Microstructure and glycosaminoglycan ratio of canine cornea after reconstructive transplantation with glycerin-preserved porcine amniotic membranes

Objective  Although amniotic membranes of canine, feline, and equine species have some advantages as corneal transplantation material in many canine ocular diseases, their softness, thinness, and low availability can pose problems. As an alternative, the more abundant porcine amniotic membranes may be used. This paper describes the use of glycerin-preserved porcine amniotic membranes in corneal transplantation in eight normal dogs.
Method  A 0.4-mm deep recipient bed in the axial cornea of the OS of all dogs was created using an 8-mm Barron radial vacuum trephine. The recipient bed was then filled with amnion, and the entire cornea was covered with another piece of the glycerin-preserved membrane. The ocular signs evaluated were corneal opacity and corneal vascularization. The dogs were euthanized on days 5, 10, 20, or 40 after surgery, and samples were collected to evaluate corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content and the glycosaminoglycan (GAG) ratio.
Results  Corneal opacity was observed immediately after surgery. Restoration of corneal transparency, regression of corneal vascularization, and visualization of the pupil and iris were noted on day 40.
Conclusions  The clinical observations were supported histologically by regained corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content, and GAG ratio, suggesting that this technique may be a novel method for the treatment of ocular surface disorders.     

Soil particles inhibit colony formation of Escherichia coli on the surface of agar media

When enumerating Escherichia coli in serial dilution of soil using selective media, soils was found to exert an inhibitory effect on colony formation on the surface of the agar plates. The inhibitory effect did not appear to be due to either soil bacteria or soluble chemicals but to the soil particles themselves. No inhibition was observed when the cells were enumerated by the most probable number (MPN) method. Some alternative plating methods, such as pour-plating or overlaying, were effective in removing the inhibitory effect. Removal of soil particles by simple sedimentation was also effective in enhancing the enumeration efficiency. These observations would be practically important for enumerating bacteria in the soil environment.     

Mycorrhizal fungi effects on nutrient composition and yield of soybean seeds

Nutrient composition and yield of soybean [Glycine max (L.) Merr] seeds are heritable traits affected by environmental factors. This study determined the effects of arbuscular‐mycorrhizal (AM) fungi on seed protein, lipid, and phosphorus (P) composition and yield in soybean grown under a high nitrogen (N) regime. Plants were grown in pot cultures without AM fungi in P‐fertilized (+P) or unfertilized (‐P) soil, or in ‐P soil inoculated with one of the AM fungi Glomus mosseae (Nicol. & Gerd.) Gerd, and Trappe (Gm), Glomus etunicatum Becker and Gerd.(Ge), or Gigaspora rosea Nicol. and Schenck (Gr). Seed yields of+AM plants, as a group, were halfway between those of the +P and ‐P plants. Seed size was highest in Gm plants. Differences in protein concentrations between Ge and Gr and the other treatments were highly significant. Seed P and protein concentrations were not significantly correlated (p=0.162), but a highly significant (r =‐0.949) negative correlation between seed P and lipid concentrations was observed. Phosphorus concentration was highest and that of lipids lowest in +AM plants. Seed yield and nutrient composition were independent of the intensity of root colonization. The seed protein/lipid ratio was highly correlated with seed P concentration and was significantly higher for +AM plants, as a group, than for both +P and ‐P ‐AM plants. Differences in seed dry weight, size, seed/ stem ratio, P content, and protein concentration among +AM plants showed mycorrhiza‐specific host responses. These responses suggest that AM fungi can modify soybean seed development and chemical composition.     

Marker-assisted selection for forelimb-girdle muscular anomaly of Japanese Black cattle

Forelimb‐girdle muscular anomaly is a hereditary disorder of Japanese Black cattle characterized by tremors and astasia caused by hypoplasia of the forelimb‐girdle muscles. The locus responsible for this disorder has been mapped on a middle region of bovine chromosome 26. In this study, we applied marker‐assisted selection to identify the carriers of this disorder. Four microsatellite markers, DIK4440, BM4505, MOK2602 and IDVGA‐59, linked to the disorder locus were genotyped in 37 unaffected offspring of a carrier sire. Transmission of the mutant or wild‐type allele of the disorder locus of the sire to the 37 offspring was determined by examining the haplotypes of these markers. The results showed that nine and 18 of the 37 animals possessed the paternally transmitted mutant and wild‐type alleles, respectively, and therefore, the nine animals with the mutant allele were identified as carriers. We concluded that the marker‐assisted selection using these four markers can be applied for the identification of the carriers of forelimb‐girdle muscular anomaly of Japanese Black cattle.     

