Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines

Phenotypic diversity and testosterone-induced normalization of mutant L712F androgen receptor function in a kindred with androgen insensitivity.

Molecular causes of phenotypic diversity in androgen insensitivity syndrome, occurring even in the same family, have rarely been identified. We report on a family with four affected individuals, three brothers (B1-3) and their uncle, displaying strikingly different external genitalia: B1, ambiguous; B2, severe micropenis; B3, slight micropenis; and uncle, micropenis and penoscrotal hypospadias. All had been assigned a male gender. We detected the same L712F mutation of the androgen receptor (AR) gene in each subject. Methyltrienolone binding on cultured genital skin fibroblasts of B2 suggested moderate impairment of the ligand-binding domain [maximal binding capacity, 38.2 fmol/mg protein (normal); Kd, 0.21 nmol/L; normal range, 0.03-0.13 nmol/L]. In trans-activation assays, the mutant 712F-AR showed considerable deficiency at low concentrations of testosterone (0.01-0.1 nmol/L) or dihydrotestosterone (0.01 nmol/L). Remarkably, this could be fully neutralized by testosterone concentrations greater than 1.0 nmol/L. Hence, the 712F-AR could switch its function from subnormal to normal within the physiological concentration range of testosterone. This was reflected by an excellent response to testosterone therapy in B1, B2, and the uncle. Taking into account the well documented individual and time-dependent variation in testosterone concentration in early fetal development, our observations clearly illustrate the potential impact of varying ligand concentrations for distinct cases of phenotypic variability in androgen insensitivity syndrome.

Gtx, an oligodendrocyte-specific homeodomain protein, has repressor activity.

Myelin, a multilamellar membrane structure that facilitates nerve conduction, is synthesized in the central nervous system (CNS) by oligodendrocytes. Gtx, a member of the homeodomain family of transcriptional factors, is a candidate regulator of myelin gene expression, because it is uniquely expressed in myelinating oligodendrocytes in postnatal rodent brain. To analyze the regulatory activity of Gtx, we first identified the optimal Gtx-binding sequence using an in vitro DNA-binding assay. This sequence, (A/T)TTAATGA, contains a TAAT core and is similar, but not identical, to that of other homeodomain protein binding sites. When coexpressed in cultured cells along with a minimal promoter containing five tandem repeats of this optimal Gtx-binding sequence, Gtx demonstrated repressor activity, which was also present when Gtx was tethered to DNA by way of the strong GAL4 DNA-binding domain. Truncations of the GAL4-Gtx fusion identified a portable repressor domain within a relatively proline/alanine-rich region N-terminal to the Gtx homeodomain. Cotransfection of a Gtx expression vector into a variety of cell lines, including oligodendrocytes, along with constructs containing portions of the PLP, MBP, or Gtx promoters fused to a reporter gene, however, did not modulate transcription from any of these promoter constructs. These data support the notion that the oligodendrocyte-specific homeodomain protein Gtx can act as a transcriptional repressor. In addition, they suggest that interaction of Gtx with other, as yet undefined, transcriptional regulators modifies Gtx activity in oligodendrocytes.

Molecular cloning of a truncated p62Dok1 isoform, p22Dok(del).

The p21Ras GTPase activating protein-associated 62-kDa protein, p62Dok1, is an early substrate of various tyrosine phosphorylation pathways. Its recent cloning in human myeloid cells and in murine pre-B cells revealed an N-terminal pleckstrin-homology domain and a tyrosine- and proline-rich C-terminal tail in its sequence. Here, we characterized a new 1261-bp cDNA identical to that of p62Dok1, but with a central 185-bp deletion (bp 456-640). This induced a frameshift leading to a premature stop codon. The deduced protein, designated p22Dok(del), corresponded to a truncated p62Dok1 isoform of 177 amino acids that can be expressed both in vitro and in vivo with an apparent molecular mass of 22 kDa. This newly identified molecule was composed of the N-terminal PH domain of p62Dok1 followed by a new 25-amino acid C-terminal sequence containing a typical class II proline-rich motif, suggesting a specific role for p22Dok(del) in signal transduction pathways.

