ABSTRACT
Gastro-oesophageal reflux disease (GORD) has long been associated with poor asthma control without an established cause-effect relationship. 610 asthmatics (421 severe/88 mild-moderate) and 101 healthy controls were assessed clinically and a subset of 154 severe asthmatics underwent proteomic analysis of induced sputum using untargeted mass spectrometry, LC-IMS-MSE. Univariate and multiple logistic regression analyses (MLR) were conducted to identify proteins associated with GORD in this cohort. When compared to mild/moderate asthmatics and healthy individuals, respectively, GORD was three- and ten-fold more prevalent in severe asthmatics and was associated with increased asthma symptoms and oral corticosteroid use, poorer quality of life, depression/anxiety, obesity and symptoms of sino-nasal disease. Comparison of sputum proteomes in severe asthmatics with and without active GORD showed five differentially abundant proteins with described roles in anti-microbial defences, systemic inflammation and epithelial integrity. Three of these were associated with active GORD by multiple linear regression analysis: Ig lambda variable 1-47 (pâ¯=â¯0·017) and plasma protease C1 inhibitor (pâ¯=â¯0·043), both in lower concentrations, and lipocalin-1 (pâ¯=â¯0·034) in higher concentrations in active GORD. This study provides evidence which suggests that reflux can cause subtle perturbation of proteins detectable in the airways lining fluid and that severe asthmatics with GORD may represent a distinct phenotype of asthma.
Subject(s)
Asthma/complications , Asthma/metabolism , Gastroesophageal Reflux/complications , Proteomics/methods , Sputum/metabolism , Adult , Asthma/epidemiology , Asthma/psychology , Endopeptidases/metabolism , European Union/organization & administration , Female , Gastroesophageal Reflux/diagnosis , Gastroesophageal Reflux/epidemiology , Humans , Immunoglobulin lambda-Chains/metabolism , Lipocalin 1/metabolism , Male , Middle Aged , Prevalence , Prospective Studies , Protease Inhibitors/metabolism , Quality of Life , Severity of Illness IndexABSTRACT
OBJECTIVE: Low-dose transdermal opioids offer a new therapeutic option for osteoarthritis (OA). This study compared symptom relief obtained with buprenorphine patches plus oral paracetamol with that obtained with an oral codeine-paracetamol combination tablet (co-codamol) in older adults with OA. METHOD: Two hundred and twenty people (aged ≥60 years) with OA hip and/or knee pain were randomised to treatment with 7-day buprenorphine patches plus oral paracetamol (5-25 µg/h buprenorphine patches plus 1000 mg oral paracetamol q.i.d. (4 times daily); n=110) or co-codamol tablets (two 8/500-two 30/500 mg tablets q.i.d.; n=110). They entered a titration period of up to 10 weeks, during which their dose of study medication was adjusted until they reached optimum pain control. Patients who achieved optimum pain control entered a 12-week assessment period. The primary outcome was average daily pain scores recorded using the box scale-11 (BS-11) pain scale. RESULTS: Both treatments significantly reduced patient pain scores. The estimated treatment difference [95% confidence interval (CI)] was -0.02 (-0.64, 0.60) for the per protocol (PP) population. The results were similar for the full analysis population. Patients receiving 7-day buprenorphine patches plus oral paracetamol needed significantly less escape medication (ibuprofen) than those receiving co-codamol tablets (P=0.002; PP population). Less than 10% of patients in the 7-day buprenorphine patches plus oral paracetamol group were receiving the highest dose level at the end of the study, compared with 34% in the co-codamol group. Withdrawal rates were high in both groups. The incidence of adverse events (AEs) was comparable between the groups (86.4% of patients in the 7-day buprenorphine patches plus oral paracetamol group; 81.7% in the co-codamol group). Six serious AEs were reported in three patients (2.7%) in the 7-day buprenorphine patches plus oral paracetamol group and one (0.9%) in the co-codamol group. CONCLUSIONS: 7-day buprenorphine patches plus oral paracetamol were non-inferior to co-codamol tablets with respect to analgesic efficacy in older adults with OA pain in the hip/knee.
