ABSTRACT
BACKGROUND AND AIM: The management of patients with recent-onset atrial fibrillation (AF) presenting at emergency departments (EDs) varies widely. Our aim was to describe the management of patients with recent-onset (<48 hours) AF, to determine safety and efficacy of pharmacological cardioversion at the ED, and to evaluate the incidence of thromboembolism or death at 30 days. METHODS: In a prospective, observational, single-center study, 236 subjects with recent-onset AF were consecutively enrolled from January 2011 until January 2013. Follow-up information was obtained by reviewing all available clinical records. RESULTS: As first-line therapy, 45.3% (n = 107) received ibutilide, 28.8% (n = 68) vernakalant, 25% (n = 59) flecainide, and 0.8% (n = 2) amiodarone, respectively. Successful cardioversion was achieved in 72.5% (n = 171) of patients after first-line therapy. There was no significant difference between treatment groups. In univariable logistic regression analysis, age (odds ratio [OR] = 1.027; 95% confidence interval [CI], 1.003-1.052; P= .03), duration of symptoms (OR = 0.968; 95% CI, 0.938-0.999; P= .045), as well as the CHA2DS2-VASc score (1 point for Congestive heart failure, Hypertension, Age between 65 and 74 years, Diabetes, Vascular disease, Sex category if female and 2 points for previous TIA/Stroke and Age ≥ 75 years) (OR = 1.237; 95% CI, 1.01-1.515; P= .04) were associated with success of pharmacological cardioversion. Within 30 days, 1 patient suffered from fatal ischemic stroke. CONCLUSION: Pharmacological cardioversion followed by discharge after a short observation period is safe. There was no significant difference between the agents used in terms of short-term safety and efficacy. Importantly, the coherence of the ED to recent guidelines regarding first-line therapy is high.
Subject(s)
Anti-Arrhythmia Agents/therapeutic use , Atrial Fibrillation/drug therapy , Electrocardiography , Aged , Atrial Fibrillation/physiopathology , Female , Follow-Up Studies , Humans , Male , Middle Aged , Prospective Studies , Treatment OutcomeABSTRACT
BACKGROUND: In 2014, Louisiana passed a law requiring abortion providers to have hospital admitting privileges. This law is temporarily on hold while a court case challenging it continues. We aimed to describe the population who would be affected if the law goes into effect and how closures of between three and five Louisiana abortion facilities would affect the distance Louisiana women would need to travel for an abortion. STUDY DESIGN: We abstracted patient data from three of the five Louisiana abortion care facilities in the year before the law was scheduled to take effect. We then estimated distance traveled and distances women would need to travel if clinics close. FINDINGS: Half (53%) of women who had an abortion had no education beyond high school, most were black (62%) or white (30%), three fourths (73%) had a previous live birth, and most (89%) were having a first-trimester abortion. Seventy-nine percent resided in Louisiana and 15% in Texas. The parishes in which abortion patients resided had lower median income and higher percentage poverty than the Louisiana average. Abortion patients residing in Louisiana traveled a mean distance of 58 miles each way for an abortion. If all Louisiana facilities close, the mean distance women would need to travel would more than triple to 208 miles, and the proportion of Louisiana women of reproductive age who live more than 150 miles from an abortion facility would increase from 1% to 72%. CONCLUSION: The admitting privileges law will likely significantly increase the distance Louisiana women need to travel for an abortion. This burden is likely to disproportionately affect Louisiana's more vulnerable residents. IMPLICATIONS: If all Louisiana abortion facilities close due to Louisiana's hospital admitting privileges law, the mean distance women would need to travel for an abortion would more than triple from 58 to 208 miles. Louisiana's law would thus present a considerable burden on many Louisiana women, particularly those who are more vulnerable.
Subject(s)
Abortion, Induced/legislation & jurisprudence , Abortion, Legal/legislation & jurisprudence , Health Services Accessibility/legislation & jurisprudence , Travel/statistics & numerical data , Admitting Department, Hospital , Adolescent , Adult , Child , Female , Humans , Louisiana , Pregnancy , Socioeconomic Factors , Texas , Vulnerable Populations , Young AdultABSTRACT
Uterine artery embolization (UAE) represents radiological treatment of uterine fibroids. It is highly effective and safe mainly in premenopausal patients with symptomatic fibroids and represents an alternative to hysterectomy in a group of women not suitable for minimally invasive surgical treatment (LAVH) and women desiring uterus sparing therapy. The future of UAE lies in optimal selection of patients based on volume-shrinkage prediction and fertility outcome. The second group is represented by methods based on direct fibroid tissue destruction using specific energy under MRI or UZ guidance. The common aim of these two groups is the volume shrinkage as well as the symptomatic relief. The second group is represented by radiofrequency ablation, focused ultrasound surgery, interstitial laser ablation and cryotherapy. Based on their non-surgical, percutaneous approach these can be classified as minimally-invasive methods. The second group of methods is suitable only for patients with the absence of any desire for child bearing due to the absence of their long-term outcome data.