Specific gene transfer mediated by galactosylated poly-L-lysine into hepatoma cells.

Plasmid DNA/galactosylated poly-L-lysine(GalPLL) complex was used to transfer luciferase reporter gene in vitro into human hepatoma cells by a receptor-mediated endocytosis process. DNA was combined with galPLL via charge interaction (DNA:GalPLL:fusogenic peptide, 1:0.4:5, w/w/w) and the resulting complex was characterized by dynamic light scattering, gel retardation assay and zeta potential analyzer to determine the particle size, electrostatic charge interaction, and apparent surface charge. The complex was tested for the efficiency of gene transfer in cultured human hepatoblastoma cell line Hep G2 and fibroblast cells NIH/3T3 in vitro. The mean diameter of the complex (DNA:GalPLL=1:0.4, w/w) was 256+/-34.8 nm, and at this ratio, it was positively charged (zeta potential of this complex was 10.1 mV). Hep G2 cells, which express a galactose specific membrane lectin, were efficiently and selectively transfected with the RSV Luc/GalPLL complex in a sugar-dependent manner. NIH/3T3 cells, which do not express the galactose-specific membrane lectin, showed only a marginal level of gene expression. The transfection efficiency of GalPLL-conjugated DNA complex into Hep G2 cells was greatly enhanced in the presence of fusogenic peptide that can disrupt endosomes, where the GalPLL-DNA complex is entrapped with the fusogenic peptide. With the fusogenic peptide KALA, the luciferase activity in Hep G2 cells was ten-fold higher than that of cells transfected in the absence of the fusogenic peptide. Our gene transfer formulation may find potential application for the gene therapy of liver diseases.

Quantitative fluorescence cytometric analysis of Bcl-2 levels in tumor cells exhibiting a wide range of inherent Bcl-2 protein expression: correlation with Western blot analysis.

BACKGROUND: A protocol to measure a wide range of Bcl-2 protein expression using quantitative fluorescence cytometry (QFCM) in different cell types was developed for use with flow cytometry. Bcl-2 measurements obtained by flow cytometry were correlated with Western blot Bcl-2 measurements to confirm specificity of the Bcl-2-FITC staining. This protocol was applied to measure absolute levels of Bcl-2 protein in different tumor cell lines including Bcl-2-transfected breast carcinoma cell lines and in peripheral blood lymphocytes (PBL). METHODS: HL-60, K562, DOHH2, Jurkat, MDA435/LCC6, MCF7 cell lines, and PBL derived from normal donors were fixed, permeabilized, stained with anti-Bcl-2-FITC antibody and evaluated by QFCM. In parallel, the same cells were evaluated for Bcl-2 protein expression by Western blot analysis. Mitochondrial localization of anti-Bcl-2-FITC antibody inside cells was confirmed using fluorescence imaging microscopy. RESULTS: Bcl-2 expression in different cell types could be accurately quantified based on antibody-binding capacity (ABC) ranging from 12.6 x 10(3) antibody-binding sites in HL-60 cells to 1.64 x 10(6) antibody-binding sites in a Bcl-2-transfected MDA435/LCC6 clone. The data from flow cytometry analysis correlated well with Western analysis (R(2) = 0.78). Bcl-2-FITC staining colocalized with dyes specific for mitochondria. CONCLUSIONS: The Bcl-2 staining protocol described here was shown to be specific, sensitive, and it was able to provide higher resolution as well as more reproducible quantitation of Bcl-2 protein content in cells when compared with Western blot methods. Quantitation of Bcl-2 content in cells by QFCM may be useful for monitoring Bcl-2 expression in cells undergoing various treatments in vitro and in vivo.

Antisense inhibition of cyclin D1 in human head and neck squamous cell carcinoma.

