A SAS-6-Like Protein Suggests that the Toxoplasma Conoid Complex Evolved from Flagellar Components

SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.     

Human erythrocyte flickering: temperature,ATP concentration,water transport,and cell aging,plus a computer simulation

Images of human erythrocytes from a healthy donor were recorded under differential interference contrast (DIC) microscopy; they were acquired rapidly (~336 Hz) and the intensity of the centermost pixel of each cell was recorded for ~60 s (20,000 values). Various techniques were used to analyze the data, including detrended fluctuation analysis (DFA) and multiscale entropy (MSE); however, power spectrum analysis was deemed the most appropriate for metrifying and comparing results. This analysis was used to compare cells from young and old populations, and after perturbing normal conditions, with changes in temperature, adenosine triphosphate (ATP) concentration (using NaF, an inhibitor of glycolysis, and α-toxin, a pore-forming molecule used to permeabilize red cells to ATP), and water transport rates [using glycerol, and p-chloromercuriphenylsulfonic acid (pCMBS) to inhibit aquaporins, AQPs]. There were measurable differences in the membrane fluctuation characteristics in populations of young and old cells, but there was no significant change in the flickering time series on changing the temperature of an individual cell, by depleting it of ATP, or by competing with the minor water exchange pathway via AQP3 using glycerol. However, pCMBS, which inhibits AQP1, the major water exchange pathway, inhibited flickering in all cells, and yet it was restored by the membrane intercalating species dibutyl phthalate (DBP). We developed a computer model to simulate acquired displacement spectral time courses and to evaluate various methods of data analysis, and showed how the flexibility of the membrane, as defined in the model, affects the flickering time course.     

Post-Operative Plasma Osteopontin Predicts Distant Metastasis in Human Colorectal Cancer

BackgroundThe overall prognosis of colorectal cancer (CRC) patients is unsatisfactory due to cancer metastasis after operation. This study aims to investigate the clinical significance of plasma osteopontin (OPN) levels as minimally invasive, predictive, and surrogate biomarkers for prognosis of CRC patients.MethodsThis randomized study design consists of pre-operative and post-operative plasma samples from a total of 79 patients. We determined plasma levels of OPN by ELISA and examined their correlation with the clinicopathological parameters of CRC patients. The effects of endogenous and exogenous OPN on CRC metastasis were investigated by examination of the effect on regulators of epithelial to messenchymal transition and migration assay.ResultsOur findings demonstrated for the first time the clinical correlation of plasma OPN with metastasis of CRC patients. High post-operative plasma OPN level (>153.02 ng/ml) associated with development of metastasis after curative resection (p<0.001). Moreover, post-operative plasma OPN level correlated with disease-free survival of CRC patients (p=0.009) and was an independent factor for predicting development of metastasis in CRC patients after curative resection (p=0.036). Our in vitro model showed that OPN ectopic expression induced DLD1 cell migration through Snail and Twist1 overexpression and E-cadherin repression, and secretory OPN level enhanced cell migration.ConclusionsThe results of the current study suggest that post-operative plasma OPN correlated with post-operative metastasis, suggesting that it is a potential non-invasive biomarker for the development of future metastasis in CRC patients. In addition, OPN was shown to be involved in the metastatic process and thus inhibition of OPN is a potential therapeutic approach to treat CRC patients.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)     

The nuclear localization signal and the C-terminal region of FHY1 are required for transmission of phytochrome A signals

Plants use the family of phytochrome photoreceptors to sense their light environment in the red/far-red region of the spectrum. Phytochrome A (phyA) is the primary photoreceptor that regulates germination and early seedling development. This phytochrome mediates seedling de-etiolation for the developmental transition from heterotrophic to photoauxotrophic growth. High intensity far-red light provides a way to specifically assess the role of phyA in this process and was used to isolate phyA-signaling intermediates. fhy1 and pat3 (renamed fhy1-3) are independently isolated alleles of a gene encoding a phyA signal transduction component. FHY1 is a small 24 kDa protein that shows no homology to known functional motifs, besides a small conserved septin-related domain at the C-terminus, a putative nuclear localization signal (NLS) and a putative nuclear exclusion signal (NES). Here we demonstrate that the septin-related domain is important for FHY1 to transmit phyA signals. Moreover, the putative NLS and NES of FHY1 are indeed involved in its nuclear localization and exclusion. Nuclear localization of FHY1 is needed for it to execute responses downstream of phyA. Together with the results from global expression analysis, our findings point to an important role of FHY1 in phyA signaling through its nuclear translocation and induction of gene expression.     

A transgenic plant cell‐suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles

We describe a novel strategy to produce vaccine antigens using a plant cell‐suspension culture system in lieu of the conventional bacterial or animal cell‐culture systems. We generated transgenic cell‐suspension cultures from Nicotiana benthamiana leaves carrying wild‐type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot‐and‐mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co‐expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large‐scale production of immunopeptide vaccines in a cost‐effective manner using a plant cell‐suspension culture system.     

Interactions of Opioid and Chemokine Receptors: Oligomerization of mu, kappa, and delta with CCR5 on Immune Cells

Activation of opioid receptors by morphine was previously shown to specifically induce the expression of chemokine receptor CCR5, promoting simian AIDS virus entry and replication in immune cells. The present study was undertaken to determine whether these two structurally and functionally distinct G-protein-coupled receptors are in close proximity and form an oligomeric complex in the cell membrane so that the activation of one triggers the activity of the other. Both human CEM ×174 and monkey lymphocytes were used in this study and gave similar results. Immunoprecipitation experiments showed that CCR5, but not CD4 nor Na⁺/H⁺ exchanger, coprecipitates with all three subtypes (mu, delta, and kappa) of opioid receptors. A single protein band immunoreactive with antibodies against both the CCR5 and the opioid receptors was identified after electrophoresis on nondenaturing polyacrylamide gels. Chemical crosslinking experiments using glutaraldehyde or BS³ indicate that these receptors are closely situated on the cell membrane with an intermolecular distance less than 11.4Å. Functional studies revealed that a combination treatment of cells with morphine, an agonist for mu, and MIP-1β, a ligand for CCR5, suppresses the inhibitory effect of MIP-1β and increases the stimulatory effect of morphine on CCR5 expression. These results suggest that oligomerization of chemokine receptor CCR5 and opioid receptors on the cell membrane of human or monkey lymphocytes may modulate receptor functions.