Comparative analysis of the plasmids from two isolates of "Candidatus Phytoplasma australiense"

Two plasmids from the plant-pathogenic mollicute "Candidatus Phytoplasma australiense" were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of "Ca. P. australiense." The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.     

Intracellular localization of organized lipid domains of C16-ceramide/cholesterol

Lipid microdomains, also called lipid rafts, consisting of sphingolipids and cholesterol, play important roles in membrane trafficking and in signaling. Despite years of study of the composition, size, half-life and dynamic organization of these domains, many open questions remain about their precise characteristics. To address some of these issues, we have developed a new experimental approach involving the use of specific monoclonal antibodies as recognition tools. One such antibody was raised against a homogeneous, mixed, ordered monolayer phase comprised of 60:40 mol% cholesterol:C16-ceramide, and has been used previously to demonstrate the existence of C16-ceramide/cholesterol domains in the membranes of cultured cells. We now use a combination of quantitative fluorescence microscopy, immuno-transmission electron microscopy and immuno-scanning cryo-electron microscopy, optimized for the study of intracellular lipid antigens. In a variety of cultured cells, C16-ceramide/cholesterol structural domains were found at high levels in late endosomes and in the trans-Golgi network, but were not found at statistically significant levels in early endosomes, lysosomes or the endoplasmic reticulum. We discuss the relevance of these results to understanding the role of lipid lateral organization in biological membranes.     

An aqueous media based approach for the preparation of a biosensor platform composed of graphene oxide and Pt-black

The combination of Pt nanoparticles and graphene was more effective in enhancing biosensing than either nanomaterial alone according to previous reports. Based on the structural similarities between water soluble graphene oxide (GrO(x)) and graphene, we report the fabrication of an aqueous media based GrO(x)/Pt-black nanocomposite for biosensing enhancement. In this approach GrO(x) acted as a nanoscale molecular template for the electrodeposition of Pt-black, an amorphously nanopatterned isoform of platinum metal. Scanning electron microscopy (SEM) images and energy-dispersive X-ray spectroscopy (EDS) showed that Pt-black was growing along GrO(x). The effective surface area and electrocatalytic activity towards H(2)O(2) oxidation of GrO(x)/Pt-black microelectrodes were significantly higher than for Pt-black microelectrodes. When used to prepare a bio-nanocomposite based on protein functionalization with the enzyme glucose oxidase (GOx), the GrO(x)/Pt-black microbiosensors exhibited improved sensitivity over the Pt-black microbiosensors. This suggested that the GrO(x)/Pt-black nanocomposite facilitated an increase in electron transfer, and/or minimized mass transport limitations as compared to Pt-black used alone. Glucose microbiosensors based on GrO(x)/Pt-black exhibited high sensitivity (465.9±48.0nA/mM), a low detection limit of 1μM, a linear response range of 1μM-2mM, and response time of ～4s. Additionally the sensor was stable and highly selective over potential interferents.     

Paired assessment of volatile anesthetic concentrations with synaptic actions recorded in vitro

The volatile anesthetic isoflurane poses a number of experimental challenges in the laboratory. Due to its rapid evaporation, the open conditions of most in vitro electrophysiological recording systems make the determination of actual isoflurane concentrations a challenge. Since the absolute anesthetic concentration in solution is directly related to efficacy, concentration measurements are important to allow comparisons between laboratory and clinical studies. In this study we quantify the sources of isoflurane loss during experimentation and describe a method for the measurement of isoflurane concentrations using gas chromatography and mass spectrometry simultaneous to in vitro electrophysiological measurements. Serial samples of perfused bath solution allowed correlation of isoflurane concentrations with ongoing biological effects. Saturated physiological solutions contained 13.4 +/- 0.2 mM isoflurane and were diluted to desired "nominal" concentrations for experiments. The perfusion system established stable isoflurane concentrations within the bath by 2 minutes. However, bath isoflurane concentrations varied substantially and unpredictably between experiments. The magnitudes of such discrepancies in isoflurane concentrations spanned clinically important levels. Our studies suggest that, despite countermeasures, solution handling significantly impacted the isoflurane content in the tissue bath. The magnitude of these discrepancies appears to necessitate systematic direct measurement of bath isoflurane concentrations during most in vitro conditions.     

Insulin growth factor signaling mediates neuron-like differentiation of adipose-tissue-derived stem cells

Abstract Our previous study showed that adipose tissue-derived stem cells (ADSC) could be induced by isobutylmethylxanthine (IBMX) to differentiate into neuron-like cells. In the present study, ADSC were treated with IBMX in the presence or in the absence of each of eight specific inhibitors of different signaling pathways (JAK/STAT, PKA, PI3K, MEK, Wnt/Frizzled, ERK/MAPK, TGF-β, and insulin growth factor [IGF]-I). PPP, a specific inhibitor of IGF-I signaling, was the only inhibitor that showed significant inhibition of IBMX-induced ADSC neuronal differentiation, as determined by changes in cell morphology in the initial screening. Further examination by immunofluorescence staining showed that the neuronal marker, β-III-tubulin, was highly induced in IBMX-treated ADSC, and the induction was significantly suppressed by PPP. Western blotting, followed by densitometry showed that PPP suppressed IBMX-induced β-III-tubulin expression by 43%, 88%, and 84% when used to treat the cells for 1, 3, and 24 hr, respectively. Treatment of ADSC with IBMX also led to the phosphorylation of IGF-I receptor at tyrosine 1136 (Y1136), as determined by immunofluorescence staining with an antibody that reacts specifically with Y1136. This effect was also abrogated by PPP. Thus, the IBMX-induced neuron-like differentiation of ADSC is mediated by IGF signaling through the phosphorylation of IGF-IR at Y1136.