Western Blot Analysis of Proacrosin/Acrosin in Frozen Dog Sperm During In Vitro Capacitation

Acrosin is an acrosomal protease synthesized as an inactive precursor, proacrosin, which is processed via autoproteolysis into active forms alpha- and beta-acrosin. In this paper, a comparative study on the immunoreactivity of acrosin during in vitro capacitation of frozen and fresh (control) canine sperm using Western blot analysis is reported. Semen samples were obtained by digital stimulation and ejaculates processed as fresh and frozen samples and then capacitated for 0, 30, 60 and 90 min. At each time period, samples were analyzed with monoclonal antibody C5F10 by Western blot. The antibody specifically recognized, in fresh and frozen/thawed spermatozoa, a 40-, 32- and 27-kDa bands corresponding to proacrosin, alpha- and beta-acrosin, respectively, during capacitation. Western immunoblots showed that the beta-acrosin reactivity in fresh sperm was directly proportional to the time of capacitation, whereas a decreased reactivity of active form of acrosin was observed with frozen-thawed sperm (p   < 0.05). These results suggest that proacrosin is activated to beta-acrosin earlier in frozen/thawed dog spermatozoa than in fresh dog spermatozoa.     

Biological characteristics of maize dwarf mosaic potyvirus from Spain

Two isolates of maize dwarf mosaic virus (MDMV-Sp and MDMV-Spl) obtained from maize in the northeast of Spain were studied. Both isolates induced similar reactions on 6 sorghum cultivars, johnsongrass and oat (cv. Clintland), with the exception of MDMV-Sp which produced a different reaction on one sorghum cultivar. Thirty-three grass species were tested as possible hosts (16 previously untested) and 18 were found to be susceptible. Among those, eight were previously unidentified hosts for MDMV:Aegilops ovata, A. ventricosa, Avena barbata, Bromus alopecurus, B. diandrus, B. fasciculatus, Echinaria capitata andLolium rigidum. Both isolates were transmitted from maize to maize nonpersistently byRhopalosiphum maidis, R. padi, Schizaphis graminum andSitobium avenae. The virus was not seed-transmitted in Mo17 and B73 maize inbred lines. Pinwheels, scrolls and short curved laminated aggregates were observed in the cytoplasm of maize cells infected by MDMV-Sp or MDMV-Sp1. In addition, laminated aggregates were observed in cells infected by MDMV-Sp1.     

Growth response of field-grown Siratro (Macroptilium atropurpureum Urb.) and Aeschynomene american L. to inoculation with selected vesicular-arbuscular mycorrhizal fungi

Summary The effect of inoculation with a selected isolate of Glomus etunicatum Becker and Gerdemann and one of G. intraradices Schenck and Smith on the growth and nutrient content of Macroptilium atropurpureum Urb. cv. Siratro and Aeschynomene americana L., at applied P levels of 10, 30, 60, and 120 kg ha^-1, was studied under field conditions. At all P levels and for all harvests, the shoot dry mass of Siratro and A. americana were greater for the plants inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungi than the control plants. Differences between the VAM fungus-inoculated and the control plants were most marked between 30 and 90 kg ha^-1 of applied P and diminished at 120 kg ha^-1. At the first harvest of Siratro, the plants inoculated with G. etunicatum had a greater shoot dry mass than those inoculated with G. intraradices, for all levels of applied P. However, for subsequent harvest of Siratro and for the one harvest of A. americana the response of shoot dry mass to the two VAM fungi was equivocal. Fungal inoculation gave at least a 30% saving in the amount of P fertilizer required (40 kg ha^-1) for the maximum yield. The plants inoculated with VAM fungi had a greater tissue concentration and total content of P and N than the control plants at low and intermediate levels of applied P. The percentage of root colonized by VAM fungi for the inoculated plants of the two legumes increased linearly with P additions up to 60 kg ha^-1. The conclusion is that under amended (limed and fertilized) soil conditions, inoculation with selected VAM fungi can improve the establishement and growth of forage legumes in fields that contain ineffective populations of native VAM fungi.     

Characterisation of a maize-infecting potyvirus from Spain

In order to characterise and classify an unknown maize-infecting potyvirus isolated from fields in northeast Spain, the entire coat protein gene and the C-terminal twothirds of the large nuclear inclusion protein (NIb) gene were cloned and sequenced. Protein sequencing enabled the cleavage site between the two proteins to be deduced and also revealed that on storage the viral coat protein undergoes a specific degradation in which the N-terminal 39 amino acids are removed. Comparison of the nucleotide sequence of the 3 non-coding region of the viral RNA and the predicted amino acid sequence of the coat protein with the equivalent regions of other members of the potyvirus group revealed that the Spanish virus is closely related to maize dwarf mosaic virus strain A.     