Subject(s)
Acetaminophen/administration & dosage , Analgesics/administration & dosage , Buprenorphine/administration & dosage , Codeine/administration & dosage , Osteoarthritis, Hip/drug therapy , Osteoarthritis, Knee/drug therapy , Administration, Cutaneous , Administration, Oral , Aged , Aged, 80 and over , Analgesics, Non-Narcotic/administration & dosage , Analgesics, Opioid/administration & dosage , Drug Administration Schedule , Drug Combinations , Drug Therapy, Combination , Female , Humans , Male , Middle Aged , Pain Measurement , Treatment OutcomeABSTRACT
Human carbamyl phosphate synthetase I (CPSI) is an essential hepatic enzyme that initiates the urea cycle. Deficiency of this enzyme usually results in lethal hyperammonemia. CPSI is encoded by the CPSI gene located on chromosome 2q35. In the present study, we report the coding sequence and define the intron-exon structure of the human CPSI gene. These data are compared to the previously defined rat CPSI gene structure. This work was generated from direct sequence determination of human genomic DNA (35 introns) and comparison to public domain sequence of anonymous BACs (2 introns). The human CPSI gene spans >120kb of genomic DNA. CPSI has 38 exons and 37 introns, and all adhere to the consensus splicing sequences. Comparison of the human and rat CPSI genes reveals that the nucleotide sequences, amino acid sequences, and intron-exon organizations are highly similar. We report the primers and conditions for screening the human CPSI exonic and bordering intronic sequences. We also screened 100 individuals for polymorphisms in the human CPSI gene and identified 14 polymorphisms in the CPSI message. The knowledge of the CPSI gene structure and the 14 polymorphisms presented in this study will greatly facilitate future molecular studies involving the CPSI gene and the enzyme it encodes.
Subject(s)
Carbamoyl-Phosphate Synthase (Ammonia)/genetics , Animals , Base Sequence , DNA/chemistry , DNA/genetics , DNA Primers/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , Exons , Genes/genetics , Humans , Introns , Molecular Sequence Data , Polymorphism, Genetic , Rats , Sequence Analysis, DNAABSTRACT
STUDY OBJECTIVE: To determine the absolute bioavailability of cilomilast, and assess the effects of food, dosing time, and coadministration of antacid agents on its bioavailability and pharmacokinetics in healthy volunteers. SETTING: Clinical pharmacology unit. DESIGN: Five prospective pharmacokinetic studies: one single-blind, dose-escalating, placebo-controlled trial; four open-label, randomized studies. SUBJECTS: Ninety-six healthy adult volunteers who were nonsmokers. INTERVENTION: In the first study, four subjects received intravenous cilomilast 1, 2, and 4 mg. In the second study, 16 subjects received oral cilomilast 15 mg or intravenous cilomilast 4 mg. In the other three studies, a total of 76 subjects were given single oral 15-mg doses; one study compared its effects in fed versus fasted subjects, one looked for differences of morning versus evening dosing, and one examined coadministration with aluminum hydroxide-magnesium hydroxide. MEASUREMENTS AND MAIN RESULTS: After intravenous administration of cilomilast, plasma concentrations increased in an approximately dose-proportional manner; the half-life, approximately 6.5 hours, was dose independent. Cilomilast clearance and volume of distribution were small. After oral dosing, the absolute bioavailability was consistently close to 100%. Absorption was slower in fed subjects than in fasted (median 2-hr delay in time to reach maximum plasma concentration, average 39% reduction in maximum plasma concentration), but the area under the concentration-time curve from time zero to infinity (systemic availability) was unaffected. Pharmacokinetic parameters were not influenced by time of dosing or coadministration of antacid. CONCLUSION: The absolute bioavailability of oral cilomilast was 100%; it was not adversely affected by time of dosing or coadministration with food or antacid.
Subject(s)
Bronchodilator Agents/pharmacokinetics , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Administration, Oral , Adolescent , Adult , Antacids/administration & dosage , Area Under Curve , Biological Availability , Bronchodilator Agents/administration & dosage , Bronchodilator Agents/adverse effects , Carboxylic Acids , Cyclic Nucleotide Phosphodiesterases, Type 4 , Cyclohexanecarboxylic Acids , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Interactions , Drug Therapy, Combination , Female , Food , Humans , Injections, Intravenous , Male , Middle Aged , NitrilesABSTRACT
Deficiency of lysosomal acid beta-glucosidase induces glycolipid storage in the macrophages of Gaucher disease but the pathways of multisystem tissue injury and destruction are unknown. To investigate the cognate molecular pathology of this inflammatory disorder, genes that were differentially expressed in spleen samples from a patient with Gaucher disease (Gaucher spleen) were isolated. Of 64 complementary DNA (cDNA) fragments sequenced from an enriched Gaucher cDNA library, 5 encode lysosomal proteins (cathepsins B, K, and S, alpha-fucosidase, and acid lipase), 10 encode other known proteins, and 2 represent novel sequences from human macrophage cell lines. Transcript abundance of the cathepsins, novel genes, pulmonary and activation-regulated chemokine (PARC), and NMB, a putative tumor suppressor gene, was greatly increased. Immunoblotting showed increased mature forms of all 3 cathepsins found in samples of Gaucher spleens. Immunofluorescence microscopy showed strong cathepsin B and K reactions in sinusoidal endothelium and Gaucher cells. The respective means, plus or minus SD, of cathepsin B, K, and S activities were 183 +/- 35, 97 +/- 39, and 91 +/- 45 nmol/min/mg protein in 4 Gaucher spleens, and 26 +/- 4, 10.5 +/- 2, and 4.0 +/- 2.1 nmol/min/mg protein in 3 control spleens. Plasma cathepsin B, K, and S activities were also elevated in Gaucher disease plasma (P <.001), but compared with control plasma samples, neither cathepsin B nor K activities were significantly elevated in 8 patients with nonglycosphingolipid lysosomal storage diseases or in 9 patients with other glycosphingolipidoses, which suggests disease specificity. All 3 cathepsin activities were increased 2-fold to 3-fold in Gaucher sera compared with control sera. In all 6 patients treated by enzyme replacement for 16-22 months, serum cathepsin activities decreased significantly (P <.01). Longitudinal studies confirmed the progressive reduction of proteinase activities during imiglucerase therapy but in 3 Gaucher patients with mild disease not so treated, serum cathepsin activities remained constant or increased during follow-up. Enhanced expression of cysteine proteinases may promote tissue destruction. Moreover, the first identification of aberrant cathepsin K expression in hematopoietic tissue other than osteoclasts implicates this protease in the breakdown of the matrix that characterizes lytic bone lesions in Gaucher disease. (Blood. 2000;96:1969-1978)
Subject(s)
Gaucher Disease/enzymology , Adult , Aged , Blotting, Northern , Cathepsin B/metabolism , Cathepsin K , Cathepsins/blood , Cathepsins/genetics , Cathepsins/metabolism , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Female , Gaucher Disease/genetics , Gaucher Disease/therapy , Gene Expression Regulation, Enzymologic , Humans , Hydrolases/metabolism , Immunoblotting , Immunohistochemistry , Lysosomal Storage Diseases/enzymology , Male , Microscopy, Fluorescence , Middle Aged , Osteoclasts/enzymology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Spleen/enzymology , Spleen/pathology , Tissue Distribution , Up-RegulationABSTRACT
Hepatomegaly is frequent in patients with type 1 Gaucher's disease and is associated with infiltration of the liver with pathological macrophages. Most patients suffer no significant clinical consequences, but a few develop portal hypertension which may progress to parenchymal liver failure. We describe four patients with Gaucher's disease who have developed portal hypertension. We have reviewed their clinical histories and all available histological and radiological material. All had severe Gaucher's disease with multi-organ involvement, and had undergone splenectomy in childhood. Histologically, this advanced liver disease was characterized by a picture of extreme and advanced confluent fibrosis occupying the central region of the liver. This massive fibrosis is associated with characteristic radiological appearances. The liver histology in these cases is highly unusual and virtually unknown in other conditions. Our studies indicate that without specific treatment the liver disease is progressive and rapidly fatal. However, institution of enzyme replacement therapy with imiglucerase may have beneficial effects even when the condition is far advanced.
Subject(s)
Gaucher Disease/complications , Hypertension, Portal/etiology , Liver Cirrhosis/etiology , Adolescent , Adult , Enzyme Therapy , Esophageal and Gastric Varices/etiology , Esophageal and Gastric Varices/therapy , Female , Gaucher Disease/therapy , Hepatomegaly/etiology , Hepatomegaly/pathology , Hepatomegaly/therapy , Humans , Hypertension, Portal/therapy , Liver Cirrhosis/pathology , Liver Cirrhosis/therapy , Liver Function Tests , Magnetic Resonance Imaging , Male , Middle Aged , Severity of Illness Index , Splenectomy/adverse effects , Tomography, X-Ray ComputedABSTRACT
Ammonia liberated during amino acid catabolism in mammals is highly neurotoxic and is detoxified by the five enzymes of the urea cycle that are expressed within the liver. Inborn errors of each of the urea cycle enzymes occur in humans. Carbamoyl phosphate synthetase I (CPSase I; EC 6.3.4.16) is located within the inner mitochondrial matrix and catalyzes the initial rate-limiting step of the urea cycle. Unless treated, complete deficiency of CPSase I, a rare autosomal recessive disease, causes death in newborn infants. Survivors are often mentally retarded and suffer frequent hyperammonemic crises during intercurrent illness or other catabolic stresses. Biochemically, CPSase I deficiency is characterized by high levels of blood ammonia, glutamine, and alanine, with low or absent citrulline and arginine levels. As a first step toward the development of gene therapy directed to the hepatocyte, we have generated a CPSase I-deficient mouse by gene targeting. Mice with homozygous disruption of CPSase I (CPSase [-/-] mice) die within 36 hours of birth with overwhelming hyperammonemia, and without significant liver pathology. This animal is a good model of human CPSase I deficiency.
Subject(s)
Ammonia/blood , Carbamoyl-Phosphate Synthase (Ammonia)/deficiency , Urea/metabolism , Amino Acid Sequence , Animals , Animals, Newborn , Base Sequence , Brain/enzymology , Carbamoyl-Phosphate Synthase (Ammonia)/genetics , Disease Models, Animal , Gene Targeting , Genetic Vectors , Genotype , Liver/enzymology , Mice , Mice, Mutant Strains , Molecular Sequence DataABSTRACT
We have isolated and sequenced a cosmid clone from the compact genome of the Japanese pufferfish (Fugu rubripes) containing portions of three genes that have the same order as in human. The gene order is microtubule-associated protein (MAP-2), myosin light chain (MYL-1), and carbamoyl phosphate synthetase (CPS III). The intron-exon organization of Fugu CPS III is identical with that of rat CPS I, although the equivalent genomic fragments of rat and Fugu CPS span 87.9 and 21 kb, respectively. This is the first report of a piscine CPS III genomic structure and predicts a close evolutionary link between CPS III and CPS I. The 8-kb intergenic region between MYL-1 and CPS gave no clear areas of transcription factor-binding sites by pairwise comparison with shark or rat CPS promoter regions. However, there was a match with the rat myosin light chain 2 (MLC-2) gene promoter and a MyoD transcription factor-binding site 874 bp upstream of the MYL-1 gene.
Subject(s)
Chromosomes, Human, Pair 2 , Fishes, Poisonous/genetics , Genome , Amino Acid Sequence , Animals , Carbon-Nitrogen Ligases/genetics , Humans , Mice , Microtubule-Associated Proteins/genetics , Molecular Sequence Data , Myosin Light Chains/genetics , Rats , Sequence Homology, Amino AcidABSTRACT
Gaucher's disease is an inherited disorder characterized by pathological storage of glycolipid in mononuclear phagocytes: it is a multi-system disease associated with striking variation in its clinical manifestations, severity and course. Although molecular analysis of the glucocerebrosidase gene in patients with Gaucher's disease has permitted broad correlations between genotype and phenotype to be made, with few exceptions genetic variation at this locus does not allow confident prediction of clinical phenotype or prognosis. Partial deficiency of glucocerebrosidase is associated principally with parenchymal disease of the liver, spleen, bone marrow and, in severe cases, the lung, in non-neuronopathic, Type 1, Gaucher's disease: here storage material in macrophages originates from turnover of exogenous glycolipids. Severe deficiency of glucocerebrosidase caused by disabling mutations is additionally associated with neurological manifestations that in part reflect a failure to degrade endogenous neuronal glycosphingolipids, the so-called neuronopathic, Type 2 and Type 3 disease categories. Here we describe the clinical features, complications and natural history principally of Type 1 Gaucher's disease: emphasis is placed on emerging pulmonary, osseous and other manifestations of obscure pathogenesis that respond poorly to enzyme-replacement therapy.
