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     

Biochemical effects and growth inhibition in MCF-7 cells caused by novel sulphonamido oxa-polyamine derivatives

The novel polyamine derivatives sulphonamido oxa-spermine (oxa-Spm) and sulphonamido oxa-spermidine (oxa-Spd) exhibited rapid cytotoxic action towards MCF-7 human breast cancer cells with IC₅₀ values of 4.35 and 6.47 μM, respectively, after 24-h drug exposure. Neither compound is a substrate of serum amine oxidase. Both oxa-Spm and oxa-Spd caused cell shrinkage, as determined by phase-contrast microscopy. After incubation with 10 μM of either compound for 8 h, the cells underwent chromatin condensation and nuclear fragmentation. However, no clear DNA ladder was obtained by electrophoresis. The sulphonamido oxa-polyamine derivatives and especially oxa-Spd enhanced the activity of polyamine oxidase (PAO), an enzyme capable of oxidising N¹-acetylated spermine and spermidine to spermidine and putrescine, respectively, generating cytotoxic H₂O₂ and 3-acetamidopropanal as by-products. The intracellular polyamine content was only marginally reduced in response to drug treatment. In conclusion, our data show that these novel sulphonamido oxa-polyamine derivatives possess high cytotoxic activity against MCF-7 cells and indicate that induction of PAO may mediate their cytotoxicity via apoptosis. Received 17 January 2002; received after revision 22 February 2002; accepted 22 February 2002     

Akt-mediated GSK-3β inhibition prevents migration of polyamine-depleted intestinal epithelial cells via Rac1

The rapid migration of intestinal epithelial cells (IEC) is important for the healing of mucosal wounds. We have previously shown that polyamine depletion inhibits migration of IEC-6 cells. Akt activation and its downstream target GSK-3β have been implicated in the regulation of migration. Here we investigated the significance of elevated phosphatidylinositol 3-kinase (PI3K)/Akt signaling on migration of polyamine-depleted cells. Polyamine-depleted cells had high Akt (Ser473) and GSK-3β (Ser9) phosphorylation. Pretreatment with 20 μM LY294002 (PI3K inhibitor) for 30 min inhibited phosphorylation of Akt, increased migration by activating Rac1 in polyamine-depleted IEC-6 cells, and restored the actin structure similar to that in cells grown in control medium. Treatment of cells with a GSK-3β inhibitor (AR-A014418) altered the actin cytoskeleton and inhibited migration, mimicking the effects of polyamine depletion. Thus, our results indicate that sustained activation of Akt in response to polyamine depletion inhibits migration through GSK-3β and Rac1. Received 25 August 2006; received after revision 3 October 2006; accepted 16 October 2006     

An abundant, truncated human sulfonylurea receptor 1 splice variant has prodiabetic properties and impairs sulfonylurea action

An alternatively spliced form of human sulfonylurea receptor (SUR) 1 mRNA lacking exon 2 (SUR1Δ2) has been identified. The omission of exon 2 caused a frame shift and an immediate stop codon in exon 3 leading to translation of a 5.6-kDa peptide that comprises the N-terminal extracellular domain and the first transmembrane helix of SUR1. Based on a weak first splice acceptor site in the human SUR1 gene (ABCC8), RT-PCR revealed a concurrent expression of SUR1Δ2 and SUR1. The SUR1Δ2/(SUR1 + SUR1Δ2) mRNA ratio differed between tissues, and was lowest in pancreas (46%), highest in heart (88%) and negatively correlated with alternative splice factor/splicing factor 2 (ASF/SF2) expression. In COS-7 cells triple transfected with SUR1Δ2/SUR1/Kir6.2, the SUR1Δ2 peptide co-immunoprecipitated with Kir6.2, thereby displacing two of four SUR1 subunits on the cell surface. The ATP sensitivity of these hybrid ATP-sensitive potassium channels (K_ATP) channels was reduced by about sixfold, as shown with single-channel recordings. RINm5f rat insulinoma cells, which genuinely express SUR1 but not SUR1Δ2, exhibited a strongly increased K_ATP channel current upon transfection with SUR1Δ2. This led to inhibition of glucose-induced depolarization, calcium flux, insulin release and glibenclamide action. A non-mutagenic SNP on nucleotide position 333 (Pro69Pro) added another exonic splicing enhancer sequence detected by ASF/SF2, reduced relative abundance of SUR1Δ2 and slightly protected from non-insulin dependent diabetes in homozygotic individuals. Thus, SUR1Δ2 represents an endogenous K_ATP-channel modulator with prodiabetic properties in islet cells. Its predominance in heart may explain why high-affinity sulfonylurea receptors are not found in human cardiac tissue.     

