Transfected CV-1 Cells Research Articles

Examination Of The Kinesin-1 Tail Interaction With Microtubules

It is well known that the kinesin-1 tail domain contains a second microtubule binding-site independent of the binding-site located in the head domain (1,2), but the affinity and location of the tail-microtubule interaction on tubulin is not known. We have used fluorescence anisotropy to measure a Kd of kinesin-1 tail for microtubules in the submicromolar range, and we are currently performing experiments to determine the specific tubulin residues involved in forming this interaction. We hypothesize that the tail binding-site will include the extreme C-terminus of tubulin, which we will test by measuring the affinity of tail for tubulin with its C-terminal residues cleaved. Any effects that kinesin head domains or select microtubule-associated proteins may have on the affinity of the tail for microtubules will also be analyzed by fluorescence anisotropy, and the possibility for the tail to inhibit the ATPase activity of the head while bound to microtubules will be tested with an enzyme-coupled ATPase assay. These studies will test the hypothesis that the kinesin tail can fold over and simultaneously contact both the head domain and microtubules, producing a state in which both the ATPase activity of the head domain is inhibited and the kinesin molecule is anchored to the microtubule via its tail domain, as proposed by Dietrich et al (2).1) Navone F, Niclas J, Hom-Booher N, Sparks L, Bernstein HD, McCaffrey G, Vale RD. Cloning and expression of a human kinesin heavy chain gene: Interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells. J. Cell Bio. 117(6), 1263-1275 (1992).2) Dietrich KA, Sindelar CV, Brewer P, Downing KH, Cremo CR, Rice SE. The kineisn-1 motor protein is regulated by a direct interaction of its head and tail. Proc. Nat. Ac. Sci. 105(26), 8938-8943 (2008).

Molecular investigation of the functional relevance of missense variants of ICAM-1

In genome-wide studies, the intercellular adhesion molecule-1 (ICAM-1) locus has been associated with cardiovascular and inflammatory bowel diseases. To determine the functional relevance of five missense ICAM-1 variants (G241R; I316V; P352L; K469E; R478W), we generated wild-type and variant proteins [M2(241R); M3(469E); M4(352L); M5(478W); M6(316V); M7(352L/469E)] and transiently transfected CV1 cells. Reverse transcription PCR, western blot, and ELISA did not reveal any differences in mRNA and protein expression levels for any construct. Conversely, in pulse-chase experiments, compared with wild-type (90-120 min), M3 and M5 possessed a prolonged half-life of approximately 150 min, whereas M2, M4, and M7 displayed a decreased half-life of approximately 60-75 min, implying differences in protein degradation. Our results do not indicate a major impact of missense variants on ICAM-1 biological function, even if G241R and K469E were functional in pulse-chase experiments. Whether these differences in protein stability exert measurable functional consequences needs to be elucidated further.

Alkyl Chain Moieties of Polyamidoamine Dendron-Bearing Lipids Influence Their Function as a Nonviral Gene Vector

We recently developed a novel family of cationic lipids consisting of a polyamidoamine (PAMAM) dendron and two dodecyl chains. Their transfection activity increases with increasing generation of the dendron moiety [Takahashi et al. (2003) Bioconjugate Chem. 14, 764-773]. In the present study, to elucidate the effect of hydrophobic tail moieties of the dendron-bearing lipids, two kinds of PAMAM G3 dendron-bearing lipids were synthesized with different alkyl lengths, DL-G3-2C18 and DL-G3-2C12. Their functions as gene vectors were compared. Irrespective of their different alkyl chain lengths, these dendron-bearing lipids formed complexes with plasmid DNA with similar efficiency. However, their complex sizes differed markedly: DL-G3-2C18 lipoplexes exhibited much smaller diameters than DL-G3-2C12 lipoplexes. Interaction of the lipoplexes with heparin revealed that the DL-G3-2C18 lipoplexes required more heparin than DL-G3-2C12 lipoplexes to cause dissociation of plasmid DNA from the lipoplexes. Although the DL-G3-2C12 lipoplexes and DL-G3-2C18 lipoplexes transfected CV1 cells with similar efficiency in the absence of serum, only the latter retained high transfection activity in the presence of serum. These results indicate that hydrophobic interaction of alkyl chain moieties plays an important role in the increment of stability and the serum-resistant transfection activity for dendron-bearing lipid lipoplexes.

