Cholera toxin structure, gene regulation and pathophysiological and immunological aspects

Many notions regarding the function, structure and regulation of cholera toxin expression have remained essentially unaltered in the last 15 years. At the same time, recent findings have generated additional perspectives. For example, the cholera toxin genes are now known to be carried by a non-lytic bacteriophage, a previously unsuspected condition. Understanding of how the expression of cholera toxin genes is controlled by the bacterium at the molecular level has advanced significantly and relationships with cell-density-associated (quorum-sensing) responses have recently been discovered. Regarding the cell intoxication process, the mode of entry and intracellular transport of cholera toxin are becoming clearer. In the immunological field, the strong oral immunogenicity of the non-toxic B subunit of cholera toxin (CTB) has been exploited in the development of a now widely licensed oral cholera vaccine. Additionally, CTB has been shown to induce tolerance against co-administered (linked) foreign antigens in some autoimmune and allergic diseases. Received 25 October 2007; accepted 12 December 2007     

Bile Acids: Chemistry, Pathochemistry, Biology, Pathobiology, and Therapeutics

Bile acids and bile alcohols in the form of their conjugates are amphipathic end products of cholesterol metabolism with multiple physiological functions. The great variety of bile acids and bile alcohols that are present in vertebrates are tabulated. Bile salts have an enterohepatic circulation resulting from efficient vectorial transport of bile salts through the hepatocyte and the ileal enterocyte; such transport leads to the accumulation of a pool of bile salts that cycles between the liver and intestine. Bile salt anions promote lipid absorption, enhance tryptic cleavage of dietary proteins, and have antimicrobial effects. Bile salts are signaling molecules, activating nuclear receptors in the hepatocyte and ileal enterocyte, as well as an increasing number of G-protein coupled receptors. Bile acids are used therapeutically to correct deficiency states, to decrease the cholesterol saturation of bile, or to decrease the cytotoxicity of retained bile acids in cholestatic liver disease.     

Molecular and Cellular Basis of Regeneration and Tissue Repair

Planarians possess amazing abilities to regulate tissue homeostasis and regenerate missing body parts. These features reside on the presence of a population of pluripotent/totipotent stem cells, the neoblasts, which are considered as the only planarian cells able to proliferate in the asexual strains. Neoblast distribution has been identified by mapping the cells incorporating bromodeoxyuridine, analyzing mitotic figures and using cell proliferation markers. Recently identified molecular markers specifically label subgroups of neoblasts, revealing thus the heterogeneity of the planarian stem cell population. Therefore, the apparent totipotency of neoblasts probably reflects the composite activities of multiple stem cell types. First steps have been undertaken to understand how neoblasts and differentiated cells communicate with each other to adapt the self-renewal and differentiation rates of neoblasts to the demands of the body. Moreover, the introduction of molecular resource database on planarians now paves the way to renewed strategies to understand planarian regeneration and stem cell-related issues.     

The utility F-box for protein destruction

A signature feature of all living organisms is their utilization of proteins to construct molecular machineries that undertake the complex network of cellular activities. The abundance of a protein element is temporally and spatially regulated in two opposing aspects: de novo synthesis to manufacture the required amount of the protein, and destruction of the protein when it is in excess or no longer needed. One major route of protein destruction is coordinated by a set of conserved molecules, the F-box proteins, which promote ubiquitination in the ubiquitin-proteasome pathway. Here we discuss the functions of F-box proteins in several cellular scenarios including cell cycle progression, synapse formation, plant hormone responses, and the circadian clock. We particularly emphasize the mechanisms whereby F-box proteins recruit specific substrates and regulate their abundance in the context of SCF E3 ligases. For some exceptions, we also review how F-box proteins function through non-SCF mechanisms.     

