Synthesis, characterization, and biological affinity of a near-infrared-emitting conjugated oligoelectrolyte.

A near-IR-emitting conjugated oligoelectrolyte (COE), ZCOE, was synthesized, and its photophysical features were characterized. The biological affinity of ZCOE is compared to that of an established lipid-membrane-intercalating COE, DSSN+, which has blue-shifted optical properties making it compatible for tracking preferential sites of accumulation. ZCOE exhibits diffuse staining of E. coli cells, whereas it displays internal staining of select yeast cells which also show propidium iodide staining, indicating ZCOE is a "dead" stain for this organism. Staining of mammalian cells reveals complete internalization of ZCOE through endocytosis, as supported by colocalization with LysoTracker and late endosome markers. In all cases DSSN+ persists in the outer membranes, most likely due to its chemical structure more closely resembling a lipid bilayer.

Insights into the arginine paradox: evidence against the importance of subcellular location of arginase and eNOS.

Reduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes overt cardiovascular disease. Increased expression of Arginase has been proposed as a mechanism to account for diminished NO production. Arginases consume l-arginine, the substrate for endothelial nitric oxide synthase (eNOS), and l-arginine depletion is thought to competitively reduce eNOS-derived NO. However, this simple relationship is complicated by the paradox that l-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. One mechanism proposed to explain this is compartmentalization of intracellular l-arginine into distinct, poorly interchangeable pools. In the current study, we investigated this concept by targeting eNOS and Arginase to different intracellular locations within COS-7 cells and also BAEC. We found that supplemental l-arginine and l-citrulline dose-dependently increased NO production in a manner independent of the intracellular location of eNOS. Cytosolic arginase I and mitochondrial arginase II reduced eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting arginase I to disparate regions of the cell did not differentially modify eNOS activity. Arginase-dependent suppression of eNOS activity was reversed by pharmacological inhibitors and absent in a catalytically inactive mutant. Arginase did not directly interact with eNOS, and the metabolic products of arginase or downstream enzymes did not contribute to eNOS inhibition. Cells expressing arginase had significantly lower levels of intracellular l-arginine and higher levels of ornithine. These results suggest that arginases inhibit eNOS activity by depletion of substrate and that the compartmentalization of l-arginine does not play a major role.

Functional effects of cytoskeletal components on the lateral movement of individual BKCa channels expressed in live COS-7 cell membrane.

The lateral diffusion of BK(Ca) channels was previously shown to be highly 'confined' in the COS-7 cell membrane. Here we report that the diffusion coefficient and the confinement area of BK(Ca) channel were significantly increased by the treatment of latrunculin A, an actin-depolymerizing agent, but not by microtubule disruption. Site-directed mutational analyses further demonstrated that a single leucine residue in the C-terminal actin-binding motif was critical for the aforementioned effects of latrunculin A. We conclude that some BK(Ca) channels are directly associated with actin filaments and their lateral mobility can be restricted by the cytoskeletal components.

Spontaneous [Ca2+]i oscillations in G1/S phase-synchronized cells.

Ca(2+) signaling controls a wide range of cellular functions such as division, fertilization, apoptosis and necrosis. Specifically, calcium signaling is thought to play a crucial role in driving cells through the different stages of the cell-division cycle. In most cells, however, this fact is far from being established. Few studies have examined this question from a different perspective: whether cells exhibit some characteristic cell cycle-dependent intracellular calcium-signaling patterns. This approach is effective in discerning the causal relationship between Ca(2+) signaling and the cell cycle. Through synchronization of the cell cycle, flow cytometry and confocal scanning microscopic intracellular calcium ion concentration ([Ca(2+)](i)) imaging, the present study shows that the G1/S phase transition is uniquely characterized by spontaneous [Ca(2+)](i) oscillations that last for up to 40 min. Most likely, these oscillations emanate from the [Ca(2+)](i) signaling that accompanies DNA replication as the cell prepares for the next division cycle. These temporal signals further affirm the significance of Ca(2+) in the cell cycle.

PIAS1 mediates TGFbeta-induced SM alpha-actin gene expression through inhibition of KLF4 function-expression by protein sumoylation.