Alternative splice variants of phospholipase C-β2 are expressed in platelets: Effect on Gαq-dependent activation and localization

Phospholipase C (PLC) β2 plays a pivotal role in G-protein dependent signal transduction in platelets. We have previously demonstrated in platelets, leukocytes and human erythroleukemia cells the presence of transcripts of two forms of PLC-β2 generated by alternative splicing. They differ by 45 nucleotides in the carboxyl-terminal region and are designated as PLC-β2a and PLC-β2b, with and without by 15 amino acid residues (corresponding to 864–878). The presence of the two variants has not been shown at the protein level in cells. Moreover, the carboxy-terminal region of PLC-β has been implicated in Gαq activation, particulate association, and nuclear localization, suggesting that the PLC-β2 splice variants may be regulated differentially. We demonstrate for the first time that both PLC-β2 isoforms are expressed in platelets at the protein level. Studies in CV-1 cells transfected with PLC-β2a or β2b cDNAs, along with constitutively activated Gαq (Q209L), showed that inositolphosphate formation was comparable between the two variants. However, the nuclear localization of the two isoforms was different with a higher cytoplasmic to nuclear ratio for PLC-β2b compared to PLC-β2a, suggesting that a great proportion of the total PLC-β2a was in the nucleus relative to PLC-β2b. There was no difference in the relative distribution of the two variants between the cytosol and particulate fractions. Both PLC-β2 alternative splice variants are expressed at the protein level in platelets. In transfected CV-1 cells, PLC-β2a is relatively more enriched in the nuclei than PLC-β2b suggesting that the two variants may have different effects in cell proliferation and differentiation.

Ligand-Controlled Interaction of Histone Acetyltransferase Binding to ORC-1 (HBO1) with the N-Terminal Transactivating Domain of Progesterone Receptor Induces Steroid Receptor Coactivator 1-Dependent Coactivation of Transcription

Modulators of cofactor recruitment by nuclear receptors are expected to play an important role in the coordination of hormone-induced transactivation processes. To identify such factors interacting with the N-terminal domain (NTD) of the progesterone receptor (PR), we used this domain as bait in the yeast Sos-Ras two-hybrid system. cDNAs encoding the C-terminal MYST (MOZ-Ybf2/Sas3-Sas2-Tip60 acetyltransferases) domain of HBO1 [histone acetyltransferase binding to the origin recognition complex (ORC) 1 subunit], a member of the MYST acetylase family, were thus selected from a human testis cDNA library. In transiently transfected CV1 cells, the wild-type HBO1 [611 amino acids (aa)] enhanced transcription mediated by steroid receptors, notably PR, mineralocorticoid receptor, and glucocorticoid receptor, and strongly induced PR and estrogen receptor coactivation by steroid receptor coactivator 1a (SRC-1a). As assessed by two-hybrid and glutathione-S-transferase pull-down assays, the HBO1 MYST acetylase domain (aa 340-611) interacts mainly with the NTD, and also contacts the DNA-binding domain and the hinge domains of hormone-bound PR. The HBO1 N-terminal region (aa 1-340) associates additionally with PR ligand-binding domain (LBD). HBO1 was found also to interact through its NTD with SRC-1a in the absence of steroid receptor. The latter coassociation enhanced specifically activation function 2 activation function encompassed in the LBD. Conversely, the MYST acetylase domain specifically enhanced SRC-1 coupling with PR NTD, through a hormone-dependent mechanism. In human embryonic kidney 293 cells expressing human PRA or PRB, HBO1 raised selectively an SRC-1-dependent response of PRB but failed to regulate PRA activity. We show that HBO1 acts through modification of an LBD-controlled structure present in the N terminus of PRB leading to the modulation of SRC-1 functional coupling with activation function 3-mediated transcription. Importantly, real-time RT-PCR analysis also revealed that HBO1 enhanced SRC-1 coactivation of PR-dependent transcription of human endogenous genes such as alpha-6 integrin and 11beta-hydroxydehydrogenase 2 but not that of amphiregulin. Immunofluorescence and confocal microscopy of human embryonic kidney-PRB cells demonstrated that the hormone induces the colocalization of HBO1 with PR-SRC-1 complex into nuclear speckles characteristic of PR-mediated chromatin remodeling. Our results suggest that HBO1 might play an important physiological role in human PR signaling.

