Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver

A thioredoxin reductase (TrxR), named here TrxR2, that did not react with antibodies to the previously identified TrxR (now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 was a dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3'-untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and contained 36 additional residues upstream of the experimentally determined NH2-terminal sequence. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Immunoblot analysis confirmed that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to 1.6 microgram/milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 microgram/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.     

Molecular cloning and expression of the functional rat C5a receptor

The C5a receptor belongs to the superfamily of G-protein coupled receptors with seven transmembrane segments. In this study we report on the cloning of the rat C5a receptor (ratC5aR). We used a hybridization probe produced by PCR utilizing degenerate primers which corresponded to conserved parts of the human, canine and murine C5a receptor nucleotide sequences and to the published partial amino acid sequence of the rat C5a receptor to screen a rat macrophage cDNA library. We found two overlapping clones containing an open reading frame of 1056 bp, a 3'untranslated region of 683 bp and a 5'untranslated region of 27 bp. The overall nucleotide acid sequence identity, compared to the murine, human and canine C5a receptor sequences, was 85.8, 70.5 and 68.9%, respectively. The greatest diversity exists in the putative extracellular domains, especially in the aminoterminal domain which is assumed to be involved in ligand binding. An N-glycosylation site is present within the N-terminal sequence at residue 6. One of the cDNA containing the 5'untranslated region, the coding sequence and part of the 3'untranslated region was cloned into an eucaryotic expression vector and stably transfected into the rat basophilic leukemia cell line RBL-2H3. Expression of the rat C5a receptor on the surface of these cells could be demonstrated by flow cytometric analysis using FITC-labeled recombinant rat C5a (rrC5a). By measuring the release of calcium from intracellular stores after stimulation with rrC5a it could further be shown that the receptor is functionally coupled. Receptor binding assays showed that rrC5a specifically binds to the ratC5aR with a KD of 0.91 +/- 0.36 and to the human C5a receptor (huC5aR) with a KD of 7.19 +/- 1.56. The determined KD for binding of human C5a (huC5a) to the huCSaR was 2.16 +/- 0.65. No binding of huC5a to the ratC5aR could be observed although high concentrations of this ligand (> 60 nM) promoted chemotaxis of RBL cells transfected with the huC5aR.     

Molecular cloning and functional expression of feline thrombopoietin

Feline thrombopoietin (TPO) was molecularly cloned to establish a basis for cytokine therapy of thrombocytopenia in cats. cDNA clones covering the whole coding sequence of feline TPO were isolated from feline liver. The feline TPO cDNA obtained in this study contained an open reading frame encoding 349 amino acid residues. The predicted amino acid sequence of feline TPO shared 78.7, 69.9, 72.9 and 83.0% similarity with sequences of human, murine, rat and canine TPO, respectively. Four cysteine residues and two of four N-glycosylation sites that are conserved among species were also found at the corresponding positions in feline TPO. The feline TPO cDNA fragment encoding the whole amino acid coding region was recloned into an expression vector, and the resulting vector was transfected into 293T cells using the calcium phosphate method. The supernatant of the transfected 293T cells stimulated the proliferation of a human megakaryoblastic leukemia cell line (UT-7/TPO) cells in a dose dependent manner, indicating that the feline TPO cDNA obtained in this study encodes biologically active feline TPO.     

Molecular cloning and functional expression of equine interleukin-1 receptor antagonist

Equine interleukin-1 receptor antagonist (IL-1ra) was molecularly cloned to establish a basis for cytokine therapy of acute and chronic inflammatory diseases in the horse. cDNA clones encoding the whole coding sequence of equine IL-1ra were isolated from equine peripheral blood mononuclear cells (PBMC) that had been stimulated with lipopolysaccharide (LPS). The equine IL-1ra cDNA obtained in this study contained an open reading frame encoding 177 amino acid residues. The predicted amino acid sequence of equine IL-1ra shared 75.7, 75.3 and 76.3% similarity with sequences of human, murine and rabbit IL-1ras, respectively. An N-glycosylation site and five cysteine residues conserved in human, murine and rabbit IL-1ras were also found at the corresponding positions in equine IL-1ra. Recombinant glutathione S-transferase (GST)-equine IL-1ra fusion protein produced by Escherichia coli was purified. This protein was shown to inhibit the cytostatic or cytotoxic activity of IL-1 on A375S2 cells, indicating that the equine IL-1ra cDNA obtained in this study encodes biologically active equine IL-1ra.     

