Inhibition of a Golgi complex lysophospholipid acyltransferase induces membrane tubule formation and retrograde trafficking

Recent studies have suggested that formation of Golgi membrane tubules involves the generation of membrane-associated lysophospholipids by a cytoplasmic Ca2+-independent phospholipase A2 (PLA2). Herein, we provide additional support for this idea by showing that inhibition of lysophospholipid reacylation by a novel Golgi-associated lysophosphatidylcholine acyltransferase (LPAT) induces the rapid tubulation of Golgi membranes, leading in their retrograde movement to the endoplasmic reticulum. Inhibition of the Golgi LPAT was achieved by 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), a previously characterized antagonist of acyl-CoA cholesterol acyltransferase. The effect of CI-976 was similar to that of brefeldin A, except that the coatomer subunit beta-COP remained on Golgi-derived membrane tubules. CI-976 also enhanced the cytosol-dependent formation of tubules from Golgi complexes in vitro and increased the levels of lysophosphatidylcholine in Golgi membranes. Moreover, preincubation of cells with PLA2 antagonists inhibited the ability of CI-976 to induce tubules. These results suggest that Golgi membrane tubule formation can result from increasing the content of lysophospholipids in membranes, either by stimulation of a PLA2 or by inhibition of an LPAT. These two opposing enzyme activities may help to coordinately regulate Golgi membrane shape and tubule formation.     

Inhibition of calpain but not caspase activity by spectrin fragments

Calpains and caspases are ubiquitous cysteine proteases that are associated with a variety of cellular pathways. Calpains are involved in processes such as long term potentiation, cell motility and apoptosis, and have been shown to cleave non-erythroid (brain) α- and β-spectrin and erythroid β-spectrin. The cleavage of erythroid α-spectrin by calpain has not been reported. Caspases play an important role in the initiation and execution of apoptosis, and have been shown to cleave non-erythroid but not erythroid spectrin. We have studied the effect of spectrin fragments on calpain and caspase activities. The erythroid and non-erythroid spectrin fragments used were from the N-terminal region of α-spectrin, and C-terminal region of β-spectrin, both consisting of regions involved in spectrin tetramer formation. We observed that the all spectrin fragments exhibited a concentration-dependent inhibitory effect on calpain, but not caspase activity. It is clear that additional studies are warranted to determine the physiological significance of calpain inhibition by spectrin fragments. Our findings suggest that calpain activity is modulated by the presence of spectrin partial domains at the tetramerization site. It is not clear whether the inhibitory effect is substrate specific or is a general effect. Further studies of this inhibitory effect may lead to the identification and development of new therapeutic agents specifically for calpains, but not for caspases. Proteins/peptides with a coiled coil helical conformation should be studied for potential inhibitory effects on calpain activity.     

Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells

Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. Negative role of RhoA, a cytoskeleton-regulating protein, and its downstream target, Rho-associated protein kinase (ROCK) in the chondrogenesis has been studied in many different culture systems including primary chondrocytes, chondrogenic cell lines, dedifferentiated chondrocytes, and micromass culture of mesenchymal cells. To further investigate the role of RhoA and ROCK in the chondrogenesis, we examined the RhoA-ROCK-myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis.     

Trifluoperazine induces domain formation in zwitterionic phosphatidylcholine but not in charged phosphatidylglycerol bilayers

The interaction of trifluoperazine with the zwitterionic lipids dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine and with anionic dimyristoylphosphatidylglycerol was studied by means of microcalorimetry and fluorescence spectroscopy. Intercalation of drug molecules into the lipid bilayers was confirmed by the observed differential scanning calorimetry peak broadening and the decrease in chain-melting temperatures. For trifluoperazine:lipid mole ratios higher than 0.4 and 0.6 (for dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine, respectively) the deconvolution of transition profiles into two Gaussian components was possible, which suggests phase separation in the studied mixtures. Deconvolution of the thermograms was not possible for any of the drug:dimyristoylphosphatidylglycerol mole ratios studied. To confirm the existence of phase separation in trifluoperazine-phosphatidylcholine mixtures fluorescence spectroscopy experiments were performed using Laurdan as a probe. From the generalised polarisation versus excitation wavelength dependences, recorded at different temperatures, we conclude that a phase separation occurs in the gel state of the studied trifluoperazine-phosphatidylcholine mixtures. We attribute the existence of domains in the bilayer to the dissimilar interactions of two protonation forms of trifluoperazine with phosphatidylcholine molecules. Structural defects present at domain boundaries could be related to the trifluoperazine induced increase of membrane permeability and fluidity. This may partially explain the mechanism of multidrug resistance modulation by trifluoperazine.     