Functional analyses of an LXXLL motif in nuclear receptor corepressor (N-CoR)

Transcriptional repression is a major regulatory mechanism in cell differentiation, organogenesis, and oncogenesis. Two repressors of ligand-dependent transcription factors, nuclear receptor corepressor (N-CoR) and the related protein SMRT were identified as a silencing mediator for thyroid hormone receptor β and as a silencing mediator for retinoic acid and thyroid hormone receptors, respectively. Nuclear receptor coactivators such as steroid receptor coactivator-1 (SRC-1) contain multiple LXXLL motifs, which are essential and sufficient for its ligand-dependent interaction with nuclear receptors. N-CoR also has an LXXLL motif, located between repressor domains 1 and 2, and conserved between mouse and man. In contrast, SMRT lacks this motif. This paper describes functional implications of the LXXLL motif in N-CoR. A 57-amino acid portion of N-CoR containing the LDNLL sequence (N-CoR LDNLL) fused to GST interacted with retinoic acid receptor α (RARα) and thyroid hormone receptor β (TRβ) in vitro. Similarly, [ 35S-methionine]N-CoR LDNLL interacted with a RARα fusion protein. N-CoR LDNLL also bound to RARα in vivo as determined in mammalian one-hybrid system in transfected CV-1 cells and by two-hybrid assays in bacteria. The interaction with RARα in vitro and in vivo was specific as determined by mutation of the sequence LDNLL to LDNAA. Our data suggest that the LDNLL motif in N-CoR has functional significance because it mediates interaction with nuclear receptors such as RARα and TRβ.

Heparin cofactor II is more sensitive than antithrombin to secretory impairment arising from mutations introduced into its carboxy-terminal region

Introduction: Antithrombin (AT) and heparin cofactor II (HCII) are plasma glycoproteins and serpins that inhibit thrombin. We showed [Blood 86 (1995) 3461] that recombinant rabbit AT containing the Utah mutation of AT, P407L, was inefficiently secreted by transfected primate and rodent cultured cells. In the current study, the effects of P407L and related substitutions in human AT and human HCII were investigated. Materials and methods: Cultured cells were transfected transiently (COS-1) or permanently (CV-1) with AT and HCII expression vectors encoding the wild type or mutant serpins. The amount of protein secreted was determined immunologically, while RNA levels were assessed by reverse-transcription-PCR (RT-PCR). The kinetics of secretion were investigated by pulse-chase experiments, supplemented by endoglycosidase H or lactacystin treatment. Results and conclusions: The F450L, P455L, P477L, P477*, and T446* (*=stop codon) mutations reduced HCII secretion 6.6- to 24-fold, while the F402L, A404T, and P407L mutations reduced AT secretion in COS-1 cells 1.7- to 5.2-fold. Homologous mutants HCII (P455L) and AT (P407L) were transcribed at similar levels in COS-1 cells, but were secreted less rapidly and less efficiently than their wild-type counterparts. HCII (P455L) exhibited intracellular proteasomal degradation in permanently transfected CV-1 cells, while AT (P407L) secretion was unaffected in this milieu. HCII secretion is thus more sensitive than that of AT to C-terminal mutations, as shown in two primate cell lines, likely reflecting a greater tendency to misfold during synthesis. We speculate that this difference may arise due to an interstrand s1C/s4B loop that is shorter in HCII than in AT.

Transactivation properties of c-Myb are critically dependent on two SUMO-1 acceptor sites that are conjugated in a PIASy enhanced manner.