OBJECTIVE: To study the role of cyclin D1 in regulating the biological behavior of head and neck cancer. DESIGN: Squamous cell carcinoma of the head and neck (SCCHN) cells were stably transfected with an antisense cyclin D1 using lipofectin-mediated transfection. In vitro growth assays, cell cycle analyses, cytotoxicity assays, and in vivo tumorigenicity assays were performed. MATERIALS: Human SCCHN cell lines TU138, TU167, TU177, TU182, MDA183, and MDA1386 and athymic nude mice were used for this study. RESULTS: The antisense cyclin D1 transfected cells revealed decreased growth rates in vitro and decreased tumorigenicity in athymic nude mice. Furthermore, antisense cyclin D1 transfection enhanced the chemosensitivity against cisplatin. CONCLUSIONS: These studies provided evidence that overexpression of cyclin D1 may play an important role in growth rates and biological behavior of human head and neck cancer. Additionally, expression of cyclin D1 may make human head and neck cancer cells resistant to platinum-based chemotherapeutic approaches. The ability to suppress the malignant phenotype by down-regulating cyclin D1 expression may provide a new gene therapy approach for patients with head and neck cancer.

Gonadotropin-releasing hormone receptor activation of extracellular signal-regulated kinase and tyrosine kinases in transfected GH3 cells and in alphaT3-1 cells.

GH3 cells were stably transfected with the wild-type murine GnRH receptor and a clonal cell line selected on the basis of inositol phosphate production and PRL/GH release in response to GnRH. This cell line (wt28) was characterized by [125I]GnRH analog binding, [3H]inositol phosphate response to GnRH, and hormone secretion. We examined the activation of the mitogen-activated protein kinase isoforms, extracellular signal-regulated kinase 1/2 (ERK1/2) and tyrosine kinases in wt28 cells and alphaT3-1 cells (which express a native GnRH) using specific phospho-ERK1/2 and phosphotyrosine antibodies. Concentration-response and time-course data revealed that a sustained ERK1/2 response was seen only in aT3-1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in alphaT3-1 cells, but not in wt28 cells. Activators for several different signaling pathways revealed distinct differences between the cell types. Protein kinase C activation by phorbol 12,13-dibutyrate was very effective in alphaT3-1 cells at phosphorylation of both ERK1/2 and tyrosine, whereas raising cAMP levels using forskolin also strongly increased wt28 cell ERK1/2 phosphorylation. Only the tyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation in wt28 cells. The lack of sustained ERK1/2 phosphorylation in wt28 cells could be the result of minimal tyrosine kinase activation by GnRH compounded by a different pathway profile for ERK1/2 activation. When pervanadate and GnRH were combined, ERK1/2 phosphorylation was synergistic and sustained in wt28 cells, whereas the response was additive in alphaT3-1 cells. In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in alphaT3-1 and wt28 cells are distinct; thus, activating GnRH receptors in each of the two cell types leads to different sequelae of events regarding ERK1/2 activation.

The peroxisome proliferator perfluorodecanoic acid inhibits the peripheral-type benzodiazepine receptor (PBR) expression and hormone-stimulated mitochondrial cholesterol transport and steroid formation in Leydig cells.

The peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.

Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation.

Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 approximately 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 microM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.

The role of CBP in estrogen receptor cross-talk with nuclear factor-kappaB in HepG2 cells.

Functional interactions or cross-talk between ligand-activated nuclear receptors and the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) may play a major role in ligand-mediated modification of diseases processes. In particular, the cardioprotective effects of estrogen replacement therapy are thought to be due in part to the ability of ligand-bound estrogen receptor (ER) to inhibit NF-kappaB function. In the current study 17beta-estradiol-bound ERalpha interfered with cytokine-induced activation of a NF-kappaB reporter in HepG2 cells. The estrogen metabolite, 17alpha-ethinyl estradiol, and the phytoestrogen, genistein, were also effective inhibitors of NF-kappaB activation, whereas tamoxifen, 4-hydroxytamoxifen, and raloxifene were inactive. This inhibition was reciprocal, as NF-kappaB interfered with the trans-activation properties of ERalpha. Ligand-bound ERalpha did not inhibit NF-kappaB binding to DNA, but it did decrease the histone acetyltransferase activity required for NF-kappaB transcriptional activity. Coexpression of the transcription coactivator CREB binding protein (CBP), but not steroid receptor coactivator 1a, reversed the ERalpha-mediated inhibition of NF-kappaB activity. Mammalian two-hybrid experiments also revealed that ligand-bound ERalpha can interact functionally with CBP-NF-kappaB complexes. We suggest that CBP targeting by ERalpha results in the inhibition of NF-kappaB and may occur through formation of transcriptionally inert multimeric complexes that are dependent upon the nature of the ERalpha ligand.

