Role of GD3-CLIPR-59 Association in Lymphoblastoid T Cell Apoptosis Triggered by CD95/Fas

We previously found that a directional movement of the raft component GD3 towards mitochondria, by its association with microtubules, was mandatory to late apoptogenic events triggered by CD95/Fas. Since CLIPR-59, CLIP-170-related protein, has recently been identified as a microtubule binding protein associated with lipid rafts, we analyzed the role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas. To test whether CLIPR-59 could play a role at the raft-microtubule junction, we performed a series of experiments by using immunoelectron microscopy, static or flow cytometry and biochemical analyses. We first assessed the presence of CLIPR-59 molecule in lymphoblastoid T cells (CEM). Then, we demonstrated that GD3-microtubule interaction occurs via CLIPR-59 and takes place at early time points after CD95/Fas ligation, preceding the association GD3-tubulin. GD3-CLIPR-59 association was demonstrated by fluorescence resonance energy transfer (FRET) analysis. The key role of CLIPR-59 in this dynamic process was clarified by the observation that silencing CLIPR-59 by siRNA affected the kinetics of GD3-tubulin association, spreading of GD3 towards mitochondria and apoptosis execution. We find that CLIPR-59 may act as a typical chaperone, allowing a prompt interaction between tubulin and the raft component GD3 during cell apoptosis triggered by CD95/Fas. On the basis of the suggested role of lipid rafts in conveying pro-apoptotic signals these results disclose new perspectives in the understanding of the mechanisms by which raft-mediated pro-apoptotic signals can directionally reach their target, i.e. the mitochondria, and trigger apoptosis execution.     

Direct observation and modelling of embolism spread between xylem conduits: a case study in Scots pine

Xylem embolism is one of the main processes involved in drought‐related plant mortality. Although its consequences for plant physiology are already well described, embolism formation and spread are poorly evaluated and modelled, especially for tracheid‐based species. The aim of this study was to assess the embolism formation and spread in Pinus sylvestris as a case study using X‐ray microtomography and hydraulics methods. We also evaluated the potential effects of cavitation fatigue on vulnerability to embolism and the micro‐morphology of the bordered pits using scanning electron microscopy (SEM) to test for possible links between xylem anatomy and embolism spread. Finally, a novel model was developed to simulate the spread of embolism in a 2D anisotropic cellular structure. Results showed a large variability in the formation and spread of embolism within a ring despite no differences being observed in intertracheid pit membrane anatomical traits. Simulations from the model showed a highly anisotropic tracheid‐to‐tracheid embolism spreading pattern, which confirms the major role of tracheid‐to‐tracheid air seeding to explain how embolism spreads in Scots pine. The results also showed that prior embolism removal from the samples reduced the resistance to embolism of the xylem and could result in overestimates of vulnerability to embolism.     

The role of miRNA-221 and miRNA-126 in patients with benign metastasizing leiomyoma of the lung: an overview with new interesting scenarios

Molecular Biology Reports - Benign metastasizing leiomyoma (BML) is a rare disease characterized by extrauterine benign leiomyomatosis in patients with a previous or concomitant history of uterine...     

Solution structure of ApaG from Xanthomonas axonopodis pv. citri reveals a fibronectin-3 fold

ApaG proteins are found in a wide variety of bacterial genomes but their function is as yet unknown. Some eukaryotic proteins involved in protein-protein interactions, such as the human polymerase delta-interacting protein (PDIP38) and the F Box A (FBA) proteins, contain ApaG homology domains. We have used NMR to determine the solution structure of ApaG protein from the plant pathogen Xanthomonas axonopodis pv. citri (ApaG(Xac)) with the aim to shed some light on its molecular function. ApaG(Xac) is characterized by seven antiparallel beta strands forming two beta sheets, one containing three strands (ABE) and the other four strands (GFCC'). Relaxation measurements indicate that the protein has a quite rigid structure. In spite of the presence of a putative GXGXXG pyrophosphate binding motif ApaG(Xac) does not bind ATP or GTP, in vitro. On the other hand, ApaG(Xac) adopts a fibronectin type III (Fn3) fold, which is consistent with the hypothesis that it is involved in mediating protein-protein interactions. The fact that the proteins of ApaG family do not display significant sequence similarity with the Fn3 domains found in other eukaryotic or bacterial proteins suggests that Fn3 domain may have arisen earlier in evolution than previously estimated.     