Molecular cloning, expression, and functional characterization of novel mouse sulfotransferases

STUDY OBJECTIVE: The present study was performed to determine the influence of a perioperative myocardial infarction on long-term mortality in patients who have undergone elective vascular surgery. STUDY DESIGN: This was a 4-year follow-up of patients who had undergone elective vascular procedures at a Veterans Affairs Medical Center. Between January 1989 and December 1990, 115 consecutive patients underwent surgery for either an expanding abdominal aortic aneurysm (AAA) (38%) or for pain in the lower extremities (62%). RESULTS: Vital status at 4 years postsurgery was determined for all patients. Thirty-day postoperative mortality was 3%, while estimates at 1, 2, 3, and 4 years were 19%, 26%, 35%, and 39%, respectively. Of the 45 patients who died within 4 years following surgery, the major causes of death were cardiac (40%), cancer (18%), cerebrovascular (13%), and peripheral vascular disease (11%). Univariate predictors of 1-year mortality on preoperative evaluation were an abnormal ECG, moderate or greater sized exercise thallium defect and left ventricular ejection fraction < or =40%, and a perioperative myocardial infarction. Univariate predictors of 4-year mortality were non-AAA surgery and diabetes mellitus. Perioperative myocardial infarction was a marginally significant independent predictor of 1-year mortality (p=0.06), while the need for non-AAA surgery was a strong independent predictor at 4 years. CONCLUSIONS: Cardiac mortality is the major cause of late death among patients undergoing elective vascular surgery. Although preoperative indicators of symptomatic coronary artery disease and nonfatal perioperative myocardial infarction identified those individuals at increased mortality in the first postoperative year, the extent of vascular disease at presentation may be a more important determinant of long-term survival. A randomized trial in such patients is needed to assess the best strategy for treating patients with coexistent coronary artery and vascular diseases.     

Molecular cloning and functional expression of a cDNA for rat phospholipid hydroperoxide glutathione peroxidase: 3'-untranslated region of the gene is necessary for functional expression

A full-length cDNA clone for phospholipid hydroperoxide glutathione peroxidase (PHGPx) was isolated from a rat brain. The cDNA was 0.761 kb in length and encoded 170 amino acids, which included a TGA-encoded selenocysteine at residue 46. The protein has a calculated molecular mass of 19,473 Da. We succeeded in the transient functional expression of a full-length cDNA for PHGPx, which includes the 3'-UTR, in COS-7 cells at the first attempt. Deletion of the 3'-UTR prevented the expression of the PHGPx activity and the incorporation of [75Se]selenious acid into the monomeric 19.7 kDa PHGPx protein. Thus, the 3'-UTR of the cDNA for PHGPx was required for the functional expression of PHGPx. Northern blot analysis demonstrated that the mRNA for PHGPx was widely expressed in normal rat tissues, especially in the testis. The mRNA levels of PHGPx in the cultured cells such as hepatomas, neuronal cells, nephroblastoma, and mammary myo-epithelial cells were higher than those of the tissues. The ratio of PHGPx to cytosolic glutathione peroxidase (cGPx) was significantly high in the testis and relatively high in the cultured cells. The mRNA levels of PHGPx in tissues were lower than those of cGPx.     

Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes

A cDNA encoding a P450 monooxygenase was amplified from reverse transcribed rat heart and liver total RNA by polymerase chain reaction using primers based on the 5'- and 3'-end sequences of two rat pseudogenes, CYP2J3P1 and CYP2J3P2. Sequence analysis revealed that this 1,778-base pair cDNA contained an open reading frame and encoded a new 502 amino acid protein designated CYP2J3. Based on the deduced amino acid sequence, CYP2J3 was approximately 70% homologous to both human CYP2J2 and rabbit CYP2J1. Recombinant CYP2J3 protein was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells using a baculovirus expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolized arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 degrees C). Immunoblotting of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-reacted with CYP2J3 but not with other known rat P450s demonstrated abundant expression of CYP2J3 protein in heart and liver. Immunohistochemical staining of formalin-fixed paraffin-embedded rat heart tissue sections using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection localized expression of CYP2J3 primarily to atrial and ventricular myocytes. In an isolated-perfused rat heart model, 20 min of global ischemia followed by 40 min of reflow resulted in recovery of only 44 +/- 6% of base-line contractile function. The addition of 5 microM 11, 12-epoxyeicosatrienoic acid to the perfusate prior to global ischemia resulted in a significant 1.6-fold improvement in recovery of cardiac contractility (69 +/- 5% of base line, p = 0.01 versus vehicle alone). Importantly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid significantly improved functional recovery following global ischemia, demonstrating the specificity of the biological effect for the 11, 12-epoxyeicosatrienoic acid regioisomer. Based on these data, we conclude that (a) CYP2J3 is one of the predominant enzymes responsible for the oxidation of endogenous arachidonic acid pools in rat heart myocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important functional role in the response of the heart to ischemia.     

Molecular cloning and functional expression of a skeletal muscle dihydropyridine receptor from Rana catesbeiana

In skeletal muscle the dihydropyridine receptor is the voltage sensor for excitation-contraction coupling and an L-type Ca2+ channel. We cloned a dihydropyridine receptor (named Fgalpha1S) from frog skeletal muscle, where excitation-contraction coupling has been studied most extensively. Fgalpha1S contains 5600 base pairs coding for 1688 amino acids. It is highly homologous with, and of the same length as, the C-truncated form predominant in rabbit muscle. The primary sequence has every feature needed to be an L-type Ca2+ channel and a skeletal-type voltage sensor. Currents expressed in tsA201 cells had rapid activation (5-10 ms half-time) and Ca2+-dependent inactivation. Although functional expression of the full Fgalpha1S was difficult, the chimera consisting of Fgalpha1S domain I in the rabbit cardiac Ca channel had high expression and a rapidly activating current. The slow native activation is therefore not determined solely by the alpha1 subunit sequence. Its Ca2+-dependent inactivation strengthens the notion that in rabbit skeletal muscle this capability is inhibited by a C-terminal stretch (Adams, B., and Tanabe, T. (1997) J. Gen. Physiol. 110, 379-389). This molecule constitutes a new tool for studies of excitation-contraction coupling, gating, modulation, and gene expression.     