Thyroid receptor activator molecule, TRAM-1, is an androgen receptor coactivator.

An androgen receptor (AR) interacting protein was isolated from a HeLa cell complementary DNA library by two-hybrid screening in yeast using the AR DNA and ligand binding domains [amino acids (aa) 481-919] as bait. AR binding of the protein in yeast was dependent on the presence of testosterone or dihydrotestosterone (DHT). The isolated protein is identical to thyroid receptor activator molecule TRAM-1 but lacking aa 1-458. TRAM-1 is a steroid receptor coactivator-3 (SRC-3) subtype. In affinity matrix assays, 35S-labeled TRAM-1 bound the GST-AR ligand binding domain (aa 624-919) and GST-AR N-terminal and DNA binding domains (aa 1-660), but not the GST-AR DNA binding domain (aa 544-634) alone. Coexpression of TRAM-1 increased DHT-dependent AR transactivation 5-fold and constitutive activity of AR (aa 1-660) N-terminal and DNA-binding domains increased 9-fold. Full-length TRAM-1 (aa 1-1424) and the partial (aa 459-1424) were AR and GR coactivators as was SRC-1. In human testis, immunostaining of SRC-3 colocalized with AR in nuclei of Sertoli cells and peritubular myoid cells, indicating it could function as an AR coactivator in these cells. SRC-3 was also present in nuclei of spermatogenic cells where AR was not expressed, suggesting it might also be a coactivator with other nuclear receptors that regulate spermatogenesis.

Failure of membrane targeting causes the functional defect of two mutant sodium iodide symporters.

Molecular cloning of the sodium/iodide symporter (NIS) allowed identification of NIS gene mutations in patients with iodide trapping defect. Whereas various mutant human (h) NIS molecules display loss of function when expressed by transfection in mammalian cells, the precise mechanism(s) responsible for the functional abnormality of these proteins remains unknown. With the aim to explore these mechanisms in three natural hNIS mutants identified previously in patients with iodide trapping defect (Q267E, S515X, and C272X), we have prepared tools allowing direct measurement of the protein at its normal location in the plasma membrane. A COS-7 cell line was made by transfection that stably expressed high levels of wild-type hNIS. It was used to screen by flow cytometry monoclonal antibodies (mAbs) prepared from mice immunized against hNIS. Genetic immunization was performed by im injection of a wild-type hNIS complementary DNA construct, because this procedure has demonstrated the ability to produce antibodies recognizing native membrane proteins. One mAb that recognized an epitope of hNIS exposed on the extracellular side of the plasma membrane was selected for further studies. The epitope was localized on the sixth putative extracellular loop of the protein on the basis that the mAb did not recognize rat NIS, which exhibits major sequence differences in this segment. When this mAb was used to test by flow cytometry the expression of the three mutant hNIS proteins in transfected COS-7 cells, it detected similar amounts of wild-type, Q267E, and the S515X hNIS molecules in permeabilized cells. In contrast, only the wild-type hNIS was detected at the surface of nonpermeabilized cells. The C272X hNIS truncation mutant was not detected in intact or permeabilized cells. This is consistent with the absence of the mAb epitope from this mutant, which is expected to lack the sixth extracellular loop. Our data demonstrate that faulty membrane targeting is implicated in the mechanisms causing iodide trapping defect in the Q267E and S515X natural hNIS mutants.

Antisense inhibition of vascular endothelial growth factor in human head and neck squamous cell carcinoma.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent paracrine angiogenic factor involved in angiogenesis. We determined whether antisense VEGF transfection can suppress angiogenic activity of a human squamous cell carcinoma of the head and neck (SCCHN) cell line. METHODS: Human SCCHN cell lines were screened for VEGF secretion by ELISA. The highest VEGF secreting cell line was transfected with an antisense VEGF vector. Endothelial cell migration assays were performed using the conditioned medium from the transfected clones. Tumorigenicity assays of the transfectants in nude mice were also performed. RESULTS: Antisense VEGF expression exhibited a 20-fold inhibition of VEGF secretion. The addition of conditioned medium from the antisense clones resulted in 50% reduction of endothelial migration. There was no effect on in vivo tumorigenicity. CONCLUSIONS: Antisense VEGF transfection effectively down-regulated VEGF secretion from SCCHN cells that had high VEGF secretion. Targeting VEGF expression may be useful for suppressing angiogenesis in head and neck cancer.