Expression Study on D123 Gene Product: Evidence for High Positivity in Testis

A novel 44-kDa gene product (D123) has been proposed as necessary for S-phase entry of the cell cycle: a point mutation resulted in a temperature-sensitive arrest in G1-phase. From human fibrosarcoma cDNA library, we have isolated an identical gene and studied its sequence and mRNA and protein expression. Compared withD123,three nucleotide differences within the human coding sequence, plus others, result in a change of two amino acids. A partial sequence similarity has been found with a yeast gene of unknown function. The protein has several potential phosphorylation sites, is highly hydrophilic, and may be highly structured in α-helix. The mRNA is abundantly expressed by a variety of normal and transformed cells and by all tissues examined, being most highly expressed in testis. Specific antibodies, raised against a rhD123 polypeptide, recognize a major 42- to 44-kDa molecule in crude extract of various human cell lines. Immunohistochemistry reveals that D123 protein is not homogeneously expressed: it is detected, often in granular vescicles, in the cytoplasm of some epithelial, stromal, and sperm cells and in varicosities lining nervous fibers, while it appears to be absent in nuclei, endothelial, and smooth muscle cells. The precise link between cytoplasmic occurrence of D123 and cell cycle progression still remains to be clarified.     

Glucose-induced alterations of intracellular ionized magnesium in human lymphocytes

The intracellular ionic content of human erythrocytes may be altered by hyperglycaemia. Despite this, very little is known about the cellular mechanisms linking glucose and cellular magnesium homeostasis. We measured intracellular ionized magnesium in human lymphocytes, by means of a fluorimetric technique, total intracellular magnesium by means of atomic absorption spectrophotometry and intracellular ATP by means of HPLC. The incubation of lymphocytes with D-glucose in the absence of insulin was followed by a significant decrease in intracellular ionized magnesium; this effect did not occur when the cells were incubated with L-glucose. The effect of glucose on intracellular ionized magnesium was blocked by amphotericin B and the EC(50) of the effect of glucose on intracellular ionized magnesium was about 5 mmol/l of glucose. The increase of intracellular ionized magnesium in cells incubated in the absence of glucose was followed by a decrease in intracellular ATP. In a Na(+)-free medium the decrease of intracellular ionized magnesium in the presence of glucose was still present and the incubation of lymphocytes with glucose did not modify total intralymphocyte magnesium. By selective permeabilization of cell membranes, we established that glucose could not increase compartmentalized intracellular ionized magnesium. Our data supports the hypothesis that glucose per se induces a substantial decrease in intracellular ionized magnesium, which is probably due to an augmented binding of intracellular ionized magnesium to cellular ATP.     

Microvesicles as promising biological tools for diagnosis and therapy

ABSTRACT

Introduction: Shed by most cells, in response to a myriad of stimuli, extracellular vesicles (EVs) carry proteins, lipids, and various nucleic acids. EVs encompass diverse subpopulations differing for biogenesis and content. Among these, microvesicles (MVs) derived from plasma membrane, are key regulators of physiopathological cellular processes including cancer, inflammation and infection. This review is unique in that it focuses specifically on the MVs as a mediator of information transfer. In fact, few proteomic studies have rigorously distinguished MVs from exosomes.

Areas covered: Aim of this review is to discuss the proteomic analyses of the MVs. Many studies have examined mixed populations containing both exosomes and MVs. We discuss MVs’ role in cell-specific interactions. We also show their emerging roles in therapy and diagnosis.

Expert commentary: We see MVs as therapeutic tools for potential use in precision medicine. They may also have potential for allowing the identification of new biomarkers. MVs represent an invaluable tool for studying the cell of origin, which they closely represent, but it is critical to build a repository with data from MVs to deepen our understanding of their molecular repertoire and biological functions.