Biochemical and genomic analysis of neoculin compared to monocot mannose-binding lectins

Neoculin occurring in an edible tropical fruit is a heterodimeric protein which has both sweetness and a taste-modifying activity that converts sourness to sweetness. Both the primary and the overall tertiary structures of neoculin resemble those of monocot mannose-binding lectins. This study investigated differences in biochemical properties between neoculin and the lectins. Structural comparison between the mannose-binding sites of lectins and the corresponding regions of neoculin showed that there is at least one amino acid substitution at each site in neoculin, suggesting a reason for the lack of its mannose-binding ability. This was consistent with hemagglutination assay data demonstrating that neoculin had no detectable agglutinin activity. DNA microarray analysis indicated that neoculin had no significant influence on gene expression in Caco-2 cell, whereas kidney bean lectin (Phaseolus vulgaris agglutinin) greatly influenced various gene expressions. These data strongly suggest that neoculin has no lectin-like properties, encouraging its practical use in the food industry.     

Cultivation of corneal epithelial cell sheets on canine amniotic membrane

Objective To develop and assess canine corneal epithelial cell sheets cultivated from limbal stem cells on amniotic membrane. Procedures Canine corneal limbal segments were obtained from six beagle dogs. Cryopreserved denuded amniotic membranes (obtained from Miniature Dachshund and Cavalier King Charles Spaniel breeds) from which the epithelial cells were removed were used as scaffolds. The limbal segments were cultured on these amniotic membranes with 3T3 feeder cells for 2 weeks. The harvested corneal epithelial cell sheets were stained with H&E for histologic analysis. The harvested sheets were analyzed immunohistochemically using a corneal epithelium‐specific marker keratin 3(K3) and putative stem cell markers ABCG2, p63, and vimentin. Results Cultivated cells from the corneal limbal tissues reached confluency in 7–8 days. The cultivated cells adhered to the denuded amniotic membrane and formed a sheet. The cultivated cell sheet was transparent and consisted of five to eight layers. K3 was observed in all layers and ABCG2, p63, and vimentin were notably present in the basal layer of the cultivated canine epithelium by immunofluorescence. Conclusions Canine corneal epithelial cells were successfully cultivated on the canine amniotic membrane. The cultivated epithelial sheets contained putative stem cells in the basal layer and had a stratified epithelium.     

Structure, allometry, and biomass of plantation Metasequoia glyptostroboides in Japan

We quantified structural features and the aboveground biomass of the deciduous conifer, Metasequoia glyptostroboides (Hu and Cheng) in six plantations in central Japan. In order to derive biomass estimates we dissected 14 M. glyptostroboides trees into three structural components (stem wood, branch wood and foliage) to develop allometric equations relating the mass of these components and of the whole tree to diameter at breast height (DBH). We found robust relationships at the branch and whole tree level that allow accurate prediction of component and whole tree biomass. Dominant tree height was similar within five older (>40 years) plantations (27–33 m) and shorter in a 20-year-old plantation (18 m). Average stem diameter varied from 12.8 cm in the youngest stand to greater than 35 cm in the oldest stand.

Metasequoia have relatively compact crowns distributed over the top 30% of the tree although the youngest stand had the deepest crown relative to tree height (up to 38%). At the individual tree level in older stands, 87% of the aboveground biomass was allocated to the stem, 9% to branch wood and 4% to foliage. We found little difference in the relative distribution of above ground biomass among the stands with the exception of lower foliage biomass in larger diameter trees. Total aboveground biomass of the older stands varied twofold, ranging from a maximum of 450 Mg ha⁻¹ in a 42-year-old stand to a minimum of 196 Mg ha⁻¹ in a 48-year-old stand. Total above ground biomass of the 20-year-old stand was 176 Mg ha⁻¹.     

Effect of different live food on survival and growth of first feeding barber goby,Elacatinus figaro (Sazima,Moura & Rosa 1997) larvae

One of the major challenges in marine fish culture is how to provide live food of adequate size and nutritional quality for first‐feeding larvae. Commonly used live food organisms, rotifers and brine shrimp, may not always be the best option. To determine the suitability of different zooplankton in the larviculture of Elacatinus figaro, three diets were tested: RE – rotifers Brachionus sp. (10 ind mL^?1)+ciliate Euplotes sp. (10 ind mL^?1), enriched with fatty acids; RC – enriched rotifers (10 ind mL^?1)+wild copepod nauplii (10 ind mL^?1); and R – enriched rotifers (20 ind mL^?1). Survival rates were estimated 10 days after hatch (DAH) for the three test groups, and growth rates were evaluated for RE and R at 10 and 20 DAH. Although survival rate was numerically higher for the RC diet (41.1±14.2%), no significant difference was detected between groups fed RE (20.5±18.1%), RC or R (32.1±16.5%). At 10 DAH, the growth rate was significantly higher in RC (5.7±0.6 mm) than in R (4.6±0.5 mm), a trend that was also observed at 20 DAH for RC (8.6±0.5 mm) and R (5.8±0.7 mm) (P<0.05). E. figaro larvae fed on ciliates did not show satisfactory results, whereas feeding copepod nauplii enhanced growth.