Transfection efficiency increases by incorporating hydrophobic monomer units into polymeric gene carriers.

The water soluble terpolymer, poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-butylmethacrylate (BMA)) was synthesized, and its efficiency in in vitro gene transfection was evaluated. Copolymers with different compositions were synthesized by radical polymerization. For a series of copolymers containing 60 mol% of DMAEMA, the plasmid bands were retained within the gel loading slot, independent of polymer/plasmid weight ratios or BMA monomer content. In contrast, for a series of copolymers containing 20 mol% DMAEMA, plasmid bands of complexes were retarded with increasing weight ratios. For the copolymer with 10 mol% BMA content, the plasmid was completely retained within the gel loading slot. The transfection efficiency of polymer/plasmid complexes was evaluated in COS-1 cells using a pCMV-lacZ plasmid, encoding for beta-galactosidase as a reporter gene. Transfection efficiency of a series of copolymers containing 20 mol% of DMAEMA varied with BMA content. The transfection efficiency of the copolymers with 0, 2, and 5 mol% of BMA was low. The transfection efficiency of the copolymers with 10 mol% of BMA was about 2-fold higher than that of the PDMAEMA control homopolymer. The transfected cells were observed at a very wide range of polymer/plasmid weight ratios. The transfection efficiency of all copolymers containing 60 mol% of DMAEMA was lower than that of the PDMAEMA homopolymer.

Regulated expression of the rat recombinant P2X(3) receptor in stably transfected CHO-K1 tTA cells.

In this report, the regulatable expression by tetracycline of the rat recombinant P2X(3) receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X(3)-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 microM ATP evoked inward currents of 2.9+/-1.6 nA in transfected cells grown in the absence of tetracycline (tet-). The P2X(3) receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [35S]ATP gamma S yielded a pK(d) of 8.2+/-0.1 and a B(max) of 31.9+/-3.5 pmol/mg protein in tet- cell membranes and a pK(d) of 8.1+/-0.1 and a B(max) of 5.8+/-0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and alpha beta MeATP displaced the binding of [35S]ATP gamma S in tet- cell membranes with very high affinity, yielding pIC(50) values of 9.4+/-0.2 and 7.5+/-0. 2, respectively. In tet+ cell membrane, displacement of [35S]ATP gamma S by 2MeSATP and alpha beta MeATP was of much lower affinity (pIC(50) values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [35S]ATP gamma S binding in tet- and tet+ cell membranes. In other experiments, cytosolic Ca(2+) was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca(2+) were elicited by 100 nM alpha beta MeATP in tet- cells while no increases in cytosolic Ca(2+) were detected below 100 microM alpha beta MeATP in either tet+ cells or untransfected cells. These calcium responses to alpha beta MeATP had a pEC(50) of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to alpha beta MeATP yielding a pK(B) of 5.6. PPADS produced non-parallel, dextral shifts of E/[A] curves to alpha beta MeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X(3) receptor which is under regulated expression in a stably transfected mammalian cell line.

Canine thyrotropin beta-subunit gene: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and transient expression in the Chinese hamster ovary cells.

