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1.
J Thromb Haemost ; 5(11): 2243-9, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17958742

ABSTRACT

BACKGROUND: Users of progestin-only contraceptives have raised protein S (PS) levels compared with baseline. This contrasts with the reduction in PS levels observed in users of combined oral contraceptives, which contain both a progestin and an estrogen. OBJECTIVES: To determine the effect of progesterone and other progestin isoforms on the expression of PS and to describe the mechanism involved. METHODS: Promoter activity of the PROS1 gene that encodes PS was assessed in vitro using breast and liver carcinoma cell lines grown in the presence of various progestins, with and without the addition of excess progesterone receptors. An electromobility shift assay (EMSA) was also performed to identify the progesterone receptor binding element. RESULTS: PROS1 transcriptional levels were directly upregulated by 25% by progesterone via a mechanism that was progesterone receptor isoform B (PR-B)-dependent. The process was blocked by the progesterone receptor modulator RU486. Results for the EMSA demonstrated that a probe comprising nucleotides -397 to -417 of the PROS1 promoter bound to ligand-activated PR-B, suggesting that the domain is a progesterone response element (PRE). The type of progestin isoform greatly influenced the level of PROS1 promoter upregulation, with medroxyprogesterone able to stimulate a > 2-fold stronger response compared with progesterone. CONCLUSIONS: The PROS1 promoter is responsive to progesterone and other progestins via a mechanism involving PR-B interacting with a PRE. The type of progestin is important as some elicit stronger upregulatory effects than others, which may influence the choice of progestin used for hormonal contraception by PS-deficient individuals.


Subject(s)
Protein S/genetics , Up-Regulation/drug effects , Cell Line, Tumor , Contraceptives, Oral , Humans , Progesterone/pharmacology , Progestins/pharmacology , Promoter Regions, Genetic , Protein Isoforms/pharmacology , Receptors, Progesterone/metabolism , Response Elements , Transcription, Genetic , Up-Regulation/genetics
2.
Pharm Res ; 24(4): 695-704, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17372696

ABSTRACT

PURPOSE: To develop a robust radiolabeling technique to enable evaluation of difficult to radiolabel gastric retentive formulations using gamma scintigraphy. The use of a successful radiolabel will allow accurate assessment of the gastric residence time of the formulations. MATERIALS AND METHODS: The retention of two radionuclides, indium ((111)In) and samarium ((153)Sm), with and without further processing to improve radiolabel performance were evaluated in simulated gastric pH in vitro. The most successful formulation from the in vitro screening was further evaluated in preclinical and clinical studies. RESULTS: In vitro evaluation revealed significant radionuclide leakage at pH 1.5 for most radiolabeling attempts. Radionuclide leakage at pH 4.5 was less pronounced. The most successful radiolabel was formulated by adsorbing indium chloride onto activated charcoal, followed by entrapment in a cellulose acetate polymer melt. This provided the best radiolabel retention under both pH conditions in vitro. The radiolabel also proved to be successful during preclinical and clinical evaluations, allowing evaluation of gastric retention performance as well as complete gastrointestinal transit. CONCLUSION: A simple, yet robust radiolabel was developed for gastric retentive formulations to be evaluated pre-clinically or in a clinical setting by entrapping the radionuclide in an insoluble polymer through a simple polymer melt process.


Subject(s)
Gastric Emptying , Indium/administration & dosage , Isotope Labeling/methods , Oxides/administration & dosage , Radiopharmaceuticals/administration & dosage , Samarium/administration & dosage , Stomach/diagnostic imaging , Technology, Pharmaceutical/methods , Administration, Oral , Adult , Animals , Cellulose/analogs & derivatives , Cellulose/chemistry , Charcoal/chemistry , Chemistry, Pharmaceutical , Cross-Over Studies , Delayed-Action Preparations , Dogs , Drug Compounding , Drug Stability , Gamma Cameras , Gastrointestinal Transit , Half-Life , Humans , Hydrogen-Ion Concentration , Indium/chemistry , Male , Middle Aged , Oxides/chemistry , Radioisotopes/administration & dosage , Radionuclide Imaging , Radiopharmaceuticals/chemistry , Samarium/chemistry , Solubility , Time Factors
3.
J Neurol Neurosurg Psychiatry ; 77(9): 1067-9, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16914755