The transcription factor v-Myb is a potent inducer of myeloid leukemias, and its cellular homologue c-Myb plays a crucial role in the regulation of hematopoiesis. Recently, Bies and coworkers (Bies, J., Markus, J. & Wolff, L. (2002) J. Biol. Chem, 277, 8999-9009) presented evidence that murine c-Myb can be sumoylated under overexpression conditions in COS7 cells when cotransfected with FLAG-tagged SUMO-1. Here we provide independent evidence that human c-Myb is also subject to SUMO-1 conjugation under more physiological conditions as revealed by coimmunoprecipitation analysis of Jurkat cells and transfected CV-1 cells. Analysis in an in vitro conjugation system showed that modification of the two sites K503 and K527 is interdependent. A two-hybrid screening revealed that the SUMO-1 conjugase Ubc9 is one of a few major Myb-interacting proteins. The moderate basal level of sumoylation was greatly enhanced by cotransfection of PIASy, an E3 ligase for SUMO-1. The functional consequence of abolishing sumoylation was enhanced activation both of a transiently transfected reporter gene and of a resident Myb-target gene. When single and double mutants were compared, we found a clear correlation between reduction in sumoylation and increase in transcriptional activation. Enhancing sumoylation by contransfection of PIASy had a negative effect on both Myb-induced and basal level reporter activation. Furthermore, PIASy caused a shift in nuclear distribution of c-Myb towards the insoluble matrix fraction. We propose that the negative influence on transactivation properties by the negative regulatory domain region of c-Myb depends on the sumoylation sites located here.

Interaction of retinal bZIP transcription factor NRL with Flt3-interacting zinc-finger protein Fiz1: possible role of Fiz1 as a transcriptional repressor.

NRL (neural retina leucine zipper) is a basic motif leucine zipper transcription factor of the Maf-subfamily. Multiple phosphorylated isoforms of NRL are detected specifically in rod photoreceptors. NRL regulates the expression of several rod-specific genes, including rhodopsin and cGMP phosphodiesterase beta-subunit, in synergy with other transcription factors (e.g. the homeodomain protein CRX). Missense mutations in the human NRL gene are associated with autosomal dominant retinitis pigmentosa, whereas the loss of its function leads to rodless retina in Nrl-knockout mice that exhibit enhanced S-cone function. To further elucidate the molecular mechanism(s) underlying NRL-mediated transcriptional regulation, we used yeast two-hybrid screening to isolate NRL-interacting proteins in the retina and report the identification of Flt3-interacting zinc-finger protein, Fiz1. Interaction of Fiz1 and NRL-leucine zipper was validated by GST pulldown assays and co-immunoprecipitation from bovine retinal nuclear extracts. Fiz1 suppressed NRL- but not CRX-mediated transactivation of rhodopsin promoter activity in transiently transfected CV1 cells. The mRNA and the protein for both Fiz1 and its only other known interacting protein Flt3, a receptor tyrosine kinase, are expressed in the retina. Our results indicate potential cross-talk among signaling pathways in the retina and suggest that the function of NRL is modulated by its interaction with specific repressor proteins.

TACC3 expression and localization in the murine egg and ovary.

A protein spot cored from a silver-stained two dimensional (2D) gel of germinal vesicle stage immature mouse oocytes was identified as Transforming Acidic Coiled Coil containing protein (TACC3) by tandem mass spectrometry. PCR amplification revealed two alternatively spliced forms, Tacc3a and Tacc3b, in mouse ovarian cDNA libraries. TACC3a encoded a 630 aa protein with a predicted mass of 70 kDa. It contained seven 24 aa repeats at the N-terminus and two coiled-coil domains at the C-terminus. TACC3b encoded a 426 aa protein with a predicted mass of 49 kDa also containing two coiled coil domains, but lacking the 168 aa repeat region. In addition to homology to the TACC family members, murine TACC3 also showed 35.7% identity to the Xenopus protein, Maskin, a cytoplasmic polyadenylation element binding protein (CPEB)-associated factor. Northern blot analysis demonstrated that TACC3a is abundantly expressed in adult testis and spleen and is moderately expressed in the ovary, heart, and lung, suggesting a wide tissue distribution. Both myc-tagged TACC3a and TACC3b targeted to the cytoplasm of transiently transfected CV-1 cells. In situ hybridization of mouse ovarian tissue sections displayed abundant expression of TACC3 specifically in the cytoplasm of growing oocytes, but not in primordial or atretic follicles. This pattern of expression suggests that TACC3 is expressed in ovarian cells undergoing active growth and development.