Subject(s)
Gaucher Disease/complications , Gaucher Disease/diagnosis , HumansABSTRACT
An attempt was made to recover naturally processed T-cell determinants following antigen pulsing of tetanus toxin-specific human B-cell clones with microgram/ml amounts of antigen. Class II major histocompatibility complex (MHC) molecules were isolated from cells pulsed under optimal conditions and the eluted peptides displayed by reverse-phase high-performance liquid chromatography (HPLC). Antigen-pulsed and control-unpulsed cells showed virtually identical optical density (OD)215 profiles, although multiple peptides derived from the input antigen could be identified at the radiochemical level. At least four distinct HPLC fractions contained naturally processed versions of the determinant 830-844, detected using the specific T-cell clone Mix 111. Quantification of the most active fraction indicated that approximately 1 pmole of this determinant was recovered from approximately 5 x 10(9) antigen-pulsed cells. Based on the amount of antigen processed following uptake on membrane immunoglobulin, and quantification of the biologically active material recovered, it was estimated that the efficiency of determinant capture was no greater than 1-2%. Further method development and a considerable increase in the number of cells used (> 10(10)) would appear to be necessary before naturally processed determinants from exogenously pulsed antigens can be reliably and fully characterized. Finally, a theoretical analysis showed that accurate (+/- 0.01%) mass information alone could identify a limited number of candidate peptides from known or putative antigens.
Subject(s)
B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , Epitopes/analysis , Tetanus Toxin/immunology , Amino Acid Sequence , Cells, Cultured , Chromatography, High Pressure Liquid , HLA-D Antigens/analysis , Humans , Molecular Sequence Data , Peptide Fragments/immunologyABSTRACT
There have been rapid advances in the development of non-viral techniques for gene transfer. Although viruses are highly evolved to infect mammalian cells, they have several limitations. The general theme is to mimic the advantageous components of viral systems whilst separating them from their limiting functions. The systems described here are broadly divided into true non-viral techniques and viral-assisted technologies. The merits and limitations of each method are presented, and examples of their application to current developments in gene therapy are discussed. At present no preferred technique has emerged as a clear favourite for non-viral delivery. Further refinement of the technology with particular emphasis on achieving long-term gene expression is required before initiating clinical trials of non-viral gene delivery.
Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , DNA/administration & dosage , Humans , Liposomes , Plasmids , RNA/administration & dosageABSTRACT
Repair of human endometrium after menstruation and preparation of the endometrium for implantation involves profound angiogenic changes. Vascular endothelial cell growth factor (VEGF) is a recently identified growth factor with significant angiogenic properties. Four species of mRNA encoding VEGFs were identified in human endometrium and myometrium. All species were present throughout the menstrual cycle. Two species, VEGF165 and VEGF121, were present in peripheral leukocytes, indicating tissue-specific splicing of the two other VEGF transcripts. In situ hybridization of mRNA encoding VEGF was not restricted to vascular smooth muscle but was present in epithelial and stromal cells of endometrium throughout the cycle, and the distribution changed during the course of the cycle. All four species of VEGF were expressed by the endometrial carcinoma cell lines Ishikawa, HEC 1-A, and HEC 1-B. Estradiol increased steady-state levels of mRNA encoding VEGF in a dose- and time-dependent manner in HEC 1-A cells. Conditioned medium from these cells possessed angiogenic activity that was depleted by passage through a heparin affinity column. None of the cell lines demonstrated mRNA for acidic or basic fibroblast growth factor (FGF), despite previous reports of the identification of immunoreactive basic FGF in HEC 1-A and HEC 1-B cells. These findings show that VEGFs, not FGFs, are the principal angiogenic growth factors secreted by these cells and that human endometrium expresses a secreted angiogenic growth factor whose site of expression changes during the menstrual cycle.