Molecular cloning and functional expression of a human cDNA encoding translation initiation factor 6

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. In this paper, we devised a procedure for purifying eIF6 from rabbit reticulocyte lysates and immunochemically characterized the protein by using antibodies isolated from egg yolks of laying hens immunized with rabbit eIF6. By using these monospecific antibodies, a 1.096-kb human cDNA that encodes an eIF6 of 245 amino acids (calculated Mr 26,558) has been cloned and expressed in Escherichia coli. The purified recombinant human protein exhibits biochemical properties that are similar to eIF6 isolated from mammalian cell extracts. Database searches identified amino acid sequences from Saccharomyces cerevisiae, Drosophila, and the nematode Caenorhabditis elegans with significant identity to the deduced amino acid sequence of human eIF6, suggesting the presence of homologues of human eIF6 in these organisms.     

Molecular cloning and functional expression of a phorbol ester-inducible 8S-lipoxygenase from mouse skin

One of the effects of topical application of phorbol ester to mouse skin is the induction of an 8S-lipoxygenase in association with the inflammatory response. Here we report the molecular cloning and characterization of this enzyme. The cDNA was isolated by polymerase chain reaction from mouse epidermis and subsequently from a mouse epidermal cDNA library. The cDNA encodes a protein of 677 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has 78% identity to a 15S-lipoxygenase cloned recently from human skin and approximately 40% identity to other mammalian lipoxygenases. When expressed in vaccinia virus-infected Hela cells, the mouse enzyme converts arachidonic acid exclusively to 8S-hydroperoxyeicosatetraenoic acid while linoleic acid is converted to 9S-hydroperoxy-linoleic acid in lower efficiency. Phorbol ester treatment of mouse skin is associated with strong induction of 8S-lipoxygenase mRNA and protein. By Northern analysis, expression of 8S-lipoxygenase mRNA was also detected in brain. Immunohistochemical analysis of phorbol ester-treated mouse skin showed the strongest reaction to 8S-lipoxygenase in the differentiated epidermal layer, the stratum granulosum. The inducibility may be a characteristic feature of the mouse 8S-lipoxygenase and its human 15S-lipoxygenase homologue.     

Molecular cloning and functional expression of mannitol-1-phosphatase from the apicomplexan parasite Eimeria tenella

A metabolic pathway responsible for the biosynthesis and utilization of mannitol is present in the seven species of Eimeria that infect chickens, but is not in the avian host. Mannitol-1-phosphatase (M1Pase), a key enzyme for mannitol biosynthesis, is a highly substrate-specific phosphatase and, accordingly, represents an attractive chemotherapeutic target. Amino acid sequence of tryptic peptides obtained from biochemically purified Eimeria tenella M1Pase was used to synthesize degenerate oligonucleotide hybridization probes. Using these reagents, a partial genomic clone and full-length cDNA clones have been isolated and characterized. The deduced amino acid sequence of E. tenella M1Pase shows limited overall homology to members of the phosphohistidine family of phosphatases. This limited homology to other histidine phosphatases does, however, include several conserved residues that have been shown to be essential for their catalytic activity. Kinetic parameters of recombinant M1Pase expressed in bacteria are essentially identical to those of the biochemically purified preparation from E. tenella. Moreover, recombinant M1Pase is subject to active site-directed, hydroxylamine-reversible inhibition by the histidine-selective acylating reagent diethyl pyrocarbonate. These results indicate the presence of an essential histidine residue(s) at the M1Pase active site, as predicted for a histidine phosphatase.     

Molecular cloning and functional characterization of murine sphingosine kinase

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.     

Molecular cloning and characterization of rat lin-10

In Caenorhabditis elegans, the vulval induction is mediated by tyrosine kinase receptor/Ras signal transduction pathway composed of the lin-3, let-23, and let-60 products. In addition to these gene products, the lin-2, lin-7, and lin-10 products are also implicated in this pathway. Lin-2 encodes a MAGUK and lin-7 encodes a small protein with one PDZ domain. The lin-10 product has no homology to known proteins. Here, we have cloned a rat homologue of the lin-10 product and characterized it. Rat lin-10 is ubiquitously expressed in various rat tissues and distributed in both the cytosol and membrane fractions. In brain, however, rat lin-10 is distributed only in the membrane fraction and enriched in the synaptic plasma membrane and postsynaptic density fractions. These results suggest that rat lin-10 is involved at least in synaptic functions in brain.