Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination

The present study was conducted to evaluate whether supplementation of semen extender with glutathione (GSH) can maintain the quality of frozen-thawed canine spermatozoa. Eighteen ejaculates were obtained from 5 dogs and placed in extender (20% egg yolk, Tris, citric acid, lactose, raffinose, antibiotics and 6.5% glycerol) containing 0 (control), 2.5, 5, 7.5 or 10 mM GSH. The samples were cooled to 4 C and then frozen in liquid nitrogen vapor. Motility parameters of the sperm were evaluated at 0, 1, 2, 3, 4, 12 and 24 h after thawing. Sperm motility was higher in the 5 mM GSH group than in the control or 2.5 and 10 mM GSH groups; this effect was observed at 1 to 24 h after thawing (P < 0.05). The 5 mM GSH group had a higher sperm viability index at 12 and 24 h after thawing compared with the other groups (P < 0.05). Acrosome integrity, evaluated at 4 h after thawing, was greater in two of the GSH-treated groups (5 and 10 mM) compared with the control. Lipid peroxidation (LP) levels immediately after thawing were lower in the 5 and 10 mM GSH groups compared with the control, while those at 12 h after thawing did not differ significantly. Frozen-thawed semen in the 5 mM GSH group was used for transcervical insemination of 4 bitches, resulting in delivery of 5 puppies from 2 bitches. These results indicate that supplementation of semen extender with 5 mM GSH was effective in improving motility, longevity and acrosomal integrity and inhibiting LP levels in post-thaw canine spermatozoa, without any adverse impacts on full-term development after transcervical insemination.     

Isolation of antifouling substances from the Palauan sponge Protophlitaspongia aga

ABSTRACT: Fouling organisms cause serious problems by settling on ships' hulls, and other marine infrastructures. Organotin compounds have been developed as effective antifoulants. However, environmental problems are associated with these compounds. It is necessary to find antifouling substances that exhibit a lower toxicity than those substances currently used. A new pyrimidine derivative, 3,4,5,6-tetrahydro-6-hydroxymethyl-3,6-dimethyl-4-pyrimidinecarboxylic acid, (compound 1) and zooanemonin (compound 2) were isolated from the marine sponge Protophlitaspongia aga as antifouling substances against the barnacle Balanus amphitrite . We also found that α-nicotinamide ribose (compound 3) inhibited germination and the attachment of Ulva spores.     

Responses of fees used for revegetation of desert areas to iron deficiency stress

We investigated the responses to iron deficiency of four major tree species, Prosopis cineraria (local name: Ghaf), Acacia tortilis (Samar), Zizyphus spina-christi (Sidr), and Leptadenia pyrotechnica (Markh), used for revegetation of desert areas in the United Arab Emirates (UAE). The responses to iron deficiency differed among the tree species. Ghaf, Samar, and Sidr decreased the nutrient solution pH in response to iron deficiency. Markh and iron-deficient Sidr did not release protons in the CaCl₂ solution. The Fe reducing capacity of the roots of Ghaf, Samar, and Sidr was significantly enhanced by iron deficiency, whereas a smaller increase in the reducing activity of the roots of Markh was observed. In all the tree species, the amount of reductant released from iron-deficient plants was higher than that from iron-sufficient ones. Markh released a small amount of reductant under irondeficient conditions. In the present study, the reductant released by all the trees was caffeic acid. Acidification treatment enhanced the amount of reductant released irrespective of iron treatments in Ghaf, Samar, and Sidr but had no effect on the amount of reductant released in iron-deficient Markh. The activity of p-coumarate hydroxylase was higher in Sidr and Ghaf than in the other trees. This activity decreased in iron-deficient Samar. Ghaf, Samar, and Sidr showed a high capacity to lower the pH of the nutrient solution, and Ghaf markedly enhanced the effectiveness of all the examined mechanisms, while Markh showed a lower ability compared to the other tree species.     

