Assessment of genetic stability of the germplasm lines of medicinal plant <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi (Huang-qin) in long-term,in vitro maintained cultures

An approach of combining flow cytometry (FCM) analysis with morphological and chemical profiling was used to assess the genetic stability and bioactive compound diversity in a Scutellaria baicalensis Georgi (Huang-qin) germplasm collection that was clonally maintained in in vitro for a period of over 6 years. Based on the FCM analysis of nuclei samples from young shoots, the nuclear DNA content of S. baicalensis was calculated as 0.84 pg/2C. FCM analysis showed no significant variation in the nuclear DNA contents and ploidy levels in the long-term in vitro maintained germplasm lines. Germplasm lines, acclimatized to ex vitro conditions, exhibited distinctive plant growth and bioactive compound production capacities. The high level of genetic stability observed in in vitro maintained S. baicalensis lines opens up a variety of opportunities such as allowing long-term aseptic preservation and easy distribution of well-characterized germplasm lines of this medicinal plant species. This study represents a novel approach for continuous maintenance, monitoring, and production of medicinal plant tissues with specific chemistry.     

Phylogenetic relationships within an endemic group of Malagasy 'assassin spiders' (Araneae, Archaeidae): ancestral character reconstruction, convergent evolution and biogeography

The phylogenetic relationships in an endemic group of Malagasy 'assassin spiders' (Araneae, Archaeidae: Eriauchenius) called the gracilicollis group, are inferred from mitochondrial 12S, 16S and COI DNA sequence data. Archaeid spiders of Madagascar have evolved varying degrees of elongation in the cephalic area. These molecular data support the monophyly of the gracilicollis group. The evolution of the cephalic area is examined by performing an ancestral character reconstruction on this character, which reveals that the cephalic area is elongating independently. The biogeography of the gracilicollis group reveals an east-west split of the clade on Madagascar.     

Microfluidics-based diagnostics of infectious diseases in the developing world

One of the great challenges in science and engineering today is to develop technologies to improve the health of people in the poorest regions of the world. Here we integrated new procedures for manufacturing, fluid handling and signal detection in microfluidics into a single, easy-to-use point-of-care (POC) assay that faithfully replicates all steps of ELISA, at a lower total material cost. We performed this 'mChip' assay in Rwanda on hundreds of locally collected human samples. The chip had excellent performance in the diagnosis of HIV using only 1 μl of unprocessed whole blood and an ability to simultaneously diagnose HIV and syphilis with sensitivities and specificities that rival those of reference benchtop assays. Unlike most current rapid tests, the mChip test does not require user interpretation of the signal. Overall, we demonstrate an integrated strategy for miniaturizing complex laboratory assays using microfluidics and nanoparticles to enable POC diagnostics and early detection of infectious diseases in remote settings.     

Ndel1 alters its conformation by sequestering cAMP-specific phosphodiesterase-4D3 (PDE4D3) in a manner that is dynamically regulated through Protein Kinase A (PKA)

The involvement of the Nuclear distribution element-like (Ndel1; Nudel) protein in the recruitment of the dynein complex is critical for neurodevelopment and potentially important for neuronal disease states. The PDE4 family of phosphodiesterases specifically degrades cAMP, an important second messenger implicated in learning and memory functions. Here we show for the first time that Ndel1 can interact directly with PDE4 family members and that the interaction of Ndel1 with the PDE4D3 isoform is uniquely disrupted by elevation of intracellular cAMP levels. While all long PDE4 isoforms are subject to stimulatory PKA phosphorylation within their conserved regulatory UCR1 domain, specificity for release of PDE4D3 is conferred due to the PKA-dependent phosphorylation of Ser13 within the isoform-specific, unique amino-terminal domain of PDE4D3. Scanning peptide array analyses identify a common region on Ndel1 for PDE4 binding and an additional region that is unique to PDE4D3. The common site lies within the stutter region that links the second coiled-coil region to the unstable third coiled-coil regions of Ndel1. The additional binding region unique to PDE4D3 penetrates into the start of the third coiled-coil region that can undergo tail-to-tail interactions between Ndel1 dimers to form a 4 helix bundle. We demonstrate Ndel1 self-interaction in living cells using a BRET approach with luciferase- and GFP-tagged forms of Ndel1. BRET assessed Ndel1–Ndel1 self-interaction is amplified through the binding of PDE4 isoforms. For PDE4D3 this effect is ablated upon elevation of intracellular cAMP due to PKA-mediated phosphorylation at Ser13, while the potentiating effects of PDE4B1 and PDE4D5 are resistant to cAMP elevation. PDE4D long isoforms and Ndel1 show a similar sub-cellular distribution in hippocampus and cortex and locate to post-synaptic densities. We show that Ndel1 sequesters EPAC, but not PKA, in order to form a cAMP signalling complex. We propose that a key function of the Ndel1 signalling scaffold is to signal through cAMP by sequestering EPAC, whose activity may thus be specifically regulated by sequestered PDE4 that also stabilizes Ndel1–Ndel1 self-interaction. In the case of PDE4D3, its association with Ndel1 is dynamically regulated by PKA input through its ability to phosphorylate Ser13 in the unique N-terminal region of this isoform, triggering the specific release of PDE4D3 from Ndel1 when cAMP levels are elevated. We propose that Ser13 may act as a redistribution trigger in PDE4D3, allowing it to dynamically re-shape cAMP gradients in distinct intracellular locales upon its phosphorylation by PKA.     

