Endogenous inhibitors of glutathione S-transferases in house flies

The inhibition of glutathione S-transferase by endogenous compounds present in the soluble fraction of house fly homogenates was investigated. The highest inhibition was found with the female abdomen and increased with incubation time and with an increase in the tissue concentration. The correlation of increased inhibition with a parallel increase in the darkening of the soluble fraction indicated a possible association with melanization, thereby suggesting quinones as the possible endogenous inhibitiors of glutathione transferase. In vitro experiments demonstrated that quinones produced by mushroom tyrosinase did indeed inhibit glutathione S-transferase. Inhibition by quinones can be prevented by including glutathione or bovine serum albumin in the homogenization buffer. The inhibitory activity of a variety of quinones and related compounds on purified glutathione S-transferase was investigated. Oxygenated aromatics with hydroxy groups in the 1,2- or 1,4-position or ketonic carbonyls in the 1,4-position are good inhibitors of glutathione S-transferase.     

First Production of Larvae Using Cryopreserved Sperm: Effects of Preservation Temperature and Cryopreservation on European Eel Sperm Fertilization Capacity

Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20°C, or cryopreserved, following existing protocols. Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20°C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few larvae (‘cryolarvae’) were obtained 55 h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success.     

Parthenogenetic Induction of Canine Oocytes by Electrical Stimulation and Ca-EDTA

In this study, we investigated parthenogenetic induction of canine oocytes by electrical stimulation following Ca-EDTA treatment. Oocyte maturation, parthenogenetic development, and cleavage rate in canine after various electrical stimulations (1.5, 1.8, 2.1 kV/cm) for 50 μs with single DC pulse following 1 mM Ca-EDTA treatment were investigated. In oocyte activated electrically at the voltage of 1.5 kV/cm after 1 mM Ca-EDTA treatment, the rate of pronucleus and two-cell was 4.1% and 2.7%, respectively. Although electrical stimulation could parthenogenetically induce immature oocyte to cleavage stage, degeneration rate in all experimental groups was more than 60%. This means that electrical stimulation after Ca-EDTA treatment could cause canine oocytes to be degenerated. However, two-cell in canine oocyte by parthenogenesis was for the first time induced. Therefore, we suggested that electrical stimulation for canine oocytes could induce parthenogenetically early embryonic cleavage. This result can be used as a basic data for parthenogenesis study in canine. Also, to perform more developed embryonic development, further study to parthenogenesis in canine need to be developed.     

Live attenuated vaccine-based control of necrotic enteritis of broiler chickens

A vaccine for necrotic enteritis (NE) of chickens would reduce the current need to prevent or treat the disease in broiler chickens with antimicrobial drugs. The objective of this study was to understand aspects of immunity to the disease. The first experiment examined the virulence of six strains of Clostridium perfringens isolated from cases of NE in broiler chickens. Using a 5-day experimental oral infection of 2-week-old broiler chickens, four of the six strains were found to be virulent. Pulsed-field gel electrophoresis and PCR showed that virulence was not associated with a plasmid encoding the beta2 toxin gene, cpb2, since this was present in virulent and one of the two avirulent strains. In the second experiment, two virulent and one avirulent strains were tested for their ability to immunize ("infection-immunization") chickens through the oral route. The procedure used experimental infection for 5 days followed by bacitracin treatment for 9 days, and then re-challenge 2 days later with a virulent strain, CP4. Infection-immunization with the virulent isolates protected chickens from subsequent virulent challenge, whereas the infection-immunization with the avirulent isolate did not. In a third experiment, two of four alpha-toxin-negative mutants of CP4 protected birds from experimental NE after oral immunization. These two mutants were also attenuated for virulence. We conclude that it is possible to immunize chickens successfully against NE and that immunogen(s) other than alpha-toxin are important in protective immunity against oral infection.     

TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism

During fasting, increased concentrations of circulating catecholamines promote the mobilization of lipid stores from adipose tissue in part by phosphorylating and inactivating acetyl-coenzyme A carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis. Here, we describe a parallel pathway, in which the pseudokinase Tribbles 3 (TRB3), whose abundance is increased during fasting, stimulates lipolysis by triggering the degradation of ACC in adipose tissue. TRB3 promoted ACC ubiquitination through an association with the E3 ubiquitin ligase constitutive photomorphogenic protein 1 (COP1). Indeed, adipocytes deficient in TRB3 accumulated larger amounts of ACC protein than did wild-type cells. Because transgenic mice expressing TRB3 in adipose tissue are protected from diet-induced obesity due to enhanced fatty acid oxidation, these results demonstrate how phosphorylation and ubiquitination pathways converge on a key regulator of lipid metabolism to maintain energy homeostasis.