Subject(s)
Leiomyoma/therapy , Radiography, Interventional , Uterine Neoplasms/therapy , Female , Humans , Leiomyoma/diagnostic imaging , Uterine Neoplasms/diagnostic imagingABSTRACT
Ormond disease - idiopathic retroperitoneal fibrosis - is a rare condition characterized in situ by the development of fibrous plaques in the retroperitoneal space and anatomicaly dependent structures. The associated encasement of both ureters and progress to hydronefrosis of the kidney are typical clinical manifestations. Less typical manifestations are possible (for example chronic periaortitis), where clinical diagnosis is more difficult. The laboratory findings are not specific for this disease and a biopsy is not always possible for anatomical reasons. In these cases, the use of positron emission tomography/computed tomography - has been found to be the solution, specifically for patients with periaortitis. Ormond disease is generally idiopathic, and secondary - to the use of certain drugs, malignant diseases, infections. Idiopathic retroperitoneal disease is thought to result from the clinical manifestation of a systemic autoimmune disease. The purpose of this article is to present two casuistics, one of a less than usual clinical manifestation. Both positron emission tomography/computed tomography were used in the diagnostics. The treatment ofOrmond disease involves the combination of surgical and immunosuppressive treatment.
Subject(s)
Retroperitoneal Fibrosis , Adult , Female , Humans , Male , Positron-Emission Tomography , Retroperitoneal Fibrosis/complications , Retroperitoneal Fibrosis/diagnosis , Tomography, X-Ray ComputedABSTRACT
PURPOSE: To study the variation of computed tomography (CT) number from a simulator-based scanner and the effect of this variation on photon-dose calculations. METHOD AND MATERIALS: CT images of a cylindrical phantom with multiple inserts were obtained using a commercially-available simulator-CT (Ximatron: Varian, Palo Alto, CA). The linear correlation coefficient and Chi-square methods were used to determine the X-ray effective energy in a phantom. CT numbers in Hounsfield units (HU) were measured as a function of phantom size, orientation, field of view (FOV), distance from the center, and time for various inserts. The change of dose calculations due to the CT number variations was then determined using the equivalent path-length (EPL) and collapsed cone convolution methods. RESULTS AND DISCUSSION: A significant beam-hardening effect was observed for the simulator-CT. Consequently, the CT number from the sim-CT was more sensitive to the size of the phantom than those from a conventional CT. The sim-CT number is not sensitive to the locations within the phantom and is stable over a 6-week period. It is important to use the proper FOV for sim-CT studies; scanning a small polystyrene phantom using a large FOV may result in an increase of l20 HU in CT number at the center of the field. However, the dose-calculation variations, due to the CT number uncertainty, do not exceed 2-3% for 6-18 MV photon beams. CONCLUSION: The simulator CT images were acquired with patients in the treatment position, and these CT numbers are useful for CT-based dose calculations.
Subject(s)
Computer Simulation , Radiotherapy Dosage , Radiotherapy Planning, Computer-Assisted , Tomography, X-Ray Computed , Algorithms , Chi-Square Distribution , Humans , Linear Models , Phantoms, Imaging , Photons , Polystyrenes , Radiotherapy, High-Energy , Reproducibility of Results , Surface Properties , Tomography Scanners, X-Ray ComputedABSTRACT
We have isolated a cDNA encoding a 1012-amino acid polypeptide cPLA2-beta, that has significant homology with cPLA2-alpha in both the calcium-dependent lipid binding domain as well as in the catalytic domain. Transient expression of cPLA2-beta cDNA in COS cells results in an increase in calcium-dependent phospholipase A1 (PLA1) and PLA2 activities compared with vector-transfected cells. cPLA2-beta is markedly less selective for cleavage at sn-2 as compared with cPLA2-alpha and cPLA2-gamma. Northern analysis reveals a cPLA2-beta transcript of 8 kilobase pairs that is expressed in all the human tissues examined. With the identification of cPLA2-beta, the newly defined cPLA2 family now comprises three members that may have dramatically different mechanisms for regulation of expression and enzymatic activation.