Subject(s)
Adenocarcinoma/metabolism , Alternative Splicing , Endometrial Neoplasms/metabolism , Endothelial Growth Factors/genetics , Estradiol/pharmacology , Lymphokines/genetics , RNA, Messenger/analysis , Uterus/chemistry , Base Sequence , Blotting, Northern , Culture Media, Conditioned , Female , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
1. Carbamoyl-phosphate synthetase (EC 6.3.5.5.) catalyses the synthesis of carbamoyl phosphate, the immediate precursor of arginine and pyrimidine biosynthesis, and is highly conserved throughout evolution. The large subunit of all carbamoyl-phosphate synthetases sequenced to date comprises two highly homologous halves, the product of a proposed ancestral gene duplication. The sequences of the enzymes of Escherichia coli, Drosophila melanogaster, Saccharomyces cerevisiae, rat and Syrian hamster all have duplications, suggesting that this event occurred in the progenote predating the separation of the major phylae. Until now, only limited data on carbamoyl-phosphate synthetase were available for the primitive eukaryote Dictyostelium discoideum and for the Archaea Methanosarcina barkeri MS. The DNA sequence of the D. discoideum carbamoyl-phosphate gene and additional sequence for the carbamoyl-phosphate synthetase gene of M. barkeri MS have been determined, and a duplicated structure for both is clearly demonstrated. 2. Genes with ancient duplications provide unique information on their evolution. A study of the intron/exon organization of the rat carbamoyl-phosphate synthetase I gene and the carbamoyl-phosphate synthetase hamster II gene in the CAD multi-gene complex shows that at least some of their introns are very old. Evidence is provided that some introns must have been present in the ancestral precursor before its duplication. 3. The human carbamoyl-phosphate synthetase I gene has been isolated and characterized. A human liver cDNA library was constructed and probed for carbamoyl-phosphate synthetase I. A human genomic DNA cosmid library was also probed for the carbamoyl-phosphate synthetase I gene. The cDNA sequence of the human carbamoyl-phosphate synthetase I gene has been determined, and work has been initiated to confirm that at least part of this gene is contained within two cosmids spanning 46kb. This will enable future studies to be made on mutations in this gene in the rare autosomal recessive deficiency of carbamoyl-phosphate synthetase I.
Subject(s)
Biological Evolution , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Conserved Sequence , Introns/genetics , Multigene Family/genetics , Animals , Base Sequence , Cricetinae , Dictyostelium/genetics , Escherichia coli/genetics , Humans , Methanosarcina barkeri/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Rats , Sequence Alignment , Sequence Analysis, DNASubject(s)
Base Sequence , DNA, Recombinant , DNA , Polymerase Chain Reaction/methods , DNA/metabolism , Indicators and Reagents , Microchemistry/methods , Molecular Sequence Data , Nucleic Acid Conformation , Radioisotope Dilution Technique , Spectrometry, Fluorescence/methods , Sulfur Radioisotopes , Templates, Genetic , ThionucleotidesABSTRACT
We have isolated and sequenced the genomic DNA from the slime-mould Dictyostelium discoideum multi-gene (PYR1-3) encoding the carbamoyl phosphate synthetase II domain (CPSase, EC.6.3.5.5). We describe sequencing by oligo-walking directly on PCR product in the solid-phase, avoiding subcloning procedures. The 2.4 kb fragment completes the sequence of the PYR1-3 gene, has no introns, and has the same structure as the rudimentary gene of Drosophila melanogaster. Comparison with the carbamoyl phosphate synthetases (CPSase I and CPSase II) of other species supports the hypothesis that this gene has arisen by tandem duplication from a smaller common ancestral gene in the progenote.
Subject(s)
Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Dictyostelium/genetics , Amino Acid Sequence , Animals , Base Sequence , Biological Evolution , Chromosome Walking , DNA, Fungal/genetics , DNA, Protozoan/genetics , Dictyostelium/enzymology , Molecular Sequence Data , Multigene Family , Polymerase Chain Reaction , Sequence Homology, Nucleic AcidABSTRACT
An unidentified atrial natriuretic peptide (ANP)-like substance is the principal hormone regulating NaCl secretion in the shark rectal gland, an epithelial model tissue for hormone-sensitive secondary active chloride transport. Antibodies to mammalian ANP do not recognize the prohormone of marine species. The polymerase chain reaction (PCR) was used to isolate a partial cDNA encoding the shark heart natriuretic peptide. Using this partial sequence as a probe, the full-length clone [882 base pairs (bp)] was obtained from a shark heart cDNA library. Amino acids 119-135 are similar to the recently identified peptide sequences of porcine C-type natriuretic peptide (CNP) and killifish brain natriuretic peptide isolated from the brain of these species. Mature shark heart CNP terminates at the second cysteine residue and lacks a COOH-terminal extension, in contrast to cardiac ANP-like peptides of all other species. The primary amino acid sequence of the shark heart prepro-CNP is distinctly different from all other cardiac natriuretic peptides. Amplification of genomic DNA spanning the coding region produced a 1.5-kb product, indicating the presence of at least one intron. Sequencing confirmed the presence of two exons of 90 and 315 bp, separated by a 1.1-kb intron. This is the first report of a cDNA encoding in nonneuronal tissue. Elasmobranch CNP may represent a primordial form of ANP-like peptides that evolved as an adaptation to environmental osmoregulatory stress.