DIFFERENCE OF IN VIVO AND IN VITRO ANTIMINERALOCORTICOID POTENCY OF PROGESTERONE

Progesterone (P) given intramuscularly increases renal sodium excretion. We tested the in vitro capacity of P to bind to the human mineralocorticoid receptor (MR) with a reticulocyte lysate system and Ps transactivation potency in transfected CV-1 cells. Progesterone binds with higher affinity to the MR than aldosterone, but shows only low transactivation activity. This results in a very strong anti-mineralocorticoid (MC) potency of P in vitro. To test the in vivo anti-MC potency of P we infused aldosterone intravenously to hypo-MC Addison's patients, followed by increasing P infusions. During the study we measured normal aldosterone plasma concentrations and high P concentrations similar to the third trimester of pregnancy. Despite the 1000-fold higher plasma P concentrations, the in vivo anti-MC effect of P (increase of urinary sodium/potassium ratio) was rather small. We suggest that this may be due to effective MR protection mechanisms, such as conversion of P to inactive metabolites.

An Isoform of Branched-chain Aminotransferase Is a Novel Co-repressor for Thyroid Hormone Nuclear Receptors

The functions of thyroid hormone receptors (TRs) are regulated by a host of co-regulatory proteins. Tissue-specific expression of these co-regulators leads to distinct expression patterns and regulation of thyroid hormone (T3) target genes in tissues. Previously we have found that human colon carcinoma RKO cells exhibit strong T3-independent transcriptional activity. We therefore searched for co-regulatory proteins in RKO cells using a yeast two-hybrid system with the intact TRbeta1 as bait. One of the three positive clones, designated as P3, was identified to be an isoform of human mitochondria branched-chain aminotransferase (BCATm). P3 was a spliced variant of BCATm with an internal 12-amino acid deletion near the carboxyl-terminal region and was abundantly expressed in RKO cells. The expressed protein localized both to the mitochondria and the nucleus of transfected CV1 cells. P3 physically interacted with TRbeta1 in a T3-independent manner that led to the inhibition in binding of TRbeta1 to thyroid hormone-responsive element. P3 not only enhanced the repressor activity of the unliganded TR but also repressed the ligand-dependent activation of TR. This repression was reversed by treatment of cells with trichostatin A, suggesting that in addition to the inhibition of DNA binding, the repression activity of P3 on TR may also be mediated by histone deacetylase activity. Thus, unlike the currently known co-repressors, P3 is a novel ligand-independent co-repressor for TR.

Cys(9), Cys(104) and Cys(207) of simian virus 40 Vp1 are essential for infectious virion formation in CV-1 cells.

Structural studies have implicated Cys(9), Cys(104) and Cys(207) of simian virus 40 (SV40) Vp1 in disulfide bond formation. Recently, we have shown the three cysteines to be essential for disulfide linkage of Vp1 complexes in vitro. Here, the role of the three cysteines was explored during the course of SV40 infection. Single-, double- and triple-mutant Vp1 at Cys(9), Cys(104) and Cys(207) continued to localize to the nuclei of transfected CV-1 cells and to bind DNA, but showed a range of abilities to form plaques. Only mutants containing the Cys(9)-->Ser change showed defects in plaque formation. Single mutants at Cys(9) formed small plaques; mutants at Cys(9). Cys(104), Cys(9). Cys(207) and Cys(9). Cys(104). Cys(207) formed no plaques. All three isolated revertants contained back-mutations at the Vp1 Cys(9) codon. These results further confirm the involvement of the three Vp1 cysteines in protein-protein interactions during virus assembly. Cys(9) is critical for production of wild-type infectious virions, whereas Cys(104) and Cys(207) play secondary roles.

Role for peroxisome proliferator-activated receptor alpha in oxidized phospholipid-induced synthesis of monocyte chemotactic protein-1 and interleukin-8 by endothelial cells.

