest possible immobilization yield of the GOL, the highest possible activity efficiency of the IGOL, and the lowest possible leakage of the entrapped GO during the GOL immobilization. As a result, the optimal immobilization conditions were found to be as follows: the liposome composition, PC/DMPE/Chol = 65/5/30 (molar percentage); the liposome size, 100 nm; the glutaraldehyde concentration, 2% (w/v); the immobilizing temperature, 4 degrees C; and the immobilizing time, 10 h. Furthermore, the optimal IGOL prepared were characterized by its rapidly increasing effective GO activity to the externally added substrate (glucose) with increasing temperature from 20 to 40 degrees C, and also by its high stability at 40 degrees C against not only the thermal denaturation in a long-term (7 days) incubation but also the bubbling stress in a bubble column. Finally, compared to the conventionally immobilized glucose oxidase (IGO), the higher operational stability of the optimal IGOL was verified by using it either repeatedly (4 times) or for a long time (7 days) to catalyze the glucose oxidation in a small-scale airlift bioreactor.     

Low sequence variation in the gene encoding the human beta-myosin heavy chain

Over 40 different mutations in the cardiac myosin heavy chain gene (MYH7) have been associated with familial hypertrophic cardiomyopathy (FHC), but no study has analyzed variation at this locus within the normal human population. Here we determine the extent and distribution of nucleotide variation in the 5808-bp MYH7 coding sequence in 25 normal individuals without FHC. We identified six single-nucleotide polymorphisms, none of which changes the encoded amino acid. At one of these sites, the frequencies of both alleles are equal; at the other five sites, the frequency of the rarer allele varies from 0.02 to 0.08. The nucleotide diversity (pi) calculated from these data is 1.73x10(-4)+/-0.49x10(-4), which is lower than the nucleotide diversity found in most other human autosomal genes. Substitution analysis of homologous genes between human and rodent also indicates that the MYH7 sequence has evolved at a very slow rate. The rate of both synonymous and nonsynonymous substitutions, especially in the portion of the sequence that encodes the alpha-helical myosin rod, is extremely low. The low level of even silent sequence variation in MYH7 in comparisons between human sequences and between human and rodent sequences may be a consequence of strong selective pressure against mutations that cause cardiomyopathy.     

Influence of GnRH analogue (fertirelin acetate) doses on synchronization of ovulation and fixed-time artificial insemination in lactating dairy cows

The effect of using a dose of 50 micro g rather than 100 micro g fertirelin in an ovulation/fixed-time insemination protocol for Holstein-Friesian dairy cows was investigated in three experiments. In each experiment, fertirelin was administered at the beginning of the protocol followed 7 days later by 500 micro g cloprosterol. Two days later, a second dose of fertirelin was given and AI performed 16-19 h later regardless of the incidence of behavioral oestrus.The effect on conception rate was studied in experiment 1 using 114 postpartum anoestrus cows. There was no significant difference in the age, parity or number of days after parturition in each treatment groups. The conception rate did not differ between the 50 micro g fertirelin group (61.1%; n=72) and the 100 micro g group (59.5%; n=42; NS). In experiment 2, a further 12 cows at 40-60 days postpartum were treated with 100 or 50 micro g fertirelin (n=6 per dose) with treatment commencing in the follicular or luteal phase of the oestrous cycle. The plasma concentration of luteinizing hormone (LH) reached similar peaks of over 5 ng/ml 120 min after the intramuscular administration of fertirelin in both groups. There were no significant differences in LH levels between treatments or phase of the oestrous cycle when treatment commenced. Doses of 50 and 100 micro g fertirelin were compared in experiment 3 using 17 cows to study follicular wave development and synchronization by transrectal ultrasonography, conception rate and corpus luteum function. There were no significant differences between treatments for these factors.It was concluded that using a dose of 50 micro g fertirelin enabled the drug costs to be reduced without affecting the efficiency of a synchronization of ovulation/fixed-time AI protocol for dairy cows.     

Monoclonal antibody to shiga toxin 1, which blocks receptor binding and neutralizes cytotoxicity

A monoclonal antibody, 5-5B, which neutralizes Shiga toxin 1 (Stx1) cytotoxicity of Escherichia coli, was constructed. An epitope analysis indicated that Asn55 in Stx1 B subunit was an important residue. This result and our previous results using an anti-Stx2 monoclonal antibody indicate that the region around the cysteine residue of the disulfide bond might be important for the neutralization of Stx cytotoxicity, making it a potential vaccination candidate.     

Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering

To monitor the fast compaction process during protein folding, we have used a stopped-flow small-angle X-ray scattering technique combined with a two-dimensional charge-coupled device-based X-ray detector that makes it possible to improve the signal-to-noise ratio of data dramatically, and measured the kinetic refolding reaction of alpha-lactalbumin. The results clearly show that the radius of gyration and the overall shape of the kinetic folding intermediate of alpha-lactalbumin are the same as those of the molten globule state observed at equilibrium. Thus, the identity between the kinetic folding intermediate and the equilibrium molten globule state is firmly established. The present results also suggest that the folding intermediate is more hydrated than the native state and that the hydrated water molecules are dehydrated when specific side-chain packing is formed during the change from the molten globule to the native state.     

Dogs as alternative intermediate hosts of Taenia solium in Papua (Irian Jaya), Indonesia confirmed by highly specific ELISA and immunoblot using native and recombinant antigens and mitochondrial DNA analysis

Serology (ELISA and immunoblot) using native glycoproteins, affinity purified glycoproteins, and a recombinant antigen is known to be highly specific to Taenia solium cysticercosis in humans and pigs. These techniques were applied for dogs in the highly endemic area of cysticercosis in Papua (Irian Jaya), Indonesia. Analysis of dog sera by both ELISA and immunoblot revealed 7 of 64 dogs were highly positive. Examination of two sero-positive dogs revealed cysticerci of T. solium in the brain and heart of these dogs. Mitochondrial DNA analysis confirmed that they were the same as T. solium previously confirmed from pigs and biopsies from local people from Irian Jaya. It is suggested that the life cycle of T. solium may be completed not only between humans and pigs but also between humans and dogs.     

Dietary diacylglycerol suppresses high fat diet-induced hepatic fat accumulation and microsomal triacylglycerol transfer protein activity in rats

We have recently shown that the long-term ingestion of dietary diacylglycerol (DAG) mainly containing 1,3-isoform reduces body fat accumulation in humans as compared to triacylglycerol (TAG) with the same fatty acid composition. The fat reduction in this human experiment was most pronounced in visceral fat and hepatic fat. Recent animal studies have also indicated that dietary DAG induces alteration of lipid metabolism in the rat liver. In the present study, the dietary effects of DAG on high fat diet-induced hepatic fat accumulation and hepatic microsomal triglyceride transfer protein (MTP) activity were examined in comparison with those of TAG diet in rats. When the TAG oil content was increased from 10 to 30 g/100 g diet, hepatic TAG concentration, hepatic MTP activity and MTP large subunit mRNA levels were significantly increased after 21 days. However, when the dietary TAG oil (30 g/100 g diet) was replaced with the same concentration of DAG oil with the same fatty acid composition, the increase of the TAG concentration and the MTP activity in the liver were significantly less and the mRNA levels remained unchanged. The MTP activity levels correlated significantly with hepatic TAG concentration.These results showed that dietary DAG may suppress high fat diet-induced MTP activity in the liver, and indicated the possibility that hepatic TAG concentration may regulate hepatic MTP activity.