Transformation-defective v-ski induces MyoD and myogenin expression but not myotube formation.

The ski oncogene induces muscle differentiation in otherwise nonmyogenic quail embryo cells (C. Colmenares and E. Stavnezer, Cell 59:293-303, 1989). Here we report that v-ski induces both MyoD and myogenin expression, suggesting that activation of these muscle regulatory genes may be a critical step in ski-induced myogenesis. We also describe a transformation-defective mutant of v-ski (tdM5i) that fails to induce myotube formation, although it induces the expression of many muscle-specific genes, including the MyoD and myogenin genes. Therefore, if activation of MyoD and myogenin expression is a necessary component of the myogenic program triggered by ski, it is clearly insufficient to account for complete muscle differentiation.     

RANKL induces formation of avian osteoclasts from macrophages but not from macrophage polykaryons

We have shown that chick macrophages express RANK at their surface and human RANKL (hRANKL) triggers the formation of osteoclasts able to degrade dentine. As described for mammalian osteoclasts, hRANKL also stimulates the resorbing activity of chick bone-derived osteoclasts. In other hands, in culture, chick macrophages spontaneously form polykaryons sharing most of the osteoclast markers but unable to resorb bone. Since both bone-resorbing osteoclasts and macrophage polykaryons found in inflammatory tissues are multinucleated cells deriving from the fusion of macrophages, we examined whether macrophage polykaryons could be induced toward bone-resorbing osteoclasts. Long-term exposure of macrophage polykaryons to hRANKL failed to activate any resorbing activity, indicating that although deriving from the same precursors macrophage polykaryons and osteoclasts are independent cell types and polykaryons are not immature osteoclasts.     

Hypoleptinemia, but not hypoinsulinemia, induces hyperphagia in streptozotocin-induced diabetic rats

To assess the dominance between hypoinsulinemia and hypoleptinemia as factors in the development of hyperphagia in streptozotocin (STZ)-induced diabetes mellitus (STZ-DM) rodents with respect to hormone-neuropeptide interactions, changes in gene expression of agouti gene-related protein (AGRP) in the arcuate nucleus of the hypothalamus were investigated using STZ-DM rats, fasting Zucker fa/fa rats and STZ-DM agouti (STZ-DM A(y)/a) mice. AGRP mRNA and neuropeptide Y mRNA were both significantly up-regulated in STZ-DM rats, which are associated with body weight loss, hyperglycemia, hypoinsulinemia and hypoleptinemia. We proceeded to analyze whether insulin or leptin played the greater role in the regulation of AGRP using Zucker fa/fa rats. The AGRP mRNA did not differ significantly between fasted fa/fa rats, which have both leptin-insensitivity and hypoinsulinemia, and fed Zuckers, which have leptin-insensitivity and hyperinsulinemia. We further found that up-regulation of AGRP expression was normalized by infusion of leptin into the third cerebroventricle (i3vt), but not by i3vt infusion of insulin, although up-regulation of AGRP was partially corrected by systemic insulin infusion. The latter finding supports hypoleptinemia as a key-modulator of STZ-DM-induced hyperphagia because systemic insulin infusion, at least partially, restored hypoleptinemia through its acceleration of fat deposition, as demonstrated by the partial recovery of lost body weight. After STZ-DM induction, A(y)/a mice whose melanocortin-4 receptor (MC4-R) was blocked by ectopic expression of agouti protein additionally accelerated hyperphagia and up-regulated AGRP mRNA, implying that the mechanism is triggered by a leptin deficit rather than by the main action of the message through MC4-R. Hypoleptinemia, but not hypoinsulinemia per se, thus develops hyperphagia in STZ-DM rodents. These results are very much in line with evidence that hypothalamic neuropeptides are potently regulated by leptin as downstream targets of its actions.     

Inhibition of denitrification activity but not of mRNA induction in Paracoccus denitrificans by nitrite at a suboptimal pH

The influence of pH on the denitrification activity of a continuous culture of Paracoccus denitrificans was studied in relation to the presence of nitrite. After a transition from aerobic to anaerobic conditions at the suboptimal pH of 6.8, P. denitrificans was not able to build up a functional denitrification pathway. Nitrite accumulated in the medium as the predominant denitrification product. Although the nitrite reductase gene was induced properly, the enzyme could not be detected at sufficient amounts in the culture. These observations indicate that either translation was somehow inhibited, or once synthesized nitrite reductase was inactivated, possibly by the high concentrations of nitrous acid (HNO₂. Interestingly, when a P. denitrificans culture which was grown to steady-state under anaerobic conditions was then exposed to suboptimal pHs, cells exhibited a reduced overall denitrification activity, but neither nitrite nor any other denitrification intermediate accumulated.     

