Culture of Amelanchier x grandiflora in a programmable micropropagation apparatus

A micropropagation system was developed to test concepts for automation and microenvironment alteration. Amelanchier x grandiflora Rehd. Princess Diana (serviceberry) shoot cultures were grown in seven-liter polycarbonate containers. Through computer control, the apparatus intermittently applied culture medium to the plant material according to a selected schedule. Shoot growth in the programmable system was compared with four micropropagation treatments: gelled and liquid medium in baby food jars and gelled and non-cycling liquid medium in a seven-liter vessel. Plants cultured in continuous contact with liquid medium became increasingly vitrified. Significantly greater shoot number, shoot length, shoot weight, and culture weight occurred in the programmable system than in jars with gelled medium. The combination of liquid medium, 7-liter vessel, and intermittent contact with medium caused a significantly higher proliferation rate than any combination of jar/vessel and gelled/liquid medium tested.     

Iota-carrageenan: molecular structure and packing of polysaccharide double helices in oriented fibres of divalent cation salts

The Ca²⁺ and Sr²⁺ salts of i-carrageenan have isomorphous crystal structures with trigonal unit cells of dimensions a = b = 1.373 nm, c = 1.328 nm, γ = 120 °. Two kinds of fibre diffraction pattern were found for the Mg²⁺ salt: one resembling the Ca²⁺ and Sr²⁺ patterns and one with additional layer lines interleaved midway between those in the usual kind of pattern. Specimens of this second type convert to the first type on storage at 92% relative humidity. These Mg²⁺i-carrageenate diffraction patterns provide direct evidence for the double-helical nature of the carrageenan molecule.A molecular model has been derived that consists of two, identical, righthanded, 3-fold helical polysaccharide chains of pitch 2.656 nm. One chain is translated axially 1.32 nm relative to the other.A packing arrangement with up-pointing and down-pointing double helices distributed randomly among the molecular sites explains the presence of both Bragg reflections and layer line streaks. The space group of our statistical crystal structure is P3₂12. The divalent cations were found by Fourier difference syntheses to be at ($\text{2}\text{3}$, $\text{1}\text{3}$, $\text{1}\text{6}$) and symmetry-related positions. The co-ordination of each cation to sulphate groups on two different helices leads to a continuous set of cation-sulphate-cation-… interactions that accounts for the high crystallinity of these salts.The structure of the Ca²⁺ salt has been refined by constrained linked-atom least-squares methods. The structural isomorphism of the Sr²⁺ salt was confirmed by an independent refinement.     

Viability of glycerol-preserved and cryopreserved anuran skin

Summary Anurans are important animal models for studying the effects of anthropogenic chemical contamination of the environment. Two-compartment Teflon flow-through diffusion cells can be used to study percutaneus absorption of xenobiotics across harvested skin. However, such an approach currently necessitates that skin be harvested just before experimentation, a requirement that calls for the continuous growth and housing of living animals. The ability to preserve and store skin would allow more efficient use of animals and more flexibility in experimental design. To this end, we examined the viability of harvested anuran skin stored under various protocols consistent with current practices of mammalian skin preservation. Skin from the American bullfrog maintained 80–85% viability after 28 d, whereas viability of skin from the marine toad was only maintained for 7–10 d.     

Diet preferences of warblers for specific fatty acids in relation to nutritional requirements and digestive capabilities

During energy-demanding periods of the annual cycle such as migration or during cold days in winter, birds store fat comprised mostly of 16- or 18-carbon unsaturated fatty acids. In such situations, birds may feed selectively on foods with specific fatty acids that enable efficient fat deposition. We offered wild-caught yellow-rumped warblers Dendroica coronata paired choices between semi-synthetic diets that differed only in their fatty acid composition. Warblers strongly preferred diets containing long-chain (18:1; carbon atoms:double bonds) unsaturated, unesterified fatty acids to diets containing long-chain saturated, unesterified fatty acids (18:0) and they preferred diets containing mono-unsaturated fats (18:1) to diets containing poly-unsaturated fats (18:2). The preference for diets containing long-chain unsaturated fatty acids to diets containing long-chain saturated fatty acids was consistent in birds tested one week after capture at 21°C, one month after capture when cold-acclimated (1°C), and six weeks after capture at 21°C. Birds acclimated to a diet with 50% of the fat comprised of unesterified stearic acid (18:0) lost mass and reduced their food intake when we reduced ambient temperature from 21°C to 11°C over three days. We conclude that especially in energy-demanding situations there are limits to the yellow-rumped warblers' ability to assimilate some long-chain saturated fatty acids and that this digestive constraint can explain in part why yellow-rumped warblers prefer diets containing long-chain unsaturated fatty acids to diets containing long-chain saturated fatty acids.     