OBJECTIVE: TGFbeta and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We have previously shown that the protein inhibitor of activated STAT (PIAS)1 activates expression of SMC differentiation marker genes including smooth muscle (SM) alpha-actin by interacting with serum response factor (SRF) and class I bHLH proteins. Here, we tested the hypothesis that TGFbeta activates SM alpha-actin through PIAS1. METHODS AND RESULTS: An siRNA specific for PIAS1 and ubc9, an E2-ligase for sumoylation, inhibited TGFbeta-induced expression of SM alpha-actin in cultured SMCs as determined by real-time RT-PCR. Overexpression of PIAS1 increased SM alpha-actin promoter activity in a TGFbeta control element (TCE)-dependent manner. Because the TCE within the SM alpha-actin promoter could mediate repression through interaction with KLF4, we tested whether PIAS1 regulates the function of KLF4 for SMC gene expression. PIAS1 interacted with KLF4 in mammalian two-hybrid and coimmunoprecipitation assays, and overexpression of PIAS1 inhibited KLF4-repression of SM alpha-actin promoter activity. Moreover, PIAS1 promoted degradation of KLF4 through sumoylation. CONCLUSIONS: These results provide evidence that PIAS1 promotes TGFbeta-induced activation of SM alpha-actin gene expression at least in part by promoting sumoylation and degradation of the TCE repressor protein, KLF4.

The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan.

OBJECTIVE: A dramatic loss of aggrecan proteoglycan from cartilage is associated with osteoarthritis. The fate of residual G1 domains of aggrecan is unknown, but inefficient turnover of these domains may impede subsequent repair and retention of newly synthesized aggrecan. Thus, the objective of this study was to determine whether ITEGE- and DIPEN-containing G1 domains, generated in situ, are internalized by articular chondrocytes, and whether these events are dependent on hyaluronan (HA) and its receptor, CD44. METHODS: ITEGE and DIPEN neoepitopes were detected by immunofluorescence staining of bovine articular cartilage chondrocytes treated with or without interleukin-1alpha (IL-1alpha). Additionally, purified ITEGE- or DIPEN-containing G1 domains were aggregated with HA and then added to articular chondrocytes, articular chondrocytes transfected with CD44delta67, or COS-7 cells transfected with or without full-length CD44. Internalized epitopes were distinguished by their resistance to extensive trypsinization of the cell surface. RESULTS: Both ITEGE and DIPEN were visualized within the extracellular cell-associated matrix of chondrocytes as well as within intracellular vesicles. Following trypsinization, the intracellular accumulation of both epitopes was clearly visible. IL-1 treatment increased extracellular as well as intracellular ITEGE epitope accumulation. Once internalized, the ITEGE neoepitope became localized within the nucleus and displayed little colocalization with HA, DIPEN, or other G1 domain epitopes. The internalization of both ITEGE and DIPEN G1 domains was dependent on the presence of HA and CD44. CONCLUSION: One important mechanism for the elimination of residual G1 domains following extracellular degradation of aggrecan is CD44-mediated co-internalization with HA.

Binding of PLCdelta1PH-GFP to PtdIns(4,5)P2 prevents inhibition of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 by neomycin.

AIM: To investigate the effects of the pleckstrin homology (PH) domain of phospholipase C(delta1) (PLC(delta1)PH) on inhibition of phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by neomycin. METHODS: A fusion construct of green fluorescent protein (GFP) and PLC (delta1)PH (PLC(delta1)PH-GFP), which is known to bind PtdIns(4,5)P2 specifically, together with laser-scanning confocal microscopy, was used to trace PtdIns(4,5)P2 translocation. RESULTS: Stimulation of the type 1 muscarinic receptor and the bradykinin 2 receptor induced a reversible PLC(delta1)PH-GFP translocation from the membrane to the cytosol in COS-7 cells. PLC inhibitor U73122 blocked the translocation. Wortmannin, a known PtdIns kinase inhibitor, did not affect the translocation induced by ACh, but blocked recovery after translocation, indicating that PtdIns(4,5)P2 hydrolysis occurs through receptor-mediated PLC activation. Neomycin, a commonly used phospholipase C blocker, failed to block the receptor-induced PLCd1PH-GFP translocation, indicating that neomycin is unable to block PLC-mediated PtdIns(4,5)P2 hydrolysis. However, in the absence of PLCd1PH-GFP expression, neomycin abolished the receptor-induced hydrolysis of PtdIns(4,5)P2 by PLC. CONCLUSION: Although PLCd1PH and neomycin bind to PtdIns(4,5)P2 in a similar way, they have distinct effects on receptor-mediated activation of PLC and PtdIns(4,5)P2 hydrolysis.