Increased mitochondrial palmitoylcarnitine/carnitine countertransport by flavone causes oxidative stress and apoptosis in colon cancer cells

Cancer cell metabolism is characterized by limited oxidative phosphorylation in order to minimize oxidative stress. We have previously shown that the flavonoid flavone in HT-29 colon cancer cells increases the uptake of pyruvate or lactate into mitochondria, which is followed by an increase in O₂^−.. production that finally leads to apoptosis. Similarly, a supply of palmitoylcarnitine in combination with carnitine induces apoptosis in HT-29 cells by increasing the mitochondrial respiration rate. Here we show that flavone-induced apoptosis is increased more than twofold in the presence of palmitoylcarnitine due to increased mitochondrial fatty acid transport and the subsequent metabolic generation of O₂^−. in mitochondria is the initiating factor for the execution of apoptosis. Received 12 August 2005; received after revision 12 October 2005; accepted 14 October 2005     

Sterol binding by OSBP-related protein 1L regulates late endosome motility and function

ORP1L is an oxysterol binding homologue that regulates late endosome (LE) positioning. We show that ORP1L binds several oxysterols and cholesterol, and characterize a mutant, ORP1L Δ560–563, defective in oxysterol binding. While wild-type ORP1L clusters LE, ORP1L Δ560–563 induces LE scattering, which is reversed by disruption of the endoplasmic reticulum (ER) targeting FFAT motif, suggesting that it is due to enhanced LE–ER interactions. Endosome motility is reduced upon overexpression of ORP1L. Both wild-type ORP1L and the Δ560–563 mutant induce the recruitment of both dynactin and kinesin-2 on LE. Most of the LE decorated by overexpressed ORP1L fail to accept endocytosed dextran or EGF, and the transfected cells display defective degradation of internalized EGF. ORP1L silencing in macrophage foam cells enhances endosome motility and results in inhibition of [³H]cholesterol efflux to apolipoprotein A-I. These data demonstrate that LE motility and functions in both protein and lipid transport are regulated by ORP1L.     

In vitro dopaminergic neuroprotective and in vivo antiparkinsonian-like effects of Δ3,2-hydroxybakuchiol isolated from Psoralea corylifolia (L.)

Cocktail recipes containing Psoralea corylifolia seeds (PCS) are used to empirically treat Parkinson disease. A PCS isolate Δ³,2-hydroxybakuchiol (BU) can inhibit dopamine uptake in dopamine transporter (DAT) transfected Chinese hamster ovary (CHO) cells, and dopamine reuptake blockade may provide an alternative approach for ameliorating parkinsonism. Here, we assessed the potential dopaminergic neuroprotective, and antiparkinsonian-like activity of BU. BU sample size was increased by using a scale-up extraction paradigm. Pharmacologically, BU significantly protected SK-N-SH cells from 1-methyl-4-phenylpyridinium (MPP⁺) insult, produced striking inhibitory actions on dopamine/norepinephrine uptake and WIN35,428 binding in synaptosomes on in vivo administration, and significantly preventing poor performance on rotarod and dopaminergic loss in substantia nigra in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice. BU acts by protecting dopaminergic neurons from MPP⁺ injury and preventing against MPTP-induced behavioral and histological lesions in the Parkinson’s disease (PD) model, possibly by inhibiting monoamine transporters. These findings suggest that BU could be meaningful in PD treatment. Received 14 January 2009; received after revision 22 February 2009; accepted 10 March 2009     

Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages

Enterococci are commensal organisms in the alimentary tract. However, they can cause a variety of life-threatening infections, especially in nosocomial settings. We hypothesized that induction of cell death might enable these facultative pathogenic bacteria to evade the innate immune response and to cause infections of their host. We demonstrate that E. faecium when exposed to lysozyme induces cell death in macrophages in vitro and in vivo. Flow cytometric analyses of J774A.1 macrophages infected with E. faecium revealed loss of cell membrane integrity indicated by uptake of propidium iodide and decrease of the inner mitochondrial transmembrane potential ΔΨ_m. Inhibition of caspases, treatment of macrophages with cytochalasin D, or rifampicin did not prevent cells from dying, suggesting cell death mechanisms that are independent of caspase activation, bacterial uptake, and intracellular bacterial replication. Characteristics of necrotic cell death were demonstrated by both lack of procaspase 3 activation and cell shrinkage, electron microscopy, and release of lactate dehydrogenase. Pretreatment of E. faecium with lysozyme and subsequently with broad spectrum protease considerably reduced cell death, suggesting that a bacterial surface protein is causative for cell death induction. Moreover, in a mouse peritonitis model we demonstrated that E. faecium induces cell death of peritoneal macrophages in vivo. Altogether, our results show that enterococci, under specific conditions such as exposure to lysozyme, induce necrotic cell death in macrophages, which might contribute to disseminated infections by these facultative pathogenic bacteria.     

Activation of the human FP prostanoid receptor disrupts mitosis progression and generates aneuploidy and polyploidy

Studies have shown prostaglandin F_2α to be an endogenous tumor promoter in mouse models of skin carcinogenesis; however, the mechanisms by which PGF_2α affects cell cycle events remain unknown. Here we performed cell cycle analyses on HEK cells stably expressing the human FP receptor and found that treatment with PGF_2α delays mitosis and is associated with an increased expression of cyclin B1 and Cdc2 kinase activity. In addition, multipolar spindles and misaligned chromosomes were observed in a significant proportion of cells treated with PGF_2α. Defective cytokinesis was also observed which resulted in gross aneuploidy and polyploidy. Expression of dominant negative Rho attenuated the cell cycle delay and prevented the generation of micronuclei following treatment with PGF_2α. This suggests that FP receptor activation of Rho signaling by PGF_2α can interfere with nuclear division. Aneuploidy is associated with genomic instability and may underlie the tumor-promoting properties of PGF_2α. Received 7 July 2005; received after revision 22 October 2005; accepted 11 November 2005     

Overexpression of HAb18G/CD147 promotes invasion and metastasis via α3β1 integrin mediated FAK-paxillin and FAK-PI3K-Ca2+ pathways

Mechanism of HAb18G/CD147 underlying the metastasis process of human hepatoma cells has not been determined. In the present study, we found that integrin α3β1 colocalizes with HAb18G/CD147 in human 7721 hepatoma cells. The enhancing effect of HAb18G/CD147 on adhesion, invasion capacities and matrix metalloproteinases (MMPs) secretion was decreased by integrin α3β1 antibodies (p<0.01). The expressions of integrin downstream molecules including focal adhesion kinase (FAK), phospho-FAK (p-FAK), paxillin, and phospho-paxillin (p-paxillin) were increased in human hepatoma cells overexpressing HAb18G/CD147. Deletion of HAb18G/CD147 reduces the quantity of focal adhesions and rearranges cytoskeleton. Wortmannin and LY294002, specific phosphatidylinositol kinase (PI3K) inhibitors, reversed the effect of HAb18G/CD147 on the regulation of intracellular Ca²⁺ mobilization, significantly reducing cell adhesion, invasion and MMPs secretion potential (p<0.01). Together, these results suggest that HAb18G/CD147 enhances the invasion and metastatic potentials of human hepatoma cells via integrin α3β1-mediated FAK-paxillin and FAKPI3K-Ca²⁺ signal pathways. Received 5 June 2008; received after revision 16 July 2008; accepted 23 July 2008     

Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds α-tubulin and promotes α/β dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds α-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Δ mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Δ mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.     