ABSTRACT

BACKGROUND: An association between the phosphodiesterase 4D (PDE4D) gene and risk of ischaemic stroke in an Icelandic population has been suggested by the deCODE group. METHODS: A case-control study of 151 hospitalised patients with first-ever ischaemic stroke and 164 randomly selected age-matched and sex-matched community controls was conducted. PDE4D genotypes for the six single-nucleotide polymorphisms (SNPs) previously reported to be independently associated with stroke were determined, common haplotypes were inferred using the expectation-maximisation algorithm, and SNP and haplotype associations with stroke were examined. A meta-analysis of published studies examining the association between PDE4D and stroke was also carried out. RESULTS: Our study of Australian patients with stroke showed an independent association between ischaemic stroke and PDE4D SNP 89 (CC: odds ratio (OR) 5.55, 95% confidence interval (CI) 1.02 to 30.19; CA: OR 1.68, 95% CI 0.96 to 2.96; AA: OR 1 (reference)), SNP 87 (CC: OR 2.13, 95% CI 1.08 to 4.20; TC: OR 1.64, 95% CI 0.89 to 3.00; TT: OR 1 (reference)) and SNP 83 (TT: OR 2.16, 95% CI 1.08 to 4.32; TC: OR 1.37, 95% CI 0.77 to 2.43; CC: OR 1 (reference)), and between ischaemic stroke and PDE4D haplotypes at SNP 89-87-83 (A-C-C: OR 2.13, 95% CI 1.15 to 3.96; C-C-T: OR 2.25, 95% CI 1.29 to 3.92), but no association between ischaemic stroke and PDE4D SNP 56, SNP 45 or SNP 41, or with PDE4D haplotypes at SNP 56-45-41. A meta-analysis of nine case-control studies (including our current results) of 3808 stroke cases and 4377 controls confirmed a significant association between stroke and PDE SNP 87 (pooled p = 0.002), SNP 83 (0.003) and SNP 41 (0.003). However, there was statistical heterogeneity (p < 0.1) among the studies in the direction of association for each of the individual SNPs tested. CONCLUSIONS: Our results and the pooled analyses from all the studies indicate a strong association between PDE4D and ischaemic stroke. This strengthens the evidence that PDE4D plays a key part in the pathogenesis of ischaemic stroke. Heterogeneity among the studies in the direction of association between individual SNPs and stroke suggests that the SNPs tested are in linkage disequilibrium with the causal allele(s).


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/genetics , Brain Ischemia/genetics , Stroke/genetics , Aged , Case-Control Studies , Cyclic Nucleotide Phosphodiesterases, Type 3 , Cyclic Nucleotide Phosphodiesterases, Type 4 , Female , Genetic Predisposition to Disease , Humans , Linkage Disequilibrium , Male , Middle Aged , Polymorphism, Single Nucleotide
4.
J Thromb Haemost ; 3(12): 2649-55, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16359503