Involvement of filamin A and filamin A-interacting protein (FILIP) in controlling the start and cell shape of radially migrating cortical neurons.

Precisely regulated radial cell migration out of the ventricular zone is essential for corticogenesis. However, molecular mechanisms controlling the start of migration and the dynamics of migrating cell shape remain elusive. Here, we show novel mechanisms that can tether ventricular zone cells and control migrating cell shape. The novel protein Filamin A-interacting protein (FILIP) interacts with Filamin A, an indispensable actin-binding protein for cell motility, and induces its degradation in COS-7 cells. Degradation of Filamin A is indicated in the cortical ventricular zone where FILIP mRNA localizes. Furthermore, most ventricular zone cells that overexpress FILIP fail to migrate in explants. These results indicate that FILIP acts through a Filamin A-F-actin axis to control the start of neocortical cell migration from the ventricular zone. Filamin A also determines the shape of migrating neocortical neurons, which show global morphological changes and complicated behavior during that migration. Dysfunction of Filamin A, caused by a mutant Filamin A expression, prevents cells from acquiring consistent polarity toward specific direction and decreases motility in the subventricular and intermediate zones. In contrast, Filamin A overexpression, achieved by a short interfering RNA for FILIP, promotes the development and maintenance of a bipolar shape also in the subventricular and intermediate zones. These results suggest that the amount of Filamin A helps migrating neurons determine their mode of migration, multipolar or bipolar, prior to entering the cortical plate and that FILIP is responsible, at least in part, for the Filamin A content of migrating neurons.

Visualization of glucocorticoid receptor and mineralocorticoid receptor interactions in living cells with GFP-based fluorescence resonance energy transfer.

Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, including stress responses in the target neural cells via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). It has been shown that the GR and MR are highly colocalized in the hippocampus. Given the differential action of the MR and GR in the hippocampal region, it is important to elucidate how these receptors interact with each other in response to corticosteroids. We investigated the heterodimerization of the MR and GR with green fluorescent protein-based fluorescence resonance energy transfer (FRET) microscopy in living cells with spatiotemporal manner. FRET was evaluated in three ways: (1) ratio imaging; (2) emission spectra; and (3) acceptor photobleaching. FRET analysis demonstrated that cyan fluorescent protein-GR and yellow fluorescent protein-MR form heterodimers after corticosterone (CORT) treatment both in the nucleus of cultured hippocampal neurons and COS-1 cells, whereas they do not form heterodimers in the cytoplasm. The content of the GR-MR heterodimer was higher at 10(-6) m CORT than at 10(-9) m CORT and reached a maximum level after 60 min of CORT treatment in both cultured hippocampal neurons and COS-1 cells. The distribution pattern of heterodimers in the nucleus of cultured hippocampal neurons was more restricted than that in COS-1 cells. The present study using mutant fusion proteins in nuclear localization signal showed that these corticosteroid receptors are not translocated into the nucleus in the form of heterodimers even after treatment with ligand and thus allow no heterodimerization to take place in the cytoplasm. These results obtained with FRET analyses give new insights into the sites, time course, and effects of ligand concentration on heterodimersization of the GR and MR.

APECED-causing mutations in AIRE reveal the functional domains of the protein.