Subject(s)
Cytosol/enzymology , Phospholipases A/genetics , Amino Acid Sequence , Calcium/pharmacology , Cloning, Molecular , DNA, Complementary/genetics , Gene Library , Group IV Phospholipases A2 , Humans , Molecular Sequence Data , Phospholipases A/biosynthesis , Phospholipases A/drug effects , Phospholipases A1 , Phospholipases A2 , Recombinant Proteins/biosynthesis , Sequence Homology, Amino Acid , Tissue Distribution , U937 CellsSubject(s)
Cloning, Molecular/methods , DNA, Complementary/isolation & purification , Glycoside Hydrolases/genetics , Protein Sorting Signals/genetics , Base Sequence , DNA Primers , DNA, Complementary/genetics , Electroporation , Escherichia coli/genetics , Gene Library , Genetic Vectors , Oligodeoxyribonucleotides , Polymerase Chain Reaction/methods , Protein Biosynthesis , Protein Sorting Signals/biosynthesis , Saccharomyces cerevisiae/genetics , beta-FructofuranosidaseABSTRACT
We report the cloning and characterization of a novel membrane-bound, calcium-independent PLA2, named cPLA2-gamma. The sequence encodes a 541-amino acid protein containing a domain with significant homology to the catalytic domain of the 85-kDa cPLA2 (cPLA2-alpha). cPLA2-gamma does not contain the regulatory calcium-dependent lipid binding (CaLB) domain found in cPLA2-alpha. However, cPLA2-gamma does contain two consensus motifs for lipid modification, a prenylation motif (-CCLA) at the C terminus and a myristoylation site at the N terminus. We present evidence that the isoprenoid precursor [3H]mevalonolactone is incorporated into the prenylation motif of cPLA2-gamma. Interestingly, cPLA2-gamma demonstrates a preference for arachidonic acid at the sn-2 position of phosphatidylcholine as compared with palmitic acid. cPLA2-gamma encodes a 3-kilobase message, which is highly expressed in heart and skeletal muscle, suggesting a specific role in these tissues. Identification of cPLA2-gamma reveals a newly defined family of phospholipases A2 with homology to cPLA2-alpha.
Subject(s)
Phospholipases A/isolation & purification , Amino Acid Sequence , Animals , CHO Cells , COS Cells , Calcium/metabolism , Cell Membrane/enzymology , Cells, Cultured , Cloning, Molecular , Cricetinae , Group IV Phospholipases A2 , Humans , Molecular Sequence Data , Molecular Weight , Phospholipases A/chemistry , Phospholipases A/genetics , Phospholipases A2 , Protein Prenylation , Sequence AlignmentABSTRACT
We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity. The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation. Purified recombinant enzyme utilizes NADP+ as a cofactor and, like its E. coli counterpart, is inhibited by GDP-fucose, suggesting that this aspect of regulation is also conserved. We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR. Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro. This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide. Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway in bacteria.
Subject(s)
Guanosine Diphosphate Fucose/biosynthesis , Hydro-Lyases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Cricetinae , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Escherichia coli , Guanosine Diphosphate Fucose/genetics , Humans , Hydro-Lyases/metabolism , Molecular Sequence Data , Sequence Alignment , TransfectionABSTRACT
We describe a simple, rapid technique for simultaneously isolating large numbers of cDNAs encoding secreted proteins. The technique makes use of a facile genetic selection performed in a strain of Saccharomyces cerevisiae deleted for its endogenous invertase gene. A cDNA cloning vector which carries a modified invertase gene lacking its leader sequence is used in conjunction with this strain. Heterologous secreted genes fused appropriately upstream of this defective invertase provide the necessary signals to restore secretion, allowing the yeast to grow on sugars such as sucrose or raffinose. This microbial growth selection facilitates scanning cDNA libraries containing millions of clones, enabling the wholesale identification of novel secreted proteins without the need for specific bioassays. The technique is similar to one previously described (Klein et al. (1996) Proc. Natl. Acad. Sci. USA 93, 7108-7113). We describe results using a cDNA library derived from activated human peripheral blood mononuclear cells (PBMC). Genes identified from this library encoded signal sequences of proteins of diverse structure, function, and cellular location such as cytokines, type 1 and type 2 transmembrane proteins, and proteins found in intracellular organelles. In addition, a number of novel secreted proteins were identified, including a chemokine and a novel G-protein-coupled receptor. Since signal sequences possess features conserved throughout evolution, the procedure can be used to isolate genes encoding secreted proteins from both eukaryotes and prokaryotes.
