Transduction of Vero cells and bovine monocytes with a herpes simplex virus-1 based amplicon carrying the gene for the bovine herpesvirus-1 Circ protein

Herpes simplex virus-1 (HSV-1) based amplicon vectors are promising gene delivery vehicles because they have a large transgene capacity and can efficiently transduce many different cell types, including non-dividing cells, of various animal species. The Circ protein of bovine herpesvirus-1 (BHV-1) is a myristylated virion component of unknown function. Preliminary experiments with a circ gene deletion mutant indicated that Circ may influence the host's immune response by downregulating MHC-II expression in bovine monocytes. To get more insight into the function of Circ, amplicon vectors were constructed with various open reading frames (ORFs) under the control of the HSV-1 IE4/5 promoter: (i) the Circ ORF alone, (ii) a fusion ORF encoding an N-terminal Circ fused to the enhanced green fluorescent protein (eGFP), (iii) the eGFP ORF alone, and (iv) the Circ ORF in the inverted orientation. Upon helpervirus-free packaging into HSV-1 amplicon particles and transduction of Vero cells, both Circ alone and the Circ-eGFP fusion protein produced a punctate pattern within the cytoplasm, suggesting membrane association of the myristylated protein. In contrast, eGFP alone was evenly distributed over the cytoplasm of transduced cells. Upon infection of bovine buffy-coat cells, it was observed that cells of the monocyte lineage but not lymphocytes were transduced. Transgene expression reached a peak around 20h after transduction and lasted for at least 90h. Transduced monocytes underwent specific morphological changes, which may be attributed to Circ synthesis.     

Growth marks observation: preferential techniques and teeth for ageing of Spanish red deer (Cervus elaphus hispanicus)

A total of 207 teeth belonging to 53 deer (Cervus elaphus hispanicus) from Sierra Morena (South of Spain) were analysed using histological thin sections and/or polished thick sections. Growth marks were observed in dentine and cementum of incisors, molars and canines. Tooth, region of the tooth, and type of preparation giving the best performance in terms of sharpness and interpretation ease of the growth marks, when used for Spanish deer ageing, were identified. The tissue chosen for the study was the cellular cementum of the inter-radicular pad from molars and the acellular cementum of the root from incisors, both observed through longitudinal cuts. Histological sections obtained with a conventional microtome were optimal for a reliable identification of the first growth marks. In sections thicker than 15 microm the first rest line, when observed in cementum can be confused with the cementum-dentine junction; whereas in the polished sections from molars it will not appear at all. Dentine interpretation was more difficult. Its observation should only be accomplished using cross-sections of the molar root. Secondary or accessory marks were visible in every tooth, being those from the molar pad the easiest to distinguish. Canines were the less suitable teeth for this study.     

Cytotoxicity in pig hepatocytes induced by 8-quinolinol, chloramine-T and natamycin

The potential cytotoxic effects of the compounds 8-quinolinol, chloramine-T and natamycin have been studied in isolated pig hepatocytes. The relative cytotoxicity of these compounds was evaluated on the basis of the leakage of cytosolic lactate dehydrogenase (LDH), 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases, uptake of neutral red (NR) by cytosolic lysosomes, glutathion (GSH) depletion and oxidized glutathion (GSSG) efflux after 24 h exposure. Evaluation of the 20%, 50% and 80% reduced absorbance data obtained from the parameters NR20, NR50, and NR80, and MTT20, MTT50 and MTT80 enabled us to rank these compounds in decreasing order of cytotoxicity: 8-quinolinol > natamycin > chloramine-T. Also for the parameters LDH and GSH, chloramine-T appears to be less cytotoxic than natamycin and 8-quinolinol. Our study demonstrated that pig hepatocytes may be a useful model for examining cytotoxic events of drugs to be used in pigs, therefore avoiding possible extrapolation problems due to species differences.     

Apoptosis-independent Poor Morphology of Bovine Embryos Produced by Multiple Ovulation

In multiple ovulation and embryo transfer (MOET) programmes in cattle, a considerable number of morphologically poor-quality embryos continue to be produced; this is one of the limiting factors of the technique. Apoptosis has often been implicated in developmental arrest and fragmentation; these are regarded as poor traits of embryonic quality in mammalian pre-implantation embryos. In the present study, apoptosis was assessed in morphologically poor-quality embryos in comparison with good-quality embryos that were recovered from a MOET programme. Retarded embryos (two to 16 cell stage), morulae with severe fragmentation and morphologically good-quality morulae recovered from superstimulated cows at day 7 post-insemination were subjected to TdT-mediated dUTP nick-end labelling (TUNEL) and Hoechst staining. Cell nuclei that showed both TUNEL staining and apoptotic morphology were considered to be apoptotic. Apoptotic index (AI) was calculated as the percentage of apoptotic cells per embryo. Fifteen of 17 retarded embryos and 10 of 15 morphologically poor-quality morulae did not show signs of apoptosis. The mean AIs in the morphologically poor-quality embryos (two to 16 cell stage, 2.2%; poor morulae, 1.3%) were as low as that in the good-quality embryos (2.9%). These results suggest that another mode of developmental arrest and/or fragmentation that is independent of apoptosis occurs in morphologically poor-quality embryos recovered from MOET programmes.     

