BBA - Molecular Cell Research (v.1499, #3)

A highly specific method for the dissociation of protein dimers has been developed. The method involves exposure of the dimers to free leucine at a concentration ranging between 3 and 10 mM. Using this method it has been possible to dissociate goat uterine oestrogen receptor homodimers, heterodimers formed between the non-activated oestrogen receptor (naER) and the oestrogen receptor activation factor (E-RAF) of the goat uterus, c-jun homodimers derived from bovine bone marrow and also glucocorticoid receptor homodimers isolated from rat liver cytosol. The pattern of dimer dissociation by leucine clearly differentiates two classes of proteins. The first is represented by steroid hormone receptors where dimerization is apparently contributed by both coiled-coil dimerization interfaces and the conserved heptad repeats of leucine. The second is represented by oncoproteins like c-fos and c-jun which dimerize through the exclusive involvement of leucine zippers. The patterns of dissociation of these two groups of proteins from the concerned affinity columns are distinctly different. This indicates a possibility that the elution pattern may be used as a yardstick to determine whether two proteins dimerize through the exclusive involvement of leucine zippers or whether coiled-coil interfaces are also involved in the dimerization process.
Keywords: Dissociation of dimers; Leucine zipper; Coiled-coil interaction;

Establishment of variant PC12 subclones deficient in stimulation–secretion coupling by Yoko Shoji-Kasai; Mieko Morishima; Reiko Kuwahara; Shunzo Kondo; Makoto Itakura; Masami Takahashi (180-190).
Clonal rat pheochromocytoma (PC12) cells have been widely used to study the molecular mechanism of exocytosis. We have isolated variant PC12 subclones with deficiencies in stimulation–secretion coupling, by a single cell recloning, and investigated the defects. PC12-1G2 hardly released dopamine following high-K+-induced depolarization, but normal release was evoked by the Ca2+-ionophore, ionomycin. Fura-2 fluorometry indicated that a nicardipine-sensitive component of Ca2+ influx was missing, suggesting that PC12-1G2 has defects in L-type Ca2+ channel function. PC12-2B3 was not responsive to high-K+-induced depolarization and ionomycin, and voltage-dependent Ca2+ entry was identical to that of the normal clone. Electron microscopy revealed that the number of vesicles adjacent or directly attached to the plasma membrane was decreased in PC12-2B3. The expression of presynaptic proteins was analyzed by immunoblotting using a panel of antibodies. Syntaxin 1, VAMP-2, SNAP-25, Munc18, Rab3C and Sec-6 were decreased compared to the control clone and that of synaptophysin was extremely low. PC12-D60 synthesized and released dopamine normally, but had almost lost its catecholamine-uptake activity. These results show that multiple PC12 cells variants are spontaneously generated, and that recloning can select PC12 subclones useful for the study of the molecular mechanisms of neurotransmitter release.
Keywords: PC12 cell; Dopamine; Exocytosis; Ca2+ channel; SNARE protein; Catecholamine transporter;

Cloning and characterization of an inhibitor of apoptosis protein (IAP) from Bombyx mori by Qihong Huang; Quinn L. Deveraux; Susumu Maeda; Henning R. Stennicke; Bruce D. Hammock; John C. Reed (191-198).
We cloned a novel inhibitor of apoptosis protein (IAP) family member, BmIAP, from Bombyx mori BmN cells. BmIAP contains two baculoviral IAP repeat (BIR) domains followed by a RING domain. BmIAP shares striking amino acid sequence similarity with lepidopteran IAPs, SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP, suggesting evolutionary conservation. BmIAP blocks programmed cell death (apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35 deficient Autographa californica nucleopolyhedrovirus (AcMNPV). This anti-apoptotic function requires both the BIR domains and RING domain of BmIAP. In mammalian cells, BmIAP inhibits Bax induced but not Fas induced apoptosis. Further biochemical data suggest that BmIAP is a specific inhibitor of mammalian caspase-9, an initiator caspase in the mitochondria/cytochrome-c pathway, but not the downstream effector proteases, caspase-3 and caspase-7. These results suggest that suppression of apoptosis by lepidopteran IAPs in insect cells may involve inhibition of an upstream initiator caspase in the conserved mitochondria/cytochrome-c pathway for apoptosis.
Keywords: Apoptosis; Insect; Inhibitor of apoptosis protein; Caspase; Cell death; Protease inhibitor;

