BBA - Molecular Cell Research (v.1691, #2-3)

The nucleocytoplasmic transport processes are mediated by soluble transport factors constantly navigating between nuclear and cytoplasmic compartments. Our understanding about nuclear export of general ‘nuclear import factors’ that deliver the cargo to the nucleus is still fragmentary. Utilizing green fluorescent protein tagged glucocorticoid receptor (GR) and relA as our working model and with judicious use of LMB, we show in living cells that all the soluble components of the nuclear import machinery exit nucleus via exportin1/CRM1 independent pathway(s).
Keywords: Nuclear import; Nuclear export; Leptomycin; Steroid receptor;

Effects of Li+ transport and intracellular binding on Li+/Mg2+ competition in bovine chromaffin cells by C.P. Fonseca; L.P. Montezinho; C. Nabais; A.R. Tomé; H. Freitas; C.F.G.C. Geraldes; M.M.C.A. Castro (79-90).
Li+ transport, intracellular immobilisation and Li+/Mg2+ competition were studied in Li+-loaded bovine chromaffin cells. Li+ influx rate constants, k i, obtained by atomic absorption (AA) spectrophotometry, in control (without and with ouabain) and depolarising (without and with nitrendipine) conditions, showed that L-type voltage-sensitive Ca2+ channels have an important role in Li+ uptake under depolarising conditions. The Li+ influx apparent rate constant, k iapp, determined under control conditions by 7Li NMR spectroscopy with the cells immobilised and perfused, was much lower than the AA-determined value for the cells in suspension. Loading of cell suspensions with 15 mmol l−1 LiCl led, within 90 min, to a AA-measured total intracellular Li+ concentration, [Li+]iT=11.39±0.56 mmol (l cells)−1, very close to the steady state value. The intracellular Li+ T1/T2 ratio of 7Li NMR relaxation times of the Li+-loaded cells reflected a high degree of Li+ immobilisation in bovine chromaffin cells, similar to neuroblastoma, but larger than for lymphoblastoma and erythrocyte cells. A 52% increase in the intracellular free Mg2+ concentration, Δ[Mg2+]f=0.27±0.05 mmol (l cells)−1 was measured for chromaffin cells loaded with the Mg2+-specific fluorescent probe furaptra, after 90-min loading with 15 mmol l−1 LiCl, using fluorescence spectroscopy, indicating significant displacement of Mg2+ by Li+ from its intracellular binding sites. Comparison with other cell types showed that the extent of intracellular Li+/Mg2+ competition at the same Li+ loading level depends on intracellular Li+ transport and immobilisation in a cell-specific manner, being maximal for neuroblastoma cells.
Keywords: Li+ affinity; Intracellular Mg2+; Fluorescence; NMR; Atomic absorption;

Effects of autolysis on properties of μ- and m-calpain by Hongqi Li; Valery F. Thompson; Darrel E. Goll (91-103).
Although the biochemical changes that occur during autolysis of μ- and m-calpain are well characterized, there have been few studies on properties of the autolyzed calpain molecules themselves. The present study shows that both autolyzed μ- and m-calpain lose 50–55% of their proteolytic activity within 5 min during incubation at pH 7.5 in 300 mM or higher salt and at a slower rate in 100 mM salt. This loss of activity is not reversed by dialysis for 18 h against a low-ionic-strength buffer at pH 7.5. Proteolytic activity of the unautolyzed calpains is not affected by incubation for 45 min at ionic strengths up to 1000 mM. Size-exclusion chromatography shows that ionic strengths of 100 mM or above cause dissociation of the two subunits of autolyzed calpains and that the dissociated large subunits (76- or 78-kDa) aggregate to form dimers and trimers, which are proteolytically inactive. Hence, instability of autolyzed calpains is due to aggregation of dissociated heavy chains. Autolysis removes the N-terminal 19 (m-calpain) or 27 (μ-calpain) amino acids from the large subunit and approximately 90 amino acids from the N-terminus of the small subunit. These regions form contacts between the two subunits in unautolyzed calpains, and their removal leaves only contacts between domain IV in the large subunit and domain VI in the small subunit. Although many of these contacts are hydrophobic in nature, ionic-strength-induced dissociation of the two subunits in the autolyzed calpains indicates that salt bridges have an important, possibly indirect, role in the domain IV/domain VI interaction.
Keywords: Calpain; Autolysis; Calcium;

The role of p27Kip1 in maintaining the levels of D-type cyclins in vivo by Vı́tězslav Bryja; Jiřı́ Pachernı́k; Ludmila Faldı́ková; Pavel Krejčı́; Robert Pogue; Iveta Nevřivá; Petr Dvořák; Aleš Hampl (105-116).
This in vivo study employs p27-deficient mice to investigate the significance of p27 for the metabolism of D-type cyclins in differentiated cells. The absence of p27 results in decreased levels of cyclins D2 and/or D3 in some organs. As demonstrated on Leydig cells of testis, such dependency is only restricted to certain cell types including terminally differentiated ones, and the absence of p27 in these cells can interfere with their differentiation. The decrease of cyclin D caused by the absence of p27 equals the amount of cyclin D physically associated with p27 in non-mutant animals. The data indicate that it is the proportion of p27-associated cyclin D that determines the response to p27 deficiency. Cells in which the level of D-type cyclin is dependent on p27 do not up-regulate the activity of their CDK2 and CDK4 upon loss of p27, and these cells have a negligible amount of p27 bound to CDK2 and/or cyclin A/E under normal conditions. Together, the findings suggest the existence of a dual role for p27, one being a classical regulation of cell cycle via inhibition of cyclin-dependent kinases (CDK), and the other being participation in the establishment and/or maintenance of differentiated status that is realized in conjunction with D-type cyclins.
Keywords: D-type cyclin; p27; Differentiation; p27-deficient mouse; Leydig cell; Thymus; Lung;

Angiogenesis, the sprouting of new blood vessels, is tightly mediated via a myriad of endogenous factors. A pro-angiogenic alteration facilitates the formation of neovascular tumour networks, thereby providing mechanisms for uncontrolled growth. The kallikrein–kinin system is postulated to be pro-angiogenic since its components have been detected in both endothelial cells and tumour tissue. No studies have, however, focussed on the role of tissue kallikrein (TK) in human angiogenic endothelial cell–tumour interactions. This study has optimised a challenge model whereby endothelial cells are presented with neuroblastoma metabolites, and vice versa. Image analysis of immunoreactive TK revealed a dose-dependant, significant reduction of TK localisation within endothelial cells, while gene expression remained unchanged, the latter determined by in situ RT-PCR. Neuroblastoma cells, when challenged with endothelial cell metabolites, displayed no change in TK synthesis or localisation. Alterations in TK synthesis and/or storage by angiogenic endothelial cells may be mediated by tumour-released signals and possibly indicate a shift from a proteolytic to a mitogenic function of TK. The challenge model provides a relatively simple experimental system to study angiogenic factors in tumour–endothelial cell interaction, and is the first to localise both TK and its mRNA within angiogenic endothelial and tumour cells.
Keywords: Angiogenesis; Endothelial cell; Tissue kallikrein; Immuno-labelling; In situ RT-PCR; Tumour metabolite;

Alpha-chemokine-mediated signal transduction in human Kaposi's sarcoma spindle cells by Jian-Feng Wang; Zhong-Ying Liu; Appakkudal R. Anand; Xuefeng Zhang; Lawrence F. Brown; Bruce J. Dezube; Parkash Gill; Ramesh K. Ganju (129-139).
The role of chemokines and their receptors in HIV biology and Kaposi's sarcoma (KS) pathogenesis has recently gained considerable attention. It has been shown that KS-associated human herpes virus type 8 (KSHV/HHV-8) encodes functional homologues of certain chemokines and chemokine receptors. This suggests that chemokines may contribute to the growth and spread of KS seen in AIDS. We found the expression of CXCR4 in primary KS tissue by using in situ hybridization (ISH). Recently, alpha-chemokine receptors CXCR1 and CXCR2 have also been shown to be expressed by KS tissues. We further characterized the expression of these chemokines as well as the signaling events induced upon binding to their respective cognate ligands in the KS 38 spindle cell line. These cells express authentic characteristics of primary KS spindle cells and provide a useful in vitro model for these studies. We observed using RT-PCR that KS 38 cells express mRNA for the alpha-chemokine receptors CXCR1, CXCR2, and CXCR4. We also confirmed the cell surface protein expression by FACS analysis. Characterization of signaling pathways revealed that the alpha-chemokines, IL-8 and stromal cell-derived factor 1α (SDF1α/CXCL12), activated members of the mitogen-activated protein (MAP) kinase family, including Erk kinase, c-Jun amino terminal kinase (JNK)/stress-activated protein kinase (SAPK) and the p38 MAP kinase. Furthermore, using DNA protein-binding experiments, we have shown that IL-8 increased AP-1 and NF Kappa B activity in these cells. IL-8 also enhanced the chemotaxis of KS cells. These results reveal that chemokine-induced signaling pathways may mediate cell growth, transcriptional activation and cell migration in KS.
Keywords: Kaposi's sarcoma; Chemokine; Transcription factor; G protein-coupled receptor;

Brain-specific RGS9-2 is localized to the nucleus via its unique proline-rich domain by Mohamad Bouhamdan; Sharon Kay Michelhaugh; Irina Calin-Jageman; Shawn Ahern-Djamali; Michael John Bannon (141-150).
Brain-specific regulator of G protein signaling 9 (RGS9-2) is a member of a family of proteins that can function as GTPase-activating proteins for heterotrimeric G proteins. In the present study, we examined the intracellular distribution of RGS9-2 in native brain tissue and transfected cells. Immunocytochemical and immunoblot experiments revealed an unexpectedly high proportion of RGS9-2 within the nuclei of forebrain neurons. A similar intracellular distribution was seen in transfected COS-7 cells. The RGS9 binding partner Gβ5 further enhanced the nuclear localization of RGS9-2, but did not affect the strongly cytoplasmic localization of RGS9-1, the retinal form of RGS9. Deletion construct analysis revealed that the unique polyproline-rich C-terminus of brain-specific RGS9-2 contains sequences necessary and sufficient to target RGS9 to the nucleus of COS-7 cells, as well as cultured striatal neurons. Furthermore, RGS9-2 transfection increased the transcriptional activity of a neuronal gene construct normally expressed in RGS9-positive neurons, suggesting that nuclear RGS9 directly or indirectly regulates transcription in vivo. The nuclear localization of RGS9-2 suggests a heretofore-unanticipated role for this brain-specific protein in transducing signals to the nuclei of forebrain neurons.
Keywords: RGS9-2; G protein signaling; GTPase activity;

Induction of ferritin expression by oxalomalate by Rita Santamaria; Carlo Irace; Michela Festa; Carmen Maffettone; Alfredo Colonna (151-159).
Ferritin is a ubiquitous protein required for intracellular iron storage; its biosynthesis is mainly regulated by iron-regulatory proteins (IRP1 and IRP2) at post-transcriptional level. This regulation prevents iron excess from promoting the formation of reactive oxygen species (ROS). IRP1 is regulated by such factors as intracellular iron levels, the oxidants H2O2 and NO. We recently demonstrated that oxalomalate (OMA, α-hydroxy-β-oxalosuccinic acid), a competitive inhibitor of aconitase, which is an enzyme of the citric acid cycle, remarkably decreases the binding activity of IRP1. The aim of the present study was to investigate whether this molecule could affect the expression of ferritin. The RNA-binding activity of IRP1, evaluated by gel retardation assay, decreased after treatment of several cell lines with 5 mM OMA, with a maximal decrease of about 3-fold after 6 h. This effect remained almost constant up to 48 h after which it returned to basal levels. Intracellular ferritin levels, determined by Western blot analysis, increased in correlation with the OMA-induced decrease of IRP1 binding activity. Furthermore, treatment of cells with OMA caused a rise in ferritin mRNA levels. Interestingly, in cells exposed to iron challenge, OMA-induced overexpression of ferritin prevented formation of ROS and cellular lipid peroxidation. These data show that an inhibitor of aconitase, OMA, besides being involved in energetic metabolism, is able to control ferritin expression, probably through molecular mechanisms of either post-transcriptional regulation or transcriptional modulation, with advantageous consequences for the cell.
Keywords: Iron metabolism; Iron regulatory protein; Oxalomalate; Iron storage protein; Oxidative stress; Cytoprotection;

Alcohols increase calmodulin affinity for Ca2+ and decrease target affinity for calmodulin by Ichiro Ohashi; Roman Pohoreki; Kiyoshi Morita; Paul M. Stemmer (161-167).
It has been proposed that alcohols and anesthetics selectively inhibit proteins containing easily disrupted motifs, e.g., α-helices. In this study, the calcineurin/calmodulin/Ca2+ enzyme system was used to examine the effects of alcohols on calmodulin, a protein with a predominantly α-helical structure. Calcineurin phosphatase activity and Ca2+ binding were monitored as indicators of calmodulin function. Alcohols inhibited enzyme activity in a concentration-dependent manner, with two-, four- and five-carbon n-alcohols exhibiting similar leftward shifts in the inhibition curves for calmodulin-dependent and -independent activities; the former was slightly more sensitive than the latter. Ca2+ binding was measured by flow dialysis as a direct measure of calmodulin function, whereas, with the addition of a binding domain peptide, measured calmodulin–target interactions. Ethanol increased the affinity of calmodulin for Ca2+ in the presence and absence of the peptide, indicating that ethanol stabilizes the Ca2+ bound form of calmodulin. An increase in Ca2+ affinity was detected in a calmodulin binding assay, but the affinity of calmodulin for calcineurin decreased at saturating Ca2+. These data demonstrate that although specific regions within proteins may be more sensitive to alcohols and anesthetics, the presence of α-helices is unlikely to be a reliable indicator of alcohol or anesthetic potency.
Keywords: Alcohol; Calmodulin; Calcineurin; α-Helix; Protein interaction;

Expression of functional β2-adrenergic receptors in the lung epithelial cell lines 16HBE14o, Calu-3 and A549 by Getu Abraham; Carsten Kneuer; Carsten Ehrhardt; Walther Honscha; Fritz R Ungemach (169-179).
Adrenergic drugs acting through the β2-adrenoceptor (β2-AR) adenylate cyclase (AC) signal transduction system elicit a variety of responses within the mammalian airway epithelium; however, its composition of multiple phenotypically differentiated cell types complicates the understanding of the regulation cascades within this tissue. The present study evaluates β2-AR mRNA level, number, subtype and the cyclic adenosine-3′,5′-monophosphate (cyclic AMP) response to isoproterenol (iso) in the human airway epithelial cell lines 16HBE14o, Calu-3 and A549, using reverse transcriptase polymerase chain reaction (RT-PCR), radioligand binding studies, [3H]-radioimmunoassay and immunocytochemical staining.After 4–5 days in culture, all three cell types produced β2-AR mRNA and protein at a magnitude of gene expression levels Calu-3≥16HBE14o>A549, whereas control cells Cos-1 and Caco-2 were negative. The β2-AR adenylate cyclase system was highly expressed and functional in the human airway epithelial cells Calu-3 and 16HBE14o. The mean β2-AR density (B max), equilibrium dissociation constant (K D), and the percentage of β-AR subtypes assessed by radioligand binding were approximately 9908±1127 and 6423±895 binding sites/cell, 32±2.7 pM and 25±1.1 pM, and approximately 100% in Calu-3 and 16HBE14ocells, respectively. However, in the alveolar cell type A549 the cell surface β2-AR was virtually undetectable by (−)-[125I]-iodocyanopindolol (ICYP) binding. Stimulation of cultured cells with (−)-isoproterenol enhanced the basal cyclic AMP accumulation only in Calu-3 and 16HBE14o cells, which was blocked by the β2-selective antagonist ICI 118,551, but not by the β1-selective antagonist CGP 20712A, confirming functional coupling of the β2-AR to adenylate cyclase in these cells. Immunocytochemical staining localised the receptor on the cell membrane and the cytoplasm in Calu-3 and 16HBE14o cells, while it was confined to the cytoplasm only in A549 cells. In conclusion, the β2-AR expression and its functional coupling to adenylyl cyclase was very high in the human airway epithelial cells Calu-3 and 16HBE14o, but not in A549, suggesting that the cell lines Calu-3 and 16HBE14o present suitable models to study function and regulation of the β-adrenoceptor signalling in the respiratory system.
Keywords: β2-Adrenoceptor; β2-AR mRNA; Cyclic AMP; Lung epithelial cells; Cell culture;

Amphibian transition to the oxidant terrestrial environment affects the expression of glutathione S-transferases isoenzymatic pattern by Fernanda Amicarelli; Stefano Falone; Franca Cattani; Marina T Alamanou; Antonella Bonfigli; Osvaldo Zarivi; Michele Miranda; Anna M Ragnelli; Carmine Di Ilio (181-192).
It has been postulated that glutathione S-transferases (GST; EC 2.5.1.18) may play a role in protecting against oxidative stress.In previous studies, we have purified and characterised from Bufo bufo embryos a GST isoenzyme (BbGSTP1-1), which falls at very low level in the adult liver, where a novel isoform (BbGSTP2-2), starts to be highly expressed. During transition to adult life, B. bufo leaves the aquatic environment to live predominantly in the terrestrial environment, characterised by higher oxygen concentration.It has been found that BbGSTP2-2 is more efficient in scavenging from organic hydroperoxides.Therefore, the appearance of BbGSTP2-2 may respond to the necessity of providing the adult toad with a more suitable protection against oxygen toxic by-products. In this work, we performed experiments aimed at verifying if oxidative stress (hyperoxic and H2O2 treatments) could act as a modulator of BbGSTP2-2 expression. Results show that: (a) BbGSTP2 mRNA starts to be expressed in the late embryonic period, while protein appears during metamorphosis; (b) oxidative stress induces anticipation of BbGSTP2 gene expression at both transcriptional and translational levels.These findings seem to indicate that the appearance of BbGSTP2-2 is aimed at endowing the adult toad with more efficient antioxidant defence in the terrestrial atmosphere.
Keywords: Glutathione S-transferase; Amphibia; Reactive oxygen species; Oxidative stress; Antioxidant defence; Gene regulation;

Author Index (193-194).

Cumulative Contents (195-196).