Effects of receptor agonists on polyamine concentrations and spermidine/spermine N1-acetyltransferase activity in rat parotid gland

The polyamine putrescine might be formed via a degradation (catalyzed by spermidine/spermine N¹-acetyltransferase, SSAT) of the higher polyamines spermidine and spermine to putrescine. The involvement of different intracellular signal pathways in the regulation of putrescine formation was studied in explants and in cultured cells of rat parotid glands by using receptor agonists that activate separate second messenger systems, and measuring their effects on the concentrations of putrescine, spermidine and spermine and on the SSAT activity. The -adrenoceptor agonist isoprenaline, which is an activator of cAMP formation, increased the putrescine concentration and stimulated the SSAT activity. Pilocarpine, a drug that activates the muscarinic receptors and thereby enhances the phosphoinositide turnover, had no effect on either the polyamine concentrations or on the SSAT activity. Epidermal growth factor (EGF), which induces activation of a protein tyrosine kinase, had no effect on the polyamine concentrations or on the SSAT activity. The adenylate cyclase activator forskolin increased the glandular levels of putrescine. Taken together, these findings suggest that increases in putrescine concentration in cultured rat parotid gland cells are accompanied by accumulation of cAMP.     

Surface molecules regulating rolling and adhesion to endothelium of neutrophil granulocytes and MDA-MB-468 breast carcinoma cells and their interaction

The extravasation of leukocytes and tumor cells is a multi-step process with the involvement of various adhesion molecules depending on the three steps rolling, adhesion, and diapedesis. We have developed an in vitro model, by which we investigated the rolling and adhesion of neutrophil granulocytes and MDA-MB-468 human breast carcinoma cells to lung endothelial cells under physiological flow-conditions. We found that norepinephrine had an inhibitory function on the fMLP-promoted adhesion of neutrophil granulocytes due to a down-regulation of β2-integrin. Furthermore, neutrophil granulocytes serve as linking cells for the interaction of the MDA-MB-468 cells with the endothelium, which are both β2-integrin negative, but express the β2-integrin ligand ICAM-1. In addition, we show here that N-cadherin is up-regulated on the endothelial cells and on neutrophil granulocytes in response to fMLP. This up-regulation resulted in a significant increase of adherent MDA-MB-468 cells, which are also N-cadherin positive. Received 3 September 2007; received after revision 17 October 2007; accepted 22 October 2007     

Antimicrobial skin peptides and proteins

Human skin is permanently exposed to microorganisms, but rarely infected. One reason for this natural resistance might be the existence of a ‘chemical barrier’ consisting in constitutively and inducibly produced antimicrobial peptides and proteins (AMPs). Many of these AMPs can be induced in vitro by proinflammatory cytokines or bacteria. Apart from being expressed in vivo in inflammatory lesions, some AMPs are also focally expressed in skin in the absence of inflammation. This suggests that non-inflammatory stimuli of endogenous and/or exogenous origin can also stimulate AMP synthesis without inflammation. Such mediators might be ideal ‘immune stimulants’ to induce only the innate antimicrobial skin effector molecules without causing inflammation. Received 9 August 2005; received after revision 21 October 2005; accepted 16 November 2005     

Activation of CD47 receptors causes histamine secretion from mast cells

Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcɛRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the βγ dimer of a G_i protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G_i protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G_i protein complex. Received 8 December 2008; received after revision 12 January 2009; accepted 29 January 2009     

Bafilomycin A1 activates respiration of neuronal cells via uncoupling associated with flickering depolarization of mitochondria

Bafilomycin A1 (Baf) induces an elevation of cytosolic Ca²⁺ and acidification in neuronal cells via inhibition of the V-ATPase. Also, Baf uncouples mitochondria in differentiated PC12 (_dPC12), _dSH-SY5Y cells and cerebellar granule neurons, and markedly elevates their respiration. This respiratory response in _dPC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca²⁺ and ΔΨm. The response to Baf is regulated by cytosolic Ca²⁺ fluxes from the endoplasmic reticulum. Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm. Baf induces stochastic flickering of the ΔΨm with a period of 20 ± 10 s. Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels. Cells treated with Baf become more susceptible to excitation with KCl. Such mitochondrial uncoupling may play a role in a number of (patho)physiological conditions induced by Baf.     

