Advantages and limitations in the use of hairy root cultures for the production of tropane alkaloids: Use of anti-auxins in the maintenance of normal root morphology

Summary Brugmansia candida hairy roots, obtained by infection withAgrobacterium rhizogenes LBA 9402, exhibit, after subculturing in liquid media, a tendency towards dedifferentiation. It has been found that the following strategies can be applied to inhibit this dedifferentiation and preserve normal root morphology: (a) lowering both the mineral and sucrose concentration in the media employed so as to diminish osmotic stress (a condition to which these roots appear to be particularly susceptible); (b) employing antiauxins in appropriate concentrations; and (c) maintaining the hairy roots on solid media prior to use in production processes in liquid media. The first strategy suggested does not favor alkaloid productivity, but in this case a two-step method could be attempted: biomass with normal root morphology could be obtained in a first stage using low sucrose concentrations, and in a second stage, sucrose could be increased in order to achieve higher productivity. In all the clones ofB. candida obtained, alkaloid production was biased towards scopolamine.     

Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.     

Peripheral sweat gland function is improved with humid heat acclimation

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The purpose of this study was to determine if humid heat acclimation improves thermoregulatory function at the level of the eccrine sweat gland.     

Engineering disulfide bonds of the novel human beta-defensins hBD-27 and hBD-28: differences in disulfide formation and biological activity among human beta-defensins

Human beta-defensins comprise a large number of peptides that play a functional role in the innate and adaptive immune system. Recently, clusters of new beta-defensin genes with predominant expression in testicular tissue have been discovered on different chromosomes by bioinformatics. beta-Defensins share a common pattern of three disulfides that are essential for their biological effects. Here we report for the first time the chemical synthesis of the new fully disulfide-bonded beta-defensins hBD-27 and hBD-28, and compare the results with synthetic procedures to obtain the known hBD-2 and hBD-3. While hBD-27 was readily converted into a product with the desired disulfide pattern by oxidative folding, hBD-28 required a selective protective group strategy to introduce the three disulfide bonds. The established synthetic processes were applied to the synthesis of hBD-2, which, like hBD-27, was accessible by oxidative folding, whereas hBD-3 required a selective strategy comparable to hBD-28. Experimental work demonstrated that trityl, acetamidomethyl, and t-butyl are superior to other protection strategies. However, the suitable pairwise arrangement of the protective groups can be different, as shown here for hBD-3 and hBD-28. Determination of the minimum inhibitory concentration against different bacteria revealed that hBD-27, in contrast to other beta-defensins tested, has virtually no antimicrobial activity. Compared to the other peptides tested, hBD-27 showed almost no cytotoxic activity, measured by hemoglobin release of erythrocytes. This might be due to the low positive net charge, which is significantly higher for hBD-2, hBD-3, and hBD-28.     

Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy.

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy.     

Biological role and structural mechanism of twinfilin-capping protein interaction

Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin-CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics.     

Hyperoxia-induced apoptosis does not require mitochondrial reactive oxygen species and is regulated by Bcl-2 proteins

Exposure of animals to hyperoxia results in lung injury that is characterized by apoptosis and necrosis of the alveolar epithelium and endothelium. The mechanism by which hyperoxia results in cell death, however, remains unclear. We sought to test the hypothesis that exposure to hyperoxia causes mitochondria-dependent apoptosis that requires the generation of reactive oxygen species from mitochondrial electron transport. Rat1a cells exposed to hyperoxia underwent apoptosis characterized by the release of cytochrome c, activation of caspase-9, and nuclear fragmentation that was prevented by the overexpression of Bcl-X(L.) Murine embryonic fibroblasts from bax(-/-) bak(-/-) mice were resistant to hyperoxia-induced cell death. The administration of the antioxidants manganese (III) tetrakis (4-benzoic acid) porphyrin, ebselen, and N-acetylcysteine failed to prevent cell death following exposure to hyperoxia. Human fibrosarcoma cells (HT1080) lacking mitochondrial DNA (rho(0) cells) that failed to generate reactive oxygen species during exposure to hyperoxia were not protected against cell death following exposure to hyperoxia. We conclude that exposure to hyperoxia results in apoptosis that requires Bax or Bak and can be prevented by the overexpression of Bcl-X(L). The mitochondrial generation of reactive oxygen species is not required for cell death following exposure to hyperoxia.     

Functional dicer is necessary for appropriate specification of radial glia during early development of mouse telencephalon

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1^-/- dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon.