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PubMed2004

Heterologous expression of G protein-coupled receptors in U-2 OS osteosarcoma cells.

Recombinant baculoviruses, in which the insect cell-specific polyhedrin promoter has been replaced with a mammalian cell-active expression cassette (BacMam viruses), are efficient gene delivery vehicles for many mammalian cell types. BacMam viruses have been generated for expression of G protein-coupled receptors (GPCRs) and used to establish Ca2+mobilization assays in HEK-293 human embryonic kidney cells and U-2 OS human osteosarcoma cells. U-2 OS cells are highly susceptible to BacMam-based gene delivery and lack many of the endogenous receptors present on HEK-293 and other mammalian cell lines typically used for heterologous expression of GPCRs. U-2 OS cells were found to have a null background for muscarine, ADP, ATP, UTP, UDP, and lysophosphatidic acid (LPA). Consequently, U-2 OS cells transduced with BacMam constructs encoding the muscarinic acetylcholine receptors (M1, M2, M3, M4, and M5subtypes), the P2Y receptors (P2Y1, P2Y2), or the LPA receptors (EDG-2, EDG-7) were used for the establishment of whole-cell Ca2+mobilization assays, assays that cannot readily be established in HEK-293 cells. U-2 OS cells were susceptible to simultaneous expression of multiple genes delivered by BacMam vectors. In U-2 OS cells the functional expression of the Gi-coupled M2and M4receptors was dependent on co-expression of the receptor and a G protein chimera, both of which were delivered to the cells via BacMam viruses. The use of U-2 OS cells and BacMam-based gene delivery has facilitated development of whole-cell-based GPCR functional assays, especially for P2Y, muscarininc acetylcholine, and LPA receptors.

Humans↗
PubMed2004

Impact of heterologous expression of Escherichia coli UDP-glucose pyrophosphorylase on trehalose and glycogen synthesis in Corynebacterium glutamicum.

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.

Corynebacterium↗
PubMed2004

Cloning and heterologous expression of a Methanococcus vannielii gene encoding a selenium-binding protein.

The activation and incorporation of selenium into selenocysteine containing selenoproteins has been well established in an Escherichia coli model system but there is little specific information concerning the transport and intracellular trafficking of selenium in biological systems in general. A selenium transport role is a possible function of a novel 42 kDa selenium-binding protein that recently was purified from Methanococcus vannielii. The gene encoding a monomer of this protein (Sbp) has been cloned, sequenced and heterologously expressed in E. coli. The 8.8 kDa gene product contains 81 amino acids. The recombinant Sbp (rSbp) protein was shown to bind selenium from added selenite. The bound selenium appeared predominantly in dimeric and tetrameric forms of the protein. The gene encoding Sbp occurs in an operon that contains a carbonic anhydrase gene and selenocysteine-containing formate dehydrogenase genes, suggesting possible roles in selenium-dependent formate metabolism.

Amino Acid Sequence↗
PubMed1998

Heterologous expression of the bacteriocin mesentericin Y105 using the dedicated transport system and the general secretion pathway.

Two different N-terminal extensions have been identified within class II bacteriocin precursors. The first one is a two-glycine-type leader peptide associated with a dedicated ATP-binding cassette transporter. The second is a signal peptide which directs the bacteriocin precursor to the general secretion machinery. Mesentericin Y105 is a class II anti-Listeria bacteriocin produced by Leuconostoc mesenteroides Y105 via a dedicated transport system (DTS). To investigate heterologous expression systems capable of producing mesentericin Y105 in various hosts, two different secretion vectors were constructed. One of them, containing the mesentericin Y105 structural gene fused to the segment encoding the divergicin A signal peptide, was introduced into Escherichia coli, Leuconostoc subsp. and Lactococcus subsp. In E. coli, mesentericin Y105 production was linked to a putative periplasmic toxicity. To take advantage of this secretion system, the mesentericin Y105 precursor was also produced in E. coli. It was demonstrated that this pre-bacteriocin exhibited some antagonistic activity against Listeria. To allow for a comparison between the two different transport systems, mesentericin Y105 production using the vector containing the mesentericin Y105 structural gene and its DTS transporter operon was examined. The production of mesentericin Y105 was monitored by a new fast purification method followed by MS analysis. It was shown that, in Leuconostoc, the production of mesentericin Y105 is enhanced via the DTS compared to the general secretion pathway.

Anti-Bacterial Agents↗
PubMed1996

Kinetic analysis of the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by heterologously expressed human P450 enzymes and the effect of P450-specific chemical inhibitors on this activation in human liver microsomes.

The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is enzymatically activated by the hydroxylation of the alpha-methyl and alpha-methylene groups, leading to the formation of reactive species which can pyridyloxobutylate and methylate DNA, respectively. The present study examined the kinetic parameters of NNK-derived keto alcohol (alpha-methyl hydroxylation), and keto aldehyde (alpha-methylene hydroxylation) formation catalyzed by human P450s heterologously expressed by either the baculovirus-insect cell expression system (P450s 2A6, 2D6, 2E1, and 3A4) or by stable expression in CHO cells (P450s 3A4 and 2D6) and human B-lymphoblastoid cells (P450 2D6). Membrane preparations of the expressed P450s catalyzed the alpha-hydroxylation of NNK, leading to the formation of keto aldehyde and keto alcohol. Human P450 2A6 showed the lowest KM (118 microM) for the formation of keto aldehyde. A similar KM was observed for keto alcohol formation by expressed P450 2A6, but the kcat was lower than the value obtained for keto aldehyde formation. The addition of exogenous b5 increased the expressed 2A6-dependent NNK hydroxylation activity 2.5-fold for both alpha-hydroxylation products. Human P450s 2E1 and 2D6 exhibited a high capacity for keto alcohol formation; however, their KM values for this reaction were in the millimolar range. Expressed human P450 3A4 oxidized NNK to keto aldehyde also with a high KM. Ten human liver microsomal samples were each shown to activate NNK to keto aldehyde and keto alcohol. A positive correlation coefficient of 0.74 was found between keto aldehyde formation and both coumarin 7-hydroxylation (P450 2A6) and 6 beta-testosterone hydroxylation (3A4) activity in characterized human liver microsomes. Keto alcohol formation showed a significant correlation with ethoxyresorufin O-dealkylation (P450 1A2) in human liver microsomes. Both coumarin and troleandomycin, specific inhibitors of P450 2A6 and 3A4, respectively, inhibited the formation of keto aldehyde, but inhibited the formation of keto alcohol only slightly in human liver microsomes. Both furafylline, a P450 1A2 inhibitor, and N-nitrosodimethylamine, a P450 2E1 substrate, inhibited the formation of keto alcohol but not keto aldehyde in human liver microsomes. Quinidine, a specific inhibitor of P450 2D6, was not an effective inhibitor of NNK metabolism. These results demonstrate that P450s 2A6 and 3A4 may be important P450s for the activation of NNK to a DNA-methylating agent and keto aldehyde via the alpha-methylene hydroxylation pathway. P450s 1A2, 2E1, and 2D6 are shown to be selective for alpha-methyl hydroxylation of NNK leading to keto alcohol and a DNA-pyridyloxobutylating agent.

Animals↗
PubMed2006

Evidence of oral toxicity of Photorhabdus temperata strain K122 against Prays oleae and its improvement by heterologous expression of Bacillus thuringiensis cry1Aa and cry1Ia genes.

Photorhabdus temperata strain K122 exhibited oral toxicity against Prays oleae with an LC50 of 58.1 x 10(6) cells ml(-1). Recombinant P. temperata strains expressing the cry1Aa and/or cry1Ia genes of Bacillus thuringiensis have been constructed. The two cry genes, encoding delta-endotoxins, were placed under the control of the lac promoter and IPTG dependent expression in P. temperata was demonstrated. The presence of the cry genes in K122 resulted in a clear improvement of oral toxicity. This improvement was of 6.2-, 6.6-, and 14.6-fold for the strains K122(pBCcry1Aa), K122(pBScry1Ia), and K122(pBCcry1Aa + pBScry1Ia), respectively. Furthermore, determination of the Synergistic Factor between Cry1Aa and Cry1Ia showed that they act synergistically. This work demonstrates that the heterologous expression of B. thuringiensis cry genes in P. temperata can be used to improve and broaden its host range for insect control.

Animals↗
PubMed1990

Primary sequence and heterologous expression of nuclear pore glycoprotein p62.

The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in nuclear transport. We have cloned, sequenced, and expressed the full-length rat p62 cDNA. The rat p62 mRNA is 2,941 nucleotides long and encodes a protein of 525 amino acids containing 30% serine and threonine residues. The amino acid sequence near the amino-terminus contains unique tetrapeptide repeats while the carboxy-terminus consists of a series of predicted alpha-helical regions with hydrophobic heptad repeats. Heterologous expression of rat p62 in African Green Monkey Kidney COS-1 cells and CV-1 cells was detected using a species-specific antipeptide serum. When transiently expressed in COS-1 cells, rat p62 binds wheat germ agglutinin and concentrates at the spindle poles during mitosis. In CV-1 cells cotransfected with rat p62 cDNA and SV40 viral DNA, rat p62 associates with the nuclear membrane without interfering with the nuclear transport of SV40 large T antigen. The ability to express p62 in tissue culture cells will facilitate analysis of the role of this pore protein in nuclear transport.

Amino Acid Sequence↗
PubMed1992

Cloning, nucleotide sequence and heterologous expression of the protective outer-membrane protein P.68 pertactin from Bordetella bronchiseptica.

The prn gene encoding the 68 kDa protective outer-membrane protein of Bordetella bronchiseptica (P.68 pertactin) was cloned, sequenced and expressed in Escherichia coli. The gene was isolated by DNA:DNA hybridization experiments using a radioactively-labelled fragment of the homologous prn gene from Bordetella parapertussis. DNA sequence analysis reveals that the gene is capable of encoding a protein with a molecular mass of 93996 Da (P.94); this precursor molecule is processed to form the P.68 antigen on the surface of B. bronchiseptica. Heterologous expression of the full-length gene encoding P.94 in Escherichia coli results in similar processing, with the P.68 antigen targeted to the bacterial outer membrane. Comparison of P.94 with the P.93 and P.95 precursors, encoding homologous proteins from Bordetella pertussis and B. parapertussis, shows a high degree (greater than 90%) of homology. The major differences between all three proteins occur in the number of repeats of the two families (Gly-Gly-Xaa-Xaa-Pro)n and (Pro-Gln-Pro)n of reiterated sequence motifs.

Amino Acid Sequence↗
PubMed2003

Ligand-specific dose-response of heterologously expressed olfactory receptors.

Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS88, 2366-2369]. Maximal stimulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant concentrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we developed an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand-specific and dose-dependent. We thus demonstrate that a differential dose-response originates from the olfactory receptor itself, which is thus capable of efficient discrimination between closely related agonists.

Animals↗
PubMed2005

Heterologous expression, purification, crystallization, X-ray analysis and phasing of the acetyl xylan esterase from Bacillus pumilus.

Bacillus pumilus PS213 acetyl xylan esterase (AXE) acts as an accessory enzyme in the plant cell wall hemicellulose biodegradation pathway. It belongs to the carbohydrate esterase family 7 and hydrolyses the ester linkages of the acetyl groups in position 2 and/or 3 of the xylose moieties of the acetylated xylan fragments from hardwood. The enzyme displays activity towards a broad range of acetylated compounds including the antibiotic cephalosporin-C. In this study we report the heterologous expression, purification, physicochemical characterization and crystallization of the recombinant B. pumilus AXE. Remarkable improvement of the crystal quality was achieved by setting up crystallization conditions, at first established using the hanging drop vapor diffusion method, in a micro-batch experiment. Rod-like diffraction quality crystals were obtained using 10% PEG 6000, 0.1 M MES pH 6.0 and a wide range of LiCl concentrations (0.2-1.0 M) as precipitant agent. Two different crystal forms, both belonging to space group P2(1), were characterized, diffracting X-rays to 2.5 and 1.9 angstrom resolution. Successful molecular replacement showed 12 molecules in the asymmetric unit of either crystal forms that are arranged as two doughnut-like hexamers, each one encompassing a local 32 symmetry. A catalytic inactive mutant Ser181Ala of B. pumilus AXE was also engineered, expressed, purified and crystallized for functional and structural studies.

Acetylesterase↗
PubMed2000

Cloning and heterologous expression of Dalpha4, a Drosophila neuronal nicotinic acetylcholine receptor subunit: identification of an alternative exon influencing the efficiency of subunit assembly.

A neuronal nicotinic acetylcholine receptor (nAChR) subunit, Dalpha4, has been identified and cloned from the fruit fly Drosophila melanogaster, together with several alternatively spliced transcripts. Intron-exon boundaries within the gene encoding Dalpha4 (nAcRalpha-80B) have been identified by comparison of cDNA and genomic sequence data. The influence of amino acids encoded by alternatively spliced exons upon nicotinic radioligand binding and subunit-subunit co-assembly has been examined by heterologous expression in Drosophila S2 cells. The efficiency of subunit assembly has been shown to be influenced by amino acids surrounding the highly conserved 15 amino acid cysteine-loop motif within the N-terminal extracellular domain of the nAChR Dalpha4 subunit. Extensive use has been made of publicly available data determined by the Berkeley Drosophila Genome Project (BDGP). This includes expressed sequence tag (EST) data as well as whole-embryo in situ hybridisation and polytene chromosome in situ hybridisation data. BDGP in situ hybridisation data suggests that the Dalpha4 mRNA is expressed within Drosophila brain and ventral nerve cord and demonstrates that the gene encoding this nAChR subunit is located at position 80B on chromosome 3. The relationship between Dalpha4 and other previously cloned nAChR subunits has been examined and the implications for the nomenclature of insect nAChRs is discussed.

Alternative Splicing↗
PubMed1995

Streptomyces peucetius daunorubicin biosynthesis gene, dnrF: sequence and heterologous expression.

The dnrF gene, responsible for conversion of aklavinone to epsilon-rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin (Dnr) gene cluster of Streptomyces peucetius ATCC 29050, close to drrAB, one of the anthracycline-resistance genes. The dnrF gene was sequenced and should encode a protein of 489 amino acids with a molecular mass of 52 kDa. The deduced DnrF protein shows significant similarities with bacterial FAD- and NADPH-dependent hydroxylases either required to introduce hydroxyl groups into polycyclic aromatic polyketide antibiotics or involved in catabolism of aromatic compounds. Heterologous expression of dnrF in Streptomyces lividans TK23 and in Escherichia coli demonstrated that the gene encodes a NADPH-dependent hydroxylase catalysing the hydroxylation of aklavinone to yield epsilon-rhodomycinone. The enzyme is inactive on anthracyclines glycosylated at position C-7 and its activity decreases to a different extent with other substrate modifications, indicating that DnrF has a significant substrate specificity.

Amino Acid Sequence↗
PubMed2001

Vesicular monoamine transporters heterologously expressed in the yeast Saccharomyces cerevisiae display high-affinity tetrabenazine binding.

A mammalian vesicular neurotransmitter transporter has been expressed in the yeast Saccharomyces cerevisiae. The gene encoding the rat vesicular monoamine transporter (rVMAT(1)) was cloned in several expression plasmids. The transporter was expressed at detectable levels only when short sequences using codons favored by S. cerevisiae were fused preceding the start of translation of rVMAT(1). The scarce expression of the wild-type protein was, most likely, due to the fact that part of the N-terminus of the protein is encoded by codons not preferred in S. cerevisiae. Furthermore, low growth temperatures increased rVMAT(1) expression and altered its processing. Whereas at 30 degrees C the protein is not glycosylated, at lower temperatures ( approximately 16 degrees C) half of the expressed transporters undergo core glycosylation. In addition, under these conditions the levels of protein expression significantly increase. Using a functional chimeric protein composed by VMAT and the green fluorescent protein (GFP), it is shown that the punctate pattern of intracellular distribution remains invariable at the different temperatures. Using a similar fusion sequence, the bovine VMAT isoform 2 (bVMAT(2)) was also expressed in yeast. The yeast-expressed bVMAT(2) binds [(3)H]dihydrotetrabenazine ([(3)H]TBZOH) with the same characteristics found in the native protein from bovine chromaffin granules. Dodecyl maltoside-solubilized bVMAT(2) retains the conformation required for [(3)H]TBZOH binding. We exploited the robust binding to follow the transporter during purification assays on a Ni(2+)-chelating column. In this report we describe for the first time the heterologous expression of a neurotransmitter transporter in the yeast S. cerevisiae.

Affinity Labels↗
PubMed1997

Isolation, heterologous expression and functional characterization of a novel cytochrome P450 3A enzyme from a canine liver cDNA library.

A cDNA encoding a new member of the cytochrome P450 3A subfamily, P450 3A26, has been isolated from phenobarbital-induced canine liver. The sequence encodes a protein of 503 amino acids with 33 nucleotide differences conferring 22 amino acid substitutions when compared with the previously identified canine CYP3A12 enzyme. Nine of the amino acid differences are within the substrate recognition sites (SRSs) identified for P450 family 2, with five residue substitutions clustered within SRS-6. To facilitate heterologous expression in Escherichia coli, the N-terminus of 3A26 was modified. The expressed protein comigrated with a 3A-immunoreactive protein in dog liver microsomes with a slightly greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than 3A12, which suggests that 3A26 corresponds to a previously noted but never characterized 3A enzyme in dogs. Functional characterization of 3A26 was undertaken with use of progesterone, testosterone and androstenedione as substrates. Assays of expressed 3A26 and 3A12 demonstrated that 3A26 displays low steroid hydroxylase activity. Identification of an additional canine 3A enzyme should increase our understanding of xenobiotic metabolism in this important animal model. These findings also suggest that 3A26 and 3A12 may be an interesting model system for the investigation of structure-function relationships involved in steroid metabolism catalyzed by members of the cytochrome P450 3A subfamily.

Amino Acid Sequence↗
PubMed1996

Heterologous expression of the cloned guinea pig alpha 2A, alpha 2B, and alpha 2C adrenoceptor subtypes. Radioligand binding and functional coupling to a CAMP-responsive reporter gene.

Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional alpha 2-adrenoceptors (alpha 2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human alpha 2-C10, alpha 2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human alpha 2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human alpha 2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-alpha 2A, and genes containing the complete coding sequences of the guinea pig alpha 2A, alpha 2B, and alpha 2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the alpha 2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-alpha 2A, 2700 pM for gp-alpha 2B and 110 pM for gp-alpha 2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (approximately 100 nM) but that it was not selective for any of the guinea pig alpha 2-AR subtypes. Co-expression of guinea pig alpha 2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-alpha 2 A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-alpha 2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-alpha 2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig alpha 2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human alpha 2-AR subtypes.

Adrenergic alpha-Antagonists↗
PubMed2002

Selective dopaminergic neurotoxicity of isoquinoline derivatives related to Parkinson's disease: studies using heterologous expression systems of the dopamine transporter.

Endogenous isoquinoline (IQ) derivatives structurally related to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridine (MPP(+)) may contribute to dopaminergic neurodegeneration in Parkinson's disease. We addressed the importance of the DAT molecule for selective dopaminergic toxicity by testing the differential cytotoxicity of 22 neutral and quaternary compounds from three classes of isoquinoline derivatives (3, IQs; 4,3,4-dihydroisoquinolines and 15, 1,2,3,4-tetrahydroisoquinolines) as well as MPP(+) in non-neuronal and neuronal heterologous expression systems of the DAT gene (human embryonic kidney HEK-293 and mouse neuroblastoma Neuro-2A cells, respectively). Cell death was estimated using the MTT assay and the Trypan blue exclusion method. Nine isoquinolines and MPP(+) showed general cytotoxicity in both parental cell lines after 72hr with half-maximal toxic concentrations (TC(50) values) in the micromolar range. The rank order of toxic potency was: papaverine>salsolinol=tetrahydropapaveroline=1-benzyl-TIQ=norsalsolinol>tetrahydropapaverine>2[N]-methyl-salsolinol>2[N]-methyl-norsalsolinol>2[N]-Me-IQ(+)=MPP(+). Besides MPP(+), only the 2[N]-methylated compounds 2[N]-methyl-IQ(+), 2[N]-methyl-norsalsolinol and 2[N]-methyl-salsolinol showed enhanced cytotoxicity in both DAT expressing cell lines with 2- to 14-fold reduction of TC(50) values compared to parental cell lines. The rank order of selectivity in both cell systems was: MPP(+)>>2[N]-Me-IQ(+)>2[N]-methyl-norsalsolinol=2[N]-methyl-salsolinol. Our results suggest that 2[N]-methylated isoquinoline derivatives structurally related to MPTP/MPP(+) are selectively toxic to dopaminergic cells via uptake by the DAT, and therefore may play a role in the pathogenesis of Parkinson's disease.