ABSTRACT

OBJECTIVE: We aimed to determine whether adding clopidogrel to aspirin in patients at high risk of future cardiovascular events would suppress laboratory measures of the antiplatelet effects of aspirin; and have greater platelet inhibitory effects in patients with the least inhibition of platelets by aspirin. METHODS: We performed a randomized, double-blind, placebo-controlled, crossover trial, comparing clopidogrel 75 mg day(-1) versus placebo, in 36 aspirin-treated patients with symptomatic objectively confirmed peripheral arterial disease. RESULTS: The addition of clopidogrel to aspirin did not suppress platelet aggregation induced by arachidonic acid, urinary 11 dehydro thromboxane B2 concentrations, or soluble markers of platelet activation markers (P-selectin, CD40-ligand) and inflammation (high sensitivity serum C-reactive protein, interleukin-6). Clopidogrel significantly inhibited platelet aggregation induced by ADP (reduction 26.2%; 95% CI: 21.3-31.1%, P < 0.0001) and collagen (reduction 6.2%; 95% CI: 3.2-9.3%, P = 0.0003). The greatest inhibition of collagen-induced platelet aggregation by clopidogrel was seen in patients with the least inhibition of arachidonic acid induced aggregation by aspirin [lower tertile of arachidonic acid-induced platelet aggregation: 2.8% (95% CI: -0.8 to 6.3%) reduction in mean collagen-induced aggregation by clopidogrel; middle tertile: 4.0% (95% CI: 0.4-7.6%); upper tertile 12.6% (95% CI: 4.5-20.8%); P-value for interaction 0.01]. CONCLUSIONS: The greatest platelet inhibitory effect of clopidogrel occurs in patients with the least inhibition of arachidonic acid-induced platelet aggregation by aspirin. This raises the possibility that the clinical benefits of adding clopidogrel to aspirin may be greatest in patients whose platelets are least inhibited by aspirin. Confirmation in clinical outcome studies may allow these patients to be targeted with antiplatelet drugs that inhibit the ADP receptor, thereby overcoming the problem of laboratory aspirin resistance.


Subject(s)
Aspirin/administration & dosage , Peripheral Vascular Diseases/drug therapy , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Ticlopidine/analogs & derivatives , Adenosine Diphosphate , Aged , Arachidonic Acid , Aspirin/pharmacology , Biomarkers/blood , Clopidogrel , Collagen , Cross-Over Studies , Double-Blind Method , Drug Resistance , Drug Therapy, Combination , Female , Humans , Male , Middle Aged , Nephelometry and Turbidimetry , Peripheral Vascular Diseases/blood , Ticlopidine/administration & dosage , Ticlopidine/pharmacology
5.
Int J Pharm ; 300(1-2): 67-75, 2005 Aug 26.
Article in English | MEDLINE | ID: mdl-15979264

ABSTRACT

Previous work has shown that polyethylene glycol 400 (PEG 400) has an accelerating effect on gastrointestinal transit and a modulating influence on drug absorption in humans. The aim of this study was to assess the impact of various excipients, PEG 400, propylene glycol, d-alpha-tocopheryl-polyethylene glycol-1000 succinate (TPGS) and Labrasol on gastrointestinal transit and drug absorption in four beagle dogs using scintigraphy. Each dog received, on five separate occasions, water (control) or a dose of excipient equivalent to 1 g PEG 400, 2 g propylene glycol, 1 g TPGS or 2 g Labrasol dissolved in water and administered in the form of two capsules. The model drugs ampicillin (200mg) and antipyrine (100mg) were co-administered in the capsules. The capsule solutions were radiolabelled with technetium-99m to follow their transit using a dual-headed gamma camera, and blood samples were collected to determine drug pharmacokinetics. On a separate occasion, the drugs were dissolved in saline and given intravenously. The capsules rapidly disintegrated in the stomach liberating their liquid contents. The mean small intestinal transit times for the different treatments (control, PEG 400, propylene glycol, TPGS and Labarasol) were 183, 179, 195, 168 and 154 min, respectively. The corresponding mean absolute oral bioavailability figures were 36, 32, 39, 42 and 32% for ampicillin and 76, 74, 85, 73 and 74% for antipyrine, respectively. The transit and bioavailability data for the excipient treatments were not significantly different from the control. In summary, these excipients, at the doses administered, have limited influence on gastrointestinal transit and drug in beagle dogs.