The role of calcium in the mediation of the hypertrehalosaemic signal of the endogenous neuropeptide Mem-CC was investigated in vitro and in vivo in the cetoniid beetle Pachnoda sinuata. The presence of Mem-CC increases the influx of extracellular 45Ca2+ into the fat body as well as the efflux of 45Ca2+ from pre-loaded fat body into the incubation medium. Extracellular calcium is essential to exert maximal activation of the fat body glycogen phosphorylase by saturating doses of Mem-CC (0.3 nM). This effect of extracellular Ca2+ is dose-dependent: maximal activation of glycogen phosphorylase by Mem-CC is achieved at calcium concentrations of approximately 1.2 mM and the ED50 was calculated to be 0.6 mM. Both, thimerosal and thapsigargin caused a stimulation of carbohydrate metabolism in the fat body, suggesting that a release of calcium from the endoplasmic reticulum is involved in this process. However, neither entry of extracellular calcium nor the release from the endoplasmic reticulum are sufficient alone for a full activation of the phosphorylase. The results of the present study suggest that calcium from extracellular as well as from intracellular sources is part of the second messenger system for the transduction of the hypertrehalosaemic signal of Mem-CC in the fat body of P. sinuata.
Keywords: Calcium; Second messenger; Carbohydrate; Glycogen; Insect flight; AKH peptide;

Many of the effects of 1α,25-(OH)2D3 and 24R,25-(OH)2D3 on costochondral chondrocytes are mediated by the protein kinase C (PKC) signal transduction pathway. 1α,25-(OH)2D3 activates PKC in costochondral growth zone chondrocytes through a specific membrane receptor (1α,25-mVDR), involving rapid increases in diacylglycerol via a phospholipase C (PLC)-dependent mechanism. 24R,25-(OH)2D3 activates PKC in resting zone chondrocytes. Although diacylglycerol is increased by 24R,25-(OH)2D3, PLC is not involved, suggesting a phospholipase D (PLD)-dependent mechanism. Here, we show that resting zone and growth zone cells express mRNAs for PLD1a, PLD1b, and PLD2. Both cell types have PLD activity, but levels are higher in resting zone cells. 24R,25-(OH)2D3, but not 24S,25-(OH)2D3 or 1α,25-(OH)2D3, stimulates PLD activity in resting zone cells within 3 min via nongenomic mechanisms. Neither 1α,25-(OH)2D3 nor 24R,25-(OH)2D3 affected PLD in growth zone cells. Basal and 24R,25-(OH)2D3-stimulated PLD were inhibited by the PLD inhibitors wortmannin and EDS. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase), PKC, phosphatidylinositol-specific PLC (PI-PLC), and phosphatidylcholine-specific PLC (PC-PLC) had no effect on PLD activity. Thus, 24R,25-(OH)2D3 stimulates PLD, and PI 3-kinase, PI-PLC and PKC are not involved, whereas PLD is required for stimulation of PKC by 24R,25-(OH)2D3. Pertussis toxin, GDPβS, and GTPγS had no effect on 24R,25-(OH)2D3-dependent PLD when added to cell cultures, indicating that G-proteins are not involved. These data show that PKC activation in resting zone cells is mediated by PLD and suggest that a functional 24R,25-(OH)2D3-mVDR is required. The results also support the conclusion that the 24R,25-(OH)2D3-responsive PLD is PLD2, since this PLD isoform is G-protein-independent.
Keywords: Chondrocyte culture; Phospholipase D; 24R,25-Dihydroxycholecalciferol; Protein kinase C; Alkaline phosphatase; Signal transduction; Cell maturation;

Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.
Keywords: Borrelia burgdorferi; Peptide transport; Oligopeptide permease; Spirochete;