A defective form of the AIRE protein causes autoimmune destruction of target organs by disturbing the immunological tolerance of patients with a rare monogenic disease, autoimmune polyendocrinopathy (APE)-candidiasis (C)-ectodermal dystrophy (ED), APECED. Recently, experiments on knockout mice revealed that AIRE controls autoimmunity by regulating the transcription of peripheral tissue-restricted antigens in thymic medullary epithelial cells. Thus, AIRE provides a unique model for molecular studies of organ-specific autoimmunity. In order to analyze the molecular and cellular consequences of 16 disease-causing mutations in vitro, we studied the subcellular localization, transactivation capacity, homomultimerization, and complex formation of several mutant AIRE polypeptides. Most of the mutations altered the nucleus-cytoplasm distribution of AIRE and disturbed its association with nuclear dots and cytoplasmic filaments. While the PHD zinc fingers were necessary for the transactivation capacity of AIRE, other regions of AIRE also modulated this function. Consequently, most of the mutations decreased transactivation. The HSR domain was responsible for the homomultimerization activity of AIRE; all the missense mutations of the HSR and the SAND domains decreased this activity, but those in other domains did not. The AIRE protein was present in soluble high-molecular-weight complexes. Mutations in the HSR domain and deletion of PHD zinc fingers disturbed the formation of these complexes. In conclusion, we propose an in vitro model in which AIRE transactivates transcription through heteromeric molecular interactions that are regulated by homomultimerization and conditional localization of AIRE in the nucleus or in the cytoplasm.

Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-dependent prolonged GAB1 binding and sustained ERK2/MAPK1 activation.

Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation.

The steroidal analog GW707 activates the SREBP pathway through disruption of intracellular cholesterol trafficking.

Recently, a new class of lipid-lowering agents has been described that upregulate LDL receptor (LDLr) activity. These agents are proposed to activate sterol-regulated gene expression through binding to the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). Here, we show that the steroidal LDLr upregulator, GW707, induces accumulation of lysosomal free cholesterol and inhibits LDL-stimulated cholesterol esterification, similar to that observed in U18666A-treated cells and in Niemann-Pick type C1 (NPC1) mutants. Moreover, we demonstrate that induction of the NPC-like phenotype by GW707 is independent of SCAP function. We find that treatment with GW707 does not increase SREBP-dependent gene expression above that observed in lipoprotein-starved cells. Rather, we show that the apparent increase in SREBP-dependent activity in GW707-treated cells is attributable to a failure to appropriately suppress sterol-regulated gene expression, as has been shown previously for U18666A-treated cells and NPC mutant fibroblasts. We further demonstrate that cells treated with either GW707 or U18666A fail to appropriately generate 27-hydroxycholesterol in response to LDL cholesterol. Taken together, these findings support a mechanism in which GW707 exerts its hypolipidemic effects through disruption of late endosomal/lysosomal sterol trafficking and subsequent stimulation of LDLr activity.

Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca2+-ATPase.

On the basis of sequence similarities to the yeast PMR1 and hSPCA gene, the rat alternatively spliced mRNA has been suggested to be a Golgi secretory pathway Ca2+-ATPase (SPCA). Data in this report lend further support for this hypothesis in that sucrose gradient fractionation of rat liver microsomes resulted in SPCA comigrating with the Golgi calcium binding protein CALNUC, which was well resolved from the endoplasmic reticulum marker calreticulin. Also, in PC-12 cells, antibody to SPCA colocalized with an antibody to the Golgi marker alpha-mannosidase II. To study the biological effects of SPCA expression, we performed stable overexpression of SPCA in COS-7 cells. Seven clones were selected for further comparison with COS-7 cells containing an empty expression vector. Overexpression of SPCA resulted in a significant reduction of plasma membrane Ca2+-ATPase, sarco(endo)plasmic reticulum Ca2+-ATPase, and calreticulin expression in these clones. In contrast, the expression of the Golgi calcium-binding protein CALNUC increased significantly. The phosphoenzyme intermediate formed using membranes from clone G11/5 was calcium dependent, significantly more intense than in COS-7 cells, and not affected by La3+ treatment. Calcium uptake by G11/5 microsomes was ATP dependent and significantly greater than in microsomes from parent COS-7 cells. The overexpression of SPCA significantly increased the growth rate of these cells compared with COS-7 cells containing only the empty vector. These data demonstrate that overexpression of the rat SPCA results in significant changes in the expression of calcium transport and storage proteins in COS-7 cells.

Interaction of 14-3-3 proteins with the insulin-like growth factor I receptor (IGFIR): evidence for a role of 14-3-3 proteins in IGFIR signaling.