CyP40, but not Hsp70, in rabbit reticulocyte lysate causes the aryl hydrocarbon receptor-DNA complex formation

Upon ligand binding, the aryl hydrocarbon receptor (AhR) translocates into the nucleus and dimerizes with its partner aryl hydrocarbon receptor nuclear translocator (Arnt). The AhR-Arnt heterodimer binds to the dioxin response element (DRE) to regulate target gene expression. Using baculovirus expressed human AhR and Arnt, we showed that the formation of the ligand-dependent AhR-Arnt-DRE complex requires protein factors in vitro. Recently, we provided evidence that p23, an Hsp90-associated protein, is involved in the complex formation. The aim of this study was to determine whether two other Hsp90-associated proteins present in rabbit reticulocyte lysate (RRL), namely CyP40 and Hsp70, play any role in forming the AhR-Arnt-DRE complex. Fractionation and immunodepletion experiments revealed that Hsp70 is not necessary for the formation of this complex. In contrast, CyP40 is involved in forming the complex since (1) immunodepletion of CyP40 from a RRL fraction reduces the intensity of the AhR-Arnt-DRE complex by 48% and (2) recombinant human CyP40 alone causes the formation of this complex. In addition, CyP40-interacting proteins appear to be essential for the full CyP40 effect on the AhR gel shift complex.     

A conserved tyrosine residue aids ternary complex formation,but not catalysis,in phage T5 flap endonuclease

The flap endonucleases, or 5' nucleases, are involved in DNA replication and repair. They possess both 5'-3' exonucleolytic activity and the ability to cleave bifurcated, or branched DNA, in an endonucleolytic, structure-specific manner. These enzymes share a great degree of structural and sequence similarity. Conserved acidic amino acids, whose primary role appears to be chelation of essential divalent cation cofactors, lie at the base of the active site. A loop, or helical archway, is located above the active site. A conserved tyrosine residue lies at the base of the archway in phage T5 flap endonuclease. This residue is conserved in the structures of all flap endonucleases analysed to date. We mutated the tyrosine 82 codon in the cloned T5 5' nuclease to one encoding phenylalanine. Detailed analysis of the purified Y82F protein revealed only a modest (3.5-fold) decrease in binding affinity for DNA compared with wild-type in the absence of cofactor. The modified nuclease retains both structure-specific endonuclease and exonuclease activities. Kinetic analysis was performed using a newly developed single-cleavage assay based on hydrolysis of a fluorescently labelled oligonucleotide substrate. Substrate and products were resolved by denaturing HPLC. Steady-state kinetic analysis revealed that loss of the tyrosine hydroxyl function did not significantly impair k(cat). Pre-steady state analysis under single-turnover conditions also demonstrated little change in the rate of reaction compared to the wild-type protein. The pH dependence of the kinetic parameters for the Y82F enzyme-catalysed reaction was bell-shaped as for the wild-type protein. Thus, Y82 does not play a role in catalysis. However, steady-state analysis did detect a large (approximately 300-fold) defect in K(M). These results imply that this conserved tyrosine plays a key role in ternary complex formation (protein-DNA-metal ion), a prerequisite for catalysis.     

Acidic stress induces the formation of P-bodies, but not stress granules, with mild attenuation of bulk translation in Saccharomyces cerevisiae

The stress response of eukaryotic cells often causes an attenuation of bulk translation activity and the accumulation of non-translating mRNAs into cytoplasmic mRNP (messenger ribonucleoprotein) granules termed cytoplasmic P-bodies (processing bodies) and SGs (stress granules). We examined effects of acidic stress on the formation of mRNP granules compared with other forms of stress such as glucose deprivation and a high Ca2+ level in Saccharomyces cerevisiae. Treatment with lactic acid clearly caused the formation of P-bodies, but not SGs, and also caused an attenuation of translation initiation, albeit to a lesser extent than glucose depletion. P-body formation was also induced by hydrochloric acid and sulfuric acid. However, lactic acid in SD (synthetic dextrose) medium with a pH greater than 3.0, propionic acid and acetic acid did not induce P-body formation. The results of the present study suggest that the assembly of yeast P-bodies can be induced by external conditions with a low pH and the threshold was around pH?2.5. The P-body formation upon acidic stress required Scd6 (suppressor of clathrin deficiency 6), a component of P-bodies, indicating that P-bodies induced by acidic stress have rules of assembly different from those induced by glucose deprivation or high Ca2+ levels.     