Subject(s)
Excipients/pharmacology , Gastric Emptying/drug effects , Intestinal Absorption/drug effects , Ampicillin/pharmacokinetics , Animals , Antipyrine/pharmacokinetics , Biological Availability , Dogs , Female , Glycerides , Male , Organic Chemicals/pharmacology , Polyethylene Glycols/pharmacology , Propylene Glycol/pharmacology , Succinates/pharmacology , Vitamin E/analogs & derivatives , Vitamin E/pharmacology
6.
Stroke ; 36(6): 1123-7, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15879328

ABSTRACT

BACKGROUND AND PURPOSE: We aimed to determine whether A-13G or G79A polymorphisms of the protein Z gene that have been reported to be an important determinant of blood concentrations of protein Z are associated with risk of ischemic stroke in a broad range of stroke patients and controls. METHODS: We conducted a case control study of 151 hospital cases of first-ever ischemic stroke and 164 randomly selected community controls. Protein Z genotype was determined for the A-13G promoter polymorphism and the G79A intron F polymorphism, and plasma protein Z concentrations were measured during the first 7 days and at 3 to 6 months after the acute stroke event. RESULTS: Geometric mean concentrations of protein Z measured within 7 days of acute stroke were significantly higher in cases compared with controls (1.51 microg/mL versus 1.13 microg/mL; P<0.0001). Protein Z concentrations were highest among subjects with the A-13G AA genotype, intermediate among those with the AG genotype, and lowest among those with the GG genotype (1.39 microg/mL versus 1.05 microg/mL versus 0.76 microg/mL; P<0.0001); and highest among those with the G79A GG genotype, intermediate among those with the GA genotype, and lowest among those with the AA genotype (1.47 microg/mL versus 1.13 microg/mL versus 0.66 microg/mL; P<0.0001). The prevalence of A-13G and G79A genotypes was not significantly different between cases of ischemic stroke and controls. However, compared with the G79A GG genotype (reference), the odds of ischemic stroke was progressively lower for the heterozygote GA (odds ratio [OR], 0.83; 95% CI, 0.52 to 1.33) and the homozygote AA genotype (OR, 0.63; 95% CI, 0.20 to 1.98). A pooled analysis showed that compared with the G79A GG genotype (reference), the odds of ischemic stroke was progressively lower for the heterozygote GA (OR, 0.78; 95% CI, 0.57 to 1.07) and the homozygote AA genotype (OR, 0.31; 95% CI, 0.14 to 0.69). CONCLUSIONS: The consistency of the association between protein Z genotypes, blood concentrations of protein Z, and ischemic stroke, determined using 2 different methods that have different sources of bias strengthens the evidence that increased blood concentrations of protein Z concentrations are associated causally with an increased risk of ischemic stroke.


Subject(s)
Blood Proteins/genetics , Ischemia/genetics , Polymorphism, Genetic , Stroke/genetics , Aged , Blood Proteins/biosynthesis , Case-Control Studies , Female , Genotype , Humans , Introns , Male , Middle Aged , Promoter Regions, Genetic , Thrombosis/genetics , Time Factors
7.
J Thromb Haemost ; 1(5): 963-70, 2003 May.
Article in English | MEDLINE | ID: mdl-12871362

ABSTRACT

Several polymorphisms of integrin alpha2beta1 and glycoprotein (GP) VI that may modify platelet-collagen interactions or subsequent signaling have been described. We conducted a case-control study involving 180 stroke patients and 172 controls to determine whether the alpha2 C807T and GPVI Q317L polymorphisms were associated with an increased risk of ischemic stroke. We found no statistically significant differences in the distribution of alpha2 C807T and GPVI Q317L in patients and controls overall or after stratification by etiological subtype. The GPVI 317QQ genotype was found to be over-represented in a subgroup of patients >/=60 years compared to corresponding controls. However, this association did not remain significant after adjustment for other cardiovascular risk factors. Our results do not support a role for the integrin alpha2 C807T and GPVI Q317L polymorphisms in the development of first-ever ischemic stroke. However, larger studies are required to confirm this.