Subject(s)
DNA, Complementary/isolation & purification , Genetic Vectors , Protein Sorting Signals , Proteins/metabolism , Receptors, Cell Surface/genetics , Amino Acid Sequence , Chemokines/genetics , Glycoside Hydrolases/genetics , Humans , Interferon-gamma/genetics , Molecular Sequence Data , Saccharomyces cerevisiae/genetics , Selection, Genetic , Sequence Alignment , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , beta-FructofuranosidaseABSTRACT
We report the purification, molecular cloning, and expression of a novel cytosolic calcium-independent phospholipase A2 (iPLA2) from Chinese hamster ovary cells, which lacks extended homology to other phospholipases. iPLA2 is an 85-kDa protein that exists as a multimeric complex of 270-350 kDa with a specific activity of 1 micromol/min/mg. The full-length cDNA clone encodes a 752-amino acid cytoplasmic protein with one lipase motif (GXS465XG) and eight ankyrin repeats. Expression of the cDNA in mammalian cells generates an active 85-kDa protein. Mutagenesis studies show that Ser465 and the ankyrin repeats are required for activity. We demonstrate that iPLA2 selectively hydrolyzes the sn-2 over sn-1 fatty acid by 5-fold for 1,2-dipalmitoyl phosphatidylcholine in a mixed micelle. Moreover, we found the fatty acid preference at the sn-2 position to be highly dependent upon substrate presentation. However, iPLA2 does have a marked preference for 1,2-dipalmitoyl phosphatidic acid presented in a vesicle, generating the lipid second messenger lysophosphatidic acid. Finally the enzyme is able to hydrolyze the acetyl moiety at the sn-2 position of platelet-activating factor.
Subject(s)
Ankyrins/metabolism , Calcium/metabolism , Isoenzymes/metabolism , Phospholipases A/metabolism , Amino Acid Sequence , Animals , CHO Cells , Cloning, Molecular , Cricetinae , Cytosol/enzymology , Humans , Isoenzymes/chemistry , Molecular Sequence Data , Molecular Weight , Phospholipases A/chemistry , Phospholipases A2 , Sequence AlignmentABSTRACT
Cytosolic phospholipase A2 (cPLA2) associates with natural membranes in response to physiological increases in Ca2+, resulting in the selective hydrolysis of arachidonyl phospholipids. The isolation and sequence analysis of cPLA2 cDNA clones from four different species revealed several highly conserved regions. The NH2-terminal conserved region is homologous to several other Ca(2+)-dependent lipid-binding proteins. Here we report that the first 178 residues of cPLA2, containing the homologous Ca(2+)-dependent lipid-binding (CaLB) motif, and another recombinant protein containing the cPLA2(1-178) fragment placed at the COOH terminus of the maltose-binding protein (MBP-CaLB) associate with membranes in a Ca(2+)-dependent manner. cPLA2 and MBP-CaLB also bind to synthetic liposomes at physiological Ca2+ concentrations, demonstrating that accessory proteins are not required. In contrast, delta C2, a truncated cPLA2 lacking the CaLB domain, fails to associate with membranes and fails to hydrolyze liposomal substrates. However, both delta C2 and cPLA2 hydrolyze monomeric 1-palmitoyl-2-lysophosphatidylcholine at identical rates in a Ca(2+)-independent fashion. These results delineate two functionally distinct domains of cPLA2, the Ca(2+)-independent catalytic domain, and the regulatory CaLB domain that presents the catalytic domain to the membrane in response to elevated Ca2+.
