Co-localization of galectin-1 with GM1 ganglioside in the course of its clathrin- and raft-dependent endocytosis

Mammalian galectin-1 (Gal-1), a beta-galactoside-binding lectin has a prominent role in regulating cell adhesion, cell growth and immune responses. Downregulation of these biological functions may occur via internalization of Gal-1. In the present study we have investigated the mechanism and possible mediator(s) of Gal-1 endocytosis. We show that internalization occurs at a temperature higher than 22 degrees C in an energy dependent fashion. After one hour incubation Gal-1 localizes in the Golgi system within the cells, and then disappears without accumulation in degradation compartments, such as lysosomes. Based on their strong intracellular co-localization, two glycoconjugates, GM1 ganglioside and CD7 are implicated in the sorting of internalized Gal-1 into Golgi. Other known Gal-1 binding glycoproteins on T cells (CD2, CD3, CD43 and CD45) do not cointernalize with the lectin. Internalization of Gal-1 depends on its lectin activity and follows dual pathways involving clathrin-coated vesicles and raft-dependent endocytosis.     

Purinergic regulation of neutrophil chemotaxis

Chemotaxis allows polymorphonuclear neutrophils (PMN) to rapidly reach infected and inflamed sites. However, excessive influx of PMN damages host tissues. Better knowledge of the mechanisms that control PMN chemotaxis may lead to improved treatments of inflammatory diseases. Recent findings suggest that ATP and adenosine are involved in PMN chemotaxis. Therefore, these purinergic signaling processes may be suitable targets for novel therapeutic approaches to ameliorate host tissue damage.     

Mid-anaphase arrest in S. cerevisiae cells eliminated for the function of Cin8 and dynein

S. cerevisiae anaphase spindle elongation is accomplished by the overlapping function of dynein and the kinesin-5 motor proteins, Cin8 and Kip1. Cin8 and dynein are synthetically lethal, yet the arrest phenotypes of cells eliminated for their function had not been identified. We found that at a non-permissive temperature, dyn1Δ cells that carry a temperature-sensitive cin8 – 3 mutation arrest at mid-anaphase with a unique phenotype, which we named TAN (two microtubule asters in one nucleus). These cells enter anaphase, but fail to proceed through the slow phase of anaphase B. At a permissive temperature, dyn1Δ, cin8 – 3 or dyn1Δcin8 – 3 cells exhibit perturbed spindle midzone morphologies, with dyn1Δcin8 – 3 anaphase spindles also being profoundly bent and nonrigid. Sorbitol, which has been suggested to stabilize microtubules, corrects these defects and suppresses the TAN phenotype. We conclude that dynein and Cin8 cooperate in anaphase midzone organization and influence microtubule dynamics, thus enabling progression through the slow phase of anaphase B. Received 10 August 2008; received after revision 22 October 2008; accepted 27 October 2008     

Entry of herpesviruses into mammalian cells

The mechanism that herpesviruses use to enter cells is one of the most complex viral entry mechanisms studied so far. This complexity seems to mount as new participants, both cellular receptors and viral glycoproteins, are identified. Recent structural work on entry glycoproteins gD and gB from herpes simplex virus (HSV) 1 has illuminated the functional roles of these glycoproteins in the process of entry. In doing so, it provided information on the mechanism of two critical steps of HSV entry: receptor-mediated activation and membrane fusion. Remarkably, it is becoming clear that herpesviruses have a lot in common with other, simpler viruses.     

HCN channels: Structure, cellular regulation and physiological function

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated pore loop channels. HCN channels are unique among vertebrate voltage-gated ion channels, in that they have a reverse voltage-dependence that leads to activation upon hyperpolarization. In addition, voltage-dependent opening of these channels is directly regulated by the binding of cAMP. HCN channels are encoded by four genes (HCN1–4) and are widely expressed throughout the heart and the central nervous system. The current flowing through HCN channels, designated I_h or I_f, plays a key role in the control of cardiac and neuronal rhythmicity (“pacemaker current”). In addition, I_h contributes to several other neuronal processes, including determination of resting membrane potential, dendritic integration and synaptic transmission. In this review we give an overview on structure, function and regulation of HCN channels. Particular emphasis will be laid on the complex roles of these channels for neuronal function and cardiac rhythmicity. Received 22 August 2008; received after revision 22 September 2008; accepted 24 September 2008     

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.     