Genetic variation in barley of crossability with wheat and its quantitative trait loci analysis

To study genetic variation in crossability, 80 barley accessions of diverse geographic origin consisting of 50 wild barleys (H. vulgare ssp. spontaneum or ssp. agriocrithon) and 30 cultivated barleys (H. vulgare ssp. vulgare) were crossed as the male parent with a highly crossable wheat variety, Shinchunaga. Crossabilities, expressed as the percentage of pollinated florets giving embryo-containing caryopses, ranged from 0% to 68.6%. Barley accessions from East Asia had generally a low crossability, while barley accessions from other regions exhibited a wider range of crossability including highly crossable genotypes. No significant difference in mean crossability was found between wild and cultivated barleys. To estimate the number and location of barley genes controlling the crossability, doubled haploid lines derived from the cross between the barley varieties Steptoe and Morex were crossed as the male parent with wheat. Quantitative trait loci (QTL) analysis using molecular markers identified four QTL. These were mapped to the centromeric regions of chromosomes 2H, 3H and 5H and the short arm of chromosome 7H. The QTL on chromosomes 3H and 5H had larger effects than those on chromosomes 2H and 7H. The four QTL collectively explained 35.4% of the total variance under a multiple QTL model. Relationships of the QTL identified in the present study with previously reported crossability genes of barley and wheat are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transport and survival of Japanese sardine (Sardinops melanostictus) eggs and larvae via particle‐tracking experiments

Particle‐tracking experiments were performed to infer the distribution of larvae of the Japanese sardine (Sardinops melanostictus) and to detect effects of transport environment on sardine recruitment, using the output of a high‐resolution ocean general circulation model and observed data of sardine spawning grounds during 1978–2004. By the 60th day following spawning, approximately 50% of the larvae had been transported to the Kuroshio Extension (KE). Whereas the spawning period and grounds changed markedly in relation to the stock level, the proportion of larvae transported to the KE remained relatively constant and no significant correlations were found between sardine recruitment and the transport proportion. Instead, the recruitment was found to be correlated with physical parameters including the mixed layer depth and the sea surface temperature along several major transport trajectories of sardine larvae. The correlations were most significant for the trajectories in the region 0.5° south to 1° north of the Kuroshio axis (defined as the location of velocity maxima at each longitude) and for larvae spawned in February and March during the high stock period (1978–94), and for larvae spawned in March and April during the low stock period (1995–2004).     

Induction of apoptosis on K562 cell line and double strand breaks on colon cancer cell line expressing high affinity receptor for granulocyte macrophage-colony stimulating factor (GM-CSF)

BACKGROUND: Immunotoxins are comprised of both the cell targeting and the cell killing moieties. We previously established a new immunotoxin, i.e. Shiga toxin granulocyte macrophage-colony stimulating factor (StxA1-GM-CSF), comprises of catalytic domain of Stx, as a killing moiety and GM-CSF, as a cell targeting moiety. In this study, the ability of the immunotoxin to induce apoptosis and double strand breaks (DSB) on different cell lines was investigated. METHODS: The recombinant hybrid protein was expressed in bacterial expression system and purified with nickel-nitrilotriacetate acid resin. The K562 (erythroid leukemia) cell line and LS174 (colon carcinoma) were used in this study. The neutral comet assay was carried out for the detection of DSB and Hoechst staining was performed for apoptosis. RESULTS: StxA1-GM-CSF effectively induced apoptosis on K562 cell line and DNA Double Strand Break (DSB) were observed on colon cancer cell line treated with StxA1-GM-CSF. CONCLUSION: This novel action i.e. DNA damage might be a relevant mechanism of action for StxA1-GM-CSF that is designed to act as immunotoxin, although further investigation is required.     

Screening and cDNA cloning of Kv1 potassium channel toxins in sea anemones

When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of (125)I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3'RACE and 5'RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ(1.3)-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34-63% identities with the other type 1 potassium channel toxins.     