Functional interactions of meiotic recombination factors Rdh54 and Dmc1

Genetic studies in budding and fission yeasts have provided evidence that Rdh54, a Swi2/Snf2-like factor, synergizes with the Dmc1 recombinase to mediate inter-homologue recombination during meiosis. Rdh54 associates with Dmc1 in the yeast two-hybrid assay, but whether the Rdh54–Dmc1 interaction is direct and the manner in which these two recombination factors may functionally co-operate to accomplish their biological task have not yet been defined. Here, using purified Schizosaccharomyces pombe proteins, we demonstrate complex formation between Rdh54 and Dmc1 and enhancement of the recombinase activity of Dmc1 by Rdh54. Consistent with published cytological and chromatin immunoprecipitation data that implicate Rdh54 in preventing the non-specific association of Dmc1 with chromatin, we show here that Rdh54 mediates the efficient removal of Dmc1 from dsDNA. These functional attributes of Rdh54 are reliant on its ATPase function. The results presented herein provide valuable information concerning the Rdh54–Dmc1 protein pair that is germane for understanding their role in meiotic recombination. The biochemical systems established in this study should be useful for the continuing dissection of the action mechanism of Rdh54 and Dmc1.     

Slits contribute to the guidance of retinal ganglion cell axons in the mammalian optic tract

RGC axons extend in the optic tracts in a manner that correlates with the expression in the hypothalamus and epithalamus of a soluble factor inhibitory to RGC axon outgrowth. Additionally, although the RGC axons extend adjacent to the telencephalon, they do not normally grow into this tissue. Here, we show that slit1 and slit2, known chemorepellents for RGC axons expressed in specific regions of the diencephalon and telencephalon, help regulate optic tract development. In mice lacking slit1 and slit2, a subset of RGC axons extend into the telencephalon and grow along the pial surface but not more deeply into this tissue. Surprisingly, distinct guidance errors occur in the telencephalon of slit1 -/-; slit2 +/- and slit1/2 -/- embryos, suggesting that the precise level of Slits is critical for determining the path followed by individual axons. In mice lacking both slit1 and slit2, a subset of RGC axons also project aberrantly into the epithalamus, pineal and across the dorsal midline. However, many axons reach their primary target, the superior colliculus. This demonstrates that Slits play an important role in directing the guidance of post-crossing RGC axons within the optic tracts but are not required for target innervation.     

Negative regulation of beta-catenin/Tcf signaling by naringenin in AGS gastric cancer cell

Functional activation of beta-catenin/Tcf signaling plays an important role in early events in carcinogenesis. We examined the effect of naringenin against beta-catenin/Tcf signaling in gastric cancer cells. Reporter gene assay showed that naringenin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by naringenin in HEK293 cells transiently transfected with constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism was related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunofluorescence, Western blot, and EMSA. As a result, our data revealed that the beta-catenin distribution and the levels of nuclear beta-catenin and Tcf-4 proteins were unchanged after naringenin treatment. Moreover, the binding activities of Tcf complexes to consensus DNA were not affected by naringenin. Taken together, these data suggest that naringenin inhibits beta-catenin/Tcf signaling in gastric cancer with unknown mechanisms.     

Chlorothalonil-biotransformation by glutathione S-transferase of Escherichia coli

It has recently been reported that one of the most important factors of yeast resistance to the fungicide chlorothalonil is the glutathione contents and the catalytic efficiency of glutathione S-transferase (GST) (Shin et al, 2003). GST is known to catalyze the conjugation of glutathione to a wide variety of xenobiotics, resulting in detoxification. In an attempt to elucidate the relation between chlorothalonil-detoxification and GST, the GST of Escherichia coli was expressed and purified. The drug-hypersensitive E. coli KAM3 cells harboring a plasmid for the overexpression of the GST gene can grow in the presence of chlorothalonil. The purified GST showed chlorothalonil-biotransformation activity in the presence of glutathione. Thus, chlorothalonil is detoxified by the mechanism of glutathione conjugation catalyzed by GST.