Control of Blue Mold of Apples by Preharvest Application of Candida sake Grown in Media with Different Water Activity

ABSTRACT Unmodified and low water activity (a(w))-tolerant cells of Candida sake CPA-1 applied before harvest were compared for ability to control blue mold of apples ('Golden Delicious') caused by Penicillium expansum under commercial storage conditions. The population dynamics of strain CPA-1 on apples were studied in the orchard and during storage following application of 3 x 10(6) CFU/ml of each treatment 2 days prior to harvest. In the field, the population size of the unmodified treatment remained relatively unchanged, while the population size of the low-a(w)-modified CPA-1 cells increased. During cold storage, the populations in both treatments increased from 10(3) to 10(5) CFU/g of apple after 30 days, and then declined to about 2.5 x 10(4) CFU/g of apple. In laboratory studies, the low-a(w)-tolerant cells provided significantly better disease control as compared with the unmodified cells and reduced the number of infected wounds and lesion size by 75 and 90%, respectively, as compared with the non-treated controls. After 4 months in cold storage, both unmodified and low-a(w)-tolerant cells of C. sake were equally effective against P. expansum on apple (>50% reduction in size of infected wounds).     

Comprehensive assessment of the quality of commercial cranberry products. Phenolic characterization and in vitro bioactivity

Cranberry (Vaccinium macrocarpon) products have been widely recommended in traditional American medicine for the treatment of urinary tract infection (UTI). A total of 19 different commercial cranberry products from American and European markets have been analyzed by different global phenolic methods and by UPLC-DAD-ESI-TQ MS. In addition, in vitro antioxidant capacity and uropathogenic bacterial antiadhesion activity tests have been performed. Results revealed that products found in the market widely differed in their phenolic content and distribution, including products completely devoid of flavan-3-ols to highly purified ones, either in A-type proanthocyanidins (PACs) or in anthocyanins. The product presentation form and polyphenolic profile widely affected the antiadhesion activity, ranging from a negative (nulel) effect to a MIC = 0.5 mg/mL for cranberry powders and a MIC=112 mg/mL for gel capsule samples. Only 4 of 19 products would provide the recommended dose of intake of 36 mg total PACs/day. Of most importance was the fact that this dose would actually provide as low as 0.00 and up to 205 μg/g of procyanidin A2, indicating the lack of product standardization and incongruence between global and individual compound analysis.     

<Emphasis Type="SmallCaps">d</Emphasis>-Cloprostenol enhances estrus synchronization in tropical hair sheep

To compare the effects of PGF2α (dinoprost tromethamine) and d-cloprostenol in a two-dose protocol for estrus synchronization in hair sheep during breeding season in Yucatán, México, two experiments were conducted. In experiment 1, 61 cyclic hair sheep were divided into two groups: G1 (control n?=?30), two doses of 50 μg of dinoprost tromethamine IM with 12 days between applications, and G2 (n?=?31), two doses of 50 μg of d-cloprostenol IM at the same time interval. For determination of progesterone levels, 16 ewes from each group were randomly selected. In experiment 2, 70 cyclic hair sheep were assigned at the same treatments (G1 and G2, n?=?35) and 48 h after the second application, the ewes in estrus were detected by two vasectomized rams. Sheep with detected estrus were inseminated, and 45 days after, pregnant animals were identified by ultrasonography. An exact Fisher’s test was performed for the analysis of ewes in estrus (experiments 1 and 2) and number of pregnant ewes (experiment 2); for the comparison of time between end of treatment-estrus presentation, a survival analysis was used. Duration of estrus in hours was analyzed using a generalized mixed model (GLM) ANOVA whereas plasma progesterone concentrations were analyzed by non-linear regression. There were significant differences (P?<?0.05) in the proportion of ewes in estrus upon treatments (G1, 57% vs G2, 87% and G1, 37.1% vs G2, 65.7% in experiments 1 and 2, respectively), and between the end of treatment-onset estrus interval (P?<?0.01), survival curves showed the highest number of sheep in estrus between 40 and 48 h (G1, 43.7?+?8.05 h vs G2, 42.9?+?6.7 h, experiment 1). There were no significant differences (P?>?0.05) in duration of estrus (G1, 42?+?6.1 h, vs G2, 41.1?+?11.2 h, experiment 1) and pregnancy in the ewes that presented estrus, and were inseminated (G1, 38.4% vs 52.1%, experiment 2). With regard to concentrations of progesterone, significant differences (P?<?0.01) were found between treatments, and progesterone levels before the second application of d-cloprostenol were higher. In consideration of the results, it can be concluded that in a two-dose protocol of a luteolytic agent, more ewes presented estrus in response to d-cloprostenol compared to dinoprost tromethamine with similar pregnancy rates.