The expression profile of TLR9 mRNA and CpG ODNs immunostimulatory actions in the teleost gilthead seabream points to a major role of lymphocytes

The potential effects of synthetic unmethylated oligodeoxynucleotides (ODN) containing CpG motifs, mimicking bacterial DNA, has never been evaluated on the immune response in the teleost fish gilthead seabream (Sparus aurata), the most important fish species in Mediterranean aquaculture. First, binding and competition studies have demonstrated that binding is saturated and promiscuous, suggesting the participation of several receptors. Moreover, leucocyte cytotoxic (NCC) activity, production of ROIs (reactive oxygen intermediates), and expression of immune-relevant genes was greatly primed by ODNs. Focusing on the mechanism, the TLR9 gene is widely distributed in seabream tissues and differently regulated in vitro by several stimuli. Moreover, and for the first time in fish, TLR9 mRNA has been detected in lymphocytes as the main cell-source. To conclude, ODNs containing GACGTT, GTCGTT (optimal for mouse and human, respectively) or AACGTT motifs are the most potent inducers of seabream immunity, whilst the involvement of TLR9 is under debate.     

Olfactory ensheathing cells are attracted to, and can endocytose, bacteria

Olfactory ensheathing cells (OECs) have been shown previously to express Toll-like receptors and to respond to bacteria by translocating nuclear factor-kappaB from the cytoplasm to the nucleus. In this study, we show that OECs extended significantly more pseudopodia when they were exposed to Escherichia coli than in the absence of bacteria (p=0.019). Co-immunoprecipitation showed that E. coli binding to OECs was mediated by Toll-like receptor 4. Lyso-Tracker, a fluorescent probe that accumulates selectively in lysosomes, and staining for type 1 lysosome-associated membrane proteins demonstrated that endocytosed FITC-conjugated E. coli were translocated to lysosomes. They appeared to be subsequently broken down, as shown by transmission electron microscopy. No obvious adherence to the membrane and less phagocytosis was observed when OECs were incubated with inert fluorescent microspheres. The ability of OECs to endocytose bacteria supports the notion that OECs play an innate immune function by protecting olfactory tissues from bacterial infection.     

Regulation of phagocyte migration and recruitment by Src-family kinases

Src-family kinases (SFKs) regulate different granulocyte and monocyte/macrophage responses. Accumulating evidence suggests that members of this family are implicated in signal transduction pathways regulating phagocytic cell migration and recruitment into inflammatory sites. Macrophages with a genetic deficiency of SFKs display marked alterations in cytoskeleton dynamics, polarization and migration. This same phenotype is found in cells with either a lack of SFK substrates and/or interacting proteins such as Pyk2/FAK, c-Cbl and p190RhoGAP. Notably, SFKs and their downstream targets also regulate monocyte recruitment into inflammatory sites. Depending on the type of assay used, neutrophil migration in vitro may be either dependent on or independent of SFKs. Also neutrophil recruitment in in vivo models of inflammation may be regulated differently by SFKs depending on the tissue involved. In this review we will discuss possible mechanisms by which SFKs may regulate phagocytic cell migratory abilities.     

ERK activation by Ca2+ ionophores depends on Ca2+ entry in lymphocytes but not in platelets, and does not conduct membrane scrambling

Rapid Ca²⁺-dependent phospholipid (PL) reorganization (scrambling) at the plasma membrane is a mechanism common to hematopoietic cells exposing procoagulant phosphatidylserine (PS). The aim of this research was to determine whether activation of the extracellular signal-regulated kinase (ERK) pathway was required for PL scrambling, based on a single report analyzing both responses induced by Ca²⁺ ionophores in megakaryoblastic HEL cells. Ca²⁺ ionophore-stimulated ERK phosphorylation was induced in platelets without external Ca²⁺, whereas exogenous Ca²⁺ entry was crucial for ERK activation in Jurkat T cells. In both cells, membrane scrambling only occurred following Ca²⁺ entry and was not blocked by inhibiting ERK phosphorylation. Furthermore, ERK proteins are strongly phosphorylated in transformed B lymphoblastic cell lines, which do not expose PS in their resting state. Overall, the data demonstrated that ERK activation and membrane scrambling are independent mechanisms. A. Arachiche, I. Badirou: These authors contributed equally to this work. Received 18 June 2008; received after revision 24 September 2008; accepted 1 October 2008