We have extended our previous yeast two-hybrid findings to show that 14-3-3beta also interacts with the insulin-like growth factor I receptor (IGFIR) in mammalian cells overexpressing both proteins and that the interaction involves serine 1283 and is dependent on receptor activation. Treatment of cells with the phorbol ester PMA stimulates the interaction of 14-3-3beta with the IGFIR in the absence of receptor tyrosine phosphorylation, suggesting that receptor activation leads to activation of an endogenous protein kinase that catalyzes the phosphorylation of serine 1283. To investigate the role of 14-3-3 proteins in IGF signal transduction, IGFIR structure-function studies were performed. Mutation of serine 1283 alone (S1283A) (a mutation that decreases but does not abolish the interaction of the IGFIR with 14-3-3) did not affect anchorage-independent growth of NIH 3T3 fibroblasts overexpressing the mutant receptor. However, the simultaneous mutation of this residue and the truncation of the C-terminal 27 residues of the receptor (Delta1310/S1283A) abolished the interaction of the receptor with 14-3-3 and reversed the enhanced colony formation observed with the IGFIR truncation mutation alone (Delta1310). The difference between the Delta1310 and Delta1310/S1283A transfectants in the soft agar assay was confirmed by tumorigenesis experiments. These findings suggest that 14-3-3 proteins interact with the IGFIR in vivo and that this interaction may play a role in a transformation pathway signaled by the IGFIR.

VACM-1, a cul-5 gene, inhibits cellular growth by a mechanism that involves MAPK and p53 signaling pathways.

Vasopressin-activated Ca2+-mobilizing (VACM)-1 gene product is a 780-amino acid membrane protein that shares sequence homology with cullins, a family of genes involved in the regulation of cell cycle. However, when expressed in vitro, VACM-1 attenuates basal and vasopressin- and forskolin-induced cAMP production. Mutating the PKA-dependent phosphorylation site in the VACM-1 sequence (S730AVACM-1) prevents this inhibitory effect. To further examine the biological role of VACM-1, we studied the effect of VACM-1 and S730AVACM-1 proteins on cellular proliferation and gene expression in Chinese hamster ovary and COS-1 cells. Cellular proliferation of VACM-1-expressing cell lines was significantly lower compared with that of the vector-transfected cells, whereas it was significantly increased in S730AVACM-1-derived cell lines. Furthermore, expression of VACM-1 but not S730AVACM-1 protein retarded cytokinesis and prevented MAPK phosphorylation. Screening with the Human PathwayFinder-1 GEArray system and subsequent Western blot analysis demonstrated that VACM-1 induces p53 mRNA and protein expression. In summary, VACM-1 inhibits cellular growth by a mechanism that involves cAMP, MAPK phosphorylation, and p53 expression.

Activation of PPARalpha and PPARgamma by environmental phthalate monoesters.

Phthalate esters are widely used as plasticizers in the manufacture of products made of polyvinyl chloride. Mono-(2-ethylhexyl)-phthalate (MEHP) induces rodent hepatocarcinogenesis by a mechanism that involves activation of the nuclear transcription factor peroxisome proliferator-activated receptor-alpha (PPARalpha). MEHP also activates PPAR-gamma (PPARgamma), which contributes to adipocyte differentiation and insulin sensitization. Human exposure to other phthalate monoesters, including metabolites of di-n-butyl phthalate and butyl benzyl phthalate, is substantially higher than that of MEHP, prompting this investigation of their potential for PPAR activation, assayed in COS cells and in PPAR-responsive liver (PPARalpha) and adipocyte (PPARgamma) cell lines. Monobenzyl phthalate (MBzP) and mono-sec-butyl phthalate (MBuP) both increased the COS cell transcriptional activity of mouse PPARalpha, with effective concentration for half-maximal response (EC50) values of 21 and 63 microM, respectively. MBzP also activated human PPARalpha (EC50=30 microM) and mouse and human PPARgamma (EC50=75-100 microM). MEHP was a more potent PPAR activator than MBzP or MBuP, with mouse PPARalpha more sensitive to MEHP (EC50=0.6 microM) than human PPARalpha (EC50=3.2 microM). MEHP activation of PPARgamma required somewhat higher concentrations, EC50=10.1 microM (mouse PPARgamma) and 6.2 microM (human PPARgamma). No significant PPAR activation was observed with the monomethyl, mono-n-butyl, dimethyl, or diethyl esters of phthalic acid. PPARalpha activation was verified in FAO rat liver cells stably transfected with PPARalpha, where expression of several endogenous PPARalpha target genes was induced by MBzP, MBuP, and MEHP. Similarly, activation of endogenous PPARgamma target genes was evidenced for all three phthalates by the stimulation of PPARgamma-dependent adipogenesis in the 3T3-L1 cell differentiation model. These findings demonstrate the potential of environmental phthalate monoesters for activation of rodent and human PPARs and may help to elucidate the molecular basis for the adverse health effects proposed to be associated with human phthalate exposure.

Expression of a fusion protein of human ciliary neurotrophic factor and soluble CNTF-receptor and identification of its activity.

Ciliary neurotrophic factor (CNTF) has pleiotropic actions on many neuronal populations as well as on glia. Signal transduction by CNTF requires that it bind first to CNTF-R, permitting the recruitment of gp130 and LIF-R, forming a tripartite receptor complex. Cells that only express gp130 and LIF-R, but not CNTF-R are refractory to stimulation by CNTF. On many target cells CNTF only acts in the presence of its specific agonistic soluble receptors. We engineered a soluble fusion protein by linking the COOH-terminus of sCNTF-R to the NH2-terminus of CNTF. Recombinant CNTF/sCNTF-R fusion protein (Hyper-CNTF) was successfully expressed in COS-7 cells. The apparent molecular mass of the Hyper-CNTF protein was estimated from western blots to be 75 kDa. Proliferation assays of transfected BAF/3 cells in response to CNTF and Hyper-CNTF were used to verify the activity of the cytokines. The proliferative results confirmed that CNTF required homodimerization of the gp130, CNTF-R and LIF-R receptor subunit whereas Hyper-CNTF required heterodimerization of the gp130 and LIF-R receptor subunit. We concluded that the fusion protein Hyper-CNTF had superagonistic activity on target cells expressing gp130 and LIF-R, but lacking membrane-bound CNTF-R.

A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants.

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu(222)-->Gly (E222G) substitution, which removed the evolutionarily invariant Glu(222). Re-introduction of Glu(222) into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of 'humanized' aceGFP and beta-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

Direct binding of a fragment of the Wiskott-Aldrich syndrome protein to the C-terminal end of the anaphylatoxin C5a receptor.

Migration of myeloid cells towards a source of chemoattractant, such as the C5a anaphylatoxin, is triggered by the activation of a G-protein-coupled receptor. In the present study, we have used a yeast two-hybrid approach to find unknown partners of the C5a receptor (C5aR). Using the cytosolic C-terminal region of C5aR as bait to screen a human leucocyte cDNA library, we identified the Wiskott-Aldrich syndrome protein (WASP) as a potential partner of C5aR. WASP is known to have an essential function in regulating actin dynamics at the cell leading edge. The interaction was detected with both the fragment of WASP containing amino acids 1-321 (WASP.321) and WASP with its actin-nucleation-promoting domain [verprolin-like, central and acidic (VCA) domain] deleted. The interaction between C5aR and the WASP.321 was supported further by an in vitro binding assay between a radiolabelled WASP.321 fragment and a receptor C-terminus glutathione S-transferase (GST) fusion protein, as well as by GST pull-down, co-immunoprecipitation and immunofluorescence experiments. In the yeast two-hybrid assay, full-length WASP showed no ability to interact with the C-terminal domain of C5aR. This is most probably due to an auto-inhibited conformation imposed by the VCA domain. In HEK-293T cells co-transfected with full-length WASP and C5aR, only a small amount of WASP was co-precipitated with the receptor. However, in the presence of the active form of the GTPase Cdc42 (Cdc42V12), which is thought to switch WASP to an active 'open conformation', the amount of WASP associated with the receptor was markedly increased. We hypothesize that a transient interaction between C5aR and WASP occurs following the stimulation of C5aR and Cdc42 activation. This might be one mechanism by which WASP is targeted to the plasma membrane and by which actin assembly is spatially controlled in cells moving in a gradient of C5a.

Sustained high-yield production of recombinant proteins in transiently transfected COS-7 cells grown on trimethylamine-coated (hillex) microcarrier beads.

The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.