Efficacy and safety of lomefloxacin on bacterial extraocular disease in the horse

Lomefloxacin is a broad-spectrum fluoroquinolone antibiotic used for the treatment of bacterial extraocular disease. This study investigated the efficacy and safety of lomefloxacin eye drops for bacterial extraocular disease in horses. Lomefloxacin ophthalmic solution (0.3%) was instilled three times daily for 2–5 days in 65 horses diagnosed with bacterial extraocular disease based on clinical findings. Clinical observations and bacteriological examinations were performed at the start of treatment, 2 and 5 days after the start of treatment, and at the discontinuation or termination of treatment. Of the 65 horses, 64 were positive for bacteria, and 22 bacterial genera and 47 bacterial species were identified. The efficacy of lomefloxacin was evaluated in 63 horses; one horse with a negative culture and another with suspected bacterial contamination were excluded. Lomefloxacin was considered to be clinically effective in 54 horses. The major bacterial species identified were Staphylococcus aureus, Streptococcus equi subsp. zooepidemicus, Acinetobacter lwoffii, Staphylococcus xylosus, Staphylococcus vitulinus, Enterobacter agglomerans, Flavimonas oryzihabitans and Staphylococcus sciuri, with a cumulative disappearance rate of 80% or more at the termination of instillation. Excluding one horse that did not undergo a bacteriological examination, the remaining 62 horses were assessed for bacteriological outcome. Full or partial bacterial clearance was detected in 95% or more of the 62 horses. One of the 65 horses reported adverse events that had no causal relation with the eye drops. Our results showed that lomefloxacin is safe and effective for the treatment of bacterial extraocular disease in horses.     

Analysis of rice <Emphasis Type="Italic">RNA-dependent RNA polymerase 1</Emphasis> (<Emphasis Type="Italic">OsRDR1</Emphasis>) in virus-mediated RNA silencing after particle bombardment

RNA-dependent RNA polymerases (RDRs) play a key role in various RNA silencing pathways in many organisms. Using the nucleotide sequence of SGS2/SDE1/RDR6 in Arabidopsis as the search query for sequences that flank the insertions of rice retrotransposon Tos17, we selected rice mutant lines (OsRDR1). RT-PCR analysis showed that OsRDR1 mRNA was undetectable in leaves and calli of the mutants, while it was detected in wild type. RNA silencing was induced by particle bombardment to investigate any effects of OsRDR1 on RNA silencing with β-glucuronidase or green fluorescent protein DNA/RNA in the mutant lines. The results showed that RNA silencing was impaired in these mutant lines by inverted repeat (IR) DNA or in vitro transcribed double-stranded RNA. Further, the mutant lines were bombarded with Brome mosaic bromovirus (BMV, a ssRNA virus) or Wheat dwarf geminivirus (WDV, a ssDNA virus), each carrying the IR sequence of a reporter gene. As a result, RNA silencing was impaired by BMV. Interestingly, however, it was not impaired by WDV. Thus we propose that OsRDR1 is required for RNA silencing mediated by Bromovirus, but not by Geminivirus in this system.     

Furosemide loading test in a case of homozygous solute carrier family 12, member 1 (SLC12A1) mutation (g.62382825G>A,p.Pro372Leu) in Japanese Black cattle

Hydrallantois is the excessive accumulation of fluid in the allantoic cavity in a pregnant animal and is associated with fetal death. We recently identified a recessive missense mutation in the solute carrier family 12, member 1 (SLC 12A1 ) gene (g.62382825G>A, p.Pro372Leu) that is associated with hydrallantois in Japanese Black cattle. Unexpectedly, we found a case of the homozygous risk‐allele for SLC 12A1 in a calf, using a PCR ‐based direct DNA sequencing test. The homozygote was outwardly healthy up to 3 months of age and the mother did not exhibit any clinical symptoms of hydrallantois. In order to validate these observations, we performed confirmation tests for the genotype and a diuretic loading test using furosemide, which inhibits the transporter activity of the SLC 12A1 protein. The results showed that the calf was really homozygous for the risk‐allele. In the homozygous calf, administration of furosemide did not alter urinary Na⁺ or Cl^? levels, in contrast to the heterozygote and wild‐type calves in which these were significantly increased. These results demonstrate that the SLC 12A1 (g.62382825G>A, p.Pro372Leu) is a hypomorphic or loss‐of‐function mutation and the hydrallantois with this mutation shows incomplete penetrance in Japanese Black cattle.