Subject(s)
Brain Ischemia/genetics , Integrin alpha2beta1/genetics , Platelet Membrane Glycoproteins/genetics , Polymorphism, Single Nucleotide , Stroke/genetics , Aged , Brain Ischemia/etiology , Case-Control Studies , Female , Gene Frequency , Genotype , Humans , Male , Middle Aged , Risk Factors , Stroke/etiology
8.
Neuroscience ; 112(1): 183-94, 2002.
Article in English | MEDLINE | ID: mdl-12044483

ABSTRACT

It has been recognized for some years that a prolonged Ca(2+) elevation that is predictive of impending cell death develops in cultured neurons following excitotoxic insult. In addition, neurons exhibit enhanced sensitivity to excitotoxic insult with increasing age in culture. However, little is known about the processes that selectively regulate the post-insult Ca(2+) elevation and therefore, it remains unclear whether it is associated specifically with age-dependent toxicity.Here, we tested the hypothesis that a group I metabotropic glutamate receptor antagonist selectively modulates the prolonged Ca(2+) elevation in direct association with its protective effects against excitotoxicity. Rat hippocampal cultures of two ages (8-9 and 21-28 days in vitro) were exposed to a 5-min glutamate insult (400 microM in younger and 10 microM in older cultures) sufficient to kill >50% of the neurons, and were treated with vehicle or the specific group I metabotropic glutamate receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), throughout and following the insult. Neuronal survival was quantified 24 h after insult. In parallel studies, neurons of similar age in culture were imaged ratiometrically with a confocal microscope during and for 60 min after the glutamate insult. A large post-insult Ca(2+) elevation was present in older but not most younger neurons. The N-methyl-D-aspartate receptor antagonist, MK-801, blocked the Ca(2+) elevation both during and following the insult. In contrast, AIDA blocked only the post-insult prolonged Ca(2+) elevation in older neurons. Moreover, AIDA was neuroprotective in older but not younger cultures. From these results we suggest that the post-insult Ca(2+) elevation is regulated differently from the Ca(2+) elevation during glutamate insult and is modulated by group I metabotropic glutamate receptors. Further, the prolonged Ca(2+) elevation appears to be directly linked to an age-dependent component of vulnerability.


Subject(s)
Calcium/metabolism , Cellular Senescence/physiology , Glutamic Acid/pharmacology , Hippocampus/metabolism , Neurotoxins/pharmacology , Receptors, AMPA/physiology , Animals , Cells, Cultured , Excitatory Amino Acid Antagonists/pharmacology , Female , Hippocampus/cytology , Indans/pharmacology , Neuroprotective Agents/pharmacology , Pregnancy , Rats , Rats, Inbred F344 , Rats, Sprague-Dawley , Receptors, AMPA/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/physiology , Time Factors
9.
Ecotoxicology ; 10(4): 217-22, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11501431

ABSTRACT

Genetics of ecotoxicology has recently emerged as a priority research field. The advent of polymerase chain reaction and molecular population genetics has made it possible to examine the genetics in even the smallest individuals. Although a potentially powerful technique, current approaches oversimplify the relationship of change in gene frequency to contaminant exposure. Many of these approaches cannot control for random correlation or accessory abiotic factors that impinge on the system tested. Indeed, the gestalt approaches of laboratory exposure or natural field experiments may ignore significant genome-level interactions that are important within a given system. At the very least, these approaches would benefit by a biogeographic survey of genetic variation to understand geographic microevolutionary patterns, or phylogeography, within a species to reduce spurious correlations and erroneous conclusions. Other single locus approaches can be chosen to enhance this approach if genetic/environmental interactions have been characterized for laboratory populations or for other model systems.


