High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes

This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.     

Seasonal prevalence of fungi in the conjunctival fornix of healthy cows during a 2‐year study

Objective The aims of the present paper were to: (i) identify and quantify conjunctival fungi isolated from healthy cows; (ii) verify the influence of different methods of farm management on the prevalence (percentage of positive cultures for each fungal species per farm) of conjunctival fungi. Material and methods Forty Friesian and twenty Limousin female cows aged 1–10 years stabled in three farms with different managements (farm 1: cows housed strictly indoors; farm 2: cows housed outside during the day and inside the stall during the night; farm 3: cows housed strictly outdoors) were investigated for conjunctival fungal flora. Air and food were also tested. Specimens were collected every season during a 2‐year study. Identification of colonies of filamentous fungi was achieved to the genus level on the basis of macro‐ and microscopic features. Results The total number of eyes positive for fungi ranged from 85 to 100% at farm 1, from 65 to 95% at farm 2, and from 55 to 95% at farm 3. Fungi most frequently isolated from conjunctival fornix were Cladosporium spp. and Penicillium spp. Statistical analysis did not show any differences in fungal prevalences among the three farms during the same season. Some fungal species were consistently isolated while others were intermittently isolated. Conclusions Fungi found in the conjunctival fornix of cows might represent transient seeding from the environment, as suspected in other species. The prevalence of conjunctival fungal organisms is not different in cattle housed indoors vs. outdoors.     

Long-term immunogenicity and protection against Mycoplasma agalactiae induced by an oil adjuvant vaccine in sheep

The long-term protective immunity of an inactivated mineral-oil adjuvanted Mycoplasma agalactiae vaccine was evaluated in sheep. The antigen suspension was emulsified with a mixture of three mineral oils (Montanide ISA-563, Marcol-52, Montane-80 at the ratio of 30%, 63%, and 7%, respectively). Twenty-two animals were divided in 2 groups (A and B) and immunised with two doses of the vaccine (group A, n = 14) or used as unvaccinated control (group B, n = 8). Five months after the second vaccination, seven animals of group A and four animals of group B were challenged by nasal route with M. agalactiae. The remaining seven vaccinated and four control animals were challenged intranasally eight months after vaccination. The vaccine was able to induce a full-protective immunity preventing the clinical signs of contagious agalactia and the infection by M. agalactiae in all groups of animals irrespective of the time of challenge after booster administration.     

Cutaneous progressive angiomatosis on the muzzle of a dog,treated by laser photocoagulation therapy

A 10‐year old, female, cocker spaniel was presented with a bleeding lesion on the muzzle and nasal planum observed initially as a small raised nodule at 1 year of age. This became clinically more pronounced at 5 years of age, progressively enlarged thereafter and was diagnosed initially as a vascular tumour. On clinical examination, multiple confluent nodules of variable diameter (0.5–1.5 cm) deformed the outline of the nose. Histological examination revealed a progressive angiomatosis with multifocal infiltrative growth of increased dermal vascularization with different sized and type of anastomosing vessels lined by a monolayer of reactive endothelium, often arranged as papillary projections into the lumina. Partial nosectomy, the first therapeutic approach, was unsuccessful and the lesion relapsed within 6 months. However, subsequent laser photocoagulation therapy provided a good cosmetic outcome, no relapse 1 year later, and appeared to provide an effective alternative to ablation surgery.     

Purinergic receptors and regulatory volume decrease in seabream (Sparus aurata) hepatocytes: a videometric study

The response of isolated hepatocytes of Sparus aurata to hypotonic stress was studied by the aid of videometric methods with the aim to investigate the possible involvement of ATP in the regulatory volume decrease (RVD). This study confirms our previous observations showing the ability of these cells to undergo RVD. In addition, it shows that the homeostatic response was inhibited by apyrase, an ATP scavenger, thus suggesting the involvement of extracellular ATP in the RVD response. Experiments performed in the presence of ATPγS or adenosine, agonists of P₂ and P₁ receptors respectively, and in the presence of suramin or 8-PT, antagonists of P₂ and P₁ receptors respectively, suggest that ATP exerts its stimulatory effect on the homeostatic response by interacting with P₂ receptors. On the other hand, the activation of P₁ receptors by ATP metabolites produces opposite effects. In an attempt to clarify the mechanisms involved in ATP release from the cell, we performed some experiments with known inhibitors of the possible mechanisms of regulated ATP release. The results we obtained let us to suppose that the mechanism allowing the exit of ATP from the cell is verapamil sensitive suggesting the involvement of the P-glycoprotein.     

Molecular and physico-chemical evaluation of enzymatic browning of whole meal and dough in a collection of tetraploid wheats

Many studies have demonstrated the role of polyphenol oxidase (PPO; E.C. 1.14.18.1) in darkening of wheat products. Even slight browning of pasta is a major hindrance to consumer acceptance. The aim of this research was to evaluate the variability of PPO activity, browning level, and protein content in a collection of more than 100 genotypes of tetraploid wheat, including cultivars and landraces. A molecular approach was followed to evaluate the efficiency of marker PPO18, discovered and used in common wheat, in detecting tetraploid wheat genotypes with low browning level. The data showed a significant genotype influence on the activity of the PPO enzyme, which was correlated with brown index of whole meal and dough, and with protein content. On the whole, the cultivars showed lower PPO activity, brown index, and protein content than landraces, but wide variability was present. Marker PPO18 detected four different alleles for Ppo-A1. The allele Ppo-A1f identified genotypes with high PPO activity and brown index, while the Ppo-A1b and null alleles were associated with the opposite characteristics. Hence, this marker can be used to select new tetraploid wheat cultivars with low browning level.     

Sourdough bread: Starch digestibility and postprandial glycemic response

To evaluate the influence of sourdough fermentation on starch digestibility in bread, four experimental breads were obtained, prepared from two different wheat flours (whole or white) by two different leavening techniques (sourdough and with Saccharomyces cerevisiae). Products were analyzed for their starch, fiber and resistant starch (RS) content and then submitted to in vitro hydrolysis with porcine alpha-amylase. On the same breads, postprandial blood glucose was evaluated in healthy human subjects. Both sourdough fermented breads gave glycaemic responses significantly lower (p < 0.001) than the corresponding products leavened with S. cerevisiae. On the contrary, the presence of fiber did not influence the glycaemic potential of breads. RS levels were higher in the sourdough products, whereas no differences were observed either in the rate of starch hydrolysis or in the degree of polymerization of the starch residues after the in vitro hydrolysis. We may conclude that sourdough fermentation is a technique able to reduce the glycaemic response to bread and that the mechanism does not seem related to the rate of starch hydrolysis.     

New Synthetic Glucosyl-Curcuminoids,and their <Superscript>1</Superscript>H and <Superscript>13</Superscript>C NMR Characterization,from <Emphasis Type="Italic">Curcuma longa</Emphasis> L.

Turmeric extracts, among which curcumin and bis-demethoxycurcumin, are by far known for their therapeutic activities. In this study we propose easy and low cost synthetic pathways in order to obtain glucosyl-curcuminoids, safe and water soluble potential drugs and dyes, which may be implied in different fields ranging from pharmacology to food chemistry. The complete ¹H and ¹³C NMR characterization of naturally occurring curcumin, bis-demethoxycurcumin and new synthetic glucosyl-curcuminoids is reported.