The gene encoding the mature beta subunit of canine thyroid stimulating hormone (cTSH beta) was cloned, sequenced and expressed in Escherichia coli and in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against the recombinant cTSH beta purified from E. coli were generated. The gene fragment that encodes mature TSH beta was cloned from the canine genomic DNA by direct polymerase chain reaction (PCR) using primers that were designed based on the consensus sequences from other species. The resulting 891 basepairs (bp) of genomic DNA consisted of two coding exons of the canine TSH beta gene and an intron of 450 bp. The two exons, which encode the mature cTSH beta subunit, was joined together by an overlap PCR and was expressed in E. coli as 6xHis-tagged protein. The purified recombinant cTSH beta with a molecular weight of about 15 kDa was recognized by the polyclonal antibodies prepared against the native canine TSH in Western blot. Monoclonal antibodies were raised against the purified cTSH beta and subsequently characterized. For transient expression in CHO cells that are permanently transfected with the bovine common alpha gene, a 60-oligonucleotide signal peptide coding sequence was added to the 5' end of the cTSH beta gene before it was cloned into the mammalian expression vector pRSV and used to transfect CHO cells. The medium from these transfected cells, presumably containing the bovine alpha and canine TSH beta in heterodimeric confirmation, exhibited TSH bioactivity as indicated by the stimulation of cAMP production in the cultured FRTL-5 thyrocytes.

Protein kinase C(alpha) actively downregulates through caveolae-dependent traffic to an endosomal compartment.

Receptor desensitization occurs through receptor internalization and targeting to endosomes, a prerequisite for sorting and degradation. Such trafficking processes may not be restricted to membrane associated receptors but may also play an important role in the downregulation of cytoplasmic transducers such as protein kinase C (PKC). It is demonstrated here that acute TPA exposure induces the transport of activated PKC(alpha) from the plasma membrane to endosomes. This process requires PKC activity and catalytically competent PKC can even promote a similar process for a truncated regulatory domain PKC(&agr;) protein. It is established that PKC(&agr;) is targeted to the endosome compartment as an active kinase, where it colocalizes with annexin I, a substrate of PKC. Thus, PKC(alpha) downregulation shares features with plasma membrane associated receptor sorting and degradation. However, it is shown that PKC(&agr;) delivery to the endosome compartment is not a Rab5 mediated process in contrast to the well characterised internalisation of the transferrin receptor. An alternative route for PKC(alpha) is evidenced by the finding that the cholesterol binding drugs nystatin and filipin, known to inhibit caveolae mediated trafficking, are able to block PKC(alpha) traffic and down regulation. Consistent with this, the endosomes where PKC(alpha) is found also contain caveolin. It is concluded that the initial step in desensitisation of PKC(alpha) involves active delivery to endosomes via a caveolae mediated process.

Sustained elevation in inositol 1,4,5-trisphosphate results in inhibition of phosphatidylinositol transfer protein activity and chronic depletion of the agonist-sensitive phosphoinositide pool.

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) in a signal-terminating reaction. We have utilised cell lines that stably underexpress the 43 kDa 5-phosphatase, as a model system to investigate whether Ins(1,4,5)P(3) can control the rate of its own formation by regulating the resupply of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). A sustained 2.6-fold elevation in the basal concentration of Ins(1,4,5)P(3), in cell lines underexpressing the 43 kDa 5-phosphatase, correlated with a 32% reduction in the total cellular mass of PtdIns(4,5)P(2). The depletion in cellular PtdIns(4,5)P(2) was confined to a Triton-insoluble cell compartment, enriched in caveolin. In resting cells with elevated Ins(1,4,5)P(3) concentrations resulting from underexpression of the 43 kDa 5-phosphatase, phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns(4)P) were depleted by 50% and PtdIns(4,5)P(2) by 61% in the caveolin-enriched Triton-insoluble compartment. Agonist stimulation resulted in the rapid turnover of phosphoinositides in the caveolin-enriched Triton-insoluble fraction of vector-transfected cells, but not in cells with high basal Ins(1,4,5)P(3) concentrations. Depletion of phosphoinositides from the caveolin-enriched Triton-insoluble pool in cells underexpressing the 43 kDa 5-phosphatase did not result from activation of phospholipase C isoenzymes, or inhibition of PtdIns 4-kinase or PtdIns(4)P 5-kinase activities. Significant inhibition of phosphatidylinositol transfer protein (PITP) activity (up to 70%) was observed in cells with elevated basal Ins(1,4,5)P(3) concentrations; however, no reduction in PITP(&agr;) protein expression was detected. These studies indicate that chronic elevation in cellular Ins(1,4,5)P(3) concentrations decreases the PITP-mediated resupply of phosphoinositides in the caveolin-enriched agonist-sensitive pool.