Maturation and germination of walnut somatic embryos

Walnut somatic embryos were multiplied by repetitive embryogenesis on a solid basal DKW medium at 25°C in the dark. When the embryos were isolated at early cotyledonary stage (1–2 mm long) from the primary embryos and cultured on the medium for 3 weeks, they developed into mature embryos showing white, enlarged cotyledons and shoot and root apex. After transfer to light on solid germination medium, however, few mature embryos (0–5%) germinated. Germination percentage increased to about 10% when the mature embryos were pretreated by a storage at 4°C in the dark for 2 months, or by desiccation at 25°C in the dark for 3 or 5 days under an air-humidity conditioned by saturated salt solutions (Mg(NO₃)₂.6H₂O, or ZnSO₄.7H₂O). Similar results were obtained by the addition of gibberellic acid (GA₃) to the germination medium. When mature embryos were desiccated and then placed on medical cotton compresses in liquid germination medium, 45% of the embryos germinated into complete plantlets. These plantlets continued their growth after transplanting to a mixture of peat and vermiculite in pots.Abbreviations GA₃ gibberellic acid - DKW medium Driver & Kuniyuki Walnut medium     

Choline Availability Modulates the Expression of TGFβ1 and Cytoskeletal Proteins in the Hippocampus of Developing Rat Brain

Choline availability influences long-term memory in concert with changes in the spatial organization and morphology of septal neurons, however little is known concerning the effects of choline on the hippocampus, a region of the brain also important for memory performance. Pregnant rats on gestational day 12 were fed a choline control (CT), choline supplemented (CS), or choline deficient (CD) diet for 6 days and fetal brain slices were prepared on embryonic day 18 (El8). The hippocampus in these brain slices was studied for the immunohistochemical localization of the growth-related proteins transforming growth factor beta type 1 (TGF1) and GAP43, the cytoskeletal proteins vimentin and microtubule associated protein type 1 (MAP1), and the neuronal cell marker neuron specific enolase (NSE). In control hippocampus, there was weak expression of TGF1 and vimentin proteins, but moderately intense expression of MAP1 protein. These proteins were not homogeneously distributed, but were preferentially localized to cells with large cell bodies located in the central (CA1–CA3) region of the hippocampus, and to the filamentous processes of small cells in the fimbria region. Feeding a choline-supplemented diet decreased, whereas a choline-deficient diet increased the intensity of immunohistochemical labeling for these proteins in El8 hippocampus. GAP43 and NSE were localized to peripheral nervous tissue but not hippocampus, indicating that the maturation of axons and neurite outgrowth in embryonic hippocampus were unaffected by the availability of choline in the diet. These data suggest that the availability of choline affects the differentiation of specific regions of developing hippocampus.     

Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism

Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.     

Insights into interactions between the alpha-helical region of the salmon calcitonin antagonists and the human calcitonin receptor using photoaffinity labeling

Fish-like calcitonins (CTs), such as salmon CT (sCT), are widely used clinically in the treatment of bone-related disorders; however, the molecular basis for CT binding to its receptor, a class II G protein-coupled receptor, is not well defined. In this study we have used photoaffinity labeling to identify proximity sites between CT and its receptor. Two analogues of the antagonist sCT(8-32) containing a single photolabile p-benzoyl-l-phenylalanine (Bpa) residue in position 8 or 19 were used. Both analogues retained high affinity for the CT receptor and potently inhibited agonist-induced cAMP production. The [Bpa(19)]sCT(8-32) analogue cross-linked to the receptor at or near the equivalent cross-linking site of the full-length peptide, within the fragment Cys(134)-Lys(141) (within the amino terminus of the receptor, adjacent to transmembrane 1) (Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. (2004) J. Biol. Chem. 279, 6720-6729). In contrast, proteolytic mapping and mutational analysis identified Met(49) as the cross-linking site for [Bpa(8)]sCT(8-32). This site differed from the previously identified cross-linking site of the agonist [Bpa(8)]human CT (Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 31177-31182) and may provide evidence for conformational differences between interaction with active and inactive state receptors. Molecular modeling suggests that the difference in cross-linking between the two Bpa(8) analogues can be accounted for by a relatively small change in peptide orientation. The model was also consistent with cooperative interaction between the receptor amino terminus and the receptor core.     

Serological evidence of an antibody response in farmed southern bluefin tuna naturally infected with the blood fluke Cardicola forsteri

In this study, adaptive immune response was investigated in farmed southern bluefin tuna, Thunnus maccoyii, infected with a sanguinicolid Cardicola forsteri. A cohort (Cohort₂₀₀₅) of southern bluefin tuna was sampled between March 2005 and August 2006. Samples were taken at the transfer of wild caught tuna to sea cages and then at regular intervals. Parasite intensity, abundance and prevalence data were recorded. An ELISA was developed to detect and quantify an antibody response against the blood fluke in southern bluefin tuna serum. Intensity and prevalence of the blood fluke were shown to peak in May 2005 at 10.9 flukes per infected fish (SE = 1.72) and 97.5% prevalence and then decreased to low prevalence (10%) and intensity (1.0). There were no significant changes in prevalence or intensity in 2006. Antibody titres and seroprevalence increased from 1.37 U μl⁻¹ and 10% at transfer in March 2005 to reach a peak in December 2005 of 25.86 U μl⁻¹ (SE = 6.26 U μl⁻¹) and 66.66%. No significant changes were observed in antibody titres for the same cohort of fish during 2006. Parasitological and serological values from Cohort₂₀₀₅ were compared to a 2006 cohort (Cohort₂₀₀₆) in March 2006 and August 2006 to determine if prior infection in Cohort₂₀₀₅ elicited any protection against infection in 2006. Although significant differences were not observed in intensities between cohorts it was shown that Cohort₂₀₀₅ had significantly lower abundances and prevalences of blood fluke infection than Cohort₂₀₀₆. Although there was no significant difference in mean antibody titres between cohorts in March 2006, the mean antibody titre of Cohort₂₀₀₆ was significantly greater than that of Cohort₂₀₀₅ in August 2006. No significant differences were observed in seroprevalence. This is one of the few studies to demonstrate the development of acquired resistance in fish against a parasite in an aquaculture environment under natural infection conditions.