ABSTRACT
Nucleotides are involved in regulating a number of important processes ranging from inflammation to platelet aggregation. Enzymes that can modulate levels of nucleotides in the blood therefore represent important regulatory components in these physiological systems. CD39L4 is a soluble E-nucleoside triphosphate dephosphohydrolase (E-NTPDase) with specificity for nucleotide diphosphates (NDPs). In this study, stable mammalian and insect cell lines were generated expressing CD39L4 protein to purify and characterize the recombinant protein. We demonstrate that recombinant CD39L4 protein expressed in human embryonic carcinoma 293 cells is glycosylated by comparing the molecular masses before and after glycosidase treatment. Activity measurements of CD39L4 isolated from tunicamycin-treated, transiently transfected COS-7 cells indicate that glycosylation is not required for full ADPase activity. Recombinant human CD39L4 protein isolated from stable insect cells was glycosylated differently, but also demonstrated relative activity comparable to that of the mammalian protein. When denatured by SDS under nonreducing conditions, a fraction of the CD39L4 protein migrates as a 110 kDa disulfide-linked dimer. We determined that the monomer is the most active form of CD39L4 by measuring the activity of sucrose density gradient fractions of monomers and partially purified dimers. The physiological significance of the biochemical and enzymatic characterization is discussed.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/biosynthesis , Adenosine Triphosphatases/genetics , Animals , Apyrase/chemistry , Apyrase/metabolism , COS Cells , Cell Line , Dimerization , Disulfides/chemistry , Enzyme Activation/genetics , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spodoptera/genetics , TransfectionABSTRACT
The interleukin (IL)-1 family of proteins plays an important role in inflammatory and defense mechanisms. The recently characterized IL1HY1 cDNA encodes a new member of the IL-1 receptor antagonist family (IL-1ra). In this report, we describe the complete nucleotide sequence of the human IL1HY1 gene. We sequenced approximately 7,600 nucleotides and found four coding exons ranging in size from 55 to 2,288 nucleotides. The 5' untranslated region is formed by one of two alternatively used exons and one invariably present exon which also contains the region encoding the first nine amino acids of the protein. IL1HY1 and IL-1ra intron positions are well conserved within the protein-coding region, providing evidence that these genes arose from a duplication of a primordial IL-1 receptor antagonist gene.
Subject(s)
Interleukins , Proteins/chemistry , Proteins/genetics , Receptors, Interleukin-1/antagonists & inhibitors , 5' Untranslated Regions/genetics , Amino Acid Sequence , Base Sequence , Chromosomes, Human, Pair 2/genetics , Exons , Gene Duplication , Humans , Introns , Molecular Sequence Data , Tumor Cells, CulturedABSTRACT
Interleukin-1 is a potent mediator of inflammation, involved in regulating a wide variety of physiological and cellular events. We have identified and characterized a novel member of the human interleukin-1 gene family (IL1HY1). The encoded protein demonstrates significant amino acid homology to the receptor antagonist (IL-1ra) at 52%. The gene was mapped to the long arm of chromosome 2, in close proximity to the IL-1 locus. IL1HY1 message is tightly regulated being most predominantly expressed in the skin, but also detected in the spleen, brain leukocyte, and macrophage cell types. Furthermore, the message can be induced in THP-1 cells by phorbol ester (PMA) and lipopolysaccharide (LPS) treatment.
Subject(s)
Chromosomes, Human, Pair 2 , Interleukins , Proteins/genetics , Receptors, Interleukin-1/antagonists & inhibitors , Skin/immunology , Amino Acid Sequence , Base Sequence , Brain/immunology , Cell Line , Chromosome Mapping , Fetus , Gene Amplification , Gene Library , Humans , Interleukin 1 Receptor Antagonist Protein , Leukocytes/immunology , Macrophages/immunology , Molecular Sequence Data , Multigene Family , Organ Specificity , Proteins/chemistry , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Sialoglycoproteins/chemistry , Sialoglycoproteins/genetics , Skin/embryology , Spleen/immunology , Transcription, GeneticABSTRACT
The human ecto-apyrase gene family consists of five reported members (CD39, CD39-L1, CD39-L2, CD39-L3, and CD39-L4). The family can be subdivided into two groups by conservation of proposed structural domains. The CD39, CD39-L1, and CD39-L3 genes all encode hydrophobic portions in their carboxy and amino termini, serving as transmembrane domains for CD39 and potentially for the other two members. CD39-L2 and CD39-L4 genes encode hydrophobic portions in their amino termini, suggesting that they might encode secreted apyrases. We demonstrate that the CD39-L4 gene encodes the first reported human secreted ecto-apyrase. COS-7 cells transfected with a CD39-L4 expression construct utilizing the naturally occurring leader peptide express recombinant protein outside of the cells. This expression can be blocked by brefeldin A, a chemical that inhibits a step in mammalian secretory pathways. We also demonstrate expression of CD39-L4 message in macrophages, suggesting that the protein is present in the circulation. Furthermore, we show that CD39-L4 is an E-type apyrase, is dependent on calcium and magnesium cations, and has high degree of specificity for NDPs over NTPs as enzymatic substrates. A potential physiological role in hemostasis and platelet aggregation is presented.
Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphatases , Antigens, CD/metabolism , Apyrase/metabolism , Animals , Base Sequence , COS Cells , Cations, Divalent , DNA Primers , Enzyme Activation , Humans , Hydrolysis , Substrate SpecificityABSTRACT
Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.
Subject(s)
Bacterial Proteins/biosynthesis , Proteins , Ribosomes/metabolism , Alleles , Amino Acid Sequence , Amino Acid Substitution/genetics , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Base Sequence , Chromosomes, Bacterial/genetics , Codon/physiology , Escherichia coli/metabolism , Escherichia coli/physiology , Molecular Sequence Data , Mutagenesis , Peptide Chain Initiation, Translational/genetics , Peptide Chain Termination, Translational/genetics , Phenotype , Ribosomal Proteins , TemperatureSubject(s)
Breech Presentation , Labor Presentation , Cesarean Section , Extraction, Obstetrical , Female , Fetal Death/etiology , Humans , Infant, Newborn , Pregnancy , Prognosis , RiskABSTRACT
PIP: The author praised the sponsors of the French legalized abortion law, but considers abortion a failure for both doctor and patient. For doctors abortion is a failure because they have vowed to preserve life: several have become severely depressed after performing abortions. For the patient abortion is a failure because of risk of infection and sterility. The abortion-related mortality statistics due to perforation, anesthesia, coagulopathy, infection, embolism, and suicide range from .04/10,000 in Yugoslavia to 4/10,000 in Scandinavia. Immediate complications of early abortions include bleeding (1-10%), infection (1-27%), thromboembolism, and perforation. Late complications include chronic inflammation, sterility in 11-15%, repeated miscarriage and prematurity in 14-24%, and acute or chronic psychological disorders. Therefore abortion should only be done in exceptional cases of contraceptive failure.^ieng
Subject(s)
Abortion, Spontaneous , Contraception , Female , Humans , PregnancySubject(s)
Birth Injuries , Joint Dislocations , Nasal Septum/abnormalities , Humans , Infant, Newborn , Nasal Septum/surgerySubject(s)
Contraception , Adolescent , Adult , Contraceptives, Oral , Female , Humans , Middle Aged , Socioeconomic FactorsSubject(s)
Labor Presentation , Parasympatholytics/administration & dosage , Adult , Female , Humans , Pregnancy , RotationSubject(s)
Amniotic Fluid/analysis , Creatinine/analysis , Embryonic and Fetal Development , Amniocentesis , Body Weight , Female , Gestational Age , Humans , PregnancySubject(s)
Contraception , Contraceptives, Oral , Adolescent , Adult , Female , France , Humans , Middle Aged , Pregnancy , Socioeconomic FactorsSubject(s)
Embryonic and Fetal Development , Pregnancy , Animals , Birth Weight , Female , Humans , Infant, Newborn , Maternal-Fetal Exchange , Nutritional Requirements , RatsABSTRACT
PIP: An 18-year-old mother of 2 who had worn an IUD for 5 months postpartum was hospitalized for severe bleeding which was due to a spontaneous abortion of a 2.5-month gestation. The thread of her IUD was in place in the cervix, but the IUD was found by x-ray to be in the cul-de-sac of Douglas. It was removed easily by posterior colpotomy. After a brief summary of perforations reported in the literature, the authors conclude that this perforation probably occurred at the time of insertion.^ieng