The attraction, binding, and entry of monocytes into the vessel wall play an important role in atherogenesis. We have previously shown that minimally oxidized/modified LDL (MM-LDL), a pathogenically relevant lipoprotein, can activate human aortic endothelial cells (HAECs) to produce monocyte chemotactic activators. In the present study, we demonstrate that MM-LDL and oxidation products of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) activate endothelial cells to synthesize monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8). Several lines of evidence suggest that this activation is mediated by the lipid-dependent transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha), the most abundant member of the PPAR family in HAECs. Treatment of transfected CV-1 cells demonstrated activation of the PPARalpha ligand-binding domain by MM-LDL, Ox-PAPC, or its component phospholipids, 1-palmitoyl-2-oxovalaroyl-sn-glycero-phosphocholine and 1-palmitoyl-2-glutaroyl-sn-glycero-phosphocholine; these lipids also activated a consensus peroxisome proliferator-activated receptor response element (PPRE) in transfected HAECs. Furthermore, activation of PPARalpha with synthetic ligand Wy14,643 stimulates the synthesis of IL-8 and MCP-1 by HAECs. By contrast, troglitazone, a PPARgamma agonist, decreased the levels of IL-8 and MCP-1. Finally, we demonstrate that unlike wild-type endothelial cells, endothelial cells derived from PPARalpha null mice do not produce MCP-1/JE in response to Ox-PAPC and MM-LDL. Together, these data demonstrate a proinflammatory role for PPARalpha in mediation of the activation of endothelial cells to produce monocyte chemotactic activity in response to oxidized phospholipids and lipoproteins.

The Aryl Hydrocarbon Receptor Interacts with Estrogen Receptor Alpha and Orphan Receptors COUP-TFI and ERRα1

The molecular mechanisms underlying the apparent “cross-talk” between estrogen receptor (ER)- and arylhydrocarbon receptor (AHR)-mediated activities are unknown. To determine how AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may inhibit ER action and, conversely, to examine how 17-β-estradiol (E2) affects AHR activity, we examined discrete activities of each receptor, i.e., protein–protein interactions, DNA binding, and transcriptional activation. We report that AHR interacts directly with ERα, COUP-TF, and ERRα1, in a ligand-specific manner in vitro. Unoccupied or β-napthoflavone (β-NF)-occupied AHR showed stronger interaction with ERα, COUP-TF, and ERRα1 than when AHR was occupied by the partial antagonist α-naphthoflavone (α-NF), indicating a role for ligand in AHR interaction with these proteins. We also report that AHR interacts with COUP-TF in transfected CV-1 cells. In contrast, the AHR nuclear translocator protein (ARNT) did not interact with COUP-TF, ERRα1, or ERα. We next examined the interaction of either ERα or COUP-TF with a consensus xenobiotic response element (XRE). Purified ERα did not bind the consensus XRE, but COUP-TFI bound the consensus XRE, suggesting a role for COUP-TF as a AHR/ARNT competitor for XRE binding. In transiently transfected MCF-7 human breast cancer cells, overexpression of COUP-TFI inhibited TCDD-activated reporter gene activity from the CYP1A1 promoter. TCDD inhibited estradiol (E2)-activated reporter gene activity from a consensus ERE and from the EREs in the pS2 and Fos genes, and COUP-TFI did not block the antiestrogenic activity of TCDD. The specific interaction of COUP-TF with XREs and AHR together with the inhibition of TCDD-induced gene expression by COUP-TF suggests that COUP-TF may regulate AHR action both by direct DNA binding competition and through protein–protein interactions.

A truncated human peroxisome proliferator-activated receptor alpha splice variant with dominant negative activity.

The peroxisome proliferator-activated receptor alpha (PPARalpha) plays a key role in lipid and lipoprotein metabolism. However, important inter- and intraspecies differences exist in the response to PPARalpha activators. This incited us to screen for PPARalpha variants with different signaling functions. In the present study, using a RT-PCR approach a variant human PPARalpha mRNA species was identified, which lacks the entire exon 6 due to alternative splicing. This deletion leads to the introduction of a premature stop codon, resulting in the formation of a truncated PPARalpha protein (PPARalphatr) lacking part of the hinge region and the entire ligand-binding domain. RNase protection analysis demonstrated that PPARalphatr mRNA is expressed in several human tissues and cells, representing between 20-50% of total PPARalpha mRNA. By contrast, PPARalphatr mRNA could not be detected in rodent tissues. Western blot analysis using PPARalpha-specific antibodies demonstrated the presence of an immunoreactive protein migrating at the size of in vitro produced PPARalphatr protein both in human hepatoma HepG2 cells and in human hepatocytes. Both in the presence or absence of 9-cis-retinoic acid receptor, PPARalphatr did not bind to DNA in gel shift assays. Immunocytochemical analysis of transfected CV-1 cells indicated that, whereas transfected PPARalphawt was mainly nuclear localized, the majority of PPARalphatr resided in the cytoplasm, with presence in the nucleus depending on cell culture conditions. Whereas a chimeric PPARalphatr protein containing a nuclear localization signal cloned at its N-terminal localized into the nucleus and exhibited strong negative activity on PPARalphawt transactivation function, PPARalphatr interfered with PPARalphatr transactivation function only under culture conditions inducing its nuclear localization. Cotransfection of the coactivator CREB-binding protein relieved the transcriptional repression of PPARalphawt by PPARalphatr, suggesting that the dominant negative effect of PPARalphatr might occur through competition for essential coactivators. In addition, PPARalphatr interfered with transcriptional activity of other nuclear receptors such as PPARgamma, hepatic nuclear factor-4, and glucocorticoid receptor-alpha, which share CREB-binding protein/p300 as a coactivator. Thus, we have identified a human PPARalpha splice variant that may negatively interfere with PPARalphawt function. Factors regulating either the ratio of PPARalphawt vs. PPARalphatr mRNA or the nuclear entry of PPARalphatr protein should therefore lead to altered signaling via the PPARalpha and, possibly also, other nuclear receptor pathways.