Extracellular ATP induces spikes in cytosolic free Ca but not in NADPH oxidase activity in neutrophils

In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca²⁺ concentration, we simultaneously measured Ca²⁺ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast damped Ca²⁺ spikes with a period of 15 s and slower irregular spikes with a period greater than 50 s. Spikes in Ca²⁺ occurred in the absence of Ca²⁺ influx, but the amplitude was damped by inhibition of Ca²⁺ influx. Using the oxidation of hydroethidine as a cytosolic marker of oxidant production, we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca²⁺ but, despite subsequent spikes in Ca²⁺ concentration, no oscillations in oxidase activity could be detected. ATP induced spikes in Ca²⁺ in a very reproducible way and we propose that the Ca²⁺ signal is an on-switch for oxidase activity, but the activity is apparently not directly correlated with spiking activity in cytosolic Ca²⁺.     

The stable analog carbocyclic TXA2 but not platelet-released TXA2 induces osteoclast-like cell formation

Thromboxan A(2) (TXA(2)) is the main product of arachidonic acid metabolism in activated platelets. Platelet-released supernatants (PRS) can induce osteoclast-like cell formation in murine bone marrow cultures via a cyclooxygenase (COX)/receptor activator of NF-kB-ligand (RANKL)-dependent pathway. Here we investigated a possible linkage between platelet-released TXA(2) and osteoclastogenesis. The stable analog of TXA(2), carbocyclic TXA(2) (CTXA(2)) can induce the formation of tartrate-resistant acid phosphatase positive multinucleated cells in murine bone marrow cultures via a RANKL-dependent pathway and requires the presence of stromal cells. Interestingly, the platelet-released instable TXA(2) does not account for osteoclastogenic effects as: (a) PRS-induced osteoclastogenesis in the presence of the TXA(2) receptor antagonist SQ29548; (b) inhibition of platelet TXA(2) synthesis by indomethacin and acetylsalicylic acid failed to decrease the osteoclastogenic potential of the corresponding supernatants; and (c) CTXA(2)-induced osteoclast-like cell formation independent of indomethacin and the selective COX-2 inhibitor NS398.     

Inhibition of oxidative hemolysis by quercetin,but not other antioxidants

We previously reported that lipid-soluble quercetin, not water-soluble dihydroquercetin, protects human red blood cells against oxidative damage. The objectives of this study were to determine if an antihemolytic effect could be produced by other lipid-soluble antioxidants and if anti-inflammatory activity played a role in antihemolysis by quercetin. This study compared three lipid-soluble polyphenols, muscadine, curcumin and quercetin, and three lipid- (α-tocopherol and α-tocotrienol) or water-soluble (ascorbic acid) vitamins. Among the tested polyphenols, muscadine was the most potent in inhibiting superoxide and 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH)-generated peroxyl radicals, whereas ascorbic acid was the most potent inhibitor of hydrogen peroxide. Activities of the polyphenols after lipid extractions showed that curcumin inhibited superoxide production to a greater extent than quercetin and muscadine. All blood cells were tested 20 min after incubation with the selected compounds. All the polyphenols caused inhibition of N-formyl-l-methionyl-l-leucyl-l-phenylalanine-induced neutrophil oxidative bursts. Quercetin, but not other polyphenols, significantly reduced AAPH-induced oxidative hemolysis. No significant effect on neutrophil oxidative burst or oxidative hemolysis was found with any of the tested vitamins. These results suggest that quercetin enhances the resistance of membrane to destruction by free radicals. This effect of quercetin is not directly mediated through antioxidative or anti-inflammatory actions. Antioxidant or anti-inflammatory potency may not be used as a simple criterion to select polyphenols for cell protection benefits.     

Activin A induces phosphorylation of Smad2 but not complex formation of Smad2 with Smad4 in human colon cancer cell line HT-29

Western blotting coupled with immunoprecipitation showed that activin A treatment induced phosphorylation of Smad2 but not complex formation of Smad2/4 in human colon cancer-derived HT-29 cells. Because HT-29 cells expressed neither Smad4 mRNA nor Smad4 protein, it is suggested that deletion of Smad4 leads to a defect of formation of Smad2/4 complex upon activin A stimulation in HT-29 cells.     

Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa

Two metal response elements, flanking an antioxidant response element, were identified in regions upstream (-3730 bp) to copper metallothionein (CuMT) gene of Neurospora crassa. Presence of copper in culture media, but not of pro-oxidants like H2O2 or menadione, induced CuMT gene expression that could not be completely abolished by antioxidants such as N-acetyl cysteine and ascorbic acid. Gel shift assays revealed the ability of nuclear extracts from copper induced cultures to bind PCR-amplified metal response or antioxidant response elements. Similar observations could not be made with cultures exposed either to pro-oxidants or antioxidants. These results differentiate between CuMT gene induction by copper from antioxidant functions associated with the identified upstream elements.