Subject(s)
Biological Evolution , Ecology , Genetics, Population , Toxicology/trends , Animals , Genetic Markers , Genetic Variation , Geography , Humans , Polymerase Chain Reaction
10.
Evolution ; 55(6): 1167-79, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11475052

ABSTRACT

The tidewater goby, Eucyclogobius newberryi, inhabits discrete, seasonally closed estuaries and lagoons along approximately 1500 km of California coastline. This species is euryhaline but has no explicit marine stage, yet population extirpation and recolonization data suggest tidewater gobies disperse intermittently via the sea. Analyses of mitochondrial control region and cytochrome b sequences demonstrate a deep evolutionary bifurcation in the vicinity of Los Angeles that separates southern California populations from all more northerly populations. Shallower phylogeographic breaks, in the vicinities of Seacliff, Point Buchon, Big Sur, and Point Arena segregate the northerly populations into five groups in three geographic clusters: the Point Conception and Ventura groups between Los Angeles and Point Buchon, a lone Estero Bay group from central California, and San Francisco and Cape Mendocino groups from northern California. The phylogenetic relationships between and patterns of molecular diversity within the six groups are consistent with repeated, and sometimes rapid, northward and southward range expansions out of central California caused by Quaternary climate change. Plio-Pleistocene tectonism, Quaternary coastal geography and hydrography, and historical human activities probably also influenced the modern geographic and genetic structure of E. newberryi. The phylogeography of E. newberryi is concordant with phylogeographic patterns in several other coastal California taxa, suggesting common extrinsic factors have had similar effects on different species. However, there is no evidence of a phylogeographic break coincident with a biogeographic boundary at Point Conception.


Subject(s)
DNA, Mitochondrial/genetics , Fishes/genetics , Phylogeny , Animals , California , Creatine Kinase/genetics , Fishes/classification , Genetic Variation , Geography , Haplotypes/genetics , Multivariate Analysis , Seawater
11.
J Mol Endocrinol ; 25(1): 17-34, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10915215

ABSTRACT

Regulating gene expression from DNA to protein is a complex multistage process with multiple control mechanisms. Transcriptional regulation has been considered the major control point of protein production in eukaryotic cells; however, there is growing evidence of pivotal posttranscriptional regulation for many genes. This has prompted extensive investigations to elucidate the mechanisms controlling RNA processing, mRNA nuclear export and localization, mRNA stability and turnover, in addition to translational rates and posttranslational events. The regulation of mRNA stability has emerged as a critical control step in determining the cellular mRNA level, with individual mRNAs displaying a wide range of stability that has been linked to discrete sequence elements and specific RNA-protein interactions. This review will focus on current knowledge of the determinants of mRNA stability and RNA-protein interactions in the pituitary. This field is rapidly expanding with the identification of regulated cis-acting stability-modifying elements within many mRNAs, and the cloning and characterization of trans-acting proteins that specifically bind to their cognate cis elements. We will present evidence for regulation of multiple pituitary genes at the level of mRNA stability and some examples of the emerging data characterizing RNA-protein interactions.


Subject(s)
Pituitary Gland/metabolism , Proteins/metabolism , RNA, Messenger/metabolism , Animals , Base Sequence , Carrier Proteins/metabolism , Cytoplasm/metabolism , Exoribonucleases/metabolism , Gene Expression Regulation , Humans , Models, Biological , Pituitary Hormones/metabolism , Poly A/metabolism , Poly(A)-Binding Proteins , Proteins/genetics , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism
12.
Evol Dev ; 2(6): 340-7, 2000.
Article in English | MEDLINE | ID: mdl-11256378

ABSTRACT

Whether the serial features found in some molluscs are ancestral or derived is considered controversial. Here, in situ hybridization and antibody studies show iterated engrailed-gene expression in transverse rows of ectodermal cells bounding plate field development and spicule formation in the chiton, Lepidochitona cavema, as well as in cells surrounding the valves and in the early development of the shell hinge in the clam, Transennella tantilla. Ectodermal expression of engrailed is associated with skeletogenesis across a range of bilaterian phyla, suggesting a single evolutionary origin of invertebrate skeletons. The shared ancestry of bilaterian-invertebrate skeletons may help explain the sudden appearance of shelly fossils in the Cambrian. Our interpretation departs from the consideration of canonical metameres or segments as units of evolutionary analysis. In this interpretation, the shared ancestry of engrailed-gene function in the terminal/posterior addition of serially repeated elements during development explains the iterative expression of engrailed genes in a range of metazoan body plans.