Calmodulin-associated post-translational regulation of rat organic cation transporter 2 in the kidney is gender dependent

In this work, regulation of organic cation transporter type 2 from rat (rOCT2) stably transfected in HEK293 cells was investigated by microfluorimetry with 4-(4-(dimethylamino)styryl)-N-methylpyridinium as substrate. The transport mediated by rOCT2 was specifically stimulated by PKA, phosphatidylinositol-3-kinase, p56^lck tyrosine kinase, mitogen-extracellular-signal-regulated-kinase-1/2, calmodulin (CaM), and CaM-kinase-II. The regulatory pattern of rOCT2 differs markedly quantitatively and qualitatively from that of other OCT isoforms. Only CaM-dependent upregulation is conserved throughout the OCT family. For this reason, CaM regulation of rOCT2 was also investigated in isolated S3-segments (known to express only rOCT2) of male and female rat proximal tubules. Inhibition of CaM by calmidazolium significantly decreased rOCT2 activity (−49.0 ± 13.6%, n = 4) in male but not female (9.0 ± 13.0%, n = 4) rats. Real-time PCR and Western blot investigations of CaM expression in rat kidneys showed that male animals have significantly higher CaM expression. This is the first study describing post-translational gender-dependent rOCT2 regulation. Received 26 February 2009; accepted 16 March 2009     

Myosin I: From yeast to human

Myosin I is a non-filamentous, single-headed, actin-binding motor protein and is present in a wide range of species from yeast to man. The role of these class I myosins have been studied extensively in simple eukaryotes, showing their role in diverse processes such as actin cytoskeleton organization, cell motility, and endocytosis. Recently, studies in metazoans have begun to reveal more specialized functions of myosin I. It will be a major challenge in the future to examine the physiological functions of each class I myosin in different cell types of metazoans.     

The Role of the Agouti-Related Protein in Energy Balance Regulation

The Agouti-Related Protein (AgRP) is a powerful orexigenic peptide that increases food intake when ubiquitously overexpressed or when administered centrally. AgRP-deficiency, on the other hand, leads to increased metabolic rate and a longer lifespan when mice consume a high fat diet. In humans, AgRP polymorphisms have been consistently associated with resistance to fatness in Blacks and Whites and resistance to the development of type-2 diabetes in African Blacks. Systemically administered AgRP accumulates in the liver, the adrenal gland and fat tissue while recent findings suggest that AgRP may also have inverse agonist effects, both centrally and peripherally. AgRP could thus modulate energy balance via different actions. Its absence or reduced functionality may offer a benefit both in terms of bringing about negative energy balance in obesigenic environments, as well as leading to an increased lifespan.     

A novel member of the Rhomboid family, RHBDD1, regulates BIK-mediated apoptosis

Rhomboid family members are widely conserved and found in all three kingdoms of life. They are serine proteases and serve important regulatory functions. In the present study, a novel gene highly expressed in the testis, RHBDD1, is shown to be a new member of the Rhomboid family, participating in the cleavage of BIK, a proapoptotic member of the Bcl-2 family. The RHBDD1-involved proteolytic modification is upstream of the BIK protein degradation pathway. Mutagenesis studies show that the amino acid residues glycine142 and serine144 of RHBDD1 are crucial for its activity in cleaving BIK at a site located in the transmembrane region. Overexpression or knock-down of RHBDD1 in HEK 293T cells can reduce or enhance BIK-mediated apoptosis, respectively. The present findings suggest that, by acting as a serine protease, RHBDD1 modulates BIK-mediated apoptotic activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 31 July 2008; received after revision 16 September 2008; accepted 19 September 2008     

Interaction of galectin-1 with caveolae induces mouse embryonic stem cell proliferation through the Src, ERas, Akt and mTOR signaling pathways

Galectins have the potential to provide a promising alternative for unveiling the complexity of embryonic stem (ES) cell self-renewal, although the mechanism by which galectins maintain ES cell self-renewal has yet to be identified. Galectin-1 increased [³H]-thymidine incorporation as well as cyclin expression and decreased p27^kip1 expression. Src and caveolin-1 phosphorylation was increased by galectin-1, and phospho-caveolin-1 was inhibited by PP2. In addition, inhibition of caveolin-1 by small interfering RNA and methyl-β-cyclodextrin (Mβ-CD) decreased galectin-1-induced cyclin expression and [³H]-thymidine incorporation. Galectin-1 caused Akt and mTOR phosphorylation, which is involved in cyclin expression. Galectin-1-induced phospho-Akt and -mTOR was inhibited by PP2, ERas siRNA, caveolin-1 siRNA and Mβ-CD. Furthermore, mTOR phosphorylation was decreased by LY294002 and Akt inhibitor. Galectin-1-induced increase in cyclin expression and decrease in p27^kip1 was blocked by Akt inhibitor and rapamycin. In conclusion, galectin-1 increased DNA synthesis in mouse ES cells via Src, caveolin-1 Akt, and mTOR signaling pathways. Received 30 October 2008; received after revision 18 February 2009; accepted 24 February 2009     