In order to study the role of cell–matrix interactions in mammary gland function, temporal changes in α2β1 integrin, the major receptor for collagen and the influence of β-oestradiol on its level and distribution in rat mammary gland at different stages of development were studied. The level of α2β1 integrin determined by ELISA, was found to be high during different days of pregnancy, while in the lactating stage, it was significantly reduced. By immunocytochemical analysis, α2β1 integrin was found to be localized towards the luminal side of acinar cells, both in the virgin and midpregnant stage, while it was not detected in the lactating stage. The possible role of hormones in modulating the level of integrin was examined in both in vitro and in vivo experiments using β-oestradiol. Supplementing β-oestradiol to isolated mammary epithelial cells from both virgin and lactating glands caused a concentration dependent increase in the incorporation of [35S]methionine into α2β1 integrin associated with the cells. Administration of β-oestradiol to virgin and lactating glands caused about 1.4–4-fold increase in the level of α2 integrin, indicating that upregulation of integrin during pregnancy may be due to oestrogen and as the oestrogen level falls during lactating phase, downregulation of α2β1 integrin occurs. Treatment with β-oestradiol also resulted in the appearance of α2β1 integrin in the acinar region of the lactating tissue, while in the untreated controls no staining for integrin was seen. These results indicate that oestrogen, apart from directly affecting the cellular activity, can influence mammary tissue function by affecting cell–ECM interactions through the modulation of integrin receptors for matrix proteins.
Keywords: β-Estradiol; Extracellular matrix; α2β1 Integrin upregulation; Mammary gland; Lactation;

Growth condition-dependent synchronized changes in transcript levels of type II hexokinase and type 1 glucose transporter in tumor cells by Yasuo Shinohara; Mami Hino; Taro Ishida; Yasuhisa Yamanaka; Hiroshi Terada (242-248).
Transcript levels of hexokinase (HK) isozymes and glucose transporter (GLUT) isoforms in RNA samples of AH130 cells obtained from dish cultures and ascites were evaluated in a quantitative manner. In AH130 cells cultured in dishes, HKI and HKII were expressed at a similar level, but HKIII and HKIV were not. GLUT1 and GLUT3 were also expressed, and messages of these two isoforms represented 27% and 71%, respectively, of the total GLUT messages. A faint signal of GLUT2 was also observed. On the contrary, in cells grown as ascites, the transcript of HKII was dominant, and its level was about 15-fold over that of dish-cultured AH130 cells. Transcript levels of GLUT1 and GLUT3 were 4.5- and 2-fold, respectively, higher than those in dish-cultured cells. Thus, GLUT1 was more susceptible to changes in culture conditions than GLUT3. Based on these results, we concluded that the change in growth conditions caused synchronized changes in the transcript levels of HKII and GLUT1 in AH130 cells. However, such marked changes in the transcript levels of HKII and GLUT1 were not observed when AH130 cells were cultured in dishes under a hypoxic condition, indicating that the observed changes were not solely attributable to the difference in oxygen concentration between the ascites and cell culture conditions. Accordingly, other factors such as growth factors may be responsible for this difference in levels of HKII and GLUT1 between the two growth conditions.
Keywords: Hexokinase; Glucose transporter; Northern analysis; Glucose metabolism; AH130;

Increased uncoupling protein-2 gene expression in brain of lipopolysaccharide-injected mice: role of tumour necrosis factor-α? by Sı́lvia Busquets; Belén Alvarez; Martin Van Royen; Maria Teresa Figueras; Francisco J López-Soriano; Josep M Argilés (249-256).
In order to understand the role of brain localized uncoupling proteins, we have examined the UCP2 and BMCP-1 gene expression in mice brain in two different catabolic states: administration of lipopolysaccharide (LPS) (2.5 mg/kg, i.p.) and tumour burden. Administration of LPS resulted in an increased UCP2 gene expression both in brain (208%) and cerebellum (77%). An increase in UCP2 gene expression was also observed after LPS treatment in double knockout mice for tumour necrosis factor-α (TNF) receptors 1 and 2 (75% in brain and 33% in cerebellum). Tumour growth also resulted in increased brain UCP2 gene expression (80%) in mice bearing the Lewis lung carcinoma as compared with the non-tumour-bearing controls. No changes were observed in BMCP-1 mRNA levels of either LPS-injected or tumour-bearing mice. From the results presented it may be suggested that: (a) the brain may contribute significantly to the increase in energy expenditure associated with hypermetabolic states such as fever and tumour burden, and (b) the regulation of UCP2 gene expression in brain does not seem to be influenced by TNF; therefore the action of other cytokines cannot be discarded.
Keywords: Lipopolysaccharide; Brain; Tumour necrosis factor-α; Uncoupling protein-2; Brain mitochondrial carrier protein-1; Tumour;

N1E-115 mouse neuroblastoma cells express mt1 melatonin receptors and produce neurites in response to melatonin by Shannon L. Bordt; Raelene M. McKeon; Pui-Kai Li; Paula A. Witt-Enderby; Melissa A. Melan (257-264).
Melatonin, a pineal hormone that induces sleep, has become a popular over-the-counter drug. The cellular effects of melatonin, however, are only beginning to be studied. We have recently shown that stimulation of the mt1 melatonin receptor induces rapid and dramatic cytoskeletal rearrangements in transformed non-neuronal cells (Witt-Enderby et al., Cell. Motil. Cytoskel. 46 (2000) 28). These cytoskeletal changes result in the formation of structures that closely resemble neurites. In this work, we show that the N1E-115 mouse neuroblastoma cell line rapidly responds to melatonin stimulation and forms neurites within 24 h. We also demonstrate that these cells readily bind 2-[125I]iodomelatonin at levels consistent with what is noted for native tissues (B max=3.43±1.56 fmol/mg protein; K d=240 pM). Western analysis shows that these cells possess and express melatonin receptors of the mt1 subtype. Treatment with pertussis toxin eliminates neurite formation whereas treatment with the MT2 subtype-specific activator, BMNEP, does not induce neurite formation. We have previously shown that increases in MEK 1/2 and ERK 1/2 phosphorylation are correlated with the shape changes in transformed CHO cells. Western analysis of the MEK/ERK signaling pathway in N1E-115 cells shows that this pathway is most likely maximally and constitutively stimulated. This may account for the spontaneous production of neurites noted for this cell line after long culture periods. The results of this work show that melatonin receptor stimulation in a neuronal cell type results in the formation of neurites and that the receptors responsible for melatonin-induced neurite formation in N1E-115 cells are most likely of the mt1 subtype.
Keywords: Melatonin; mt1 receptor; N1E-115 cell; Neurite; Neuronal development; Signal transduction;

Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process by Rafat A. Siddiqui; Laura J. Jenski; Kristiana Neff; Kevin Harvey; Richard J. Kovacs; William Stillwell (265-275).
Docosahexaenoic acid (DHA) is an omega-3 fatty acid under intense investigation for its ability to modulate cancer cell growth and survival. This research was performed to study the cellular and molecular effects of DHA. Our experiments indicated that the treatment of Jurkat cells with DHA inhibited their survival, whereas similar concentrations (60 and 90 μM) of arachidonic acid and oleic acid had little effect. To explore the mechanism of inhibition, we used several measures of apoptosis to determine whether this process was involved in DHA-induced cell death in Jurkat cells. Caspase-3, an important cytosolic downstream regulator of apoptosis, is activated by death signals through proteolytic cleavage. Incubation of Jurkat cells with 60 and 90 μM DHA caused proteolysis of caspase-3 within 48 and 24 h, respectively. DHA treatment also caused the degradation of poly-ADP-ribose polymerase and DNA fragmentation as assayed by flow cytometric TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. These results indicate that DHA induces apoptosis in Jurkat leukemic cells. DHA-induced apoptosis was effectively inhibited by tautomycin and cypermethrin at concentrations that affect protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) activities, respectively, implying a role for these phosphatases in the apoptotic pathway. Okadaic acid, an inhibitor of protein phosphatase 2A, had no effect on DHA-induced apoptosis. These results suggest that one mechanism through which DHA may control cancer cell growth is through apoptosis involving PP1/PP2B protein phosphatase activities.
Keywords: Docosahexaenoic acid; Jurkat cell; Apoptosis; Caspase-3; Protein phosphatase;