Dynein is required for polarized dendritic transport and uniform microtubule orientation in axons

Axons and dendrites differ in both microtubule organization and in the organelles and proteins they contain. Here we show that the microtubule motor dynein has a crucial role in polarized transport and in controlling the orientation of axonal microtubules in Drosophila melanogaster dendritic arborization (da) neurons. Changes in organelle distribution within the dendritic arbors of dynein mutant neurons correlate with a proximal shift in dendritic branch position. Dynein is also necessary for the dendrite-specific localization of Golgi outposts and the ion channel Pickpocket. Axonal microtubules are normally oriented uniformly plus-end-distal; however, without dynein, axons contain both plus- and minus-end distal microtubules. These data suggest that dynein is required for the distinguishing properties of the axon and dendrites: without dynein, dendritic organelles and proteins enter the axon and the axonal microtubules are no longer uniform in polarity.     

Contribution of Copper Ion Resistance to Survival of Escherichia coli on Metallic Copper Surfaces

Bacterial contamination of touch surfaces poses a serious threat for public health. The use of bactericidal surface materials, such as copper and its alloys, might constitute a way to aid the use of antibiotics and disinfectants, thus minimizing the risk of emergence and spread of multiresistant germs. The survival of Escherichia coli on metallic copper surfaces has been studied previously; however, the mechanisms underlying bacterial inactivation on copper surfaces have not been elucidated. Data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems were slightly more sensitive than was the wild type. Preadaptation to copper enhanced survival rates upon copper surface exposure. This study constitutes a first step toward understanding the reasons for metallic copper surface-mediated killing of bacteria.     

TRPV1 antagonist with high analgesic efficacy: 2-Thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides

A series of 2-thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Among them, compound 24S showed stereospecific and excellent TRPV1 antagonism of capsaicin-induced activation. Further, it demonstrated strong anti-allodynic in a rat neuropathic pain model. Consistent with its action in vitro being through TRPV1, compound 24S blocked capsaicin-induced hypothermia in mice. Docking analysis of 24S with our hTRPV1 homology model was performed to identify its binding mode.     

Interactions of Archaeal Chromatin Proteins Alba1 and Alba2 with Nucleic Acids

Background

Architectural proteins have important roles in compacting and organising chromosomal DNA. There are two potential histone counterpart peptide sequences (Alba1 and Alba2) in the Aeropyrum pernix genome (APE1832.1 and APE1823).

Methodology/Principal Findings

These two peptides were expressed and their interactions with various DNAs were studied using a combination of various experimental techniques: surface plasmon resonance, UV spectrophotometry, circular dichroism–spectropolarimetry, gel-shift assays, and isothermal titration calorimetry.

Conclusions/Significance

Our data indicate that there are significant differences in the properties of the Alba1 and Alba2 proteins. Both of these Alba proteins can thermally stabilise DNA polynucleotides, as seen from UV melting curves. Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT). Surface plasmon resonance sensorgrams for binding of Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 to DNA oligonucleotides show different binding patterns. Circular dichroism indicates that Alba2 has a less-ordered secondary structure than Alba1. The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures. Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs.     

Multiple Delivery of siRNA against Endoglin into Murine Mammary Adenocarcinoma Prevents Angiogenesis and Delays Tumor Growth