Subject(s)
Biological Evolution , Homeodomain Proteins/genetics , Mollusca/growth & development , Mollusca/genetics , Transcription Factors , Animals , Mollusca/anatomy & histology , Polymerase Chain Reaction
13.
Biotechniques ; 27(5): 1032-9, 1042, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10572651

ABSTRACT

Considerable interest has recently focused on defining the mechanisms involved in the regulation of gene expression at the level of mRNA stability and translational efficiency. However, the assays used to directly investigate interactions between RNA and cytoplasmic proteins have been difficult to establish, and methods are not widely available. Here, we describe a robust method for RNA electrophoretic mobility shift and UV cross-linking assays that allows rapid detection of cytoplasmic RNA-protein interactions. For added convenience to new investigators, these assays use mini-gels with an electrophoresis time of 15-20 min, enabling a high throughput of samples. The method works successfully with many different probes and cytoplasmic extracts from a variety of cell lines. Furthermore, we provide a system to optimize characterization of the RNA-protein complex and troubleshoot most assay difficulties.


Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , RNA, Messenger/analysis , RNA-Binding Proteins/analysis , Animals , Breast Neoplasms , Cross-Linking Reagents , ErbB Receptors/genetics , Ferritins/genetics , Gene Expression Regulation, Neoplastic , Heparin/metabolism , Humans , Ribonuclease T1/metabolism , Thyrotropin/genetics , Tumor Cells, Cultured , Ultraviolet Rays
14.
Endocrinology ; 139(3): 1093-100, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9492042

ABSTRACT

Thyroid hormone (T3) negatively regulates TSH beta-subunit (TSHbeta) messenger RNA (mRNA) gene expression in whole rat pituitary, in part at the level of mRNA stability. However, the regulation of TSHbeta mRNA turnover by T3 in pure populations of thyrotropes and in other species is unknown. To further investigate this, we used murine thyrotropic TtT97 tumor cells. Using primary cultures of TtT97 cells, T3 down-regulated TSHbeta mRNA to approximately 35% of the control level by 8 h. Actinomycin D chase revealed that T3 destabilized TSHbeta mRNA, reducing the half-life from approximately 24 to 7 h, and was accompanied by a decrease in TSHbeta mRNA size. Ribonuclease H analysis revealed that this T3-induced decrease in size was due to a shortening of poly(A) tail from approximately 160 to approximately 30 nucleotides and was specific for TSHbeta mRNA. Cycloheximide mimicked the poly(A) tail effect observed with T3. In the absence of T3, actinomycin D deadenylated TSHbeta mRNA without inducing rapid decay. We conclude that T3 reduces the steady state half-life of TSHbeta mRNA in murine TtT97 thyrotropic tumor cells accompanied by a reduction in poly(A) tail length. However, in the absence of T3, deadenylation alone is not sufficient to induce TSHbeta mRNA decay. Together with the high degree of sequence conservation in the 3'-untranslated region of murine and rat TSHbeta mRNA sequences and the similarities of the T3 effect, these data provide the first evidence for a highly conserved posttranscriptional mechanism operative across species. We propose a model in which T3 coordinately regulates shortening of the poly(A) tail and the activity of a transacting RNA-binding protein and/or an exonuclease to accelerate TSHbeta mRNA turnover.


Subject(s)
Gene Expression Regulation/drug effects , RNA, Messenger/metabolism , Thyroid Hormones/pharmacology , Thyroid Neoplasms/metabolism , Thyrotropin/genetics , Animals , Cells, Cultured , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Mice , Poly A/metabolism , Rats , Species Specificity
15.
Mol Biol Evol ; 14(8): 867-74, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9254925