Simulated biosynthesis of anahygrine

Zusammenfassung Kondensation von N-Methyl-2-hydroxypyrrolidin undΔ ¹-Piperidein mit Acetondicarbons?ure liefert Anahygrin und vier verwandte Alkaloide.      

Nephrin in human lymphoid tissues

When nephrin, the protein product of NPHS1, was cloned, it was proposed to be specific for the kidney glomerular podocytes. Recently, however, new reports have emerged verifying additional nephrin expression sites, particularly the insulin-producing β cells of the pancreas, as well as the central nervous system. In this study, we demonstrate nephrin expression in lymphoid tissues, specifically the tonsil, adenoid and lymph node. Nephrin mRNA expression levels were 4-fold higher in tonsils and adenoids than in thymus or B lymphocytes, and 20-fold higher than in T lymphocytes or monocytes, as shown by quantitative RT-PCR analysis. Anti-nephrin antibodies recognised a specific 165-kDa band in lysates of tonsil and adenoid. In immunofluorescence and immunohistochemichal stainings of adenoid and lymph node sections, nephrin-positive cells were detected in the germinal centres of the lymphoid follicles in a staining pattern typical for interdigitating cells. These results indicate a definite and additional presence of nephrin in lymphoid tissue. Received 17 October 2005; received after revision 22 November 2005; accepted 20 December 2005     

Functionally independent subunits in the oligomeric structure of the GABA cotransporter rGAT1

We have combined structural and functional approaches to investigate the role of oligomerization in the operation of the GABA transporter rGAT1. Xenopus laevis oocytes were induced to express, either separately or simultaneously, the wild-type form of rGAT1 and a mutated (Y140W) form, unable to translocate GABA and to generate transport currents, although its intramembrane charge movement properties are only slightly affected. These characteristics, together with the insensitivity of Y140W to the blocking action of SKF89976A, were used to study the possible functional interaction of the two forms in an heteromeric structure. The electrophysiological data from oocytes coexpressing wild-type and Y140W rGAT1 were consistent with a completely independent activity of the two forms. Oligomerization was also studied by fluorescence resonance energy transfer (FRET) in tsA201 cells expressing the transporters fused with cyan and yellow fluorescent proteins (ECFP and EYFP). All combinations tested (WT-ECFP/WTEYFP, Y140W-ECFP/Y140W-EYFP and WT-ECFP/ Y140W-EYFP) were able to give rise to FRET, confirming the formation of homo- as well as heterooligomers. We conclude that, although rGAT1 undergoes structural oligomerization, each monomer operates independently. Received 18 July 2005; received after revision 21 September 2005; accepted 6 October 2005     

Disruption of clc-5 leads to a redistribution of annexin A2 and promotes calcium crystal agglomeration in collecting duct epithelial cells

Mutations in CLCN5, which encodes the voltage-dependent Cl⁻/H⁺antiporter, CLC-5, cause Dent’s disease. This disorder is characterized by low molecularweight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis. Using a collecting duct cell model (mIMCD-3) in which endogenous clc-5 is disrupted by antisense clc-5 or overexpression of truncated clc-5, we demonstrate altered expression of the crystal adhesion molecule, annexin A2. Endogenously expressed annexin A2 is intracellular with limited plasma membrane localization. Following clc-5 disruption, there is both a marked increase in plasma membrane annexin A2 and an increase in cell surface crystal retention and agglomeration, which may be attenuated using pretreatment with anti-annexin A2 antibodies or wheat germ agglutinin lectin but not by concanavalin A. We hypothesize that in Dent’s disease, endocytic failure leads to an accumulation at the plasma membrane of crystal-binding molecules that include annexin A2 leading to retention of calcium crystals and ultimately nephrocalcinosis and nephrolithiasis. Received 22 October 2005; received after revision 26 November 2005; accepted 2 December 2005