Animals↗
PubMed2006

Mechanism of cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel activation by isoflurane in a heterologous expression system.

BACKGROUND: Activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channel during metabolic stress initiates cellular events that preserve cardiac performance. Previous studies showed that halogenated anesthetics prime KATP channels under whole cell voltage clamp and act in intracellular pH (pHi)-dependent manner on KATP channels in excised membrane patches. However, it is not known how halogenated anesthetics interact with these channels. METHODS: The authors evaluated the effect of pHi and isoflurane on the KATP channel subunits, the pore-forming inward rectifier Kir6.2, and the regulatory sulfonylurea receptor SUR2A, using HEK293 cells as a heterologous expression system. Single channel activity was recorded in the inside-out patch configuration. RESULTS: At pHi 7.4, isoflurane had negligible effect on activity of wild-type Kir6.2/SUR2A, but at pHi 6.8, the channel open probability was increased by isoflurane (0.177 +/- 0.077 to 0.364 +/- 0.164). By contrast, the open probability of truncated Kir6.2DeltaC26, which forms a functional channel without SUR2A, was attenuated by isoflurane at both pHi 7.4 and pHi 6.8. Coexpression of Kir6.2DeltaC26 with SUR2A restored pHi sensitivity of channel activation by isoflurane. Site-directed mutagenesis within the Walker motifs of SUR2A abolished isoflurane activation of KATP channel at pHi 6.8. In addition, the pancreatic-type channels expressing sulfonylurea receptor SUR1 could not be activated by isoflurane. CONCLUSIONS: The nucleotide binding domains of SUR2A play a crucial role in isoflurane facilitation of the KATP channel activity at moderately acidic pHi as would occur during early ischemia. These findings support direct and differential interaction of isoflurane with the subunits of the cardiac sarcolemmal KATP channel.

ATP-Binding Cassette Transporters↗
PubMed1996

Heterologous expression of the spiny dogfish shark (Squalus acanthias) cytochrome P450c17 (17 alpha-hydroxylase) in Escherichia coli.

Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue. This protein possesses two enzymatic activities. The 17-alpha-hydroxylase activity introduces a hydroxyl group into the steroid nucleus. The resultant 17-alpha-hydroxylated, C(21) pregnenes can be converted to a C(19) androgen by the C(17,20)-lyase activity. The cDNA encoding the spiny dogfish shark (Squalus acanthias) testicular form of cytochrome P450c17 was used to direct heterologous expression in Escherichia coli. The wild-type P450c17 protein was not conducive to expression in E. coli using either a pET21 or a pCwori+ vector. However, modification of the amino terminus permitted overexpression of inactive shark protein with the pFT21 vector. This protein was fused with a hexahistidinyl peptide to facilitate purification by column chromatography using a Ni(2+)-chelated resin. Transformed bacteria yielded an average of 1.8 mg of purified, concentrated P450c17 protein per 100-ml culture. This modified protein was used to raise antisera in rabbits and the resultant antisera was used at a working titer of 1:10,000 for Western blot analysis. Culture conditions that result in the accumulation of bioactive recombinant bovine P450c17 failed to demonstrate expression of either the native or the modified form of shark P450c17 using the pCWori+ and pET21 vectors. These results suggest that the amino terminus of the native shark P450c17 was not conducive to synthesis in E. coli. However, modifications of the amino terminus permitted synthesis of an inactive protein that was protected from degradation within inclusion bodies.

Amino Acid Sequence↗