Subject(s)
Dogfish/metabolism , Myocardium/chemistry , Nerve Tissue Proteins/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , DNA/genetics , Molecular Sequence Data , Natriuretic Peptide, C-Type , Nerve Tissue Proteins/genetics , Polymerase Chain ReactionABSTRACT
The presence of mRNA for epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) was demonstrated in small fragments of human endometrium and decidua by use of the technique of reverse transcriptase-polymerase chain reaction with nested oligonucleotide primers. The presence of mRNA encoding EGF and TGF alpha has not been shown in human endometrium previously. Other studies using conventional techniques, such as Northern blot or in-situ hybridization, showed the presence in low copy number of EGF but not TGF alpha in murine endometrium. Messenger RNA for EGF was not present in peripheral leukocytes or platelets, suggesting an endometrial source for the message. Messenger RNA for TGF alpha was found in these blood components, thus preventing confirmation of the source of TGF alpha mRNA.
Subject(s)
Decidua/chemistry , Endometrium/chemistry , Epidermal Growth Factor/genetics , RNA, Messenger/analysis , Transforming Growth Factor alpha/genetics , Base Sequence , DNA , Female , Humans , Molecular Sequence Data , Polymerase Chain Reaction , RNA-Directed DNA Polymerase/metabolismABSTRACT
F protein is found predominantly in the liver and is of unknown function. The protein has been of interest to immunologists in the areas of self tolerance and the immunogenetics of the anti-F protein response. In the mouse there are two alleles (F1 and F2), and although mice are completely tolerant to the self form of the protein, mice of responder strains make a good antibody response to immunization with the non-self form. This response cross-reacts with the self form, implying firstly, that autoreactive B cells are present and that tolerance is therefore maintained at the T cell level, and secondly, that the difference between the two allelic products defines a T cell epitope. Primers based on the published sequence for rat F protein were used in the polymerase chain reaction to amplify the cDNA for the two mouse alleles. Subsequent sequencing shows a high degree of sequence identity between the rat and mouse cDNA. The two mouse cDNA are identical apart from a single A to G base change which predicts an asparagine (F1 protein) to aspartate (F2 protein) amino acid residue change. Using allele-specific oligonucleotide probes we confirmed that this base change has the same strain distribution as the previously determined F protein type. Isoelectric focusing shows that F1 protein migrates in a more basic position than F2 protein, as predicted by the asparagine to aspartate change. Finally, a synthetic peptide from the allovariable site of F2 protein will successfully restimulate T cells in vitro from an F1 type mouse primed in vivo with whole F2 protein, whereas the corresponding peptide from F1 protein will not. This is evidence that, as predicted, the allovariable site does indeed define a T cell epitope. Peptides covering the rest of the F2 protein molecule were not stimulatory.
Subject(s)
Alleles , Epitopes/analysis , Oxidoreductases , Proteins/genetics , T-Lymphocytes/immunology , Amino Acid Sequence , Animals , Base Sequence , DNA/analysis , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Molecular Sequence Data , Polymerase Chain Reaction , Proteins/immunology , Proteins/physiologyABSTRACT
Cultured pig and bovine endothelial cells are capable of synthesizing endothelin-1 (ET-1). Thus the observation that the kidney contains a large number of binding sites for ET distributed in close proximity to endothelial cells suggests that ET-1 may be released from the endothelium to act locally on these receptors. In support of this hypothesis, using the technique of reverse transcription with specific amplification of cDNA, we report here that ET-1 mRNA is expressed in the rat kidney. The partial sequence of the amplified rat ET-1 cDNA confirms that the mature rat peptide is identical to that of the mouse, man and pig, but with some differences in codon usage.
Subject(s)
Endothelins/genetics , Gene Expression , Kidney/metabolism , Animals , Base Sequence , Codon , Humans , Male , Molecular Sequence Data , Nucleic Acid Hybridization , Polymerase Chain Reaction , Rats , Rats, Inbred Strains , SwineABSTRACT
In this paper we describe a rapid method for the direct generation of DNA sequencing templates from phage or bacteria. Sequencing of these PCR products can be performed by radioactive and fluorescent methods. The non-radioactive method has been used to sequence a total of approximately 100 kb of human DNA fragments generated by digestion with HpaII and subsequent cloning. The method depends on direct small scale amplification using a biotinylated primer, and the binding of the product to streptavidin coated magnetic beads. All the procedures are carried out in a microtitre plate thus facilitating the handling of large numbers of clones and has potential for automation.