Cell-specific nuclear import of plasmid DNA.

One factor limiting the success of non-viral gene therapy vectors is the relative inability to target genes specifically to a desired cell type. To address this limitation, we have begun to develop cell-specific vectors whose specificity is at the level of the nuclear import of the plasmid DNA. We have recently shown that nuclear import of plasmid DNA is a sequence-specific event, requiring the SV40 enhancer, a region known to bind to a number of general transcription factors (Dean DA, Exp Cell Res 1997; 230: 293). From these studies we developed a model whereby transcription factor(s) bind to the DNA in the cytoplasm to create a protein-DNA complex that can enter the nucleus using the protein import machinery. Our model predicts that by using DNA elements containing binding sites for transcription factors expressed in unique cell types, we should be able to create plasmids that target to the nucleus in a cell-specific manner. Using the promoter from the smooth muscle gamma actin (SMGA) gene whose expression is limited to smooth muscle cells, we have created a series of reporter plasmids that are expressed selectively in smooth muscle cells. Moreover, when injected into the cytoplasm, plasmids containing portions of the SMGA promoter localize to the nucleus of smooth muscle cells, but remain cytoplasmic in fibroblasts and CV1 cells. In contrast, a similar plasmid carrying the SV40 enhancer is transported into the nuclei of all cell types tested. Nuclear import of the SMGA promoter-containing plasmids could be achieved when the smooth muscle specific transcription factor SRF was expressed in stably transfected CV1 cells, supporting our model for the nuclear import of plasmids. Finally, these nuclear targeting sequences were also able to promote increased gene expression in liposome- and polycation-transfected non-dividing cells in a cell-specific manner, similar to their nuclear import activity. These results provide proof of principle for the development of cell-specific non-viral vectors for any desired cell type.

Quantity of partial agonist activity for antiglucocorticoids complexed with mutant glucocorticoid receptors is constant in two different transactivation assays but not predictable from steroid structure

An unsolved question in steroid hormone action is why the amount of agonist activity displayed by antisteroids is not constant but varies with the assay conditions. Receptor mutations have provided insight into hormone action, presumably due to changes in the tertiary structure of the receptor that alter its interaction surfaces with the transcriptional machinery or/and co-factors. We have now employed two mechanistically different induction assays to determine whether disparate transactivation processes are similarly altered by receptor mutations. The two activation assays studied were (i) the standard induction of GREtkLUC in transiently transfected CV-1 cells and (ii) a novel modulation of endogenous receptor activity by transiently transfected receptors in HeLa cells. Five different mutations in the ligand binding and DNA binding domains of the rat glucocorticoid receptor (CS1, CS1/CD, 451/9, C656G, and R732Q) and seven steroids of varied structures (five antagonists and two agonists) were selected for use. The results in both induction assays were the same. However, no generalizations regarding steroid structure and activity emerged. Neither of two potent glucocorticoids were active with GR-CS1, or GR-CS1/CD, while RU 486 was the only antisteroid with appreciable agonist activity. With the GR-451/9 mutant, three antagonists afforded partial agonist activity. We confirmed that the C656G mutant is both “super-sensitive” and “super-selective” for transactivation. In contrast, the R732Q mutation caused significant decreases in activity with both antagonists and subsaturating concentrations of agonists. This inability to generalize about the behavior of any class of steroids with mutant receptors may reflect an induced fit for each receptor–steroid complex. Nevertheless, the activity of a given steroid appeared to be constant in two different transactivation assays for a given mutant receptor. Thus, disparate transactivation processes may utilize identical receptor surfaces, even in the expression of partial agonist activity for specific antiglucocorticoids.

Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p.

Kinetoplastid protozoa confine large parts of glycolysis within glycosomes, which are microbodies related to peroxisomes. We cloned the gene encoding the second most abundant integral membrane protein of Trypanosoma brucei glycosomes. The 24 kDa protein is very basic and hydrophobic, with two predicted transmembrane domains. It is targeted to peroxisomes when expressed in mammalian cells and yeast. The protein is a functional homologue of Pex11p from Saccharomyces cerevisiae: pex11Delta mutants, which are defective in peroxisome proliferation, can be complemented by the trypanosome gene. Sequence conservation is significant in the N- and C-terminal domains of all putative Pex11p homologues known, from trypanosomes, yeasts and mammals. Several lines of evidence indicate that these domains are oriented towards the cytosol. TbPex11p can form homodimers, like its yeast counterpart. The TbPEX11 gene is essential in trypanosomes. Inducible overexpression of the protein in T.brucei bloodstream forms causes growth arrest, the globular glycosomes being transformed to clusters of long tubules filling significant proportions of the cytoplasm. Reduced expression results in trypanosomes with fewer, but larger, organelles.

Vesicular acetylcholine transporter (VAChT) protein: A novel and unique marker for cholinergic neurons in the central and peripheral nervous systems

Acetylcholine (ACh) is synthesized in nerve terminals from choline and acetyl coenzyme A by the cytoplasmic enzyme choline acetyltransferase (ChAT). The neurotransmitter is thereafter transported into synaptic vesicles, where it is stored until release. cDNA clones encoding a vesicular ACh transporter (VAChT) were recently isolated. In this paper, we report on the generation of highly specific goat polyclonal antisera to the rat VAChT protein by using a synthetic carboxy-terminal 20-amino-acid peptide sequence as an immunogen. Characterization of the antisera revealed recognition of VAChT, but not vesicular monoamine transporter (VMAT) protein, in transfected CV-1 cells. VAChT immunoreactivity was also detected in cells that endogenously express the protein, such as in PC12 cells and in primary cultures of spinal motoneurons. Absorption controls showed that the VAChT antisera could be completely blocked at the 10(-5) M concentration by cognate peptide used for immunization. The antisera cross-reacted with the VAChT protein in rat and mouse but not in guinea pig, rabbit, or cat. Immunohistochemistry and confocal laser microscopy, using the goat VAChT antisera, showed strong immunoreactivity in discrete fibers and neuronal cell bodies of the central and peripheral nervous systems. Within cell bodies and axonal nerve terminals, as well as in dendrites, the staining appeared granular, presumably representing labeling of synaptic vesicles containing ACh. In the rat central nervous system, VAChT-positive cell bodies were demonstrated in the cerebral cortex, striatum, septum, nucleus basalis, medial habenula, mesopontine complex, cranial, and autonomic and spinal motor nuclei and in the intermediomedial region near the central canal. High densities of VAChT-immunoreactive axonal fibers were encountered in areas such as the olfactory bulb, cerebral cortex, striatum, basal forebrain, amygdala, thalamus, hypothalamus including median eminence, hippocampal formation, superior colliculus, interpeduncular nucleus, and pedunculopontine and laterodorsal tegmental nuclei. In cranial and spinal motor nuclei, particularly large varicosities were seen in close proximity to the motoneuron cell somata and their proximal dendrites. In the peripheral nervous system, VAChT immunoreactivity was also detected in motor endplates of skeletal muscle as well as in fibers of sympathetic and parasympathetic abdominal ganglia, heart atrium, respiratory tract, gastrointestinal tract, pancreas, adrenal medulla, male genitourinary tract, and salivary and lacrimal glands. Direct double labeling revealed colocalization of VAChT and ChAT immunoreactivity in neurons. The results show that VAChT antisera represent novel and unique tools for the study of cholinergic neurons in the central and peripheral nervous systems.