Cryphonectria hypovirus 1 (CHV‐1) survey reveals low occurrence and diversity of subtypes in NE Spain

Cryphonectria parasitica (Murrill.) Barr is a necrotrophic fungus that affects European chestnut populations. In Catalonia (NE Spain), this fungus is widely spread affecting chestnut stands. In addition to vegetative compatibility (VC) types of the fungus, knowledge of the Cryphonectria hypovirus (CHV) occurrence is important to implement a biocontrol programme. We confirmed the presence of hypoviruses in several chestnut subpopulations of Catalonia, and we determined the VC types of CHV‐infected isolates. We also studied the nucleotide sequences of these hypoviruses from a variable region located in the hypovirus ORF‐A. Our results show a low occurrence of hypoviruses throughout C. parasitica populations in Catalonia, except for a few localized stands. From 312 sampled cankers, we obtained 179 white, pale orange or rickety isolates. In 35 of them, we detected CHV‐1. Infected isolates belonged to 5 of the 7 sampled subpopulations. We found 12 CHV‐1 haplotypes, based on the nucleotide sequence analysis. Most of the hypoviruses have the same nucleotide sequence or show high homology at nucleotide level with isolates previously included in CHV‐1 subtype‐I. However, we found that 3 haplotypes share at least 97% of their ORF‐A nucleotide sequence with CHV‐1 subtype‐E. Phylogenetic analysis grouped these 3 isolates in a different cluster than the other hypoviruses. These results suggest that multiple introductions of CHV‐1 have occurred recently in Catalonia. Dominant VC types of hypovirus‐infected isolates are EU‐1, EU‐2 and EU‐5, which are similar to those previously reported in this region.     

Hydroxytyrosol prevents oxidative deterioration in foodstuffs rich in fish lipids

Hydroxytyrosol, a natural phenolic compound obtained from olive oil byproduct, was characterized as an antioxidant in three different foodstuffs rich in fish lipids: (a) bulk cod liver oil (40% of omega-3 PUFAs), (b) cod liver oil-in-water emulsions (4% of omega-3 PUFAs), and (c) frozen minced horse mackerel ( Trachurus trachurus) muscle. Hydroxytyrosol was evaluated at different concentration levels (10, 50, and 100 ppm), and its antioxidant capacity was compared against that of a synthetic phenolic, propyl gallate. Results proved the efficiency of hydroxytyrosol to inhibit the formation of lipid oxidation products in all tested food systems, although two different optimal antioxidant concentrations were observed. In bulk oil and oil-in-water emulsions, a higher oxidative stability was achieved by increasing the concentration of hydroxytyrosol, whereas an intermediate concentration (50 ppm) showed more efficiency, delaying lipid oxidation in frozen minced fish muscle. The endogenous depletion of alpha-tocopherol and omega-3 polyunsaturated fatty acids (omega-3 PUFAs) was also inhibited by supplementing hydroxytyrosol in minced muscle; however, the consumption of the endogenous total glutathione was not efficiently reduced by either hydroxytyrosol or propyl gallate. A concentration of 50 ppm of hydroxytyrosol was best to maintain a longer initial level of alpha-tocopherol (approximately 300 microg/g of fat), whereas both 50 and 100 ppm of hydroxytyrosol were able to preserve completely omega-3 PUFAs. Hydroxytyrosol and propyl gallate showed comparable antioxidant activities in emulsions and frozen fish muscle, and propyl gallate exhibited better antioxidant efficiency in bulk fish oil.     

Efficiency of natural phenolic compounds regenerating alpha-tocopherol from alpha-tocopheroxyl radical

Benzoic acid-derived phenolics (p-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, vanillic acid, syringic acid, and gallic acid) and the polyphenols epicatechin and epigallocatechin gallate (EGCG) were evaluated for their efficiency in regenerating alpha-tocopherol from alpha-tocopheroxyl radical in comparison with ascorbyl palmitate, which is known to regenerate alpha-tocopherol. Ethanolic solutions of phenolic compounds were added to a homogeneous hexane medium containing alpha-tocopheroxyl radical generated by reaction of alpha-tocopherol in molar excess with 1,1-diphenyl-2-picrylhydrazyl radical, and the alpha-tocopheroxyl radical was monitored by electron spin resonance spectroscopy. p-Hydroxybenzoic acid, vanillic acid, and syringic acid (400 microM) did not exhibit a significant effect on alpha-tocopheroxyl radical concentration (0.6-0.7 microM). In contrast, 3,4-dihydroxybenzoic acid and gallic acid were able to reduce the concentration of alpha-tocopheroxyl radical by 16 and 64%, respectively. Epicatechin showed a reduction of alpha-tocopheroxyl radical similar to gallic acid, and EGCG and ascorbyl palmitate were the most effective, reducing alpha-tocopheroxyl radical completely at a much lower phenolic concentration (66.7 microM). The moles of alpha-tocopheroxyl radical reduced per mole of ascorbyl palmitate (0.93), EGCG (0.066), gallic acid (4.3 x 10(-4)), and epicatechin (4.5 x 10(-4)) were determined, and the logarithm of these stoichoimetric ratios showed a negative linear relationship with the bond dissociation enthalpies of the O-H bond of the phenolics. The relative capacity to reduce alpha-tocopheroxyl radical was found to be ascorbyl palmitate (2142) > EGCG (151) > gallic acid (1) approximately epicatechin (1).