Endoglin is a transforming growth factor-β (TGF- β) co-receptor that participates in the activation of a signaling pathway that mediates endothelial cell proliferation and migration in angiogenic tumor vasculature. Therefore, silencing of endoglin expression is an attractive approach for antiangiogenic therapy of tumors. The aim of our study was to evaluate the therapeutic potential of small interfering RNA (siRNA) molecules against endoglin in vitro and in vivo. Therapeutic potential in vitro was assessed in human and murine endothelial cells (HMEC-1, 2H11) by determining endoglin expression level, cell proliferation and tube formation. In vivo, the therapeutic potential of siRNA molecules was evaluated in TS/A mammary adenocarcinoma growing in BALB/c mice. Results of our study showed that siRNA molecules against endoglin have a good antiangiogenic therapeutic potential in vitro, as expression of endoglin mRNA and protein levels in mouse and human microvascular endothelial cells after lipofection were efficiently reduced, which resulted in the inhibition of endothelial cell proliferation and tube formation. In vivo, silencing of endoglin with triple electrotransfer of siRNA molecules into TS/A mammary adenocarcinoma also significantly reduced the mRNA levels, number of tumor blood vessels and the growth of tumors. The obtained results demonstrate that silencing of endoglin is a promising antiangiogenic therapy of tumors that could not be used as single treatment, but as an adjunct to the established cytotoxic treatment approaches.     

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.     

Integrin-dependent Control of Translation: Engagement of Integrin αIIbβ3 Regulates Synthesis of Proteins in Activated Human Platelets

It has been proposed that the plasma membrane of many cell types contains cholesterol-sphingolipid-rich microdomains. Here, we analyze the role of these microdomains in promoting oligomerization of the bacterial pore-forming toxin aerolysin. Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore. We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution. Our observations indicate that it is not merely the number of receptors on the target cell that is important for toxin sensitivity, but their ability to associate transiently with detergent resistant microdomains. Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.     

Diagnostic utility of sialosyl-Tn in discriminating carcinomatous cells from benign mesothelium in body cavity effusions

OBJECTIVE: Detecting malignant cells in the setting of reactive mesothelium can be difficult. Several techniques have been tried but without widespread acceptance. Sialosyl-Tn (STn) is an aberrantly glycosylated precursor of the MN blood group antigen frequently expressed in carcinomas and dysplastic epithelium. We investigated the STn monoclonal antibody for its clinical utility as an isolated stain to discriminate benign mesothelium from malignant cells. STUDY DESIGN: Cell block material from 72 cases of body cavity fluids were immunostained for STn using the avidin-biotin complex method without antigen retrieval. Slides were incubated overnight at 4 degrees C in a humidified chamber. RESULTS: Strong immunoreactivity was noted in 31/40 (77%) carcinomatous cases. Only moderate staining was noted in 1 of 28 (4%) benign effusions and weak staining in 5 (18%) additional benign cases. Specificity was 100%, sensitivity 78%, positive predictive value 100% and negative predictive value 76%. No staining was noted in four noncarcinomatous malignant effusions. CONCLUSION: STn may have diagnostic value in this cytologic setting as part of a diagnostic panel but not as an isolated stain.     

Stopped-flow dynamic light scattering as a method to monitor compaction during protein folding

Kinetic dynamic light scattering is a useful tool to follow compaction during protein folding. In contrast to measurements of the formation of secondary structure and side chain ordering, kinetic measurements of compactness are not well established up to now. This work describes the adaptation of a stopped-flow system (SFM-3) to a dynamic light scattering apparatus, which allows one to monitor the compaction of protein molecules by measuring the hydrodynamic Stokes radius R. The feasibility of such investigations was demonstrated by measuring R and the integrated scattered intensity I during refolding of ribonuclease A and phosphoglycerate kinase from yeast. Refolding was initiated by rapid mixing of protein solutions containing high concentrations of guanidine hydrochloride with buffer. Between 20 and 50 mixing events were performed in these experiments. Measuring both R and I in one and the same experiment is important to distinguish between true folding of individual molecules and cases where folding is accompanied by the appearance of transient oligomers or associated misfolded structures. On refolding of ribonuclease A (0.6 M GuHCl, 25 °C), after a fast phase the Stokes radius decreased from 2.26 nm to 1.95 nm with a time constant of 27 s without detectable aggregates. By contrast, transient and stable oligomers have been observed during the more complex folding of phosphoglycerate kinase. In general, the time-resolution of the method is of the order of 1 s. It can be extended to the subsecond time-range if the number of shots is not limited by the amount of protein available. Received: 8 August 1996 / Accepted: 18 October 1996