Vaults and the major vault protein: Novel roles in signal pathway regulation and immunity

The unique and evolutionary highly conserved major vault protein (MVP) is the main component of ubiquitous, large cellular ribonucleoparticles termed vaults. The 100 kDa MVP represents more than 70% of the vault mass which contains two additional proteins, the vault poly (ADP-ribose) polymerase (vPARP) and the telomerase-associated protein 1 (TEP1), as well as several short untranslated RNAs (vRNA). Vaults are almost ubiquitously expressed and, besides chemotherapy resistance, have been implicated in the regulation of several cellular processes including transport mechanisms, signal transmissions and immune responses. Despite a growing amount of data from diverse species and systems, the definition of precise vault functions is still highly complex and challenging. Here we review the current knowledge on MVP and vaults with focus on regulatory functions in intracellular signal transduction and immune defence. Received 27 June 2008; received after revision 25 July 2008; accepted 30 July 2008     

The antioxidant function of Bcl-2 preserves cytoskeletal stability of cells with defective respiratory complex I

Human thyroid carcinoma XTC.UC1 cells harbor a homoplasmic frameshift mutation in the MT-ND1 subunit of respiratory complex I. When forced to use exclusively oxidative phosphorylation for energy production by inhibiting glycolysis, these cells triggered a caspase-independent cell death pathway, which was associated to a significant imbalance in glutathione homeostasis and a cleavage of the actin cytoskeleton. Overexpression of the anti-apoptotic Bcl-2 protein significantly increased the level of endogenous reduced glutathione, thus preventing its oxidation after the metabolic stress. Furthermore, Bcl-2 completely inhibited actin cleavage and increased cell adhesion, but was unable to improve cellular viability. Similar effects were obtained when XTC.UC1 cells were incubated with exogenous glutathione. We hence propose that Bcl-2 can safeguard cytoskeletal stability through an antioxidant function. Received 28 May 2008; received after revision 8 July 2008; accepted 29 July 2008     

Regulation of chondrocyte differentiation by ADAMTS-12 metalloproteinase depends on its enzymatic activity

ADAMTS-12, a metalloproteinase that belongs to ADAMTS family, is strongly upregulated during chondrogenesis and demonstrates prominent expression in the growth plate chondrocytes. ADAMTS-12 potently inhibits chondrocyte differentiation, as revealed by altered expression of both early and later genes critical for chondrogenesis. In addition, ADAMTS-12-mediated inhibition of chondrogenesis depends on its enzymatic activity, since its point mutant lacking enzymatic activity completely loses this activity. Furthermore, the C-terminal four thrombospondin motifs known to bind COMP substrate is necessary for its full proteolytic activity and inhibition of chondrocyte differentiation. Mechanism studies demonstrate that ADAMTS-12 induces PTHrP, whereas it inhibits IHH during chondrogenesis. Furthermore, PTHrP induces ADAMTS-12 and ADAMTS-12 is hardly detectable in PTHrP-/-growth plate chondrocytes. Importantly, knocking down ADAMTS-12 mRNA levels or blocking ADAMTS-12 activity almost abolishes the PTHrP-mediated inhibition of type X collagen expression. Collectively, these findings demonstrate that ADAMTS-12, a downstream molecule of PTHrP signaling, is a novel regulator of chondrogenesis. X. H. Bai, D.W. Wang: These two authors contributed equally to this work.     

Effects of ulapualide A and synthetic macrolide analogues on actin dynamics and gene regulation

Several marine macrolide toxins act as potent and specific actin-severing molecules. Recent elucidation of their stereochemistries and modes of interaction with actin has allowed the syntheses of bioactive analogues. Here we used synthetic analogues in a structure-function analysis of ulapualide A, a trisoxazole-based macrolide. Ulapualide A harboured potent actin-depolymerising activity both in cells and in vitro. Its synthetic diastereoisomer was three orders of magnitude less active than the natural toxin and synthetic macrolide fragments lacked actin-capping/ severing activity altogether. Modulation of serum response factor (SRF)-dependent gene expression, as described for other actin-binding toxins, was also examined. Specific changes in response to ulapualide A were not observed, primarily due to its profound effects on cytoskeletal integrity and cell adhesion. Several synthetic fragments of ulapualide A also had no effect on SRF-dependent gene expression. However, inhibition was observed with a molecule corresponding to the extended aliphatic side chain of halichondramide, a structurally related macrolide. These findings indicate that side-chain derivatives of trisoxazole-based macrolides may serve to uncouple gene-regulatory events from actin dynamics. E. Vincent and J. Saxton: These two authors contributed equally Received 27 September 2006; received after revision 30 November 2006; accepted 8 January 2007