Subject(s)
Calcium/metabolism , Lipid Metabolism , Phospholipases A/chemistry , Phospholipases A/metabolism , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Catalysis , Cell Line , Chickens , Cloning, Molecular , Cricetinae , Cytosol/enzymology , Fishes , Humans , Mice , Molecular Sequence Data , Phospholipases A/genetics , Phospholipases A2 , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
CD70 is a surface Ag found on activated but not resting T and B lymphocytes. The biologic activity of this Ab-defined cell surface molecule on lymphocytes has not been established. Therefore, in an effort to understand the function of the CD70 protein, a mAb defining the CD70 Ag was used to isolate by expression cloning the cDNA responsible for the CD70 molecule. The predicted protein product is a type II transmembrane protein. Bioassays demonstrated that the CD70 cDNA clone expressed in African green monkey kidney cells would induce the proliferation of PHA-costimulated T cells. Comparison with known sequences indicates identity with the CD27 ligand. Therefore the molecule defining the CD70 Ag is identical to the recently defined ligand for CD27.
Subject(s)
Antigens, CD/chemistry , Membrane Proteins/chemistry , Amino Acid Sequence , Antigens, CD/biosynthesis , Antigens, CD/genetics , Base Sequence , CD27 Ligand , Cell Line , Cloning, Molecular , Humans , Lymphocyte Activation , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Molecular Sequence Data , T-Lymphocytes/immunology , Tetradecanoylphorbol Acetate/pharmacology , Tumor Necrosis Factor Receptor Superfamily, Member 7/metabolismABSTRACT
A wide range of growth and differentiation processes are regulated by the signalling of receptor tyrosine kinases (RTKs). We have developed a nested polymerase chain reaction (PCR) procedure with degenerate primers, and used it to identify RTKs expressed in murine fetal thymus. A novel RTK, called FLT4, and the murine homologue of FLT were found, and their PCR fragment sequences were used to isolate larger cDNA clones spanning the complete coding regions of these receptors. FLT4 was found to contain an extracellular region similar to the corresponding sequences of FLT and Flk-1, containing seven immunoglobulin domains.
Subject(s)
Cloning, Molecular , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Receptors, Cell Surface/genetics , Amino Acid Sequence , Animals , Base Sequence , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Polymerase Chain Reaction , Proto-Oncogene Proteins/chemistry , RNA, Messenger/analysis , Vascular Endothelial Growth Factor Receptor-1ABSTRACT
We evaluated 148 patients with allergic conjunctivitis in a double-masked, paired comparison clinical trial comparing ketorolac 0.5% ophthalmic solution with vehicle. Patients received one drop of each study medication in preassigned eyes, four times a day, for seven days. Both treatments showed significant changes from baseline in the signs and symptoms associated with allergic conjunctivitis. Evaluations at the final visit (day 7 or 8) showed that ketorolac-treated eyes had a significant treatment response when compared to vehicle-treated eyes for conjunctival inflammation (p = 0.010), ocular itching (p = 0.006), swollen eyes (p = 0.002), discharge/tearing (p = 0.021), foreign body sensation (p = 0.035), and conjunctival injection (p = 0.016). Mean scores evaluating the overall therapeutic effect of the study treatments at the completion of the study were higher for ketorolac-treated eyes than for vehicle-treated eyes as rated by investigators (p = 0.004) and study patients (p < 0.001). Results of this study confirmed the trends of a previous study showing that ketorolac 0.5% ophthalmic solution applied topically is an effective therapy for allergic conjunctivitis.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Conjunctivitis, Allergic/drug therapy , Tolmetin/analogs & derivatives , Tromethamine/administration & dosage , Adolescent , Adult , Aged , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Double-Blind Method , Drug Administration Schedule , Drug Combinations , Drug Evaluation , Female , Humans , Ketorolac Tromethamine , Male , Middle Aged , Ophthalmic Solutions , Pharmaceutical Vehicles , Tolmetin/administration & dosage , Tolmetin/adverse effects , Tromethamine/adverse effectsABSTRACT
The partial sequence of a novel PtdIns-specific phospholipase C of the beta subfamily (PtdIns-PLC beta 3) is described. Based upon the predicted protein sequence, monospecific antibodies have been raised and used to identify a suitable source for purification of the protein. Fractionation of HeLa S3 cells revealed that immunoreactive PtdIns-PLC beta 3 is membrane associated; purification (approximately 1000-fold) from this fraction yielded a single immunoreactive protein of 158 kDa, with a specific activity of 136 mumol.min-1.mg-1, with PtdIns 4,5-bisphosphate as substrate. Substrate specificity and Ca2+ dependence of this purified PtdIns-PLC are characteristic of the PtdIns-PLC beta subfamily.