ABSTRACT

Numerous complete mitochondrial DNA sequences have been determined for species within two arthropod groups, insects and crustaceans, but there are none for a third, the chelicerates. Most mitochondrial gene arrangements reported for crustaceans and insect species are identical or nearly identical to that of Drosophila yakuba. Sequences across 36 of the gene boundaries in the mitochondrial DNA (mtDNA) of a representative chelicerate. Limulus polyphemus L., also reveal an arrangement like that of Drosophila yakuba. Only the position of the tRNA(LEU)(UUR) gene differs; in Limulus it is between the genes for tRNA(LEU)(CUN) and ND1. This positioning is also found in onychophorans, mollusks, and annelids, but not in insects and crustaceans, and indicates that tRNA(LEU)(CUN)-tRNA(LEU)(UUR)-ND1 was the ancestral gene arrangement for these groups, as suggested earlier. There are no differences in the relative arrangements of protein-coding and ribosomal RNA genes between Limulus and Drosophila, and none have been observed within arthropods. The high degree of similarity of mitochondrial gene arrangements within arthropods is striking, since some taxa last shared a common ancestor before the Cambrian, and contrasts with the extensive mtDNA rearrangements occasionally observed within some other metazoan phyla (e.g., mollusks and nematodes).


Subject(s)
Arthropods/classification , DNA, Mitochondrial/genetics , Horseshoe Crabs/genetics , Amino Acid Sequence , Animals , Arthropods/genetics , Base Composition , Base Sequence , Drosophila/genetics , Genes , Genes, Regulator , Molecular Sequence Data , Nucleic Acid Conformation , Phylogeny , Proteins/genetics , RNA, Ribosomal/genetics , RNA, Transfer/genetics , Sequence Alignment , Species Specificity
16.
Mol Mar Biol Biotechnol ; 6(2): 141-3, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9200840

ABSTRACT

We report the first polymerase chain reaction (PCR) amplification of the complete mitochondrial genomes of a nemertean and a sipunculan worm in one piece using a recently published two-polymerase protocol for long and accurate DNA amplification. Successful amplification was achieved from nanogram quantities of both purified mitochondrial DNA (nemertean) and crude total DNA (sipunculan). This technique allows the rapid generation of sufficient quantities of entire mitochondrial DNAs for cloning and restriction fragment length polymorphism (RFLP) analyses, and thus will facilitate comparative studies of metazoan mitochondrial genomes.


Subject(s)
DNA, Mitochondrial/genetics , Invertebrates/genetics , Polymerase Chain Reaction/methods , Animals , Species Specificity
17.
Immunol Cell Biol ; 73(1): 23-32, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7768541

ABSTRACT

Stress proteins (SP) are major immunogens in a number of microbial infections and have been implicated in some autoimmune diseases. The aetiology of sarcoidosis, a non-caseating granulomatous disease, remains unknown, but mycobacteria as well as autoimmunity have been considered. In the present study, patients diagnosed with sarcoidosis and other interstitial lung diseases (ILD), as well as healthy volunteers were studied to determine: (i) the level of expression of SP in alveolar macrophages and blood monocytes; (ii) the serum levels of antibodies specific for mycobacterial SP65 and SP70; and (iii) the reactivity of peripheral blood and alveolar lymphocytes to mycobacterial SP65. Our results suggest that SP are expressed constitutively at high levels in alveolar macrophages, retrieved by bronchoalveolar lavage, from all individuals regardless of health status. In contrast, freshly isolated blood monocytes express low levels of SP, which are, however, readily upregulated following exposure to IFN-gamma and TNF-alpha. Lymphocyte reactivity and presence of antibodies against mycobacterial SP may reflect the current state of in vivo inflammation rather than the cause of inflammation.


Subject(s)
Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/immunology , Lung Diseases, Interstitial/immunology , Sarcoidosis, Pulmonary/immunology , Antibodies, Monoclonal/immunology , Bronchoalveolar Lavage Fluid/cytology , Cells, Cultured , Humans , Immunoblotting , Immunoenzyme Techniques , Interferon-gamma/analysis , Interleukin-2/analysis , Lymphocyte Activation/immunology , T-Lymphocytes/immunology
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