Human Cytomegalovirus-Induces Cytokine Changes in the Placenta with Implications for Adverse Pregnancy Outcomes

Human cytomegalovirus (CMV) infection of the developing fetus can result in adverse pregnancy outcomes including death in utero. Fetal injury results from direct viral cytopathic damage to the CMV-infected fetus, although evidence suggests CMV placental infection may indirectly cause injury to the fetus, possibly via immune dysregulation with placental dysfunction. This study investigated the effects of CMV infection on expression of the chemokine MCP-1 (CCL2) and cytokine TNF-α in placentae from naturally infected stillborn babies, and compared these changes with those found in placental villous explant histocultures acutely infected with CMV ex vivo. Tissue cytokine protein levels were assessed using quantitative immunohistochemistry. CMV-infected placentae from stillborn babies had significantly elevated MCP-1 and TNF-α levels compared with uninfected placentae (p = 0.001 and p = 0.007), which was not observed in placentae infected with other microorganisms (p = 0.62 and p = 0.71) (n = 7 per group). Modelling acute clinical infection using ex vivo placental explant histocultures showed infection with CMV laboratory strain AD169 (0.2 pfu/ml) caused significantly elevated expression of MCP-1 and TNF-α compared with uninfected explants (p = 0.0003 and p<0.0001) (n = 25 per group). Explant infection with wild-type Merlin at a tenfold lower multiplicity of infection (0.02 pfu/ml), caused a significant positive correlation between increased explant infection and upregulation of MCP-1 and TNF-α expression (p = 0.0001 and p = 0.017). Cytokine dysregulation has been associated with adverse outcomes of pregnancy, and can negatively affect placental development and function. These novel findings demonstrate CMV infection modulates the placental immune environment in vivo and in a multicellular ex vivo model, suggesting CMV-induced cytokine modulation as a potential initiator and/or exacerbator of placental and fetal injury.     

Cysteine cathepsins S and L modulate anti-angiogenic activities of human endostatin

Human endostatin, a potent anti-angiogenic protein, is generated by release of the C terminus of collagen XVIII. Here, we propose that cysteine cathepsins are involved in both the liberation and activation of bioactive endostatin fragments, thus regulating their anti-angiogenic properties. Cathepsins B, S, and L efficiently cleaved in vitro FRET peptides that encompass the hinge region corresponding to the N terminus of endostatin. However, in human umbilical vein endothelial cell-based assays, silencing of cathepsins S and L, but not cathepsin B, impaired the generation of the ～22-kDa endostatin species. Moreover, cathepsins L and S released two peptides from endostatin with increased angiostatic properties and both encompassing the NGR sequence, a vasculature homing motif. The G10T peptide (residues 1455-1464: collagen XVIII numbering) displayed compelling anti-proliferative (EC(50) = 0.23 nm) and proapoptotic properties. G10T inhibited aminopeptidase N (APN/CD13) and reduced tube formation of endothelial cells in a manner similar to bestatin. Combination of G10T with bestatin resulted in no further increase in anti-angiogenic activity. Taken together, these data suggest that endostatin-derived peptides may represent novel molecular links between cathepsins and APN/CD13 in the regulation of angiogenesis.     

ATP sensitive bi-quinoline activator of the AMP-activated protein kinase

The AMP-activated protein kinase (AMPK) regulates cellular and whole-body energy balance in response to changes in adenylate charge and hormonal signals. Activation of AMPK in tissues such as skeletal muscle and liver reverses many of the metabolic defects associated with obesity and Type 2 diabetes. Here we report a bi-quinoline (JJO-1) that allosterically activates all AMPK αβγ isoforms in vitro except complexes containing the γ3 subunit. JJO-1 does not directly activate the autoinhibited α subunit kinase domain and differs among other known direct activators of AMPK in that allosteric activation occurs only at low ATP concentrations, and is not influenced by either mutation of the γ subunit adenylate-nucleotide binding sites or deletion of the β subunit carbohydrate-binding module. Our findings indicate that AMPK has multiple modes of allosteric activation that may be exploited to design isoform-specific activators as potential therapeutics for metabolic diseases.