Subject(s)
Phosphoric Diester Hydrolases/isolation & purification , Amino Acid Sequence , Base Sequence , Calcium/pharmacology , Cell Line , DNA/chemistry , DNA/isolation & purification , Fibroblasts/enzymology , HeLa Cells/enzymology , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Substrate SpecificityABSTRACT
Receptor tyrosine kinases mediate a range of growth and differentiation processes in multiple biological systems. In this work, we report the identification of a novel tyrosine kinase-related molecule, nyk-r, and the molecular cloning of its complete cDNA. Its extra-cellular domain bears no apparent homology with other receptor families, but its intracellular kinase-related region has considerable similarity with members of the insulin-receptor family such as c-met and trk B. Also, the nyk-r gene is expressed in a wide range of tissues and cell lines.
Subject(s)
Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases , Receptors, Cell Surface/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides/genetics , Polymerase Chain Reaction , Protein-Tyrosine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-met , Receptors, Cell Surface/chemistry , Sequence Homology, Amino Acid , Tumor Cells, Cultured , c-Mer Tyrosine KinaseABSTRACT
cDNAs corresponding to a previously uncharacterized phospholipase C were isolated from an HL-60 cell cDNA library. The cDNAs encodes a putative polypeptide of 1181 amino acids with a calculated molecular mass of 133,700 daltons. Comparison of the amino acid sequence of the predicted protein with those of five mammalian phospholipase C isoforms (PLC-beta 1, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2) revealed that the new enzyme is most closely related to PLC-beta 1 with an overall amino acid sequence identity of 48%. Thus, the new phospholipase C was named PLC-beta 2. The least similarity between PLC-beta 1 and PLC-beta 2 is apparent in the carboxyl-terminal 450 amino acids. Both PLC-beta 1 and PLC-beta 2 were purified from extracts of HeLa cells that had been transfected with vaccinia virus containing the corresponding cDNAs. Like other mammalian PLC isoforms, including PLC-beta 1, the catalytic activity of PLC-beta 2 was entirely dependent on Ca2+, and PLC-beta 2 preferred phosphatidyl-inositol 4,5-bisphosphate to phosphatidylinositol as substrate. Recently, the alpha subunit of the pertussis toxin-insensitive G-protein alpha q has been shown to activate PLC-beta 1 but not PLC-gamma 1 and PLC-delta 1. When alpha q purified from bovine brain was reconstituted with PLC-beta 1 or PLC-beta 2, no stimulation of PLC-beta 2 was observed in the presence of either AlF4- or guanosine 5-O-(3-thiotriphosphate) (GTP gamma S), whereas PLC-beta 1 activity was enhanced markedly in the presence of AlF4- and less markedly but significantly in the presence of GTP gamma S. These results suggest that the receptor-dependent stimulation of PLC-beta 1 and that of PLC-beta 2 may require different G-protein alpha subunits. (see also accompanying article (Lee, C. H., Park, D., Wu, D., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 16044-16047).
Subject(s)
GTP-Binding Proteins/metabolism , Isoenzymes/metabolism , Type C Phospholipases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Calcium/pharmacology , Cell Line , Chromatography, High Pressure Liquid , Cloning, Molecular , Enzyme Activation , HeLa Cells , Humans , Isoenzymes/genetics , Molecular Sequence Data , Molecular Weight , Oligodeoxyribonucleotides , Rats , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Nucleic Acid , Type C Phospholipases/genetics , Type C Phospholipases/isolation & purificationABSTRACT
We report here the nucleotide sequence of a full-length Chinese hamster genomic proviral element, CHIAP34. CHIAP34 is 6,403 bp long with long terminal repeats of 311 bp at each end. The genetic organization of CHIAP34 was determined by comparison with intracisternal A particle (IAP) genetic elements from the mouse and Syrian hamster. Extensive homology at the nucleotide and deduced amino acid sequence levels was observed between CHIAP34 and the mouse and Syrian hamster IAP elements. CHIAP34 may represent a defective Chinese hamster IAP genetic element. The gag gene consists of 837 codons, of which 558 codons are in a single long open reading frame followed by several frameshifts. The pol gene begins with a -1 frameshift and consists of a long open reading frame of 753 codons followed by a short open reading frame of 103 codons. The putative env region contains multiple termination codons in all reading frames. CHIAP34 is representative of the predominant retroviral elements in the Chinese hamster ovary cell genome present at around 80 copies per haploid genome.